SIP online sharing

dPBL Package 2

2008-01-25T01:35:00.000+08:00

Problem Statement

There are outbreaks of viral, fungal and protozoa diseases among platoons of army soldiers in Indonesia. Soldiers reported sick after 2 weeks of jungle warfare training. It is of concern to the ministry that there are also sporadic reports of avian flu in the nearby villages. In view of these outbreaks, you have been tasked to conduct a pre-mission briefing with blogs and poster to educate future batches of soldiers.
Your group needs to keep a blog containing information about a variety of pathogens that cause problems during jungle training so that the soldiers are made aware of the dangers and could take the necessary precautions.

Indonesia -

located in South East Asia

experience tropical climates year-long (warm and humid + rainy seasons)

Army conditions -

jungle (may be warm and humid or damp pending weather changes)

warfare (exposed to soil, air, water - living conditions for pathogens)

soldiers (army of soldiers: many people; cluster)

exposure to wildlife animals including birds (avian)

harsh living conditions with improper sanitation

shortage of food/water (may lead to consumption of contaminated food/water)

Possible Protozoa Agents

2008-01-24T17:54:00.000+08:00

Entamoba

Transmission
Ingestion of cysts that are transmitted primarily by the fecal-oral route in contaminated food or water.

Pathogenesis
The ingested cysts differentiate into trophozoites in the ileum but tend to colonize the cecum and colon. Trophozoites invade the colonic epithelium and secrete enzymes that cause localized necrosis. Little inflammation occurs at the site. As the lesion reaches the muscularis layer, a typical flasked-shaped ulcer forms that can undermine and destroy large areas of the intestinal epithelium. Progression into the submucosa leads to invasion of the portal circulation by the trophozoites.

Clinical Syndrome
Dysentery
Lower abdominal discomfort
Flatulence
Tenesmus
Diarrhea
Weight loss
Fatigue
Amebic abscess of liver
Asymptomatic infections

Laboratory Diagnosis
Detecting trophozoites in diarrheal stools or cysts formed in stools.

Epidemiology
Infection occurs worldwide but occurs most frequently in tropical countires, especially in areas with poor sanitation. It is widely prevalent in male homosexuals.

Treatment
Metronidazole or tinidazole

Prevention and Control
Avoiding fecal contamination of food and water and observing good personal hygiene. Purification of water and avoid using human waste to fertilize crops.

Giardia

Transmission
Ingestion of cysts that are transmitted primarily by the fecal-oral route in contaminated food or water.

Pathogenesis
Encystation takes place in the duodenum, where the trophozoites attaches to the gut wall but does not invade. The trophozoites causes inflammation of the duodenal mucosa, leading to malabsorption of protein and fat.

Clinical Syndrome
Watery, foul-smelling diarrhea
Nausea
Anorexia
Flatulence
Abdominal cramps (week to months)

Laboratory Diagnosis
Detecting trophozoites or cysts in diarrheal stools or cysts formed in stools. ELISA test detects a Giardia cyst wall antigen.

Epidemiology
It occurs worldwide. Half of those infected excrete cysts in the stool, which then contaminates water sources. Giardia infection is common in homosexual males, children in day-care centres and patients in mental hospitals.

Treatment
Metronidazole or quinacrine hydrochloride.

Prevention and Control
Drinking boiled, filtered or iodine-treated water in endemic areas and while hiking. No prophylactic drug or vaccine is available.

Cryptosporidium

Transmission
Acquired by fecal-oral transmission of oocysts from either human or animal sources.

Pathogenesis
The oocysts excyst in the small intestine, where the trophozoites attach to the gut wall. Invasion does not occur. The jejunum is the site most heavily infested. The pathogenesis of diarrhea is unknown.

Clinical Syndrome
Watery, nonbloody diarrhea causing large fluid loss and malnutrition

Laboratory Diagnosis
Finding oocysts in fecal smears when using a modified Kiyoun acid-fast stain.

Epidemiology
Cryptosporidia cause diarrhea worldwide. Large outbreaks of diarrhea caused by cryptosporidia in several cities in the United States are attributed to inadequate purification of drinking water.

Treatment
Paromomycin, Nitazoxanide for children aged 1 to 11 years old.

Prevention and Control
Purification of the water supply, including filtration to remove the cysts, which are resistant to the chlorine used for disinfection, can prevent cryptosporidiosis.

Plasmodium

Transmission
Anopheles mosquito

Pathogenesis
Red blood cells are destroyed by the release of merozoites and by the action of the spleen to first sequester the infected red cells and then to lyse them.

Clinical Syndrome
Sudden fever
Chills
Headache
Myalgias
Arthralgias
Nausea
Vomiting
Abdominal pain
Splenomegaly
Anemia
Hepatomegaly in 1/3 of infections

Laboratory Diagnosis
Microscopic examination of thick and thin Giemsa-stained smears. PCR test for Plasmodium nucleic acids. ELISA test for a protein specific P. Falciparium.

Epidemiology
More than 200 million people worldwide have malaria, and more than 1 million die of it each year, making it the most common lethal infectious diseas. IT occurs primarily in tropical subtropical areas, especially in Asia, Africa and Central and South America. Malaria in the United States is seen in Americans who travel to areas of endemic infection without adequate chemoprophylaxis and in immigrants from areas of endemic infection. It is not endemic in the United States. Certain regions in SEA, South America, and east Africa are particularly affect by chloroquine-resistant strains of P. falciparium. People who have lived or traveled in areas where malaria occurs should seek medical attention for febrile illnesses up to 3 years after leaving the malarious area.

Treatment
Chloroquine, Malarone and Mefloquine.

Prevention and Control
Chemoprophylaxis consisting of mefloquine or doxycyline for travelers going to areas where chloroquine-resistant P. falciparium is endemic. Choloroquine used for travelers to areas where other plasmodia are found.

Images

http://www.google.com/ > Images > entamoeba

http://www.google.com/ > Images > Giardia

http://www.google.com/ > Images > Cryptosporidium

http://www.google.com/ > Images > Plasmodium

Desmond Heng

0503179D

TG02

Possible Viral Agents Part 3

2008-01-24T10:41:00.000+08:00

1) Adenoviruses- Serotypes 3, 4, 7
What are adenoviruses?
Adenoviruses can replicate and produce disease in the respiratory, gastrointestinal and urinary tracts and in the eye. Many adenovirus infections are subclinical and may persist in the host for months.

Mode of transmission:

Direct contac
Respiratory droplet
Fecal-oral route

Pathogenesis: Adenoviruses infect and replicate in epithelial cells of the respiratory and gastrointestinal tracts, urinary bladder and liver. They usually do not spread beyond the regional lymph nodes.

Clinical syndrome:

Respiratory diseases: cough, nasal congestion, fever and sore throat
Eye infections: acute conjunctivitis
Gastrointestinal disease: gastroenteritis

Laboratory diagnosis:
Polymerase Chain Reaction (PCR) assays in which primers from a conserved viral sequence can detect all serotypes of adenovirus infections in tissue samples or body fluids.
Serological methods such as the Complement Fixation (CF) test can be used to detect complement-fixing antibodies to adenovirus group antigens.

Epidemiology: Adenoviruses exist in all parts of the world. They are present year-round and usually do not cause community outbreaks of disease. Respiratory disease due to types 3, 4 and 7 is common among military recruits.

Treatment: No specific treatment

Prevention and control:

Careful hand washing
Disinfect environment surfaces with sodium hypochlorite
Use of paper towels

2) Hepatitis viruses- Hepatitis A virus (HAV)
What are HAV?
HAV is a distinct member of the picornavirus family.

Mode of transmission:

Fecal-oral route
Close contact

Pathogenesis: The virus replicates in the hepatocytes and interferes with normal liver function. The individual’s immune system is then activated to produce a specific reaction to combat the infectious agent. As a result, the liver becomes inflamed.

Clinical syndrome:

Jaundice
Fever
Fatigue
Gastrointestinal symptoms: abdominal pain, loss of appetite, nausea, vomiting and vomiting

Laboratory diagnosis:
Immune electron microscopy is used for the detection of HAV in stool and liver preparations.
Sensitive serological assays such as ELISA and PCR methods are used to measure specific HAV antibodies.

Epidemiology: HAV is widespread throughout the world. Outbreaks of type A hepatitis are common in families, institutions, summer camps, day care center, neonatal intensive care units and among military troops.

Treatment: Viral vaccines and protective immune globulin preparations against HAV

Prevention and control:

Hands washing after using the bathroom, changing a diaper, and before preparing and eating food.
Use own personal products

3) Poxviruses- Monkeypox virus
What is monkey virus?
Monkeypox virus is a species of Orthopoxvirus. The disease was first recognized in captive monkeys in 1958.

Mode of transmission: Direct contact with primary hosts (eg. rodents. Monkeys)

Pathogenesis:Monkeypox virus replicates in lymphoid tissue and localizes in mononuclear phagocytic cells. It is then release into the bloodstream, and then localizes again in skin cells.

Clinical syndrome: Similar to that of smallpox with the exception of enlargement of the cervical and inguina lymph nodes.

Laboratory diagnosis:
Direct examination of clinical materials under electron microscope is used for rapid identification of virus particles.
Antibody assays such as ELISA or RIA can be used as confirmatory tests.

Epidemiology: The disease is a rare zoonosis that has been detected only in remote villages in tropical rain forests.

Treatment: Vaccination with vaccinia

Prevention and control: Vaccinia vaccination is recommended for laboratory workers who directly handle cultures or animals infected with monkeypox virus.

4) Paramyxoviruses- Mumps virus
What is Mumps virus?
Mumps virus mostly causes a mild childhood disease, but in adults complications including meningitis and orchitis are fairly common. More than one-third of all mumps infections are asymptomatic.

Mode of transmission:

Direct contact
Air-borne droplets

Pathogenesis: Primary replication occurs in nasal or upper respiratory tract epithelial cells. Viremia then disseminates the virus to the salivary glands and other major organ systems.

Clinical syndrome:

Swell and pain of the paratid glands
Fever
Headache
Loss of appetite

Laboratory diagnosis:
Immunofluorescence using mumps-specific antiserum can detect mumps virus antigens as early as 2-3 days after inoculation of cell cultures in shell vials.
ELISA is useful because it can detect mumps-specific IgM antibody or mumps-specific IgG antibody.

Epidemiology: Cases appear throughout the year in hot climates. Epidemics may occur in army camps.

Treatment: No specific therapy

Prevention and control: Immunization with attenuated live mumps virus vaccine

5) Herpes Simplex Viruses- Epstein-Barr Virus (EBV)
What is EBV?
EBV is the causative agent of acute infections mononucleosis and lymphoproliferative disorders in immunodeficient individuals.

Mode of transmission:

Respiratory secretions
Infected saliva

Pathogenesis: Viral replication occurs in epithelial cells of the pharynx and salivary glands. Infected B cells spread the infection from the oropharynx throughout the body. Following primary infections, the virus persists in a latent form in the B lymphocytes of the host. Periodic reactivation of the virus is associated with shedding of virus in saliva.

Clinical syndrome:

Infectious mononucleosis: headache, fever, malaise, fatigue and sore throat
Oral hairy leukemia: wart like growth on the tongue
Lymphoma

Laboratory diagnosis:
Nucleic acid hybridization is the most sensitive means of detecting EBV in patient materials.
Common serologic procedures for detection of EBV antibodies include ELISA tests, immunnoblot assays and indirect immunofluorescence tests using EBV-positive lymphoid cells.

Epidemiology: EBV is common in all parts of the world. In industrialized nations, more than 50% of EBV infections are delayed until late adolescence and young adulthood.

Treatment:
Acycloir reduces EBV shedding from the oropharynx during the period of drug administration.
Adoptive transfer of EBV-reactive T cells shows results of acceptable treatment for EBV-related lymphoproliferative disease.

Prevention and control: No EBV vaccine available

Pictures:
1) Adenovirus- under ELectron Microscopy

http://www.yahoo.com> image search> adenovirus

2) Hepatitis A virus (HAV)- under Electron Mircoscopy

http://www.yahoo.com> image search> hepatitis A virus

3) Monkeypox virus

http://www.yahoo.com> image search> monkeypox virus

4) Mumps virus

http://www.yahoo.com> image search> mumps virus

5) Epstein-Barr virus (EBV)- in the nucleus of a mammalian cell

http://www.yahoo.com> image search> epstein-barr virus

References:
1) Geo.F.Brooks, Janet S.Butel, Stephen A.Morse. (2004). Medical Microbiology. Asia: The McGraw-hill Companies.

2) Centers for Disease Control and Prevention. Viral Hepatitis A. (2007). Retrieved 22nd January 2008, from http://www.cdc.gov/ncidod/diseases/hepatitis/a/fact.htm

3) Stanford Education Group. Monkeypox virus. (2000). Retrieved 22nd January 2008, from http://www.stanford.edu/group/virus/pox/2000/monkeypox_virus.html

4) Medicine Net. Mumps. (2007). Retrieved 22nd January 2006, from http://www.medicinenet.com/mumps/article.htm

Tham Wan Jin June
TG02
0505073G

Possible Fungal Agents

2008-01-24T10:07:00.000+08:00

1. Dermatophytes (Epidermophyton, Microsporum, and Trichophyton species)
Transmission: Direct contact with the skin scale
Pathogenesis: Fungi only grow in the superficial keratinized layer of the skin. Presences of lesions are due to the inflammatory response to the fungi. Frequency of infection is enhanced by moisture and warm.
Clinical Symptoms: May present as asymptomatic, usually mild itching lesion with a scaling, inflammation usually manifest as a vesicular or bullous disease.
Laboratory Diagnosis: Skin scale is examined microscopically in a KOH preparation for the presence of hyphae. It is identified by the appearance of its mycelium and it asexual spore on SDA plates.
Treatment: Topical anti-fungal agents such as miconazole, clotrimazole or tolnaftate are used
Prevention: Practise personal hygiene such as chamging sock frequently, wiping area between toes to keep it dry.

2. Histoplasma capsulatum
Transmission: Inhalation of airborne sexual spores. Mould grows preferentially in soil enriched with bird dropping. It also grows well in a warm and moist environment, prevalent in Americas, India and southeastern Asia.
Pathogenesis: Microcondia (spores) enter the lungs and differentiate into yeast cells. The yeast cells are ingested by the alveolar macrophages and multiply within them.
Clinical Symptoms: Non-specific respiratory symptoms such as cough and flu. Enlargement in spleen, adrenal can be seen in severe infection.
Laboratory Diagnosis: Sputum can be examined microscopically and cultured on SDA plate. Presence of tuberculate chylamydospores in culture at 25 degree Celsius is diagnostic.
Treatment: Amphotericin B for disseminated disease; itraconazole for pulmonary disease
Prevention: No vaccine is available

3. Coccidioides Immitis
Transmission: Inhalation of airborne arthrospores. It grows well in the soil of arid regions.
Pathogenesis: Arthrospores differentiate into spherules in the lungs. Spherules rupture, releasing endospores that form new spherules, spreading the infection within the body.
Clinical Symptoms: Fever, mild respiratory distress, rashes
Laboratory Diagnosis: Sputum should be examined microscopically for spherules and cultured on SDA plate.
Treatment: Amphotericin B or itraconazole for disseminated disease
Prevention: No vaccine is available

4. Crytococcus neoformans
Transmission: Inhalation of airborne yeast cells. This heavily encapsulated yeast grows well in soil enriched by bird dropping (especially pigeon droppings).
Pathogenesis: Yeast cells spread via the bloodstream to the meninges. Reduced cell-mediated immunity pre-disposes to severe disease, cryptococcal meningitis occur in immunocompetent people.
Clinical Symptoms: May present as asymptomatic, usually pneumonia-like symptoms such as flu and fever, unusual sweating at night, skin rash, chest pain, confusion, nausea
Laboratory Diagnosis: Gram stain is not preferred due to reliable result. Stain such as methenamine-silver, periodic acid-Schiff will allow the organism to be visualized. Colonies are presented as mucoid due to presence of large amount of capsular polysaccharide. Serologic test such as cryptococcal antigen test can be used to determined organism presence.
Treatment: Combined treatment with amphotericin B and flucytosine is used in disseminated disease and meningitis.
Prevention: No known specific mean of prevention.

5. Aspergillus fumigatus
Transmission: Inhalation of airborne spore (conidia)
Pathogenesis: It exists as an opportunistic pathogen, invasive in immunocompetent patient. The organism invades the blood vessels, causing thrombosis and infarction. “Fungal ball” may be developed in tuberculosis patient and allergic brochopulomonary asperigillosis may developed in allergic patient.
Clinical Symptoms: Fever, weakness, chest pain, aches, blood in urine.
Laboratory Diagnosis: Examined microscopically for invasive septate hyphae. Form characteristic mycelium when cultured on SDA plate.
Treatment: Amphotericin B for invasive asperigillosis. Lesion such as fungal ball can be surgically removed. Steroid therapy is recommended for allergic brochopulomonary asperigillosis
Prevention: No vaccine or prophylactic drug is available

Reference:
1) WARREN LEVINSON, MD, PhD (2006) REVIEW OF Medical Microbiology and Immunology. NINTH EDITION; McGraw-Hill company
2)wikipedia.org> search> organism name
3) MMIC lectures notes

Yeo Ching Wei
0503288C

Possible Viral Agents Part 2

2008-01-24T00:48:00.000+08:00

Photos/Images

Poliomyelitis Viruses

A microscopic image shows poliomyelitis viruses, which enter the body through the nose and mouth and destroy nerve cells by multiplying rapidly inside of them. It can cause permanent paralysis. An effective vaccine have developed and poliomyelitis has been nearly eliminated in developed countries.

Taken from http://encarta.msn.com/media_461522427_761572271_-1_1/Poliomyelitis_Viruses.html

Hepatitis A

Hepatitis A magnified 225,000 times

Taken from http://www.healthofchildren.com/G-H/Hepatitis-A.html

Hepatitis E

Hepatitis E
Taken from http://images.google.com.sg/imgres?imgurl=http://patric.vbi.vt.edu/organism/overview/images/7.jpg&imgrefurl=http://patric.vbi.vt.edu/organism/overview.php%3ForganismId%3D7&h=288&w=430&sz=35&hl=en&start=3&tbnid=7K3Rf5je4gVGCM:&tbnh=84&tbnw=126&prev=/images%3Fq%3DHepatitis%2BE%26gbv%3D2%26svnum%3D10%26hl%3Den

Yellow Fever Virus
An electron micrograph of Yellow Fever Virus virions. Virions are spheroidal, uniform in shape and are 40-60nm in diameter. The name "Yellow Fever" is due to the ensuing jaundice that affects some patients. The vector is the Aedes aegypti or Haemagogus spp. mosquito

Taken from http://www.lib.uiowa.edu/hardin/md/cdc/2176.html

Schematic illustration of the relation between climate and the transmission of tick-borne diseases in humans. In this study, the different direct and indirect climate-dependent interactions, here shown within the yellow field, have been treated as a black box.

Taken from http://www.ecologyandsociety.org/vol2/iss1/art5/

References

1) Geo, F.B., Janet, S.B., Stephen, A.M. (2001). Medical Microbiology, Twenty Third Edition: The McGraw-Hill Companies

2) cdc.gov>search>virus name

3) lecture notes from medical microbiology

Lin Juexiu
0503151C
TG02

Possible Viral Agents Part 1

2008-01-23T23:06:00.000+08:00

Please click to enlarge image

Rabies virus

http://www.yahoo.com>image search>Rabies virus

Influenza virus

http://www.yahoo.com/ >image search>influenza virus

Dengue virus

http://www.yahoo.com> image search> Dengue virus

Chikungunya virus

http://www.yahoo.com/>image search>Chikungunya virus

Arbovirus

http://www.yahoo.com> image search>Arbovirus

References

1) Geo, F.B., Janet, S.B., Stephen, A.M. (2001). Medical Microbiology, Twenty Third Edition: The McGraw-Hill Companies.

2) Medical Microbiology Lecture Notes

Done by Sim kai lin (0503211E)

Possible Protozoa Agents

2008-01-23T21:06:00.000+08:00

1. Entamoeba histolytica
Transmission: Contaminated food/water, person to person, fecal exposure during sexual contact, rodents (vectors for transmitting E. histolytica cysts)
Pathogenesis: Following ingestion, the cysts pass through the stomach, where exposure to gastric acid stimulates release of the pathogenic trophozoite in the duodenum. The trophozoites divide and produce extensive local necrosis in the large intestine. The basis of tissue destruction may be attributed to the production of a cytotoxin. Necrosis requires direct contact with the amoeba, so lysosomal enzymes (phospholipase A2) may be important.
Epidemiology: Worldwide distribution. Its incidence is highest in tropical and subtropical regions that have poor sanitation and contaminated water.
Clinical syndromes: Intestinal or extraintestinal amebiasis (symptoms: abdominal pain, cramping, colitis with diarrhea, bloody stools, hepatomegaly)
Lab diagnosis: Identification of E. histolytica trophozoites and cysts in stools and trophozoites in tissue under microscope, specific serologic tests, immunological tests for detection of fecal antigen and DNA-probe assays for E. histolytica nucleic acids.
Treatment: Treated with metronidazole followed by iodoquinol
Prevention and control: Introduction of adequate sanitation measures and education about the routes of transmission, water should be boiled and fruits/vegetables should be thoroughly cleaned before consumption

Picture of Entamoeba histolytica and its life cycle (http://www.google.com/ > image > Entamoeba histolytica)

2. Giardia lamblia
Transmission: Contaminated food/water, person to person, fecal-oral or oral-anal routes
Pathogenesis: Infection with G. lamblia is initiated by ingestion of cysts. Gastric acid stimulates excystation with release of trophozoites in the duodenum and jejunum, where organisms multiply by binary fission. Trophozoites can attach to intestinal villi by a prominent ventral sucking disk.
Epidemiology: Worldwide distribution
Clinical syndromes: Result in either asymptomatic carriage or symptomatic disease, ranging from mild diarrhea to a severe malabsorption syndrome (symptoms: watery diarrhea, abdominal cramps, flatulence)
Lab diagnosis: Stool specimens should be examined for cysts and trophozoites under microscopy, specific serologic and immunological tests.
Treatment: Quinacrine, metronidazole
Prevention and control: Avoidance of contaminated food/water and high-risk sexual behaviour, public health efforts to identify reservoir of infection and proper functioning filtration systems in water supplies
Picture of Giardia lamblia (http://www.google.com/ > image > Giardia lamblia)

3. Balantidium coli
Transmission: Swine and monkeys, fecal-oral route, contaminated water, person to person
Pathogenesis: Similar to amebiasis as organisms elaborate proteolytic and cytotoxic substances that mediate tissue invasion and intestinal ulceration
Epidemiology: Worldwide distribution
Clinical syndromes: Result in either asymptomatic carriage or symptomatic disease (symptoms: abdominal pain and tenderness, tenesmus, nausea, anorexia, watery stools with blood and pus, ulceration of intestinal mucosa)
Lab diagnosis: Microscopic examination of feces for trophozoites and cysts
Treatment: Tetracycline, iodoquinol and metronidazole
Prevention and control: Appropriate personal hygiene, maintenance of sanitary condtions and careful monitoring of pig feces

Picture of Balantidium coli (http://www.google.com/ > image > Balantidium coli)
4. Isospora belli
Transmission: Contaminated food/water, oral-anal sexual contact
Pathogenesis: Both sexual (gametogony) and asexual (schizogony) reproduction in the intestinal epithelium can occur. The end product of gametogenesis is the oocyst.
Epidemiology: Worldwide distribution
Clinical syndromes: May be asymptomatic carriers or gastrointestinal disease ranging from mild to severe. Disease most commonly mimics giardiasis, with a malabsorption syndrome characterized by loose, foul-smelling stools.
Lab diagnosis: Careful examination of concentrated stool sediment and special staining with either iodine or a modified acid-fast procedure will reveal the parasite.
Treatment: Trimethoprim sulfamethoxazole, combined pyrimethamine-sulfadiazine
Prevention and control: Maintain personal hygiene, high sanitary conditions and avoidance of oral-anal sexual contact

Picture of Isospora belli (http://www.google.com/ > image > Isospora belli)

5. Cryptosporidium parvum
Transmission: Zoonotic spread from animal reservoirs to humans, person to person, fecal-oral and oral-anal routes
Pathogenesis: Typical of coccidians. In contrast to Isospora, Cryptosporidium is found within the brush border of the intestinal epithelium. The coccidian attach to the surface of the cells and replicate by a process that involves schizogony.
Epidemiology: Worldwide distribution
Clinical syndromes: May be asymptomatic carriers or mild self-limiting enterocolitis characterized by watery diarrhea without blood
Lab diagnosis: Stool examination, modified zinc sulfate centrifugal flotation technique or by Sheather’s sugar flotation procedure, specimens may be stained with modified acid-fast method or by an indirect immunofluorescence assay
Treatment: No broadly effective therapy
Prevention and control: Due to the widespread distribution of organism in humans and animals, preventing infection is difficult. Maintain personal hygiene, high sanitary conditions and avoidance of oral-anal sexual contact.
Picture of life cycle of Cryptosporidium parvum (http://www.google.com/ > image > Cryptosporidium parvum)

6. Plasmodium species (P. vivax, P. falciparum, P. ovale, P. malariae)
Transmission: Via bite of female Anopheles mosquito vector
Pathogenesis: Infectious plasmodia sporozoites are introduced after a bite through the saliva of the Anopheles mosquito. The sporozoites are carried to the hepatocytes where asexual reproduction occurs. Some species (P. vivax and P. ovale) can establish a dormant hepatic phase in which hypnozoites do not divide. The hepatocytes eventually rupture, liberating merozoites which attach to specific RBC receptors and invade them, initiating the erythrocytic cycle. Asexual replication progresses through a series of stages (ring, trophozoite, schizont) that culminates in the rupture of the RBC, releasing more merozoites. Some merozoites also develop within RBCs into male and female gametocytes.
Epidemiology: Worldwide distribution depending on the type of species
Clinical syndromes: Fever, chills, rigors, muscle and joint pains, anaemia and convulsions. Severe malaria (usually caused by P. falciparum) may cause cerebral malaria, blackwater fever which could lead to coma, death.
Lab diagnosis: Thick and thin blood smears stained with Giemsa stain and observed under microscopy, rapid diagnostic kits
Treatment: Chloroquine, primaquine, mefloquine, quinine, doxycyline (however, there are increasing multidrug-resistant P. falciparum strains)
Prevention and control: Chemoprophylaxis and prompt eradication of infections, control of mosquito breeding and protection of individuals by screening, netting, protective clothing and insect repellents.

Picture of life cycle of Plasmodium species (http://www.google.com/ > image > Plasmodium)

7. Toxoplasma gondii
Transmission: Ingestion of improperly cooked meat from animals serving as intermediate hosts or infective oocysts from cat fecal contamination, transplacental infection
Pathogenesis: Organisms develop in the intestinal cells of the cat, as well as during an extraintestinal cycle with passage to the tissues via bloodstream. The organisms from the intestinal cycle are passed in cat feces and mature in the external environment within 3 to 4 days into infective oocysts. These oocysts can be ingested by mice and other animals (including huamsn) and produce acute and chronic infection of various tissues.
Clinical syndromes: May be asymptomatic carriers or symptomatic disease (symptoms: chills, fever, headaches, myalgia, lymphadenitis)
Lab diagnosis: Serologic tests, ELISA test for detecting IgM antibodies, biopsy specimens
Treatment: Depends on the nature of both the infectious process and the immunocompetence of the host
Prevention and control: Avoidance of consumption and handling of raw or undercooked meat and exposure to cat feces

Picture of Toxoplasma gondii (http://www.google.com/ > image > Toxoplasma gondii)

8. Leishmania species
Transmission: Via sandflies (Phlebotomus or Lutzomyia)
Pathogenesis: The hemoflagellates are flagellated insect-transmitted protozoa that infect blood and tissues. The diseases are distinguished by the ability of the organism to infect deep tissues (visceral leishmaniasis) or replicate only in cooler superficial tissues (cutaneous or mucocutaneous leishmaniasis). The reservoir hosts and geographical distribution differ for the 3 species. There are two morphological forms in the cells: promastigote (with an anterior flagellum) in the insect host, and amastigote (without flagella) in the vertebrate host.
Clinical syndromes: Visceral (fever, swelling of spleen and liver, anaemia), cutaneous/mucocutaneous (skin reactions)
Lab diagnosis: Amastigote stafe can be demonstrated in tissue biopsy, bone marrow examination, lymph node aspiration and through examination of properly strained smears. Culture of blood, bone marrow and other tissues often demonstrates the promastigote stage. Specific serologic testing is also available.
Treatment: Stibogluconate
Prevention and control: Protection from sandflies by screening and use of insect repellents

Picture of Leishmania species (http://www.google.com/ > image > Leishmania)

9. Wuchereria bancrofti and Brugia malayi
Transmission: Via bite of Anopheles, Aedes and Culex mosquitoes
Pathogenesis: The infective larvae migrate from the location of bite to the lymphatics, where growth to adulthood occurs. For 3 to 12 months after the initial infection, the adult male worm fertilizes the female, who in turn produces the sheathed larval microfilariae that find their way into the circulation. Both species have a periodicity in production of microfilariae (nocturnal periodicity) which results in greater numbers of microfilariae in blood at night.
Epidemiology: W. bancrofti occurs in both tropical and subtropical areas and is endemic in central Africa and many parts of Asia with no animal reservoir identified. B. malayi is found primarily in parts of Asia, while cats and monkeys are recognized as animal reservoirs.
Clinical syndromes: Fever, lymphanigitis, filarial elephantiasis of extremities
Lab diagnosis: Eosinophilia present, thick and thin blood smears stained with Giemsa stained under microscopy, anticoagulated blood and urine specimens, specific serological testing
Blood specimens are recommended to be taken between 10pm – 4am.
Treatment: Diethylcarbamazine
Prevention and control: Education regarding filarial infections, mosquito control, use of protective clothing and insect repellents, treatment of infections to prevent further transmission

Picture of microfilariae (http://www.google.com/ > image > microfilariae)

References:
- Murray, P.R., Kobayashi, G.S., Pfaller, M.A., Rosenthal, K.S. (1994). Medical Microbiology Second Edition. London: Mosby-Year Book, Inc
- Medical Microbiology Notes
- http://www.wikipedia.org/ > search

Researched by: Alex Tan 0503222B

Biochemical tests for bacteria indentification-Group post 2

2007-12-10T00:52:00.001+08:00

Biochemical tests for gram negative bacteria identification

1)Indole

-measure the ability to hydrolyse and deaminate tryptophan

-Klesiella-enterobacter-salmonella-serratia are mostly negative

-positive-red colour

2)Methyl red

-methyl red, a pH indicator with a range between 4.4(red) and 6.0(yellow)

-only species that produce suffiicient acids can maintian the pH at below4.4 against the buffer system of the test medium

-most species of Enterobacteriaceae produce strong acids. Enterobacter-serratia do not produce enough acids

-positive-stable red colour in the surface layer of the medium

3)Voges-proskauer reaction test

-this test is based on the conversion of acetoin to a red coloured complex through the action of KOH, atmospheric 02 and alpha napthol

-Klesiella-enterobacter-serratia is able to perform this pathway

-red colour at the surface of the medium after 15 mins following the addition of reagents

4)Citrate utilisation test

-some bacteria have the ability to utilize citrate as the sole carbon sourc and turn the medium allkaline due to production of ammonia

-Escherichia-Edwardisella-shigella-salmonella cannot utilise citrate as the sole source of carbon

-positive-from colour green to blue

5)Urease test

-some species posses the enzyme urease and able to hydrolyze urea with the release of ammonia and carbon dioxide

-this is used mainly to differentiate urease positive Proteus species from other member of Enterobacteriaceae

-positive-yellowish orange to pink

Biochemical tests for gram positive bacteria

1)Oxidase test

-this is to differentiate those that possess the enzyme cytochrome oxidase c from those that lack of the enzyme

-useful in screening for bacteria species which belong to the Enterobacteriaceae or the Pseudomonas genus

-positive-development of purple colour

2)Coagulase test

-the coagulase test is used to differentiate staphylcoccus aures from other staphylcoccus species

-positive-clot forms

2007-12-10T00:02:00.000+08:00

Particulars of patient

Name:Khong Fay Seah

Sex:Female

Age:27

Clinical Diagnosis

Complaints: fever,chills,dysuria

Diagnosis:urinary tract infection

Antibiotic treatment: if any (nil)

Possible Microorganisms	Description	Investigations
Escherichia coli(Most common cause of UTI)	-Belongs to family of Enterobacteriaceae(Escherichia) -Clinical findings: UTI (Pyelonephritis and Cystitis), diarrhea disease -Is a member of normal intestinal flora	-Microscopy and gram staining: Gram negative rod -Culture: Haemolyic colonies on blood agar, lactose fermenting colonies (Red/Pink) on MacConkey agar, green metallic sheen on EMB agar -Biochemical tests: -Indole positive -Methyl red positive - Voges-Proskauer (VP) negative -Urease negative -Citrate negative -Catalase positive -Oxidase negative -TSI: acidic slant/acidic deep/no H2S -Susceptibility testing: ampicillin, cephalosporin, aminoglycosides, sulfonamides
Staphylococcus saprophyticus(Second most common cause of UTI)	-Belongs to family of staphylococci -Clinical findings: Urinary tract infection -Present in the urinary tract and bladder of sexually active females	-Microscopy and gram staining: Gram positive cocci in clusters -Culture: Pale colonies on blood agar -Biochemical tests: -Catalase positive -Coagulase negative -Susceptibility testing:Quinilone
Klebsielle Pneumoniae	- Belongs to family of Enterobacteriaceae(Klesbsiella) - Clinical findings: urinary tract infection -Present in the respiratory tract and feces of about 5% normal individuals	-Microscopy and gram staining: Gram negative rod -Culture: Pink, mucoid colonies on MacConkey agar -Biochemical tests: -Indole negative - Methyl red negative -Voges-Proskauer reaction positive - Urease negative -Citrate positive - TSI: acidic slant/acidic deep/no H2S -Susceptibility testing:Cephalosporin
Enterobacter aerogenes	- Belongs to family of Enterobacteriaceae(Enterobacter) - Clinical findings: urinary tract infections and sepsis - Present in the intestinal tract-	-Microscopy and gram staining: Gram negative rod -Culture:Pink to purple colonies on EMB agar -Biochemical tests: -Indole negative -methyl red negative -VP positive -urease negative - Citrate positive -TSI: acidic slant/acidic deep/no H2S Susceptibility testing: cephalosporin
Proteus mirabilis	- Belongs to family of Enterobacteriaceae(Proteus) - Clinical findings: urinary tract infections and produce bacteremia, pneumonia	-Microscopy and gram staining: Gram negative rod -Culture: Pale colonies on MacConkey agar, swarming growth on blood agar -Biochemical testing: -Indole negative -Methyl red positive -VP positive/negative -Urease positive - Citrate positive -Oxidase negative -TSI: alkaline slant/acidic deep/H2S -Susceptibility testing: penicillin, cephalosporins, quinolones
Enterococcus faecalis	- Belongs to family of Streptococci -Clinical findings: abdominal abscess, urinary tract infection ((bladder infection, Pyelonephritis) -Present in colon	-Microscopy and gram staining: Gram positive cocci in chains -Culture: Red dot colonies on MacConkey agar -Biochemical testing: -Decolourise the litmus milk, -Indole negative -Catalase negative -Susceptibility testing: Vancomycin,ampicillin
Pseudonomas aeruginosa	- Belongs to family of Pseudomonads -Clinical findings: Typically infects the pulmonary tract, urinary tract(Pyelonephritis) -Distributed in nature and is commonly present in moist environments in hospital	-Microscopy and gram staining: Gram positive spherical -Culture: Greenish pyocyanin pigment on MacConkey agar -Biochemical testing: -Indole negative -Urease negative -Oxidase positive, -TSI: allaline slant/alkaline deep -Susceptibility testing: Ciprofloxacin,imipenam

PICTURES OF THE POSSIBLE CAUSATIVE AGENTS

E Coli ( red dot colonies on Mac agar)

K. Pneumonia (red/pink colonies on Mac)

Staphyloccocus Saprophyticus

Pseudomonas aeruginosa

Enterococcus faecalis

Enterobacter aerogenes

Proteus mirabillis

References

http:www.yahoo.com>image search> Proteus mirabilis
http:www.yahoo.com>image search> Enterobacter Aerogenes
http:www.yahoo.com>image search>Enterococcus faecalis
http:www.yahoo.com>image search>Pseudomonas aeruginosa
http:www.yahoo.com>image search>Staphyloccocus Saprophyticus
http:www.yahoo.com>image search>K. Pneumonia
http:www.yahoo.com>image search> E Coli
http://www.mc.maricopa.edu/~johnson/labtools/Dbiochem/emb.html>EMB agar
http://en.wikipedia.org/wiki/MacConkey_agar> MacConkey Agar
http://en.wikipedia.org/wiki/Blood_agar#Blood_agar_types> blood agar

Book
Tony Hart, Paul Shears (2004).Color Atlas of Medical Microbiology: Elsevier’s Health Sciences Department

kai lin
0503211E

MMIC dPBL Case 3 (Maisy Hong - 2nd posting cont)

2007-12-09T23:11:00.000+08:00

Pictures of possible causative agents of case 3

Pseudomonas aeruginosa growing on XLD agar Acinetobacter baumanii as seen under microscopy
Serratia marcescens growing on XLD agar
Candida albicans as seen under microscopy

Staphylococcus saprophyticus growing on nutrient agar Enterococcus faecalis as seen under scanning electron microscopy
Escherichia coli as seen under microscopy
Proteus mirabilis growing on XLD agar

Klebsiella pneumoniae as seen under microscopy
References:

All images are credited to

1) www.yahoo.com > image search > "microorganism name"

2) www.wikipedia.org > "microorganism name" > search

Information are credited to

3) Murray, P.R., Kobayashi, G.S., Pfaller, M.A., Rosenthal, K.S. (1994). Medical Microbiology Second Edition. London: Mosby-Year Book, Inc

4) Medical Microbiology Notes

5) www.yahoo.com > "microorganism name"

6) www.wikipedia.org > "microorganism name"

Tan Yi Wei Alex Tg02 0503222B

MMIC dPBL Case 3 (Maisy Hong - 2nd posting)

2007-12-09T22:50:00.000+08:00

Learning issues:

1) Identify suspected microbial agents
2) Conduct self-directed learning based on specific case and symptoms
3) Propose relevant tests according to suspected causative agents
4) Propose treatment to suspected causative agents

Keywords: Female, 67 years, Fever, Chills, Bladder distension, Indwelling catheter, Urinary tract infection, Urine specimen

Patient's Particulars

Name: Maisy Hong
Age: 67 years old
Sex: Female

Clinical Diagnosis

Complaints: Fever, chills, bladder distension (inability to urinate)
Diagnosis: Urinary tract infection (UTI)
Note: On indwelling catheter (a urinary catheter is a plastic tube which is inserted through a patient's urinary tract into their bladder)

Diagnostic approach to UTI in adults

1) Patient symptomatic? Yes, go to 2a. No, go to 2b.
2a) Complicating factors? Yes, go to 3a. No, go to 3b.
2b) Asymptomatic bacteriuria
3a) Complicated UTI
3b) Recurrent episode? Yes, go to 4a. No, go to 4b.
4a) Recurrent UTI
4b) Symptoms of upper tract involvement? Yes, go to 5a. No, go to 5b.
5a) Pyelonephritis
5b) Consider cystitis, urethritis or vaginitis (lower tract)

Usually, before proceeding to microbial identification tests, a urinalysis and chemical dipstick testing will be performed on the urine sample and important factors of UTI such as leucocytes, erythrocytes, pH as well as presence of certain chemicals like nitrite are things to look out for. As we've covered this in clinical chemistry, I will just talk about the medical microbiological portion. :)

Below is a table of possible causative agents, characteristics, proposed tests for agent identification and antibiotic susceptibility testing (treatment).

(Please click the pictures for enlarged images)

Tan Yi Wei Alex Tg02 0503222B

2007-12-09T21:38:00.000+08:00

CASE STUDY 4

Particulars of Patient
Name: Tong Wei Hong
Sex: Male
Age: 68 years old

Clinical Diagnosis
Signs and symptoms: Fever, chills, excessive phlegm, breathing problems Diagnosis: Bronchitis
Specimen collected: Sputum

Description of Bronchitis
Bronchitis is an inflammation of the bronchi (medium-size airways) in the lungs. Acute bronchitis is usually caused by viruses or bacteria and may last several days or weeks. Acute bronchitis is characterized by cough and sputum (phlegm) production and symptoms related to the obstruction of the airways by the inflamed airways and the phlegm, such as shortness of breath and wheezing. Diagnosis is by clinical examination and sometimes microbiological examination of the phlegm.

Chronic bronchitis is not necessarily caused by infection and is generally part of a syndrome called chronic obstructive pulmonary disease (COPD); it is defined clinically as a persistent cough that produces sputum (phlegm) and mucus, for at least three months in two consecutive years.

Possible Causative Agents

1. Adenovirus
Non-enveloped double-stranded linear DNA
Icosahedral nucleocapsid with a fiber protruding from each of the 12 vertices
Causes Bronchitis in the lower respiratory tract

2. Bordetella
Small, coccobacillary, encapsulated gram negative rod
Restricted to the respiratory tract (negative blood culture)

3. Haemophilus influenzae
Non-motile gram-negative coccobacillus
Found in the upper respiratory system of humans
Major cause of lower respiratory tract infections, associated with pneumonia

4. Moraxella catarrhalis
Gram-negative, aerobic, oxidase-positive diplococcus
May colonise and cause respiratory tract-associated infection in humans
Known to cause Bronchitis

5. Streptococcus pneumoniae
Gram positive lancet-shaped cocci
Arranged in pairs or short chains
Higher mortality in persons aged 65 and above

Investigations
The colour of the sputum may indicate the kind of microorganism in the sputum; yellow to green may indicate bacteria while white may indicate viruses. The sputum should be cultured to isolate the microorganisms listed above. In addition,

Biochemistry
-Gram staining
-Citrate test
-Catalase test
-Oxidase test
-Urease test

Microbiology
-Culture aerobically on blood agar plates

Antibiotic Suspectibility testing

Serology (Virus detection)
-Enzyme-Linked ImmunoSorbent Assay (ELISA)
-Haemagglutination

References
www.wikipedia.org > English > search > Bronchitis
www.wikipedia.org > English > search > Adenovirus
www.wikipedia.org > English > search > Bordetella
www.wikipedia.org > English > search > Haemophilus influenzae
www.wikipedia.org > English > search > Moraxella catarrhalis
www.wikipedia.org > English > search > Streptococcus pneumoniae

Desmond Heng
0503179D

Follow-up of case 6

2007-12-09T20:31:00.000+08:00

Above is the procedure that i will carry out during laboratory to determine the identity of the microoraganism present in the vaginal swap.

Materials Needed:
1) Blood Agar
2) MacConkey Agar
3) Gram stain Reagent
4) Caoagulase Reagent
5) Oxidase Reagent
6) Catalase Reagent
7) Commercial ID Testing Kits
8) Biochemical Tests Reagents
9) Antibody lines
Patient: Ong Fei Fei
Age: 37 Years old
Complaints: Fever, pain during urination, virginal discharge
Diagnosis: UTI

1)Enterococcus Faecalis
Labortatory Diagnosis:
(i) Alpha, beta or non hemolytic colonies on blood agar
(ii) Grow in 6.5% NaCl and hydrolyzes esculin on the presence of 40% bile
(iii)Catalase Negative

2)Eschericahia coli
Labortatory Diagnosis:
(i) Lactose fermenting on macConkey agar
(ii) TSI show acid slant, acid butt with gas but no H2S
(iii)Indole +
(Iv) Motile

3)Enterobacter-Klebsiella-Serrtia Family
Labortatory Diagnosis:
(i) Lactose fermenting on macConkey agar
(ii) Appear as mucoid colonies on agar plate

4)Proteus-Providencia-Morganella Family
Labortatory Diagnosis:
(i) Non-lactose fermentor on Macconkey
(ii) Distinct swarming on blood agar
(iii)P.vulgaris and P.mirabilis produce H2S that blacken the butt of TSI agar
(iv) P.mirabilis is indole negative whereas the other 2 are indole positive
(v) Urease Positive

Antibiotic suceptibility Testing will be carried out on antibodies mention in previous posting after bacterial is identified.

Yeo Ching Wei
0503288C

MMIC Blog Post 2

2007-12-09T17:45:00.000+08:00

Patient’s Particulars:
Name: Kwan Siew Yan
Sex: Female
Age: 29 years

Clinical Diagnosis:
Complaints: Diarrhea
Diagnosis: Entercolitis
Antibiotic Treatment: Nil

Possible organisms:
1) Vibrio species:
V. parahaemolyticus- Facultative anaerobes
Microscopic features:
· “Curved”, comma-shaped rods
· Motile with polar flagella
Laboratory investigations:
· Gram staining: Negative
· Oxidase: Positive on blood agar
· TSI: acid slant/acid deep without H2S production
· 0/129 (Vibriostat) Disk: Susceptible
· Nutrient Broth with 0% or 6-8% NaCl: Requires 1-7% NaCl
· Decarboxylase: Positive
· Culture media: TCBS as green colonies due to absence of sucrose fermentation
Antibiotic-susceptibility test:
· Tetracycline

2) Campylobacter species:
C. jejuni- Microaerophilic
Microscopic features:
· “S” shaped rods
· Darting motility
Laboratory investigations:
· Gram staining: Negative
· Oxidase: Positive
· Catalase: Positive
· TSI: Alkaline slant/alkaline deep
· Hippurate hydrolysis: Positive
· Culture media: Campylobacter agar incubated under microaerophilic conditions at 42oC as colourless or gray colonies
Antibiotic-susceptibility test:
· Erythromycin

3) Clostridium species:
C. perfringens- Obligate anaerobes
Microscopic features:
· Large, motile rods
Laboratory investigations:
· Gram staining: Positive
· Hemolysis test: Beta-hemolysis on blood agar as flat, spreading, translucent colonies
· Nagler test: Lecithinase positive
· Thioglycolate medium: Various sugars present
· Action on milk: Turned acid or digested
· Culture media: Egg yolk agar as colonies surrounded by precipitates with absence of spores
Antibiotic-susceptibility test:
· Penicillin

4) Escherichia species (Diarrheal diseases) – Facultative anaerobes
Microscopic features:
· Rod-shaped
Laboratory investigations:
· Gram staining: Negative
· TSI: Acidic slant/acidic deep with gas
· Indole: Positive
· Methyl red: Positive
· Voges-Proskauer: Negative
· Citrate: Negative
· Urease: Negative
· Culture media: EMB agar as greenish metallic sheen or MAC agar as red or pink colonies
Antibiotic-susceptibility test:
· Ampicillin

5) Salmonella species- facultative anaerobes:
Microscopic features:
· Rod-shaped
· Motile
Laboratory investigations:
· Gram staining: Negative
· TSI: Alkaline slant/acidic deep with H2S production
· Indole: Negative
· Methyl-red: Positive
· Voges-Proskauer: Negative
· Citrate: Positive
· Urease: Negative
· Culture media: EMB agar as translucent colonies or MAC agar as uncoloured colonies (non-lactose fermenting colonies)
Antibiotic-susceptibility test:
· Ampicillin

6) Shigella species- facultative anaerobes:
Microscopic features:
· Slender, rod-shaped
· Non-motile
Laboratory investigations:
· Gram staining- Negative
· TSI: Alkaline slant/acidic deep without gas or H2S production
· Indole: Negative
· Methyl-red: Positive
· Voges-Proskauer: Negative
· Citrate: Negative
· Urease: Negative
· Culture media: Culture media: EMB agar as translucent colonies or MAC agar as uncoloured colonies (non-lactose fermenting colonies)
Antibiotic-susceptibility test:
· Ampicillin

Pictures of possible organisms:
1) V. parahaemolyticus on TCBS agar

2) C. jejuni

3)Enteroinvasive E. coli (EIEC)

EMB agar

MAC agar

4) Salmonella species

EMB agar

5) Shigella species

MAC agar

References
1) http:www.yahoo.com>image search> Vibrio parahaemolyticus
2) http:www.yahoo.com>image search> Campylobacter jejuni
3) http:www.yahoo.com>image search> Enteroinvasive E. coli
4) http:www.yahoo.com>image search> Salmonella
5) http:www.yahoo.com>image search> Shigella

Tham Wan Jin June
TG02
0505073G

Flowchart of Identity Testing - Group post 1

2007-12-09T16:42:00.000+08:00

7 Biochemical Identity Tests

(1) Kligler Iron Agar Test
It is used primarily to determine the carbohydrate fermentation and hydrogen sulphate production.
Slant
Butt
Acid Yellow
Acid Yellow
Alkaline Red
Alkaline Red

Red slant/ yellow Butt = Glucose fermented
Yellow slant/ yellow butt = Glucose fermented + lactose fermented
Red slant/ red butt = neither of them fermented

(2) Simon Citrate Agar Test
It is used to determine if an organism is capable of using citrate as sole source carbon for metabolism and growth
Positive
Growth with/ without an intense blue colour on the slant
Negative
No growth, no colour change

(3) Motility Test
It is used to determine if a microorganism is motile.

(4) Urea Hydrolysis Test
It to detect urease activity of both rapidly urea positive organism as well as a number of enterobacteriacae
Positive
Pink colour on the slant
Negative
No change in colour (pale yellow)

(5) Phenylalanine Deaminase Test
It is to determine the ability of organism to deaminate phenylalanine to phenylpyruvic acid
Positive
Light to dark green on the slant
Negative
No change in colour (yellow)

(6) Malonate Test
It is to determine the ability of organism to use malonate as a source sole of carbon
Positive
Light Blue to deep blue throughout the medium
Negative
No change in colour

(7) Indole Test
It is to determine the ability of organism to hydrolyze and deaminate tryptophan with the production of indole, pyruvic acid and ammonia
Positive
Red halo ring seen
Negative
No change in colour (yellow)

MMIC 2ND POSTING: CASE 5

2007-12-09T13:20:00.000+08:00

CASE STUDY 5

Particulars
Patient: Wong Fei Hong

Sex: Male

Age: 37

Clinical Diagnosis

Complaints: Fever, swelling around operation wound

Diagnosis: wound infection

Antibiotic treatment (if any): Nil

Specimen Particulars

Specimen: Specimen 5 (Swab)

Date of collection: 26/11/2007

Time of Collection: 9 am

Result of First and Second Investigation through Gram Staining and Culturing

Biochemical Tests and Antibiotics Susceptibility Testing

Pictures of the Listed Microorganisms

Staphylococcus aureus

Left: Staphylococcus aureus gram stain
Right: Staphylococcus aureus on blood agar

Streptococcus pyogenes

Left: Streptococcus pyogenes gram stain
Right: Streptococcus pyogenes on blood agar

Enterococci

Left: Enterococci gram stain
Right: Enterococci on blood agar

Clostridium perfringens

Left: Clostridium perfringens gram stain
Right: Clostridium perfringens on blood agar

Pseudomonas aeruginosa

Left: Pseudomonas aeruginosa gram stain
Right: The soluble blue pigment pyocyanin is produced by many strains of Pseudomonas aeruginosa

References

Book:
Medical Microbiology, 23rd Edition Warren Levinson

Website:
http://keprice.myweb.uga.edu/pseudomonas_files/image002.jpg

http://www.bmb.leeds.ac.uk/mbiology/ug/ugteach/dental/tutorials/images/blood/staphaureus.jpg

http://www.textbookofbacteriology.net/pseudomonas.html

http://www.textbookofbacteriology.net/normalflora.html

http://www.mgm.ufl.edu/~gulig/mmid/mmid-lab/labimage/spy1.jpg

http://biomarker.cdc.go.kr:8080/pathogenimg/plate/Streptococcus_pyogenes_blood_agar(colony-1).JPG

http://www.textbookofbacteriology.net/clostridia.html
http://www.genomenewsnetwork.org/gnn_images/news_content/01_02/Clostridium_seq/clost2.jpg

By:

Lin Juexiu

0503151C

2007-12-03T00:36:00.000+08:00

Particulars of Patient
Name: Tong Wei Hong
Sex: Male
Age: 68 years old

Clinical Diagnosis
Signs and symptoms: Fever, chills, excessive phlegm, breathing problems Diagnosis: Bronchitis
Specimen collected: Sputum

Description of Bronchitis
Bronchitis is an inflammation of the bronchi (medium-size airways) in the lungs. Acute bronchitis is usually caused by viruses or bacteria and may last several days or weeks. Acute bronchitis is characterized by cough and sputum (phlegm) production and symptoms related to the obstruction of the airways by the inflamed airways and the phlegm, such as shortness of breath and wheezing. Diagnosis is by clinical examination and sometimes microbiological examination of the phlegm.

Chronic bronchitis is not necessarily caused by infection and is generally part of a syndrome called chronic obstructive pulmonary disease (COPD); it is defined clinically as a persistent cough that produces sputum (phlegm) and mucus, for at least three months in two consecutive years.

Possible Causative Agents
1. Adenovirus
-Non-enveloped double-stranded linear DNA
-Icosahedral nucleocapsid with a fiber protruding from each of the 12 vertices
-Causes Bronchitis in the lower respiratory tract

2. Bordetella
-Small, coccobacillary, encapsulated gram negative rod
-Restricted to the respiratory tract (negative blood culture)

3. Chlamydia pneumoniae
-Obligate intracellular bacteria
-Require host cells for growth

4. Moraxella catarrhalis
-Gram-negative, aerobic, oxidase-positive diplococcus
-May colonise and cause respiratory tract-associated infection in humans
-Known to cause Bronchitis

5. Streptococcus pneumoniae
-Gram positive lancet-shaped cocci
-Arranged in pairs or short chains
-Higher mortality in persons aged 65 and above

Desmond Heng

0503179D

TG02

MMIC POST: CASE 5

2007-12-03T00:33:00.000+08:00

MEDICAL MICROBIOLGY: CASE STUDY 5

Particulars
Patient: Wong Fei Hong
Sex: Male
Age: 37

Clinical Diagnosis
Complaints: Fever, swelling around operation wound
Diagnosis: wound infection
Antibiotic treatment (if any): Nil

Specimen Particulars
Specimen: Specimen 5 (Swab)
Date of collection: 26/11/2007
Time of Collection: 9 am

Step 1
Microscopy Examination
Gram stain is to be order.
This is to identify the gram reaction (purple-blue indicate gram-positive; pink indicate gram-negative) and morphology (shape: cocci, rods, bacilli and others) of the bacteria.

Step 2
Culture
Two types of culturing condition: aerobic and anaerobic
Select appropriate culture media
For wound infection as there is common media used.

Step 3
Biochemical tests which is to identify the microorganisms family
Tests to use will depend on the morphology of the bacteria.

Step 4
Antibiotic Susceptibility
Choose the appropriate antibiotic for choosing the correct antibiotic for the patient.

Possible Microorganisms

Antiobiotics Used

Lin Juexiu

0503151C

2007-12-02T21:39:00.000+08:00

MMIC PBL BLOG POST

Particular of patient
Name: Khong Fay Seah
Sex: Female
Age: 27years old

Clinical diagnosis
Complaints: fever, chills and dysuria
Diagnosis : Urinary tract infection
Antibiotic treatment: NIL

Description of urinary tract infection

A urinary tract infection (UTI) is a bacterial infection that affects any part of the urinary tract. Urine is sterile and contain no bacteria. When bacteria gets into bladder or kidney, it cause UTI. There are three kinds of UTI which are cystitis, pyelonephritis and urethritis. Cystitis is infection of bladder, pyelonephritis is infection of kidney and Urethritis is the infection of urethra.

Possible causative agents

1)Escherichia coli (Most common cause of UTI)

Belongs to family of Enterobacteriaceae(Escherichia)
Clinical findings: UTI(Pyelonephritis and Cystitis), diarrhea disease
Is a member of normal intestinal flora
Treatment: ampicillin, cephalosporin, aminoglycosides, sulfonamides

2)Staphylococcus saprophyticus(Second most common cause of UTI)

Belongs to family of staphylococci
Clinical findings: Urinary tract infection
Present in the urinary tract and bladder of sexually active females
Treatment:Quinilone

3)Klebsielle Pneumoniae

Belongs to family of Enterobacteriaceae(Klesbsiella)
Clinical findings: urinary tract infection
Present in the respiratory tract and feces of about 5% normal individuals
Treatment : Cephalosporin

4)Enterobacter aerogenes

Belongs to family of Enterobacteriaceae(Enterobacter)
Clinical findings: urinary tract infections and sepsis
Present in the intestinal tract
Treatment : cephalosporin

5)Proteus mirabilis

Belongs to family of Enterobacteriaceae(Proteus)
Clinical findings: urinary tract infections and produce bacteremia, pneumonia
Treatment: penicillin, cephalosporins,quinolones

6)Enterococcus faecalis

Belongs to family of Streptococci
Clinical findings: abdominal abscess, urinary tract infection((bladder infection, Pyelonephritis)
Present in colon
Treatment: vancomycin,ampicillin

7)Pseudonomas aeruginosa

Belongs to family of Pseudomonads
Distributed in nature and is commonly present in moist environments in hospital
Clinical findings: typically infects the pulmonary tract, urinary tract(Pyelonephritis)
Treatment : ciprofloxacin,imipenam

Reference

1)http://en.wikipedia.org/wiki/Urinary_tract_infection>urinary tract infection

2)Geo.F.Brooks,Janet S.Butel,Stephen A.Morse (2004).Jawetz,Melnick&Adelberg’SMedical Microbiology,twenty third edition: Lange Medical Books/ McGraw-Hill Medical Publishing Division

Sim kai lin

0503211E

2007-12-02T19:52:00.000+08:00

MMIC PBL Blog Post 1

Particulars of Patient:
Name: Kwan Siew Yan
Sex: Female
Age: 29 years

Clinical Diagnosis:
Complaints: Diarrhea
Diagnosis: Entercolitis: Inflammation of large and small intestines
Antibiotic treatment: NIL

Possible organisms:
1) Vibrio species:
· V. parahaemolyticus
2) Campylobacter species:
· C. jejuni
3) Bacilius species:
· B. cereus
4) Clostridium species:
· C. difficile
· C. perfringens
5) Escherichia species:
· Enteroinvasive E. coli (EIEC)
6) Salmonella species
· S. enteritidis
· S. typhimurium
7) Shigella species
· S. dysenteriae
· S. flexneri
· S. boydii
· S. sonnei

1) Vibrio species
V. parahaemolyticus
Definition: Curved, rod-shaped, gram-negative bacterium that is found in saltwater
Possible disease: Gastroentritis
Symptoms: Watery diarrhea, nausea, vomiting, abdominal cramping, fever and chills
Cause: Ingestion of bacteria in raw or uncooked seafood
Treatment: Not necessary since the disease is self-limiting. The most common ones are Tetracycline and erythromycin.

2) Campylobacter species
C.jejuni
Definition: Curved, rod-shaped, gram-negative bacterium that produces a cholera-like enterotoxin
Possible disease: Gastroentritis
Symptoms: Diarrhea, abdominal pain and fever
Cause: Ingestion of incorrectly prepared meat and poultry and drinking of contaminated water
Treatment: No antibiotics are given as the disease is self-limiting. Erythromycin may be given for serious cases.

3) Bacillus species
B. cereus
Definition: Rod-shaped, gram-positive bacterium that can produce protective endospores in addition to enterotoxin
Possible disease: Foodborne illnesses
Symptoms: Diarrhea, gastrointestinal pain, nausea and vomiting
Cause: Ingestion of improperly cooked food or improperly refrigerated food
Treatment: No specific treatment is needed. Vancomycin and gentamycin may be given for symptomatic treatment.

4) Clostridium species
C. difficile
Definition: Rod-shaped, spore-forming, gram-positive bacterium that are capable of producing enterotoxin and cytotoxin
Possible disease: Pseudomembraneous colitis
Symptoms: Diarrhea and abdominal pain
Cause: Ingestion of antibiotics that alters the normal intestinal flora, causing the bacterium to grow and produce toxins.
Treatment: No treatment needed for mild cases. Anti-clostridial agents like metronidazole may be used.

C. perfringens
Definition: Rod-shaped, spore-forming, gram-positive bacterium that produces heat-resistant enterotoxin
Possible disease: Foodborne illnesses
Symptoms: Diarrhea, gastrointestinal pain
Cause: Ingestion of poorly prepared meat and poultry
Treatment: Only symptomatic treatment is given.

5) Escherichia species
Enteroinvasive E. coli (EIEC)
Definition:
Possible disease: Human diarrheal illness
Symptoms: Watery diarrhea, abdominal cramping and fever
Cause: Ingestion of contaminated food and water
Treatment: The infection can be treated with TMP-SMX (trimethoprim-sulfamethoxazole).

6) Salmonella species
S. enteritidis
S. typhimurium
Definition: Rod-shaped, gram-negative bacteria infects without causing visible disease
Possible disease: Gastroenteritis
Symptoms: Diarrhea, fever and abdominal cramps
Cause: Ingestion of foods that are not properly cooked
Treatment: No antibiotic treatment is required as the disease is self-limiting.

7) Shigella species
S. dysenteriae
S. flexneri
S. boydii
S. sonnei
Definition: Rod-shaped, non-spore forming, gram-negative bacteria, closely related to Salmonella and Escherichia coli species
Possible disease: Human diarrheal illness
Symptoms: Diarrhea, fever and stomach cramps
Cause: Ingestion of contaminated food, poor hygienic practices
Treatment: No treatment is needed in mild cases. Ampicillin may be used for severe ones.

Tham Wan Jin June
TG02
0505073G

MMIC

2007-12-02T18:59:00.000+08:00

Name: Ong Fei Fei
Sex: Female
Age: 37 years old
IC NO: S210334X
Complaints: Fever, pain during urination, virginal discharge
Diagnosis: UTI

Possible Causative Agents:
1) Enterococcus Faecalis
Characteristics:
1. Appeared as gram positive bacteria in chains
Catalase negative
2. May appear as alpha, beta or non-hemolytic on blood agar
Treatment: Penicilin, gentamicin
2) Escherichia Coli
Characteristiccs:
1. Appeared as gram negative rod
2. Lactose Fermentor
3. Triple Sugar Iron test show acid slant, acid butt with gas
Treatment: Ampicillin, sulfonamides
3) Enterobacter-Klebsiella-Serrtia Family
Characteristics:
1. Appeared as gram negative rod
2. lactose fermentor
Treatment: Gentamicin, cefotaxime
4) Proteus-Providencia-Morganella
Characteristics:
1. Appeared as gram negative rod
2. lactose non-fermentor
3. Appear as swarming on blood agar
Treatment: Ampicillin, cefotaxime

5) Pseudomonas
Characteristics:
1. Appear as gram negative rod
2. Glucose Non-fermentor
3. Metallic sheen growth on the surface of TSI agar
4. Oxidase positive
Treatments: Gentamicin, amikacin
6) Candida albican
Characteristics:
1. Appear as gram positive
2. Typical yeast colonies can be observed on culture plate
Treatments: Clotrimazole troches, nystatin
7) Trichomonas
Characteristics:
1. Urogenital protozoan
2. Pear shaped
3. Flagellated trophozoities
Treatments: Metronidazole
Yeo Ching Wei
0503288C

MMIC dPBL Case 3

2007-12-02T18:29:00.000+08:00

Learning issues:
1) Identify suspected microbial agents
2) Conduct self-directed learning based on specific case and symptoms
3) Propose relevant tests according to suspected causative agents

Keywords:
Female, 67 years, Fever, Chills, Bladder distension, Indwelling catheter, Urinary tract infection, Urine specimen

Patient's Particulars
Name: Maisy Hong
Age: 67 years old
Sex: Female

Clinical Diagnosis
Complaints: Fever, chills, bladder distension (inability to urinate)
Diagnosis: Urinary tract infection (UTI)
Note: On indwelling catheter (a urinary catheter is a plastic tube which is inserted through a patient's urinary tract into their bladder)

UTI can be grouped as either lower or upper UTI. Common symptoms of lower UTI (e.g cystitis, urethritis) in adults include back pain, hematuria, cloudy urine, inability to urinate despite the urge, fever, frequent urination, malaise and dysuria. Symptoms that indicate upper UTI (e.g pyelonephritis) in adults include chills, high fever, nausea, pain below the ribs and vomitting.

Possible causative bacterial agents:
Gram-positive: Staphylococcus sp. (Staphylococcus saprophyticus), Enterococci sp.
Gram-negative: Enterobacteriaceae sp. (Escherichia coli, Proteus sp. , Providencia sp. , Morganella sp. , Serratia sp.) , Pseudomonas sp. (Pseudomonas aeruginosa), Acinetobacter sp.

Catheter-associated urinary tract infections are caused by a variety of pathogens, including Escherichia coli, Klebsiella, Proteus, Enterococcus, Pseudomonas, Enterobacter, Serratia, and Candida. E. coli appears to be the most common cause of UTI whereas S. saprophyticus is the second most frequent causative organism of uncomplicated UTI, though more commonly seen in young, sexually active women.

Laboratory investigations:
- Enterobacteriaceae
Culture: On MacConkey, Eosin Methylene Blue agar
Identification: Gram-negative; Various biochemical identification
- Enterococci
Culture: On sheep's blood agar (showing gamma-hemolysis)
Microscopy: Gram-positive cocci
- Staphylococcus saprophyticus
Culture: On sheep's blood agar, culture with 7.5% NaCl and mannitol, non-selective media (aerobically & anaerobically)
Microscopy: Gram-positive cocci (cluster)
Identification: Positive urease, beta-galactosidase, acetoin production, novobiocin resistance
- Pseudomonas
Culture: On sheep's blood, MacConkey agar under aerobic incubation
Microscopy: Gram-negative bacilli
Identification: Positive oxidase reaction

References
- www.google.com > "urology" > sponsored links: Urologist in Singapore
- www.google.com > "indwelling catheter + urinary tract infection" > Guideline for Prevention of Catheter-associated Urinary Tract Infections
- www.wikipedia.org
- Murray, P.R., Kobayashi, G.S., Pfaller, M.A., Rosenthal, K.S. (1994). Medical Microbiology Second Edition. London: Mosby-Year Book, Inc.

Tan Yi Wei Alex
TG02 0503222B

Ans to synovial fluid

2007-11-09T18:52:00.000+08:00

Sorry for the late replying as I need to ask my colleagues.

Dorothy:

1.) Blood-stained synovial is hemorrhage. It bleeds into the joint, the blood is usually evenly distributed throughout the fluid. It is caused by traumatic tap. Any time bleeding occurs into the joint fluid, fibrinogen is introduced and will permit clot formation. In the event that fibrinogen is thought to be introduced, a tube containing sodium heparin should be available as part of the collection process. Non-clotted SyF is necessary for the microscopic examination.

2) In normal circumstances, the total protein of synovial fluid will be about 1/3 that of blood plasma. There are a variety of joint disorders that are characterized by elevated total protein levels (examples are rheumatoid and septic arthritis, crystal-induced synovitis, and hemorrhagic problems. It has been found that protein determinations of Synovial fluid does not assist in the differentiating of joint disorders, hence is not reliable. Protein determinations are usually not requested.

Ming Boon:

Traumatic tap is refer to bleeding into the subarachnoid space at the puncture site

For the differentiation of the exudate and transudate fluid is two type of category acording to the Light's criteria. Which i am trying to load the table. The table is the below:

Elaine:

Another limitation will be the fluid cell count must be done immediately as cell will broken down on prolong standing thus fluid must be examine as soon as possible.

Juexiu

TG02

Nature and Pathogenesis of Infection

2007-11-02T11:34:00.000+08:00

Pathogen: Any organism that is capable of invading the body and cause disease

Parasite: An organism that live in another organism, deriving benefit from it but providing nothing in return.It may or may not be an pathogen. E.g E.histolytica is capable of evading the bowel wall, causing colitis and abcess in liver and other tissue BUT entamoeba. coli live in human gut without causing any disease.

Infection: Refers to a disease caused by a pathogen. It is furtehr defined by the presence of replicating organism in association with tissue damage.

Normal floral may developed into an infection. Usually, it compete with potential pathogen for attachment site by producing anti-microbial substance and compete with nutrient. Under special circumstance, it may become a pathogen, e.g C.difficile altered by antibody therapy produce toxin, causing pseudominate colitis.

Intoxification: It is DIFFERENT from infection, simply mean posioning. E.g adult botulism caused by C.botulinum, develop when food is ingested in which organism has grown and produce a neutraoxin.

Communicable disease: An infection capable of spreading from person to person.
NOTE: Not all disease are communicable.
Transmission may be by direct person to person, respiratory, sexual or mucosal contact and by insect vector.

Pathogenicity: It is the ability to cause disease E.g neisseria gonorrahoae is the causative agent. Some strain have pili while some don't. Those that lack pili are non-pathogenicity. Mechanism of pathogenicty are numerous.

Virulence: It is the power to cause severe disease. It is affected by virulence factors possessed by organism. However virulence may not alway be assoicated with pathogenicity.

Infectiousness: It is the ease which pathogen can spread in a population. E.g measle is highly infectious whereas mump is less so. It can be measure by IRR ( intrinsic reproduction rate).

The behaviour of a pathogen in a population/community depend heavily upon the interaction bewteen the host, agent and enviroment.

1) Host: It affect the chance of explosure to a pathogen and individual response to infection. E.g Travel, sexual behaviour, hygenic, occupation etc

2) Agent factor: It simply refers to the infectiousness, pathogenicity, viruluence and ability to survive in human host and under different enviroment factors. E.g the ability to produce resistance against accine, immune response etc

3) Enviroment factor: Temperature, dust and humidty, use of anitbody and pesticide affect survival of pathogen outside the host.

One case study example:

Malaria
Host factor affect the transmission of malaria. People who live in endemic area develop partial immunity as a result of repeated explosure. Sickle-cell trail individual has a low rate of parasitaemia as parasite caanot derived effective nutrition from haemglobin.

Agent of p.falciparium malaria has disease resistance to increasing range of prophylactic drug. Ths inevitable mean that traveller now have to search for new vaccine to protect themselves.

Enviroment affect transmission adversely. Malaria diesease predominate in tropical zone especially during rainy season.

Reservoir of infection: It refers to the human/animal population or enviroment in which the pathogen exist and from which it can transmitted.

1) Horizontal spread: It is between individual in the same population E.g Cough

2) Vertical spread: It is from mother to fetal. Many pathogen can cross placenta but few can cause fetal damage.

3) Zoonosis: It is animal disease which is later spread to human.

Outbreak is the occurence of a disease clearly in excess of normal expantancy.

1) Point-source outbreak: Group of individual expose to a single source of infection at a defined point of time. E.g Wedding guest who consumed contaminated food developed food poisoning shortly afterward.

2) Common source outbreak: Group of individual expose to a single source but not at a same time. E.g tattoo palour using contaminated equipment resulting in the spread of Hepatitis B.

3) Person to person outbreak: No common source but maintain by chain of transmission between infected individual. E.g measle outbreak in the school

Hopefully all these information will help you to understand the terminalogy used in M.Mic.

Ching Wei
0503288C

SIP blogging

2007-10-29T21:54:00.000+08:00

Hello to all, this week I will be blogging on body fluids for examination. Body cavity fluids other than blood or urine are referred as extravascular fluids and they are:
1.) Cerebrospinal (around the brain and spinal cords)
2.) Synovial (around the joints)
3.) Pleural (around the lungs)
4.) Peritoneal (around the abdominal and pelvis cavities)

This blog i going to talk about synovial fluid.

Normal synovial fluid is clear straw-coloured or pale yellow fluid found in small amount in joints and tendons sheaths. It is an ultrafiltrate of plasma plus a mucopolysaccharide, produced by the lining synovial cells which make the fluid thick and viscous. Normal volume of fluid in knee joints is about 3.5ml.

Function of synovial fluid is to lubricate the joint space, transport nutrients to the articular cartilage, remove waste and debris from joint and medium for leucocytes to circulate and phagocytize debris.

Clinical significance for synovial fluid analysis is in the differential diagnosis of swollen joint: distinguish crystal-inducing disease from septic joints.

Sample Collection/Preparation
Synovial fluid is collected by aspiration of fluid from the joint space with a needle. Due to cells are easily destroyed and glucose may also undergo glycolysis on standing, thus the analysis of fluid must be done immediately once received.

Laboratory Analysis of Synovial Fluid
1. Appearance

First, state the appearance and clarity of the fluid received. Synovial fluid can be described as:
- Straw colour or pale yellow and clear is the normal appearance of synovial fluid
- Turbid or purulent sample is seen in bacterial infection or high leucocytes count.
- Blood-stained
- Chylous fluid refer to having a characteristics milky, opaque appearance which remains in the supernatant after centrifugation, which is caused by lymphatic leakage or obstruction.
If the fluid is blood stained or turbid, spun it down and state the appearance of the supernatant.

2. Cell Count and WBC Differential

A cell-count with differential would aid in making the diagnosis as bacterial infections will have a predominance of neutrophils while viral, fungues and mycobacterial infections may have a predominance of lymphocytes or show a mixed inflammatory response.

If the effusion is predominantly neutrophils, an acute inflammatory process is the cause and differential count showing essentially all lymphocytes suggests a chronic process.

3. Glucose

Fluid glucose is reduced due to bacterial, increased WBCs or infiltration with malignant cells the reduction is the result of the metabolic requirement of the infecting organisma and of the inflammatory cells as well as the tumour cells.

4. Total Protein

Total protein is abnormally raised in: infection, malignant infiltration, chronic inflammatory conditions or traumatic tap (false positive)

Differentiation of exudate and transudate fluid

5. Crystals

The 3 most common types of crystals present in the joint fluid are:
- Monosodium urate
- Calcium pyrophosohate
- Cholesterol

Monosodium Urate Crystals(MSU):
These crystals are needle shaped, double refractile of 8-10um long, negatively birefringent and soluble in water. They are associated with gout and may be intracellular. MSU crystals are negatively birefringent and when aligned parallel to the compensator will show a yellow colour and when turned perpendicular to the compensator the colour change to blue.

Calcium Pyrophosphate Dihydrate Crystals(CPPD):
These are associated with pseudogout, which most frequently involves the wrists and knees. These crystals have various shapes but usually rhomboid in shape and are positively birefringent. They are broader than uric acid crystals and up to 25um long, have a line running through them. The crystals are blue in colour when parallel to the compensator and yellow when perpendicular to it.

Cholesterol Crystal:
Cholesterol crystals have a characteristics notched-plate and birefringent. They are present in chronic inflamed joints such as rheumatoid arrthritis. In pseudogout, serum uric acid is normal while serum uric acid is high in gout. Cholesterol crystals may be found in any chronic effusion.

6. Summary:

Normal Synovial Fluid
- Clear , yellow fluid which does not clot spontaneously
- UP to 200 WBC/uL of which less than 25% are neutrophils
- No Crystals
- Total protein: 18g/L
- Glucose level is similar to serum glucose level

Juexiu
tg02

2007-10-19T19:19:00.000+08:00

SIP- Histopathology

Hey everyone! I shall touch on the 2 special stains that I did when I was attached to Special Staining. They are namely the Liver Orcein stain and the Victoria Blue stain.

Liver Orcein Stain

Function: Demonstrates Hep B surface antigen and elastic tissues

Principle: The disulphide bridges in the elastic tissue are broken down into its anionic derivatives during oxidation. These derivatives can then be stain by Orcein stain. Virus inclusion bodies can be stain by Orcein after oxidation.

Control: A known Hep positive liver tissue

Fixative: 10% buffered neutral formalin

Chemicals required: Orcein solution
0.5% potassium permanganate
1% oxalic acid
3% sulphuric acid

Procedures:
1) Dewax (removal of wax) and bring tissue section to water
2) Place in acidified potassium permanganate solution for 5 mintues (section will appear brown in colour)
3) Wash in water
4) Bleach section till colourless in 1% oxalic acid
5) Wash in water
6) Stain in Orcein solution for 4 hours at room temperature or overnight in fridge
7) Wash quickly in 2 changes of absolute alcohol
8) Dehydrate, clear then mount

Results: Coarse elastic fibers- reddish brown
Fine elastic fibers- dark brown
Hep B surface antigen- dark brown

Victoria Blue Nuclear Fast Red

Function: Stain elastic fibers

Principle: Elastic fibers are highly cross-linked by disulphide bridges. Following oxidative treatment, these bridges may convert into anionic sulphuric acid derivatives. These derivatives are strongly basophilic and capable of selective reactions with the basic dyes.

Control: Skin and artery for elastic fibers demonstration. Inflamed skin for mast cells demonstration.

Fixative: 10% buffered neutral formalin

Chemicals required: 0.5% potassium permanganate
3% sulphuric acid
4% sodium bisulphate
VB solution
Nuclear Fast Red

Procedures:
1) Dewax and bring section to water
2) Stain with acidified potassium permanganate solution for 5 minutes (section will appear brown in colour)
3) Bleach with 1% sodium bisulphite until colourless
4) Wash in running water
5) Dry on hotplate
6) Leave in VB solution for 24 hours
7) Differentiate in 70% alcohol
8) Wash in running water
9) Stain with Nuclear Fast Red for 5 minutes
10) Rinse in running water
11) Dehydrate, clear then mount

Results: Elastic fibers, lipofuchsin and mast cells- blue
Cytoplasm and nuclei- red

This is the end of my posting. Thanks for reading! Enjoy the rest of your SIP! =)

*I want to make an important note. According to my colleague, the Liver Orcein Stain will be used to stain the HepB antigen while the VB Stain is only used for elastic fibers demonstration.

Tham Wan Jin June
TGo2
0505073G

SIP Week 16 Blog Posting - Special Stains in Histopathology

2007-10-14T22:47:00.000+08:00

Hey poly peeps, Desmond here. It's week 16 and just another 4 more weeks before we go back to campus. Having covered Cytopathology and Histopathology routine (embedding and microtomy) in my 2 previous posts, I've decided to touch on some of the special stains performed in Histopathology.

GRAM STAIN

Function:
This stain differentiates Gram positive and Gram negative bacteria

Principle:
Both bacteria’s, positive and negative cell wall is composed of peptidoglycan, (the gram positive has a thicker wall) and both will take up the crystal violet. The gram-negative has a layer of lipopolysaccharide external to the peptidoglycan wall, which is disrupted in the acetone rinse, allowing the crystal violet to be differentiated out. This allows the gram-negative bacteria to take up the fuchsin stain.

Control:
An infected appendix, or any tissue containing both negative and positive gram rods.

Fixative:
1. 10% Buffered Neutral Formalin

Reagents required:
1% aqueous crystal violet solution
1% Basic fuchsin
Iodine
Potassium iodide
Formalin 37-40%
Glacial acetic acid
Picric acid
Acetone
Xylene

Reagent Preparation:
1. Gram iodine solution
Iodine 1g
Potassium iodide 2g
Type II water 300ml
Allow iodine and potassium iodide to dissolve completely. Mix before use. Solution is stable for 6 months at room temperature.

2. Gallego’s differentiating solution
Type II water 100ml
Formalin 37-40% 2ml
Glacial acetic acid 1ml
Mix before use. Solution is stable for 6 months at room temperature

3. Picric acid-acetone solution
Picric acid 1g
Acetone 100ml
Solution is stable at room temperature for 6 months.

4. Acetone-Xylene solution
Equal parts of acetone and xylene.
Solution is stable at room temperature for 6 months.

5. 1% Crystal violet
Crystal violet 1g
Type II water 100ml
Filter into bottle. Solution is stable for 1 year at room temperature.

6. 1% Basic Fuchsin
Basic Fuchsin 1g
Type II water 100ml
Filter into bottle. Solution is available for 6 months at room temperature.

Staining Procedure:
1. Dewax and bring section to water.
2. Place in 1% crystal violet solution for 1 minute.
3. Rinse in tap water.
4. Place in Gram iodine solution for 1 minute
5. Rinse in tap water.
6. Decolourize in acetone until background is clear.
7. Immediately wash in tap water.
8. Place in 1% Basic Fuchsin solution for 5 minutes.
9. Rinse in tap water.
10. Place in Gallego’s differentiating solution, 2 changes, 1 minute each.
11. Rinse in tap water.
12. Transfer to a staining dish.
13. Treat with acetone for 30 seconds.
14. lace in picric acid-acetone solution for 2-3 minutes.
15. Place in acetone-xylene solution for 2 changes.
16. Clear in xylene, 2 changes.
17. Mount in DPX.

Results:
Gram positive - Blue
Gram negative - Red
Background - Yellow

Ziehl Neelsen Stain

Function:
To demonstrate acid fast bacteria belonging to the genus mucobacterium

Principle:
The lipid capsule of the acid-fast organism takes up carbol-fuchsin and resists decolourization with a dilute acid rinse. The lipoid capsule of the mycobacteria is of such high molecule weight that it is waxy at room temperature and is not stained by bluing solutions such as methylene blue.

Control:
Any tissue containing acid-fast organisms

Fixative:
1. 10% Buffered Neutral Formalin

Reagents required:
Commercial TB stains (Merck)
Loeffler’s Methylene Blue
1% Potassium Hydroxide
1% acid alcohol

Reagent Preparation:
1. Stock Loeffler’s Methylene Blue solution
Methylene Blue 1gm
95% Alcohol 100ml
2. 1% Potassium Hydroxide
Potassium Hydroxide 1gm
Type II water 100ml
3. Working solution
1% Loeffler’s Methylene Blue 30ml
Type II water 99ml
1% Potassium Hydroxide 1ml
4. 1% Acid Alcohol
Concentrated Hydrochloric Acid 20ml
70% Alcohol 1980ml

Staining Procedure:
1. Dewax and bring sections to water.
2. Commercial TB colour – 5 minutes (or hot carbol fuchsin solution for 30 mins)
3. Differentiate with 1% alcohol
4. Wash in water
5. Couterstain with 1% Loeffler’s methylene blue.
6. Wash in water and go straight to 95% alcohol to control blue colour.
7. Dehydrate clear and mount in DPX.

Results:
Tubercle bacilli - Red
Background - Blue

LENNERT GIEMSA STAIN

Function:
Stains Bone marrow lymph node. Differentiates cells present in hematopoietic (lymph nodes).

Principle:
The neutral dyes combining the basic dyes methylene blue and dye eosin, give a wide colour range when staining. The more acid pH levels give more selective chromatin staining and less cytoplasmic basophilla. Less acid pH levels give denser nuclei and increased cytoplasmic basophilla.

Control:
Spleen

Fixative:
1. 10% Buffered Neutral Formalin

Reagents required:
Commercial Giemsa Stain

Reagent Preparation:
Working Giemsa solution (for 5 slides or less)
Giemsa Solution 3ml
Type II water 12ml
Mix and use in plastic slides mailer. Discard after use.

Staining Procedure:
1. Dewax and bring section to water.
2. Rinse section in Type II water
3. Place sections in working Giemsa solution for 1 hour at room temperature.
4. The sections are removed from the Giemsa solution and put into 100ml Type II water, to which 3-4 drops of undiluted glacial acetic acid have been added. The sections are agitated gently in this solution for a few seconds, slightly differentiated and then immediately put into 96% ethyl alcohol, in which they are differentiated further until the desired staining is achieved (microscopic control).
5. Differentiation is stopped and, at the same time, dehydration is achieved by dipping in 3 changes of isopropanol for 2 minutes each.
6. Dehydrate, clear and mount with DPX.

Results:
RNA, DNA - Blue (Basophillic)
Acidophilic substances - Pink or reddish orange
Acid mucopolysaccharides - Reddish violet

JENNER’S GIEMSA STAIN

Function:
Stains Bone marrow and gastric biopsies. To demonstrate helicobacter. To differentiate cells present in hematopoeietic tissue (lymph nodes). Use in blood smears.

Principle:
The neutral dyes combining the basic dyes methylene blue and dye eosin, give a wide colour range when staining. The more acid pH levels give more selective chromatin staining and less cytoplasmic basophilla. Less acid pH levels give denser nuclei and increased cytoplasmic basophilla. Methylene blue in an alkaline pH solution stains metachromatic.

Control:
Stomach or colon tissue containing helicobacter. Skin for mast cells.

Fixative:
1. 10% Buffered Neutral Formalin

Reagents required:
Commercial Jenner solution
Commercial Giemsa solution
1% acetic acid

Reagent Preparation:
1. Jenner working solution
Stock Jenner Solution 2ml
Type II water 2ml
Mix and use. Discard after use.
2. Giemsa working solution
Stock Giemsa solution 2ml
Type II water 40ml
Mix and use. Discard after use.

Staining Procedure:
1. Dewax and bring sections to water.
2. Place slides in 2 changes of methyl alcohol for 3 minutes,
3. Dip direct into working Jenner solution for 5 to 6 minutes.
4. Transfer direct to working Giemsa solution for at least 45 minutes.
5. Rinse in distilled water. Check under microcope and monitor differentiation
6. Differentiate in 1% acetic acid.
7. Dip in 95% alcohol for 5 to 6 times.
8. Dehydrate clear and mount in DPX.

Results:
Cytoplasm - Pink
Nuclei - Blue
Erythrocytes - Red
Mast Cell granules - Purple
Bacteria - Blue
Malaria Parasite - Blue

Desmond Heng
0503179D
TG02

Student Internship Programme (SIP) HAEM

2007-10-06T11:56:00.000+08:00

Neutrophil Alkaline Phosphatase (NAP) stain

Intended use
NAP score is used to distinguish Chronic Granulocytic Leukaemia from other myeloproliferative disorders and Polycythaemia Rubra Vera from secondary polycythaemia.

Principle

At an alkaline pH, NAP hydrolyses substrate naphthol AS-BI phosphate to liberate napthol-AS. The liberated naphthol couples with a diazonium salt, Fast Blue BB to produce an insoluble complex which precipitates at the site of enzyme activity. The sites of alkaline phosphatase activity will appear as blue granules. The NAP score is the sum of the rating of 100 consecutive segmented and band neutrophils using a scale of 0 to 4+ according to the appearance and intensity of the precipitated dye.

Specimen collection and handling conditions

1. Fresh capillary/venous blood preparation, not anticoagulated is required.
2. EDTA blood is not recommended as enzyme activity is inhibited.

Preparation of stock substrate solution

a) Dissolve 30mg naphthol AS phosphate in 0.5ml N,N-dimethylformamide. CAUTION: N,N-dimethylformamide is toxic by inhalation. To be prepared in fume hood.
b) Add 0.2M tris buffer, pH 9.1 to bring volume to 100ml.

c) Store at 4-10 deg celcius. Stable for 2 months.

Preparation of working substrate

a) Add 10mg Fast Blue BB to 10ml stock substrate solution. Mix well. CAUTION: Fast Blue BB is carcinogenic and a possible mutagen. To be prepared in fume hood.

b) Prepare fresh when needed.

Quality control

A normal control smear must be included with every batch of stain and results should fall within tolerance limits.

Procedures

1. Fix air-dried smear in buffered 60% Acetone in Citrate brought to room temperature for at least 5 mins.

2. Rinse with tap water and dry slide.

3. Using filter paper No.41, filter the freshly prepared working substrate directly onto slides. Stain for 15 mins at room temperature. Discard working solution into the toxic waste container.

4. Wash in tap water.

5. Counterstain with 0.5% neutral red for 1 min.

6. Wash and dry.

7. Count under oil immersion 100 neutrophils. The slides are examined and counted by 2 technologists and an average of 2 results is reported.

Interpretation

Sites of enzyme activity are represented by discrete bright blue granules of varying sizes. Nucleus is stained red. The enzyme is present predominantly in segmented neutrophils. Cytoplasmic granules of eosinophils do not stain while basophils cannot be differentiated from other granulocytes.

One hundred segmented or band forms of neutrophils are rated. The sum of 100 cell ratings give a score with a possible range from 0-400.

Normal range: 30-100

Clinical significance
Chronic Granulocytic Leukaemia and Paroxysmal Nocturnal Haemoglobinuria are associated with abnormally low or absent staining. Conversely, leukaemoid reactions, polycythaemia vera and myelofibrosis are associated with markedly elevated NAP scores. High scores can also be obtained from neonates, pregnant women or women taking oral contraceptives.

Notes
1. Do not make the smear too thick to avoid inadequate fixation.
2. Fixed smears should be stored at -20 deg celsius if staining delayed for >5-6 hours.
3. Scoring of enzyme activity should be made in areas of slide with optimal cell morphology.

Answers to possible questions

Why must the working solution be prepared fresh before every staining?
Precipitation will occur over time once the Fast Blue BB is added to the stock substrate solution that will be indicated by a colour change from bright yellow to dull brown. Therefore it is important that the working solution be mixed and applied fast.

What are the various illnesses stated?
Please visit Wikipedia or other search engines to administer your enquiries. I'm sure you will learn more than what I'll be able to explain this way. :)

I've also taken some pictures and will seek to post them as soon as possible once I get relevant approval. Hope you've learnt something!

Alex Tan 0503222B TG02

2007-09-29T21:24:00.000+08:00

Hello everyone!! It’s my turn to blog for the week. I am currently at coagulation lab. The test which I am going to share with you guys is 50%PT/APTT. I suppose you should be familiar with PT and APTT which we had done before in during haematology lab session. Does it ring a bell? To refresh your memory…

PT is used to screen for abnormalities of those factors that are involved in the extrinsic pathway (factor 5,7,10) and prothrombin and fibrinogen.

APTT is sensitive to deficiencies of factor II,V,VIII,IX,X,XI,XII( Factor 2,5,8,9,10,11,12) involved in the intrinsic pathway.

Test: PT

1) PT reagent is incubated in water bath at 37 degrees for 3mins
2) 0.1ml of test plasma is incubated in 37 degrees water bath for 3mins
3) 0.2ml of PT reagent was pipette into the 0.1ml of test plasma after the incubation period
4) start the stopwatch simultaneously and shake the test tube to mix the contents well and observed for clot

Test:APTT

1) 0.1ml of APTT reagent and 0.1ml of calcium chloride was incubated in water bath at 37 degrees for 5mins
2) 0.1ml of test plasma was added to 0.1ml of APTT reagent. Remember to shake the tube to mix the contents well
3) the tube was placed at 37 degree water bath for 4mins
4) 0.1ml of calcium chloride was pipette into the plasma and APTT reagent
5) start the stop watch simultaneously and observed for clot

Test: 50%PT and APTT correction test

Intended use:
It is used to screen for the presence of inhibitors in patient’s plasma.

Principle:

50% addition of normal plasma is sufficient to restore the deficient factor. Complete or partial correction time suggests a clotting factor deficiency, whereas failure to correct the clotting time suggest the presence of inhibitors. Normal plasma contains all coagulation factors and is mixed with patient’s plasma to detect the presence of circulating inhibitors in the patient’s plasma. If a factor deficiency is responsible for the abnormal clotting time addition of normal plasma should provide the deficient factors and correct the PT and APTT time. However if abnormal clotting time is caused by a circulating inhibitors in the patient’s plasma, the clotting time will remain prolonged even when normal plasma is added. The inhibitor inhibits factors in normal plasma as well as in the patient’s plasma.

Example: if the patient’s PT is 18secs
Normal plasma is 12secs
Average is (18+12)/2=15secs
PT is corrected if 50%PT <15secs>

that's all! feel free to ask if in doubt..

kai lin

0503211E

SIP posting

2007-09-16T21:22:00.001+08:00

For this past few weeks I was attached to Haematology

At haematology section, I have learn various principle of the tests such as retic count, Dengue testing.

RETIC COUNT
For retic count, the analyzer used is CELL-DYN Ruby. Reticulocytes are defined as transitional red cells between nucleated red cells and the mature erythrocytes. The reticulocyte assay enables determination of the percentage of reticulocytes using a whole blood specimen. The method used is based on light scatter measurement of stained cells.

Staining Procedure:
1. Label one tube of CELL-DYN Reticulocyte Reagent(Phosphate buffered saline solution contain New Methylene Blue N) for one specimen.
2. Verify the whole blood specimen have no clotting and mixed well.
3. Pipette 20 micro-litre of whole blood specimen into the tube of reticulocyte reagent.
4. Incubate the stained reticulocyte specimen for 15 mins at room temperature.
5. Ready for sampling in CELL-DYN Ruby

Principle of Procedure:
Reticulocytes contain ribosomal RNA, this RNA can be seen by certain dyes that simultaneously stain and precipitate the polyanion to form a reticulum. The CELL-DYN Ruby reticulocyte method uses the thiazine dye New Methylene Blue N. Sample preparation is done by diluting a small volume of blood into pre-measured staining solution and incubate for at room temperature for 15 mins for the staining of reticulum to complete. The stained sample could be tested using CELL-DYN Ruby. The stained sample will be aspirated in the analyzer and diluted with the Reagent(WBC Lyse). Once diluted, the RBCs sphere due to the influence of the nonionic detergent incorporated into the staining solution. Sphering is necessary to eliminate optical orientational noise that would otherwise be introduced into the scatter measurement. The usual lytic action of the Reagent is prevented by electrolytes contained in the staining solution and the lack of the usual incubation period used in this channel during WBC analysis. Also the high New Methylene Blue concentration in the staining reagent exerts a stabilizing effects on RBCs.

During data acquisition, 0,10 and 90 degrees scatter is collected. The 0 degrees threshold is set high enough to exclude most platelets. Histogram data is used to differentiate reticulocytes, mature RBCs, platelet clumps and nucleated cells. Reticulocytes have similar 10 degree scatter to mature RBCs but differ them by exhibiting greater 90 degree scatter.

Dengue Testing using DENGUE DUO CASSETTE
The Dengue Duo Cassette is for the qualitative presumptive detection fo IgM and IgG Ab to dengue virus in human serum, plasma and whole blood. The assay can be used for the presumptive differentiation between primary and secondary infection. Positive result are presumptive and must be confirmed by virus isolation, paired serum analysis, Ag detection by immunochemistry or viral nucleic acid detection for confirmation of dengue virus infection.

Dengue, a flavivirus is found in large areas of the tropics and subtropics. Transmission is by mosquito, principally Aedes aegypto and Aedes albopictus. Dengue virus infection causes a spectrum of clinical manifestation ranging from asymptomatic to fatal haemorrhagic disease.

In the Dengue Duo Cassette, IgM and IgG are determined simultaneously using a single addition of serum, plasma or whole blood. Thus, a differentiation between primary and secondary infection can be made by a single application of serum, plasma or whole blood. In primary infections, serum IgM Ab can be detected from dengue patients as early as 3-5 days after onset of fever, generally persisting for 30-90 days, although detectable levels may be present for 8 months post-infection.

Secondary infection is characterised by high IgG levels that may or may not be accompanied by elevated IgM levels. The sensitivity of this assay has been set so that in patients with primary dengue, IgM is positive while IgG is negative. In contrast, patient with secondary infections will have a positive IgM result.

Assay Procedure
1. Add 10 micro-litre of whole blood, serum or plasma to the cirular well and allow the sample to be absorb into the specimen pad within the circular well.

2. Hold the buffer bottle vertically and 1 cm above the square well and add 2 drops of the buffer to the square well at the base of the cassette.

3. Read the result exactly after 15 mins after adding the buffer.

Princple
When present in the patient sample, dengue-specific IgM or IgG Ab bind to the anti-human IgM or IgG Ab immobilized in two lines across the cassette membrane. Colloidal gold complexes containing recombinant dengue 1-4 Ag are captured by the bound patient's IgM or IgG to give visible pink line. A procedural control is included to indicate that the assay has been performed correctly.

Interpretation of Result

Primary infection

Pink bands appear in the IgM and Control regions

The test is positive for IgM Ab and is suggestive of primary dengue infection.

Secondary infection

Pink bands appear in the IgM, IgG and Control regions.

The test is positive for the IgM and IgG Ab and is suggestive of a secondary dengue infection.

Secondary infection

Pink bands appear in the IgG and Control region.

The test is positive for IgG Ab and is suggestive of secondary infection.

Negative

A pink band appears in the control region only.

No detectable IgG and IgM Ab to dengue. The result does not exclude dengue infection. Retest in 3-4 days if dengue infection is suspected.

Invalid

No pink band appear in the control region.

The test is invalid and should be repeated.

This are the 2 tests I have learn. Feel free to ask question.
Juexiu TG02

2007-09-09T09:56:00.000+08:00

SIP- Cytology

This month, i'm scheduled to the Cytology department in which the bulk of the specimens it deals with are mainly PAP smears, urine and body fluids (such as peritoneal, pleural and pericardial fluids, CSF).

Scope of Cytology:

Diagnosis of malignancy
Identify premalignant conditions
Identify infections and associated organisms
Evaluation of hormonal effects of estrogen and progesterone

PAP (Papanicolaou) Smearing and Staining

Purpose: Diagnosis of cervix cancer (causative agent: Human Papillomavirus).

Procedures:

After smearing on slides, specimems will be sent to the Cytology lab.
Check to make sure the patients' data on the request log tallies with that on the slides.
Load them into a rack then into the automated staining machine which has been programmed to perform PAP staining.
After staining, the slides are mounted for microscopic examination.

PAP Staining (The Regressive Method- Nuclei Overstained)

Principle: Exhibit differences in cellular morphology, maturity and metabolic activity. As intact cells in a cytologic smear tend to appear in 3-D configurations and overlap, PAP allows resultant transparency and clear definition of nuclear details.

Procedures:

Fix the cells in 95% ethanol for 15 minutes to allow the cells to absorb the dyes in the subsequent staining procedures which are fully automated.
Unacidified haematoxylin
Blue in water (nuclear staining)
Dilute hydrochloric acid (remove background staining caused by excess haematoxyin)
Rinse in water(3 rounds to set the haematoxylin)
95% alcohol (3 rounds to remove all water as subsequent staining reagents are alcohol-based and water cannot mix with alcohol)
OG (Orange-G) stain (stains keratin orange)
95% alcohol (2 rounds)
EA Polychrome stain (stains mature cells- mostly cancerous cells red and immature cells green)
95% alcohol (2 rounds to provide clearer view of overlapping cells)
100% alcohol (2 rounds for final dehydration)
Xylene (2 rounds for clearing to allow microscopic examination)

Results: Nuclei- blue or black

Cytoplasm (non-keratinised)- green or blue

Cytoplasm (keratinised)- pink or orange

Red blood cells- orange

Quality Control of PAP Staining

Checking of nuclear staining

Staining is crisp and sharp
Parachromatin is clear (not muddy)

2. Cytoplasmic counterstains

Sharp and clean
3 distinct colours: red (Eosin-Y); orange (OG); green (EA)

Troubleshooting Guidelines for PAP Staining

Problems with absorption of haematoxylin due to inadequate removal of carbowax resulting in irregular and spotty staining (poor quality).

Soak in 95% ethanol for complete fixation to enable the cells to have better absorption of haematoxylin

2. Lack of contrast in nuclear staining due to poor quality of haematoxylin.

Replace water rinses used for blueing purposes
Tap water should be slightly alkaline. Acidic tap water will result in nuclear fading while heavily chlorinated tap water will bleach out the haematoxylin.

3. Problems with cytoplasmic stains due to improper rinsing of stains after OG and EA

Do not allow the slides to sit in alcohol solution following OG and EA stains as it can wash the stains away
Rotate ethanol rinses as ethanol nearest the dye becomes discoloured

4. Water droplets seen on slides due to water in xylene (xylene will appear milky)

Change the xylene
Add Silica-Gel pellets to absolute alcohol to minimize the possibility of water contamination of xylene

That's all! Enjoy your SIP! Take care! ^_^

June Tham

TG02

0505073G

Embedding and Microtomy

2007-09-03T22:27:00.000+08:00

Embedding

Introduction
After the completion of the processing cycle, the tissues are removed from the tissue processing machines to the blocking room for embedding. Each batch of blocks is tallied against the listing of the blocks for the corresponding batch to ensure that no blocks are missing before embedding.

Principles
The tissue is placed in molten paraffin (56* melting point) such that after the paraffin cools, the tissue and paraffin will form a block of sufficient consistency to cut thin sections

Materials
Embedding machine
Embedding moulds
Forceps

Embedding technique
1. Open the processing cassette carefully and view the tissue.
2. Select a mould that best corresponds to the size of the tissue.
3. Partially fill the mould with paraffin.
4. With warm forceps remove the tissue from the cassette and place it at the bottom of the mould – refer to specimen orientation below.
5. Gently press the surface of the tissue against the solidifying wax to hold it in the desired position.
6.Ensure no tissue is stuck on the lid then discard the lid of the cassette and place the bottom of the cassette firmly on the top of the mould.
7. Fill the combined mould and cassette with paraffin.
8. Place the mould on ice to solidify the wax, thus separating embedding tissue-cassette from the mould.
9. The tissue and cassette forms a paraffin block ready for sectioning.

Specimen orientation
1. Tissue sections are embedded flat to ensure that complete section is obtained.
2. Orientation should be such that the resistance the tissue offers the knife proceeds from the lesser amount towards the greater amount as the block is sectioned. This prevents the harder tissue from compressing the softer tissues and produces a smoother section.
3. There should be an adequate margin of embedding medium surrounding all sides of the tissue for maximum cutting support.
4. Tubular structures such as vas deferens, veins, arteries and fallopian tubes must be embedded such that the knife cuts across the lumen. These should be placed vertically in the mould.
5. Tissues with epithelial surfaces such as skin, intestine, gallbladder, urinary bladder and uterus must be positioned such that the plane of the section is across all tissue layers. The epithelial surface should be placed such that the plane of section is perpendicular.
6. Multiple specimens should be placed side by side close together so that call pieces can be sectioned.
7. Rectangular tissues should be orientated parallel to each other and with their long axis perpendicular to the plane of section.
8. Small bisected cysts should be embedded with the cut surface down and ensure that no air bubbles are trapped in the paraffin.
9. Muscle biopsy in 2 pieces should be embedded with one piece in a longitudinal and the other in a vertical position.

Microtomy

Introduction
Paraffin blocks from the blocking room are brought to the cutting room for sectioning

Principles
Microtomy is the use of a microtome to make thin sections for microscopy. Rotary microtomes are used in the laboratory

Materials
Room Temperature floatation bath
Warm floatation bath – 48 plus-minus 4 degrees Celsius thermostatically controlled
Cryoplate
Disposable microtome blade
Microscope glass slides
Rotatry microtome
Soft pencil

Solution preparation
1. Type II Water 2000ml

Allow the water in the floatation bath to reach 48 plus-minus 4 degrees Celsius on a thermostat before sectioning. Technologist must record this temperature on the temperature chart before sectioning

2. 1% alcohol floatation bath
95% alcohol 5ml
Type II Water 500ml

This is used to float the tissue section prior to transferring on the heated floatation bath above. It is an alternative when folds on the tissue are difficult to get rid, as alcohol having low vapour pressure will increase the surface tension when transferring the tissue onto the heated floatation bath.

Microtomy Technique
1. Rough cutting – Secure the paraffin block in the block holder of the microtome. Adjust it to ensure that it clears the knife. Readjust the block holder screws to place the block parallel to the knife if necessary. During the rough-cut, while being manually advanced, the block is repeatedly sectioned at 20 microns thickness per slice. Sectioning stops when the entire surface of the tissue is exposed. The block is then removed from the holder.
2. All blocks should be rough cut before sectioning because:

a.Dense and hard tissue that may cause nicks and blunt the knife more rapidly can be identified e.g. bone, cervix and thyroid. Such blocks could be separated from the rest and appropriately pre-treated before sectioning. These blocks should be sectioned last as they may still cause nicks and score lines to the blade.
b. Sutures and staples attached to the surgical specimens may not have been removed and can be detected during rough cutting.
c. Hard bone is identified and additional surface decalcification can be done, by placing the block face down in decalcifying agent e.g. RDO for further decalcification prior to sectioning.
d. Fibrous tissue e.g. fibroids can be softened in mollifex or 10% fabric softener for 5 minutes. The paraffin blocks are washed in water after the appropriate treatment porior to sectioning.
3. Chilling – the blocks are then placed face down on the cryoplate to chill the block to facilitate fast sectioning. This renders the block sufficiently hard for thin sections.
4. Sectioning – secure and adjust the block in a similar manner as rough cutting. Using the handwheel, section the block at 3-4 microns, by allowing the block to advance automatically. Some tissue biopsies are sectioned at different thickness.
5. Flotation- gradually lower the section or sections if a ribbon is cut, onto the water bath. If difficulty is encountered in spreading of the tissue, float the section on the alcohol bath first so as to increase the surface tension before transferring onto the water bath. Allow the section to remain on the water bath until it has spread sufficiently. Transfer the section onto a glass slide. The corresponding biopsy number is written on the frosted end of the glass slide.
6. Separation of slides – Slides for routine HE stain, special stains and unstained sections should be separated on different racks.

Serial Sections
First submission
Tissues less than 0.5cm in size
Gastric biopsies
i. 1 HE slide with 6 serial sections
Liver biopsies
i. Slide 1 – 2 consecutive complete sections for HE
ii. Slide 2 – 1 section for MT
iii. Slide 3 – 1 section for Ret
iv. Slide 4 – 2 consecutive complete sections for HE
Bone marrow
i. Section at 2um thick
ii. 1 HE slide with 6 serial sections for small specimen or 3 HE sections on 3 slides labelled VL1, VL2, VL3 for larger specimens and 1 slide for Retic
Renal biopsies
i. Section at 2um thick
ii. 10 slides with 3 sections each as follows:
1. HE on slide 2, 5 and 8
2. Pas on slide 1m 6 and 9
3. PaAg on slide 4
4. PgMT on slide 3, 7 and 10
TBLB – Trans bronchial lung bx
i. Section at 5um thick
ii. 10 slides with 2 sections each as follows:
1. HE on slide 2, 5 and 8
2. TB stain on slide 10 for TB cases only
Endomyocardial bx
i. Section at 5um thick.
ii. 10 slides with 2 sections each as follows:
1. HE on slide 4, 6, 10
2. MT on slide 7
Prostatic needle bx
i. 1 HE slide with 6 serial sections
Tissue biopsies less than 0.5cm in size not specificed above
i. 1 HE slide with 6 serial sections
Larger tissue biopsies
i. 1 HE slide
Request for variable levels
For small tissue approximately less than 0.5cm in size
i. Repeat 6 serial sections on 1 slide labelled VL 1 for HE
For larger tissue approximately more than 0.5cm in size
i. Repeat 3 serial sections on 3 slides labelled VL1, VL2 and VL3 respectively for HE
Request for special stains
Addition sections will be cut and stained as requested
For additional levels of routine biopsy specimens or irregular number of sections required, serial sections will be cut according to the request of the pathologist.
All personnel receiving verbal or phone orders must read back the entire order to verify accuracy and transcription.

Desmond Heng
TG02
0503179D

Student Internship Programme (SIP) HAEM

2007-08-24T22:28:00.000+08:00

Are you surprised that haematology department also performs urinalysis? I hope you do, because I, for one, certainly did. The difference between the urine examinations in various departments lies mainly with the type of tool used for analysis. The principles are similar in that common findings such as RBCs, WBCs, epithelial cells (EC) are counted and reported. Kova glasstic 10 has 10 chambers[able to charge 10 different urine samples] and in each of the chamber, there lies 81 small grids.

Name of Test: Urine microscope examination using Kova glasstic 10 with grid slide

Principle of Test: Undiluted and uncentrifuged urine is drawn into the Kova slide 10 chamber for microscopic examination and identification

Type of Specimen: "Early morning" or random urine, midstream clean catch samples

[Morning-voided sample preferred as it is more concentrated than random samples during the day as urine could be diluted due to increased fluid consumption; giving false picture of a patient's health]

Procedures[FYI]:

1. Check that name on form tally with sample.

2. Assign a lab number and stamp for WBC, RBC, EC grid on request form.

3. Mix urine thoroughly by swirling several times before opening.

4. Record the appearance(colour/turbidity) of sample.

5. Charge 9µl urine on the Kova glasstic slide chamber.

6. By capillary action, 6.6µl of the urine will be drawn into the chamber resulting in a homogenous suspension of the sediment. (you may ask how, but there is a tiny compartment under the chamber where excess urine goes and only the stipulated amount of urine will be held in the chamber)

7. Allow 10 minutes for the cells to settle. (as urine continues to flow within the chamber)

8. Examine for urinary sediments and quantitate casts/crystals at low power 100x

9. Quantitate all cells at high power 400x. Count the cells within the lines of the small 0.33mm square grid.

a) For low cell count sample: count the total cells of each specific type contained in 36 small grids of the counting grid. Write the values beside the stamp 'WBC', 'RBC' and EC accordingly and write 36 beside the stamp grid.

b) For high cell count sample: count the total cells of each specific type contained in 10 small grids of the counting grid. Write the values beside the stamp 'WBC', 'RBC' and EC accordingly and write 10 beside the stamp grid.
c) For very high cell count sample: count the total cells of each specific type contained in 1 small grid. Write the values beside the stamp 'WBC', 'RBC' and EC accordingly and write 1 beside the stamp grid.

10. Enter the reported values and the LIS system will compute the resules and convert the values to per µl.

11. Transfer the computed values to the request form on the appropriate space.

Reference ranges:

WBC: 0-6/ µl

RBC: 0-3/ µl

-Alex Tan Tg02 0503222B

2007-08-24T20:00:00.000+08:00

To dorothy

Flow cytometry is a technology that stimulataneously measures and then analyses multiple physical characteristics of single particles, usually cells as they flow in a fluid stream through a beam of light. The properties include the particle's size, granularity and fluorescence intensity. samples are incubated with monoclonal antibodies conjugated with flurochrome which are excited by laser.Physical properties are measured as the flurochrome tagged cells pass through the laser light.

reference range of CD4 : 28.2-50.7%
reference range of CD8: 12.5-38.5

To kangting

here's the ans

reference range if CD4 : 28.2-50.7
reference range of CD8:12.5-38.5

To phuiyuen

This test can be performed after renal transplant to see whether is there any sign of rejection by the recipient. CD4 level should be dropping and CD8 level on the rise.

To zahirah

CD4 T-cells are a type of lymphocyte (which is one type of white blood cell). They initiate the immune system’s response to viruses such as HIV. CD4 cells can be infected and killed by HIV, so the CD4 count generally declines. CD8 T-cells, on the other hand, are the immune system’s killers of abnormal or infected body cells. As HIV infection worsens, the number of CD8 cells present in the body tends to increase.

Flurochorme labeled antibodies are used such as FITC and PE.

To jiahao

Ya. it is also possible for leukaemia.

Take b-cell type Chronic lymphocytic leukaemia as an example, the cell markers are CD5, CD20,CD52,CD23,CD19 and CD37.

To Michelle

In this case, i am talking about CD4 and CD8, these two surface markers. CD4 is for T- helper cells and CD8 for T-supressor cells which both have their respectively specific functions which i mentioned in my post. As for the subgroups, CD4 and CD8 are actually subgroups of mature T lymphocytes and the surface marker for it is CD3. CD4 and CD8 are subsets of CD3.

To Yvonne

well, this test is not a confirmatory test for HIV. other tests are needed to carry out to confirm the diagnosis.

2007-08-18T14:46:00.000+08:00

HELLO!

section posted: flow cytometry lab

Name of test: CD 4 and CD 8 count

Purpose of the test : it is used as a diagnostic and prognostic tool for clinical classification of immunodeficiency states such as autoimmune state and immune monitoring of responses to therapy such as to monitor progression of AIDS by measuring CD4 and CD 8 levels.
CD4 is expressed by T-Helper cells (collaborate with B cells promoting their proliferation and promote t cytotoxic cells development.)
CD8 is expressed by T- suppressor cells (suppress immune responses)

Principle of the test: flow cytometry has been commonly used for the measurement of surface marker antigens by fluorescent labeled monoclonal antibody which will pass through laser beam generating scattered and fluorescent signal that can be processed by computers. These surface makers are related to specific functions and subgroups.

Clinical significance: CD4 and CD 8 measurement are used to monitor progression of AIDS as CD4 cells are depleted by HIV infection whereas CD8 cells persist.

well, that's all! have fun for sip..

kai lin
0503211E
tg02

Rountine Laboratory

2007-08-12T14:38:00.000+08:00

I was posted to the routine laboratory which deal with all body fluid, these include urine, peritoneal fluid, CSF and semen as well. Today i will be posting about semen analysis which is a pretty interesting topic itself.

Semen analysis is the testing for male infertility problems. This test provide information about the quality and quantity of the sperms. The semen sample is analysed for volume, viscosity, pH and colour of the ejaculate, sperm concentration, motility and morphology. It is also examine for presence of RBC or WB which is often an indicator of infection, inflammation.

The ejaculate should be obtained after at least 3 days of abstinence from sexual intercourse and is best produced by masturbation. The entire ejaculater is collected in a clean sterile container. Condom must NOT be used as it is spermicidal. The sample must be examined within 1-2 hours after collection.

Procedure:
a. Measure the volume received
b. Note the appearance; record and abnormalities such as change in colour, decreased in turbidity or presence of blood.
c. Note the change in viscosity.
d. SQA 11 C- P will provide information on progressive motility and normal morphology
e. Aggluntination- exmaine under microscope for clumping of spermatoza
f. viability:
1) Add a drop of seminal fluid to 1 drop of eosin stain in a 75x 10 mm test tube
2) Mix and stand for 5 minutes
3) Examine under 40 x objective for viable spermatoza. Dead spermatoza will be stained pink
g. pH - Dip a pH indicator strip into the seminal fluid and record the result using the standarh chart
h. Cell count (density)
1) Mix the seminal fluid thoroughtly and dilute fluid to a 1:20 concentration
2) Mix and count in KOVA slide
i. % motility -Mix specimen well. Place a drop into the glass slide and read microscopically. ( Note the quality of motility and progression)

Interpretation
a. Volume: norma volume ranges between 1.5-6.0ml
b. Appearance:
1) Colour- normal semen appear grey to white opalescent fluid. Different colour may indicate problem
2) Viscosity - hyper viscosity may impair sperm motility
3) Liquefaction - Normal time frame is 10-30 minutes.
c. Normal sperm count is > 20 x 10^6sperm/ml
d. Motility - sperm motility study identifies the number if motile sperm seen in an ejaculate specimen.
e. Morphology - Nomal sperm have oval head shapes, an intact central and an uncoiled single tail.
f. Aggluntination - presence of aggluntination can be caused by antisperm antibody.
g. pH - normal pH should range from 7.2-8.0

Ching Wei
0503288C

SIP online sharing

2007-08-05T21:46:00.000+08:00

Hihi to all. Sorry for the late posting as I couldnt log in due to the cookies. It is the 6th week of SIP, time really fly. For the past 6 weeks I been went to Processing, Biochemistry and Immunoassy Section. For the biochemistry and immunoassay section, all of the tests is done by analyser. While processing is understandable is to order entry

For this sharing, I will talk more about immunoassay section. I will be sharing some guideline on HIV screening & confirmation tests and BDvacutainer uses.

In the immunoassay section, the analyser use is the Architect ci2000. This machines uses the chemiluminescent microparticle immunoassay technololgy to determine the presence of Ag, Ab and analytes in the sample.

The BDVacutainer System

Cap Colour: Red
Additives: None
Effects on Specimen: Blood clots, and the serum is separated by centrifugation
Uses: Chemistries, Immunology and Serology, Blood Bank (Crossmatch)

Cap Colour: Gold
Additives: Separating gel and clot activator
Effects on Specimen: Serum separator tube (SST) contains a gel at the bottom to separate blood from serum on centrifugation
Uses: Serology, endocrine, immunology, including HIV

Cap Colour: Light green
Additives: Plasma Separating Tube (Na Heparin)
Effects on Specimen: Anticoagulants with lithium heparin; Plasma is separated with PST gel at the bottom of the tube
Uses: Chemistries

Cap Colour: Lavender/Purple
Additives: EDTA (liquid form)
Effects on Specimen: Forms calcium salts to remove calcium to prevent clotting and platelet clumping
Uses: Hematology

Cap Colour: Light blue
Additives: Sodium citrate (Na Citrate)
Effects on Specimen: Forms calcium salts to remove calcium
Uses: Coagulation tests (PT, PTT)

Cap Colour: Dark green
Additives: Sodium heparin or lithium heparin
Effects on Specimen: Inactivates thrombin and thromboplastin
Uses: Ammonia, lactate, HLA typing

Cap Colour: Dark Blue
Additives: Sodium heparin
Effects on Specimen: Forms calcium salts. Tube is designed to contain no contaminating metals
Uses: Toxicology and trace element testing (zinc, copper, lead, mercury) and drug level testing

Cap Colour: Light Gray
Additives: Sodium fluoride and potassium oxalate
Effects on Specimen: Antiglycolytic agent preserves glucose
Uses: For lithium level, use sodium heparin. Glucose test.

Guideline to interpretation of result of HIV Screening and Western blot confirmation tests

HIV Screen: Non-reactive

Interpretation: A non-reactive HIV Ab screening test does not necessary exclude the possibility of infection with HIV. If exposure to HIV is suspected and thus serum is taken less than 3 months after this exposure, this should be retested after that time.

HIV Screen: Reactive

HIV Ab Confirmation: Negative

Interpretation: This result is commonly due to the presence of non-specific Ab, but occasionally may be seen in very early HIV infection. If exposure to HIV is suspected, 10ml blood in EDTA is send in 2-4 weeks time for repeat screening. For female patients in late pregnancy, the repeat sample is sent immediately.

(Rationale: HIV Ag is detected 1 to 2 weeks earlier than HIV Ab, thus making HIV screening test more sensitive than the western blot in the window period)

HIV Screen: Reactive
HIV Ab Confirmation: Indeterminate
Interpretation: This reactivity pattern commonly occurs in uninfected individuals due to the presence of non-specific Ab but may also be observed in early HIV infection. If exposure to HIV is suspected, 10ml blood in EDTA is send in 2-4 weeks time for repeat screening. Otherwise, another serum sample taken 6 weekslater will permit diagnosis in the majority cases.
Note: If a second sample is taken after 6 weeks and the western blot remain unchanged(still indeterminate), the following will be the interpretation applies:
- This indeterminate western blot profile has remain unchanged over 6 weeks. This may indicate a negative HIV Ab status associated with the lack of clinical signs and symptoms and/or supported by a negative history of exposure. If repeated exposure is suspected after the first sample is collected, send another sample for repeating screening.

HIV Screening: Reactive
HIV Ab Confirmation: Inconclusive
Interpretation: This is an indeterminate profile that include Ab to envelope and/or polymerase genes, which one often more sensitive indicators of earlu seroconversion. This profile may however also be due to non-specific Ab. 10ml blood in EDTA is send in 2-4weeks time to resolve the result.

HIV Screening: Reactive
HIV Ab Confirmation: Positive
Interpretation: Patient is consider HIV Ab positive

To confirm whether HIV positive or negative take a long procedure. And HIV Ab confirmation is negative does not mean negative too. As to test for HIV Ab confirmation take a period of time after exposure.

Feel free to ask qn but pls be mercy.

Juexiu Tg02

SIP- Histopathology

2007-07-28T09:37:00.000+08:00

Hey everyone! I'm attached to Histopathology for the whole of 20 weeks. This week, i'm scheduled to do special staining for one month. Most specimens will undergo routine H&E (Haematoxylin & Eosin) stanining while some will need special stains for diagnosis.

After fishing of specimens onto slides, excess water must be drained out before placing on the hotplate for 3 minutes to melt off the wax. Then, it can be loaded into a rack for staining.

H&E staining
Principle: The purpose is to demonstrate the tissue constituents in contrasting colours. Hamatoxylin gis oxidised to haematein in the presence of an oxidising agent. Haematein is taken up by nucleic acids present in the nuclues in the presence of a mordant which are usually metallic salts that is able to chelate the haematein dye to the tissue component. Eosin stains the cytoplasm and connective tissue pink.
For routine H&E staining, the process is fully automated with the help of this machine (Leica Autostainer XL):

Extracted from:http://www.spencerscicorp.com/html/slidestain.html

This is how it works: After loading the rack into the machine, press LOAD and check that the machine is programmed to routine H&E staining. After that, the whole staining process will be carry out by the machine in this format:

1) Xylene--> 2 minutes

2) Xylene--> 2 minutes

* Steps 1 & 2- remove wax that has not melt off as wax are not water soluble.

3) Abs alcohol--> 1 minute

4) 95% alcohol--> 1 minute

5) 70% alcohol--> 1 minute

*Steps 3-5- remove xylene and rehydrate the specimens

6) Water--> 1 minute

*As haematoxylin (Hx) is aqueous based, must wash with water first

7) Hx--> 4 minutes

8) Hx--> 3 minutes

* Steps 7 & 8- stain the whole specimen 9)

9) Water--> 1/2 minute

10) 0.5% acid alcohol--> 2 dips

* Differentiation- remove excess Hx
11) Water--> 1 minute

12) Lithium carbonate--> 2 minutes

* An alkali which blue the specimen when place in water

13) Water--> 2 miuntes

* Blue in water

14) Water--> 3 minutes

15) Eosin--> 1/2 minutes

* Stains cytoplasm

16) 70% alcohol--> 1 minute

17) 95% alcohol--> 1 minute

18) 4 rounds of abs alcohol--> each for 1 minute

* Steps 16-18- dehydration as water cannot mix with xylene)

19) 3 rounds of xylene--> each for 1 minute

* Remove alcohol as depex used for mounting is xylene-based and also to raise refractive index of tissue

Results:

Nucleur component- blue
Cytoplasmic component- red

After the above process, the specimens will be send for mounting which is also automated.

Special staining

Principle: Some cases need special stains for diagnosis. H&E is not able to demonstrate the desired components.

Types of special stains:

Periodic Acid Schiff

GMS Fungus

Reticullum ll

Alcian Blue

Iron

Giemsa

Congo Red

Gram

Procedures:
1) Print barcode label(s) (according to biopsy number assgined to the specimen)
2) Paste lable(s) on the frosted end of the slide
3) Dewax and hydrate slide
4) Load the slide covered with special wash solution to prevent the specimen from drying up into the instument
5) Load appropriate stain kit into the reagent carousel (depending on the type of stains)
6) Select Run
* Pre-checklist will appear. Ensure all slides and reagent kit are in place. Cap of reagent kit must be open.
7) Enter number of slides and hit the Run button
The machine will now initiate the staining by reading the slide and reagent barcode labels. After the barcodes are read, information for the staining will be 'downloaded' from the NexES computer to the staining module and the run will resume. When the run has completed, click 'SIGN OFF' and remove the slides from the instument. Drain slides then load into rack and place in 95% alcohol to remove residual liquid before dehydration, clearing and mounting.

Manual Staining
Principle: The Ventana NexES machine are not able to carry out some special stains. An example is the Ziehl Neelsen staining. (I only perform this manual staining so far)

Tuberculosis Test/ Ziehl Neelsen:
Principle:To demomonstrate acid fast bacteria belonging to the genus mycobacterium responsible for causing tuberculosis. (diagnosis test for tuberculosis)

Control: Any tissue containing acid fast organism.

Reagents required:

Commercial TB colour carbo reagent--> stains tubercle bacilli red
Loeffler's Methylene Blue--> background staining
1% potassium hydroxide
1% acid alcohol

Procedures:
1) Dewax and bring sections to water
2) Stain with commercial TB colour carbo reagent for 5 minutes
3) Wash in running water
4) Differentiate with 2 rounds of 1% acid alcohol until colourless (patient is negative), light pink (patient is positive)
5) Wash in water
6) Counterstain with 1% Loeffler's methylene blue for 10-15 seconds
7) Wash in water and go to 95% alcohol to control intensity of the blue colour
*if very blue, go to 70% alcohol (the more diluted the alcohol, the greater the differentiation)
8) Check under microscope, if too much blue has gone off due to differentiation, repeat steps 6&7
9) Dehydrate in absolute alcohol, clear in 3 rounds of xylene and mount

Results:
Positve- red tubercle bacilli on blue background
Negative- sky blue

Finally, its the end of my long posting. LOL. Feel free to ask me any easy questions. Hope you guys continue to learn more from SIP! Take care.=)

June Tham
TG02
0505073G

Cytology Pictures

2007-07-21T20:09:00.000+08:00

Centrifugers

Safety Cabinet Level 2 b

Safety Cabinet Level 2 b

Virkon solution

Autostainer

0.5% Sodium Hypochloride

Shandon reagent

Oven

Cytocentrifuge funnel, clamp, filter paper and labelled glass slides

Cytocentrifuge funnel, clamp, filter paper and labelled glass slides ready for cytocentrifugation

Disclaimer: These pictures were taken by me with permission from my supervisor; the latter has seen each picture and allowed me to use them to enhance the learning process.

Student Internship Programme - Cytology

2007-07-21T17:51:00.000+08:00

Hey people, Desmond here. I've been attached to the cytology for 4 weeks now and have been doing non-gynaecological procedures for the first 3. In this posting, I will guide you through what is done from the time a urine specimen is recieved right until it is "reported" to the cytologist.

I will be posting pictures soon and feel free to ask any EASY questions =].

Subject title
Pathology (Cytology)

Aims
Cytology testing of Urine for malignancy

Introduction
The principle of cytology testing of urine is to differentiate the nucleus and cytoplasm of the cells to detect malignancy by comparing the Nucleus: Cytoplasm ratio (N:C ratio). The Papanicolaou staining method is used for this. It is a polychrome staining reaction (staining the cytoplasm of different cells different colours) designed to exhibit differences in cellular morphology, maturity and metabolic activity. Because intact cells in a cytological smear tend to overlap and some appear in 3D configurations, the greatest value of the Pap staining method are the resultant transparency of he cells and clear definition of nuclear detail.

Urine however has small number of cells; Cytocentrifugation is needed to pull the cells into a confined/defined region on the glass slide.

Materials
Glass slides
Depex
Xylene
Autostainer
Centrifuge
Cytocentrifuge
Cytocentrifuge funnel and clamp
10ml centrifuge tube
22mm by 22mm coverlips
Labels/Stickers
Shandon reagent
0.5% sodium hypochloride
Virkon solution

Methods
(a) Receiving Specimens (Urine)

1. Scan the time onto the patient’s form
2. Fill in the type of specimen and initials of the person receiving the specimen (e.g. DH)
3. Label the patient’s form with pre-made stickers which are chronologically numbered and bar-coded.
4. Assign a cytologist to the case and record details like case number and the cytologist assigned to the case.

(b) Processing specimen (urine)

1. Enter Biosafety Cabinet Level 2.
2. Remove the container with the patient’s urine from the biohazard bag
3. Compare IC numbers on patient form and the container
4. Label the container and a 10ml centrifuge tube with the pre-made stickers (e.g. NG1234/07)
5. Indicate on the patient’s form the following:
Volume of urine
Condition of urine (e.g. Colourless, bloody or yellowish)
Initials of the person processing the urine
Date
Number and type of stains required (in this case 2 Pap stains)
6. Gently mix the urine by overturning the container
7. Aliquot about 10ml of urine into the 10ml centrifuge tube
8. Centrifuge the urine for 10 minutes at 2000rpm.
9. Prepare 2 labelled glass slides for cytocentrifugation
10. Decant the supernatant into 0.5% Sodium Hypochloride
11. Add 6 drops of Shandon reagent to the cell pellet.
12. Mix by pipetting up and down
13. Add 3 drops of the mixture of cells and Shandon into the cytocentrifuge funnel.
14. Cytocentrifuge for 6 minutes at 800rpm
15. After cytocentrifugation, fix the glass slides in 95% alcohol.

(c) Staining

1. After fixation for 20 to 30 minutes, load the glass slides onto a staining rack.
2. Load the rack into the autostainer
3. Set the autostainer to the Papanicolaou staining programme.

(d) Mounting

1. After staining, mount the 2 glass slides.
2. Dip the glass slide into xylene
3. Add a drop of depex to the stained side of the glass slide
4. Place a coverlip on the Depex
5. Press out any air bubbles
6. Place the glass slides into an oven (45 degrees Celsius) to let the Depex solidfy (5 to 10 minutes)

(e) Microscopic observation and reporting

1. Observe under microscope to determine if there is an adequate number of cells or staining for the cytologist to detect any abnormalities/malignancy

2. Report the case including the patient’s form and glass slides to the cytologist to detect abnormalities (malignancy)

Results
(Reference)
Nuclei – Blue
Acidophillic cells – Red
Basophilic cells – Blue-green
Erythrocytes – Orange-red

(Urine tested)
Nucleus appears very large when compared to cytoplasm. There are multinucleated cells and the nuclei appear to be intensively stained blue-green.

Discussion

The patient’s Cells are malignant. There is nuclear enlargement without an increase in the overall size of the cell, giving a decreased cytoplasm to nuclear ratio. There is also hyperchromasia due to increased amounts of DNA. The nuclear outline appears irregular and there is a variation in size and shape. Moreover, nucleoli increase in size and number. Abnormal cell division results in multinucleation. Finally, there is uneven distribution and variation in size and chromatin particles.

Desmond Heng Chih Pheng

0503179D

TG02

Long-awaited answers!

2007-07-19T23:09:00.000+08:00

....

Despite my despairing pleas, everyone didn't heed them and -still- posed tough questions for me! sigh.. and here goes,

Phui Yuen: hahaha yeah countless PBFs done, but there's still room for improvement! regarding your qns 'under what disease/clinical presentation is ESR done? or that ESR is just a general routine test done?', ESR isn't considered a general routine test but a miscellaneous(single) test ordered separately by the consultant himself/herself. ESR, as stated, is a nonspecific screening test whose results do not confirm a diagnosis of diseases. for eg, a patient can have a normal ESR readings and still have a problem. so yeah, basically it's up to the consultant to decide if he/she wants the test done to aid in diagnosis but that will be more towards clinical aspect.

Azhar: 'Since you said that the erythrocyte sedimentation process can be affected by a number of factors, is there any special equipment that is used to minimise these factors?'

hm actually there isn't any special equipment. perhaps the special and most important one is the ESR stand to support the tube in a vertical position for a more reliable and accurate reading. if you had noticed in the factors that i've stated, most of them would largely lean towards human variation(errors). also, the ESR stand is secured at the middle of the table and not near the ends where the alignment may not be equal and certainly not near any centrifuges.

Eugene & Nisha: hahaha. 'what's hairy cells?' they are cells with 'hairs'. ok lame :P the image is taken from a previous control slide but the presence of hairy cells in the blood basically shows that the patient is suffering from hairy cell leukemia(hcl), and hcl is classified under chronic lymphoid leukemia and a b-cell disease. the abnormal b-cell will have hairlike cytoplasmic projections on its surface. i dare not go into details for i'm not too sure myself!

Doreen: yeah my lab uses hematek2000 stainer but occasionally, manual staining using leishman stain. that's during time constraints. as for FBC report reflecting low platelet count, the follow-up actions will be to check for fibrin clot in the sample using wooden applicator sticks, any presence of giant platelets or platelet clumps in the blood film diagnosis which will be screened by the senior med techs and after which, they can proceed by doing manual differential count when necessary and report significant RBC and platelet findings.

Yeng Ting: to your first qns 'Rouleaux formation is the stacking of erythrocytes? Is the settling of erythrocytes equal to rouleaux formation?' yes, rouleaux formation is the stacking of RBCs atop one another. in ESR, the basic principle of the test is for the RBCs to form rouleaux and as the complex gets 'heavier', it generally settles down into what we call the packed cell volume. to your second qns 'In what ways can the ESR results be affected?' let me put this simply with a patient with sickle cell anemia as an example, sickle cells unlike normal RBCs are shaped like crescents. these sickle cells will still settle down but unable to form a typical rouleaux like normal RBCs probably with 'gaps' between them. so, going back to the principle of ESR, results will be affected by the structural variations of cells and in this case, give a decreased value.

Ying Ying: hahaha. i didn't see this coming. 'Why perpendicular and not vertical?' define perpendicular and it has the meaning of being vertical and upright in the sense that the 90-degree angle is created by the table surface and the ESR tube. so, simply put it this way, perpendicular in this case equals vertical!

Eunice: !!! 'Why are there 2 reference ranges for females?' the reference ranges listed were obtained locally based on results of healthy individuals. there being 2 ranges for females divided at about 50 years are probably due to normal physiology of females like menopause etc. there are significant changes of the mean result of females at <50>51 years during the tabulation of data and so the reference ranges were divided as so for females.

Lizzie: really? your place received over a thousand samples for ESR?! wow. hahaha.

ESR here are done manually. whether it is done before or after FBC totally depends on the amount of blood sample received. ideally, it would be done before FBC due to the waiting time of an hour but if there aren't enough blood in the sample to run both FBC and ESR, running FBC will be the priority.

Chaur Lee: yeah there certainly will be interference of results after 60 minutes as RBCs will still continue to settle down beyond the time frame resulting in false high readings. here, the count-down timer is a med tech's best friend. the reading is taken plus/minus 1 minute of the 60 minutes when the alarm sounds, so there is virtually a remote possibility that these results are not used. however, it's human to err so you wouldn't want to discount human errors if it happens? and yup, variations in RBC affect ESR results but in this case, ESR being a miscellaneous test is ordered by the consultant so maybe it's fair to say that we do our jobs by performing the test and report the results. :P

Wing Fat: 'What is platelet satellitism?' i must admit this is a new term to me when i saw the control slide and took a pic to share with you guys! how could you do this to me.. but yeah after some research(thanks ah), platelet satellitism is characterized by platelets forming around polymorphonuclear leucocytes(neutrophils) and are seen in blood films stained by Wright's stain prepared from EDTA-anticoagulated blood samples and not seen in other anticoagulants like sodium citrate, heparin etc. wouldn't go into details as this is only what i've learnt! thanks ah wing fat.

Jiaxin: hahaha. i suppose if the company is paying for your living, it is a must to learn to do everything. us as interns, if the supervisor deems you fit as to run patient samples, you are being treated like one of them and will be good to help around to your abilities and learn at the same time. so, the answer to your question is DO EVERYTHING!

Juexiu: for panic alarms such as excessively abnormal levels of platelets, haemoglobin and total white, we have to inform the wards immediately. while for general abnormal results from tests, we report the results and include comments with it.

Ci Liang: yeah, variations in RBC sizes affect the ESR results. i asked a senior med tech and she told me ESR for children were done mostly done in the western countries and it had been done here in the past, but not at present. but i've also found a reference range for children of 0-10mm/hr developed at Baptist Memorial Health Care Corporation, Memphis, Tennessee. hopefully it helps. :D

Peishan: this question got me thinking for quite a while, and i actually set up an experiment today just for you! it is definitely important to mix the ESR tube thoroughly before setting the test up to achieve a state of homogeneity between the components of blood. if blood is not mixed well, RBCs will settle at the bottom and form 2 clear distinct layers of plasma and RBCs. from what i've observed from my experiment, setting up the ESR test on blood that was not mixed(tube 1) will therefore allow the plasma region to be catapulted right to the top of the tube in place of homogenized blood from the control(normal ESR/tube 2). from the start, in tube 1, it can be observed that it isn't a fair test due to the segregation of plasma and RBC layer giving a reading of about 8mm following by the RBC layer that is unevenly spread while in tube 2, the reading is at the zero mark. after 60 minutes, the readings which i recorded were 85mm/hr and 32mm/hr in tubes 1 and 2 respectively. this clearly shows that the tube without mixing gives a false high result. after discussing on the findings with a senior med tech, it can be said that in the tube 1, some areas of the red cell layer had already settled down which MAY had formed rouleaux. with this uneven distribution of weight in the vertical ESR tube, say at the centre, it may force the RBCs further down along with gravitational pull which will give a high reading come the end of 60 minutes. these are of course, based on my observations and discussion, i really hope you did understand what i'm trying to bring. perhaps mr alvin poh[if he does read] can explain why?

Joan: both primary and secondary controls are commercial controls, given by the manufacturer of the automation. in the morning, the 3 levels of control (low/medium/high) is run in the machine before running patient samples to ensure that results fall within the manufacturer's given reference ranges. running secondary control also utilises the commercial controls but the difference is that only 1 of the 3 levels are used at the given time intervals. this is part of internal quality control and it also functions to keep patients' results in check within the reference ranges and that results are quality-assured.

Sharifah: hahaha in the lab, the ESR station is secluded in the middle of a single table. only materials involved in ESR are placed there, namely ESR stand, ESR tubes, timer, record book etc. the station is usually left alone after setting the ESR tests and no other materials known to cause vibrations are placed there. the only vibrations then, can only be due to people treating the table like a drum! but trust me, why play at the station? it's no fun! :P

AHHHHHHHHH! and trust me, answering questions are no fun! but of course, end of the day, i hope i did help in easing those lingering doubts that you once had and learnt something from my experience!

case closed! *wide grins :D* till my next post.. have fun everyone!

Peek-tures

2007-07-14T12:07:00.000+08:00

Mixing of blood to prevent clotting

Patient with chronic granulocytic leukaemia

(Note the huge amounts of total white cells

as well as different stages of differentiating cells)

Hairy cells

Platelet satellitism
(Clumps of platelets adhering to neutrophils)

ESR station

Images released with the permission of company supervisor

- Alex

Student Internship Programme (SIP) HAEM

2007-07-13T23:12:00.000+08:00

Howdy my coursemates!

I've been scheduled to the routine lab of the haematology department for the first 4 weeks (not first6weeks :D). It's not named the routine lab for nothing and surely there is always going to be a general routine flowchart to follow. So, here goes (a brief one)!

1) Starting up the automation at the start of operating hours (8am), daily maintenance check, run quality control (QC)
2) Run commercial controls a.k.a primary controls (low/medium/high) during startup and secondary controls (either one of low/medium/high) at specific intervals, say 10am, 12noon, 2pm
3) Collect patient samples and test request forms at the reception and run various tests
4) File unverify automatch results
5) Delta check prompt? (it means a significant difference in current and previous results spotted by the LIS in any tests done in the same individual patient)
6) Screen blood films (BF)
7) Full blood count (FBC) result tally with BF diagnosis?
8) Enter and verify results in LIS

In case you're wondering what sort of tests are done in the routine lab, there are full blood count, miscellaneous tests like

- Retics
- Haemoglobin (Hb)
- Malarial parasite (MP)
- Erythrocyte sedimentation rate (ESR)
- Platelet
- White blood cell (WBC)
- HbH test etc..

The test that I've chosen to elaborate will be the ESR.

Name of Test: Erythrocyte Sedimentation Rate (ESR)

Principle of Test: ESR test measures the settling of erythrocytes in human plasma over a specific time period, usually 60 minutes. The reported numerical value is derived from measuring the distance (millimeters) from the bottom of the surface meniscus to the top of the erythrocyte sediment in a column of anticoagulated blood that has remained perpendicular in a special-purpose pipette (solely for ESR usage) for 60 minutes.

A typical sigmoid curve resulting from the descent of the plasma-red interface plotted against time distinguishes three phases of ESR. The lag phase (initial portion of curve) reflects the period where individual erythrocytes form rouleaux. During the decantation phase (second portion), the plasma-red interface falls more rapidly. In the final phase, the cell aggregate pile up on the bottom of the tube but may not always be evident within the 60 minutes time frame. The final ESR test result, read at 60 minutes, will therefore include varying contributions from the three phases.

The erythrocyte sedimentation process can be affected by a number of factors such as
1) incorrect amount of blood for dilution, 2) air bubbles in ESR tube, 3) inadequate mixing of sample before and after dilution, 4) failure to ensure vertical alignment of tubes and 5) vibration.

Conditions such as variations in erythrocyte shape for eg, sickle-cell formation or acanthocytosis (mixed erythrocyte shapes) will inhibit the ability of erythrocytes forming rouleaux and hence, ESR results will be affected.

Test Results with reference range:
Male - 1-10 mm/hr
Female (up to age 50 years) - 3-15 mm/hr
Female (age 51 years and above) - 3-20 mm/hr

Clinical interpretation: ESR is a nonspecific test used to detect inflammatory, neoplastic, infectious and necrotic processes which are associated with a modification of the plasma protein (primarily fibrinogen, globulins and albumin). It is not diagnostic for any particular disease or injury. It is a fairly reliable indicator of the course of disease and therefore can be used to monitor disease therapy.

I hope I've put up a fair recollection of what's happening around me and share a thing or two with everyone.

Have mercy please. Don't kill me with all the questions! :/

- Alex Tg02 0503222B

Haematology laboratory

2007-07-07T18:54:00.001+08:00

Second week of SIP

Hello everyone… I was posted to haematology department for the whole of 20 weeks. This week I was scheduled to thalassaemia lab and will be there for the next two weeks. Well, I had learnt quite a lot at the lab dealing with the blood samples that were sent in for Hb electrophoresis. Firstly, I will collect patient’s blood samples at the reception sent to them by telelift, transporter or pneumatic tubes. After which, I will do labeling on the samples and check that the patient’s data tally with the request form. Next, key the specimen no. into LIS.

Test performed: Cellulose acetate alkaline electrophoresis

Type of specimen: a minimum of 3.0ml of EDTA blood
EDTA preferred but heparin is acceptable.
Reject clotted blood and lyse specimen except for clotted cord blood (very difficult to get the blood)

Purpose: it is used as the initial procedure to screen for hemoglobin variants. The technique is sensitive to blood samples throughout the human life span (from newborns, cord blood, to old age)

Principle: Hb has a net negative charge at pH8.6 and move in an electrical filed towards the anode (positive) due to the variation in the amino acid content off different Hb, the net charge of each Hb types varies and this will determine their rate of mobility. Various supporting media such potato starch, paper and polyacrylamide gel have been used in electrophoresis. Cellulose acetate is used because it is easily available and provides a sharp resolution of the Hb bands in a short time; allow cleaning, densitometric quantitiation and permanent storage of the transparent film.

Procedures:

1) Pipette 200microlitires of blood to tubes and wash 2x
2) Pipette 25microlitres of washed packed red cells into the tube.
3) Add 150microlitres of hemolystate reagent and allow standing for 20 minutes.
4) Label the plate with specimen no. using water proof marker and place in carrying rack. ( the other side of the plate is the cellulose acetate)
5) Slowly, lower the rack into heme buffer and soak for 5 minutes. ( it must be done slowly to prevent any air bubbles)
6) Pipette 10microlitres of the patient’s blood and hemolystate into each sample well. Control is pipette into the first well. Control used is ESFA. E and S are abnormal variants. F and A represent fetal and adult respectively.
7) Prime the applicator by depressing gently into samples well 1 to 2 times and blot on a piece of filter paper.
8) Remove the wetted cellulose acetate and blot dry
9) Place the plate in the aligning base with cellulose acetate side up
10) Transfer the applicator to the aligning base
11) Apply samples to plate (on the cellulose acetate) by the contacting the tips (“teeth”) of the applicator to the plate and hold for 5 seconds.
12) Place the plate in electrophoresis chamber with cellulose acetate side down
13) Electrophoresis the plate for 25 minutes at 350volts

Staining of bands

1) The plate is stained in ponceau S stain for 5 minutes
2) Destain in 3 successive washes of 5% acetic acid (2 minutes for each wash)

Evaluation of bands

For qualitative evaluation, the haemoglobin is inspected visually for the presence of abnormal haeomglobin bands. EFSA control provides a marker for band identification

For quantative evaluation, the relative percentage of each haemoglobin band is determined by scanning the plate in the densitometer using 525nm filter.

Record results

taken from http://www.dokkyomed.ac.jp/dep-k/cli-path/a-super/h25.html

Test performed: sickling test

Purpose: used to demo the sickling phenomenon in test samples and a confirmatory test following alkaline cellulose acetate electrophoresis

Procedure:

1) 25microlitres of reagent is added to 5microlitres of EDTA blood used the pipette tip to mix to a coin shaped
2) cover with cover glass and left dry
3) seal with grease and incubate at 37degrees for about 2 hours
4) examine microscopically
5) record results

Quality control: include a negative and positive control to check that reagents are okay.

That’s all… have fun for SIP yea :)

kai lin

0503211E

tg02

Microbiology Laboratory

2007-07-01T12:02:00.000+08:00

I was posted to microbiology laboratory for 2 week. Read and understood the laboratory safety manual and general protocol.

Was sent to the processing bench for the first 2 days. I learnt how to clocked in the various samples that was sent to the microbiology laboratory to verify the time that the samples were received. I was taught how to differentiate the various nature of the specimen so as to allocate them to the various benches for tests. The blood bottles was first incubated in an incubator for to check for sign of mircoorganism growing in the bottle. The type of test is denoted by the colour of the cap. Blue represent aerobic culture, golden represent anaerobic culture while red represent fungal culture. Urine specimen regardless of catheter or mid-stream urine was first culture in a blood and macConkey spilt-well agar. It is noted that streaking must be done on blood agar first before macConkey so as to avoid carry-over effect from macConkey agar to blood. MacConkey agar will encourage growth of gram-negative bacteria. Specimen that were not urine or blood were labeled as miscellenous item. All swab of superficial wound will be culture on blood and macConkey agar, while addition culture on PEA plate to be carried out if swab of site is below waist. PEA agar encourage growth of gram-positive bacteria. Type of culture to be carried out depend on appearance of stool. If it is a normal stool, culture will be carried out on macConeky, Salmonella-shigella agar and selenite broth. Addition culture will be carried out on watery stool. They are TSA agar with 5% sheep agar, thiosulphate Citrate Bile Salt sucrose agar and alkanline peptone water. These agar will help to detect the presence of Vibro which are usually associated with watery stool.

Supervisor then allocated me to the microscopy bench on the third day to observe the various procedure. I was taught to differentiate the appearance for the respiratory specimen. It may be mucoid, plurelent, watery or blood-stain. Depending on what type of stain the doctor request, the medical techologist will then process the request. Usually gram staining is carried out. Slide is stained with crystal violet for one minute and then washed. Slide is then stained with iodine for 2 minutes follow by washing and rapid decolourising by 100% acetone. Slide is lastly counter-stained with safranin for 1 mintues and then read under microscope. I was taught to identify blastoconidia and mycellium under the microscope. If acid-fast stain is request, it must be read under an immuno-fluroscent microscope in the dark room to identify mycobacellium.

I was then allocated to the urine bench on the fourth and fifth day to observe interpretration of the urine culture that was cultured on the blood and macConkey spilt-well plate. Colour and appearance of the culture will be noted to aid in the interpretattion. Usuaaly, if there is green colour on the blood agar culture, it implies culture is alpha hemolytic, and if there is pink colour culture on the macConkey agair, it implies culture is a lactose fermenter. Addition information such as whther culure is in swamps will aids in the identifcation.If culture contain less than 10^5 and is not a pure culture, no further testing will be carried out as this is usually due to contamination. Addition tests that are carried out may be radID one procedure, staph grouping kit, indole test etc.

Yeo Ching Wei
0503288C
TG02

Miscellaneous - Pictures

2007-05-23T00:25:00.000+08:00

Image 1: Borrelia burgdorferi under light microscopy (1000x with oil)

Miscellaneous - Pictures

2007-05-23T00:24:00.000+08:00

Image 2: Single Borrelia burgdorferi under transmission electron microscopy

Miscellaneous - Pictures

2007-05-23T00:23:00.000+08:00

Image 3: Life cycle of lyme disease ticks

Miscellaneous - Pictures

2007-05-23T00:22:00.000+08:00

Image 4: Pictures of ‘bulls-eye’ rashes significant of Lyme disease

Miscellaneous - Pictures

2007-05-23T00:21:00.001+08:00

Image 5: Microscopic view of Rickettsia typhi, causative agent of murine typhus

Miscellaneous - Pictures

2007-05-23T00:19:00.000+08:00

Image 6: Transmission vector of murine typhus, flea

Miscellaneous - Pictures

2007-05-23T00:15:00.000+08:00

Image 7: Transmission cycle of Rickettsia typhi from vectors to human

Miscellaneous - Picture links

2007-05-22T11:11:00.000+08:00

Image 1: Extracted from http://acpcommunity.acp.edu/Facultystaff/moon/Biology1/Bacteria

Image 2: Extracted from http://www.cdc.gov/ncidod/dvbid/lyme/ld_Borreliaburgdorferi.htm

Image 3: Extracted from http://textbookofbacteriology.net/Lyme.html

Image 4: Extracted from http://textbookofbacteriology.net/Lyme.html

Image 5: Extracted from http://www.hgsc.bcm.tmc.edu/projects/microbial/microbial/graphics/rtyphi.jpg

Image 6: Extracted from http://www.the-travel-doctor.com/typhus.htm

Image 7: Extracted from http://www.cdc.gov/ncidod/eid/vol4no2/azad.htm

Final diagnoses

2007-05-20T20:51:00.000+08:00

1) Murine typhus

Causative microorganism: Rickettsia typhi

Transmission vehicles: Lice or fleas

How we deduced: It is highly probable as the patient is living in unhygienic conditions and there is also an infestation of fleas. Moreover, the patient feeds stray dogs and cats which could have fleas carrying Rickettsia typhi.

Sample(s) taken: Blood

Collection via: Venipuncture (venous blood)

Preliminary testing: Full blood count, Blood culture

Confirmatory testing: Direct Fluorescent Antibody (DFA), Skin rash biopsy

2) Lyme disease

Causative microorganism: Borrelia burgdorferi

Transmission vehicles: Rodent ticks

How we deduced: The infestation of rats in his premise makes the probability of contracting lyme disease a reality for the patient.

Sample(s) taken: Blood

Collection via: Venipuncture (venous blood)

Preliminary testing: Physical investigations for visible rash, ELISA to detect lyme antibodies

Confirmatory testing: Lyme disease testing (Anti-borrelia burgdorferi IgM/IgG), Western Blot, Polymerase Chain Reaction (PCR)

MMIC-case study one part 2

2007-04-25T18:46:00.000+08:00

6 illnesses that we would like to further investigate and associate with regards to the old man's symptoms are:

1) Typhus: The old man lives in unsanitised condition (soiled mattress with the presence of rodents and stray animals).

2) Rat-bite fever: The old man lives in areas infested with rodents and stray animals.

3) Cellulitis: The old man was feeding stray cats and dogs and may have been scratched or bitten by them without knowing.

4) Bubonic plague: The old man has the following symptoms listed in the case study such as fever, rashes and weakness. This disease is spread among rodents by flea bites while the old man may have been infected when he came in contact with the infected animals (stray cats, dogs) and their feces.

5) Toxoplasmosis: The old man may have come in contact with the stray cats' feces which may have the protozoan intracellular parasite Toxoplasma gondii.

6) Lyme disease: As the premise of the old man is playing host to the infestations of pests and rodents such as cockroaches, fleas and rats, the disease is very much a suspected possibility. The transmission vehicles for lyme disease are through mice ticks and the symptoms for the disease are also similar to what the old man has presented (fever, rash and general weakness)

We would also like to consider the fact that the old man's age (85 years) may play a part in reduced immunity levels making him more susceptible to various possible infectious diseases.

MMIC-Case study one

2007-04-25T11:15:00.000+08:00

List of possible diseases

1) Bubonic plague
http://www.nlm.nih.gov/ > MedlinePlus > Medical Encyclopedia > Bl-Bz > Plague
http://www.nri.org/ > Project Websites > The prevention of sanitary risks.. > Rodents as Carriers of Disease
2) Toxoplasmosis
http://www.nlm.nih.gov/ > MedlinePlus > Medical Encyclopedia > To-Tz > Toxoplasmosis
http://www.nri.org/ > Project Websites > The prevention of sanitary risks.. > Rodents as Carriers of Disease
http://www.wrongdiagnosis.com/ > A - Z listing of Diseases > T > Toxoplasmosis
3) Rheumatic fever
http://www.wrongdiagnosis.com/ > A - Z listing of Diseases > R > Rheumatic fever
4) Trichinosis
http://www.wrongdiagnosis.com/ > A - Z listing of Diseases > T > Trichinosis
5) Ebola haemorrhagic fever
http://www.who.int/ > WHO sites > M > Media centre > Fact sheets > E > Ebola haemorrhagic fever
6) Rat-Bite fever
http://www.cdc.gov/ > Morbidity and Mortality Weekly Report (MMWR) > Health Topics A-Z > R > Rat-Bite Fever
http://www.cdc.gov/ > Morbidity and Mortality Weekly Report (MMWR) > Weekly Report > Past Volumes > Volume 53 (2004).. > Fatal Rat-Bite Fever..
http://www.ratfanclub.org/ > Helpful Info > Rat-Bite Fever
7) Plague
http://www.cdc.gov/ > P > Plague > Plague (Site)
8) Cellulitis
http://www.medicinenet.com/ > Diseases and conditions > C > Cellulitis > Cellulitis Main Article
9) Dengue fever
http://symptoms.wrongdiagnosis.com/ > Fever > Rash (Add symptom) > Weakness (Add symptom) > Dengue Fever (Possible Causes)
10) Typhus
http://www.pathlights.com/ > Natural Remedies Encyclopedia > 16 - Poisons > Transmitted Diseases
11) Lyme disease
http://www.nlm.nih.gov/ > MedlinePlus > Medical Encyclopedia > Lo-Lz > Lyme disease

MMIC Tutorials 1 to 4: A Simulated Case

2007-04-25T10:25:00.000+08:00

A 85-year-old male was found to be staying alone in one-room flat. It is found that his mattress was soiled with human excreta and urine. The premise is infested with cockroaches, fleas and rats. It is known to the neighbors that the elderly man has the habit of feeding stray dogs and cats in the neighborhood.

The elderly man was referred to a nursing home and presented with high fever, rash and general weakness.

How would you approach this situation in order to provide final diagnosis of the suspected microorganisms[s]?

Symptoms presented : High fever, rash, general weakness

Approaching the situation :
- Symptoms
- Suspected diagnosis
- Transmission vehicles [ >1 ]
- Causative microbes [ >1 ]

Group's Expectations

2007-04-25T09:59:00.000+08:00

GPA for this Semester: 3.5 and above

SIP learning trip: To have fun and at the same time, be exposed to the reality of working life so as to gain experience.

Projects for the first 6 weeks: To accomplish what is expected of us and at the same time, gain knowledge. Ultimately, to achieve a grade of at least B+.

Attitude: To remain positive and upbeat no matter what happens; doing whatever it takes to get the job done without compromising who we are.

Punctuality: No procrastination and a high level of self-discipline at all times