MMIC PBL 2 (VIRUSES)

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.

Possible Viral Pathogens:

Images of Viruses:

Hepatitis Virus

Adenovirus

Enterovirus

Dengue virus

References:
1. Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company
2. http://www.google.com/ > image search > "microorganism name"


By Doreen Ng
TG01

Images of Viruses:

Influenza Virus

Measles Virus

Mumps Virus

RSV

Parainfluenza Virus

Nipah Virus

Hendra Virus

Rubella (German Measles) Virus

References:
1. Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company
2. http://en.wikipedia.org/wiki/Main_Page > "microorganism name"
3. http://www.google.com/ > image search > "microorganism name"


By Dorothy Png
TG01

MMIC PBL 2 (PROTOZOA)

Possible Protozoal Pathogens:

Images of Protozoa:

Entamoeba histolytica

Acanthamoeba spp.

Giardia lamblia

References:
1) Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company
2) http://www.tulane.edu/~wiser/protozoology/notes/free.html
3) http://www.ncbi.nlm.nih.gov/books/bv.fcgi?rid=mmed.section.4267
4) http://www.google.com/ > image search > "microorganism name"

By Chong Wing Fat
TG01

Images of Protozoa:

Leishmania tropica

Toxoplasma gondii

Pneumocystis

Blastocystis hominis

Taenia solium (worms)

References:
1) http://en.wikipedia.org/wiki/Main_Page > Cutaneous_leishmaniasis
2) http://www.dpd.cdc.gov/dpdx/HTML/Blastocystis.htm
3) Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company
4) http://www.google.com/ > image search > "microorganism name"

By Khoo Boon Ching
TG01

MMIC PBL 2 (FUNGI)

Possible Fungal Pathogens:

Images of Fungi:

Histoplasma capsulatum

Sporothrix schenckii

Trichophyton rubrum

Cryptococcus neoformans

Aspergillus fumigatus

References:
1) Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company
2) http://en.wikipedia.org/wiki/Main_Page > Histoplasmosis
3) http://en.wikipedia.org/wiki/Main_Page > Sporotrichosis
4) http://en.wikipedia.org/wiki/Main_Page > Athletes_foot
5) http://en.wikipedia.org/wiki/Main_Page > Ringworm
6) http://en.wikipedia.org/wiki/Main_Page > Jock_itch
7) http://en.wikipedia.org/wiki/Main_Page > Cryptococcosis
8) http://en.wikipedia.org/wiki/Main_Page > Aspergillosis
9) http://www.google.com/ > image search > "microorganism name"


By Soong Ci Liang
TG01

Type of Fungi	Pathogenesis & pathology	Symptoms	Laboratory Diagnosis	Preventive measures
Ascomycota	Mammalian lung parasite causing Pneumocystis pneumonia. Ability to consume collogen, an abundant structural protein in animals as food sources. The disease attacks indiviuals with decreased CD4 at the interstitial, fibrous tissue of the lungs, with marked thickening of the alveolar septa and alveoli leading to significant hypoxia, which can be fatal if not treated aggressively.	- Non-productive cough - shortness of breath - weight loss and night sweats	- characteristic appearance of the chest x-ray shows widespread pulmonary infiltrates - causative organism in induced sputum or bronchial wash - low arterial oxygen level	Maintains high levels of CD4 by avoiding immuno-supressents.
Phythium	Occurrence in wet environments and summer months- Infection can also be acquired after contact with soil and grass containing Phythium insidiosum. Organism is acquired through traumatic implantation and remains localized or spreads to infect other tissues, especially arteries. Pythiosis is the disease which results from an infection of the fungal like organism Pythium insidiosum.rmation of subcutaneous lesions and the invasion of the main arteries. If not treated the infection is fatal.	Inflammation of affected extremity.	- Culture, serology, and histopathology - Hyphae of this oomycete are present in the infected tissues - Serological test such as ID and ELISA have proved to be of value for its early diagnosis.	- Amputation in the affected extremity. - Vaccines are being researched on currently.
Fusarium	Widely distributed in soil and in association with plants.- Some species produce mycotoxins in cereal crops that can affect human and animal health if they enter the food chain. Opportunistic infections in humans may occur at nails and in the cornea. In immunosupressed individual, aggressive fusarial infections penetrating the whole body and bloodstream may occur.	- Abdominal pain - Diarrhea - Vomiting - Prostration - Fever - Chills - Myalgias - Bone marrow depression with granulo-cytopenia - Pharyngeal - gastro-intestinal ulceration -vaginal bleeding -bloody diarrhea - hematuria	- FBC with low WBC suggest infection - Bacterial corneal ulcer - Tissue from corneal scraping is cytologically diagnostic.	Natamycin drug therapy

References:
1) Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company

By Douglas Yew
TG01

MMIC PBL 2 (VIRUSES)

Types of Viruses	Possibility? (Y/N)	Reasons
Herpesvirus - Herpes Simplex virus	No	- No animal reservoirs or vectors are involved with the human viruses - Transmission is by contact with infected secretions
Poxviruses - Orthopoxvirus - Parapoxvirus - Molluscipoxvirus - Yatapoxvirus	No	- Control & eradication of smallpox - Human infections are rare
Hepatitis viruses - Hepatitis A (HAV) - Hepatitis B (HBV) - Hepatitis C (HCV) - Hepatitis D (HDV) - Hepatitis E (HEV)	Yes for HAV No for HBV, HCV, HDV	Transmission of hepatitis viruses: - Person to person (HAV) - Fecal-oral (HAV & HEV - Perinatal (HBV) - Blood & blood products (HBV, HCV, HDV)
Orthomyxoviruses (Influenza virus) - Influenza A (Inlcuding H5N1 avian influenza) - Influenza B - Influenza C	Yes	- Transmission through respiratory droplets and contact with contaminated hands - Transmitted avian virus to humans through pigs or through physical contact with infected birds - Influenza A: Circulated in birds population, domestic poultry population - Onset of symptoms: Varies from 1 to 4 days
Paramyxoviruses - Measles (Rubeola) virus - Mumps virus - Respiratory syncytial virus (RSV) - Parainfluenza virus - Newcastle disease virus (avian paramyxovirus) - Nipah virus - Hendra virus - Rubella (German Measles Virus)	Yes for Measles (Rubeola) virus, Mumps virus, RSV, Parainfluenza virus, Nipah virus, Hendra virus, Rubella (German Measles Virus) No for Newcastle disease virus (avian paramyxovirus)	Measles (Rubeola) virus - Highly infectious disease - Incubation period/onset of symptoms: 8 to 12 days - Transmission via respiratory route - No animal reservoir Mumps virus - Contagious; Transmitted by direct contact, airborne droplets, or formites contaminated with saliva or urine. - Appear in hot climates and temperate climates Respiratory Syncytial virus - Spread by large droplets and direct contact - Survive on environmental surfaces for up to 6 hours Parainfluenza virus - Transmitted by direct person-to-person contact or by large-droplet aerosols - Onset of illness: About 1 week Newcastle disease virus (avian paramyxovirus) - Transmission through contact with infected birds - Eradication of Newcastle disease virus Nipah virus - Human-to-human transmission - Transmission from fruit bats/flying foxes to human through infected pigs Hendra virus - Transmission via exposure to bat urine or birthing fluids Rubella (German Measles Virus) - Affects children and young adults - Onset of symptoms: 7 to 9 days - Transmitted by respiratory route - Present in the urine, faeces and on the skin.
Picornaviruses - Enteroviruses 1. Polioviruses 2. Coxsackie viruses A & B 3. Echoviruses - Rhinoviruses - Aphthovirus	Yes for coxsackie-viruses, echoviruses, rhinoviruses & aphthovirus No for polioviruses	Coxsackieviruses - Transmitted primarily via the fecal-oral route and respiratory aerosols Echoviruses - Transmitted through ingestion of fecally contaminated materials Rhinoviruses - Transmitted to susceptible individuals by direct contact or by aerosol particles Aphthovirus - Transmission via contact or ingestion Poliovirus - Humans are the only known reservoir of infection with poliovirus. - Nowadays, most people have become immune early in life.
Arboviruses	Yes	- Transmission vector via mosquito Aedes aegypti
Human Immunodeficiency Virus (HIV)	No	- Transmission of HIV: - Blood & blood products - Sexual contact Perinatal (mother to child)
Other Human Viruses - Adenovirus - Norwalk virus - Calicivirus - Filovirus	Yes	Adenovirus - Spread by direct contact, fecal-oral route, respiratory droplets or by contaminated fomites Norwalk virus and calivirus - Both spread by fecal-oral route, food-borne, or person-to-person transmission Filovirus - Transmission via human/physical contact with infected bodily fluids although droplet infection can occur

References

1. Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company

2. http://en.wikipedia.org/wiki/Main_Page > "microorganism name"

By Doreen & Dorothy
Tg01

MMIC PBL2 (FUNGI)

List of Fungi	Possibility? (Y/N)	Reasons
Asomycetes	Yes	Decomposers which break down organic materials as dead leaves, twigs, fallen trees that are commonly present in jungle.
Basidomycetes(Cryptococcus spp.)	Yes	- Soil contaminated with pigeon droppings- Eucalyptus trees- Decaying wood forming hollows in living trees
Rhizoctomia	No	Infects and pathogenic against plants only
Phythium	Yes	- Occurrence in wet environments and summer months- Infection can also be acquired after contact with soil and grass containing Phythium insidiosum.
Fusarium	Yes	- Widely distributed in soil and in association with plants.- Some species produce mycotoxins in cereal crops that can affect human and animal health if they enter the food chain
Phytophtora	No	Infects and pathogenic against plants only

By Douglas
Tg01

MMIC PBL 2 (FUNGI)

Types of Fungal	Possibility (Y/N)	Reasons
Histoplasma capsulatum	Yes	-Soil-borne, dimorphic fungus; grows preferentially in soil enriched with bird droppings [1] -Found throughout the world but is most prevalent in countries favoring a warm, moist, and humid climate [2] -Transmitted by inhalation of airborne asexual spores [1]
Sporothrix schenckii	Yes	-Habitat is soil or vegetation -Transmission through small cuts and abrasions in the skin [1]
Trichophyton rubrum	Yes	-Habitat is human skin -Transmission of T. rubrum : - Skin-to-skin contact - Contact with contaminated items - Person to person [1]
Cryptococcus neoformans	Yes	-Habitat is soil, especially where enriched by pigeon droppings -Transmitted by inhalation of airborne yeasts cells [1]
Aspergillus fumigatus	Yes	-Habitat is soil -Transmitted by inhalation of airborne spores [1]
Coccidioides immitis	No	-Habitat is soil of arid regions like southwestern United states and Latin America [1]
Blastomyces dermatitidis	No	-Habitat is rich soil, especially in the upper midwestern region of United States [1]
Paracoccidioides brasiliensis	No	-Thermally-dimorphic fungus distributed in Brazil and South America [2]

References

1)Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company

2)http://en.wikipedia.org

Soong Ci Liang TG01

MMIC PBL2 (PROTOZOA)

Types of Protozoa	Possibility (Y/N)	Reasons
Malaria	Yes	-Widespread in tropical and subtropical regions -Possible transmission via female anopheles mosquitoes -Symptom of flu
Leishmania tropica	No	-Althought forest rodent are their main reservoirs through sandflys, it occurs mainly in Middle East, India and africa
Toxoplasma	Yes	-Possible transmission through ingesting oocytes by uncooked meat or unwashed vegetables -Symptom of flu
Pneumocystis	No	-Happens in immunocompromised individuals -Animals not a reservoirs for humans infection
Blastocystis hominis	Yes	-Possible transmission through drinking water or eating food contaminated with feces from an infected human or animal
Taenia solium (worms)	Yes	-Possible transmission through by eating raw or undercooked pork containing the larvae

Published by boonching.

Reference:

Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company

MMIC PBL2 (PROTOZOA)

Types of Protozoa	Possibility (Y/N)	Reasons
Naegleria fowleri	No	-Transmitted via inhalation -Able to enter the body of an individual from the mucous membranes while he/she is swimming -The organism can be found in fresh water lakes and soil -But infection to humans are rare
Acanthamoeba spp.	Yes	-Transmitted via inhalation into the respiratory track -Can also be transmitted via open wound -Found in soil or warm water lake -Capable of causing keratitis in individual who are using contact lens
Balamuthia mandrillaris	No	-Human infections are rare
Entamoeba histolytica	Yes	-Transmitted by ingestion of cysts by the fecal-oral route in contaminated food and water
Giardia lamblia	Yes	-Transmission occurs by ingestion of the cyst in fecally contaminated food and water
Trypanosoma such as: T. cruzi T. gambiense	No	-T. cruzi transmitted by Reduviid bug -T.gambiense transmitted by tsetse fly -Reduviid bug and tsetse fly can only be found in africa

Reference:
-Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company

Wing Fat

TG01

MMIC PBL2

Possible Pathogens

Possible Viral Diseases Found in Jungle Training

Case 1 Posting 2

Subject: MMIC
Case: Case 1 - Khong Fay Seah
Complaints: Fever, chills, dysuria,
Diagnosis: Urinary Tract Infection

List of Organisms	Tests Required to Identify	Expected Test Results	Treatment
Escherichia Coli	Culture Gram stain and Microscopy Biochemical test test – TSI, IMViC (Indole, Methyl red, Voges-Proskauer, citrate), motility	Lactose-fermenting (Yellow colonies in CLED); Flat, grey colonies on BAP Gram-negative bacilli Biochemical test TSI - Acid slant acid butt with gas but no H2S Indole – Positive (Bright red interface) Methy red – Positive (Red) Voges-Proskauer - Negative Citrate – Negative (Remains green)Motility – Motile (Turbidity out of line of inoculation stab)	Trimethoprim-sulfamethoxazole ampicillin
Klebsiella spp.	Culture Gram stain and Microscopy Catalase test Biochemical test – IMViC (Indole, Methyl red, Voges-Proskauer, citrate), motility	Lactose-fermenting (Yellow colonies in CLED); Mucoid, convex, white colonies on BAP Gram-negative bacilli Positive catalase test (Effervescence) 1) Biochemical test Indole – Negative (No colour change) Methy red – Negative (Yellow) Voges-Proskauer – Positive (Colour change) Citrate – Positive (Deep blue)Motility – Nonmotile (Growth along stab line)	Cephalosporins Aminoglycosides
Streptococcus spp.	Culture Gram stain and Microscopy Catalase test	Lactose-fermenting (Yellow colonies in CLED); Small, white colonies on BAP Haemolysis: α-haemolytic: eg. Streptococcus pneumoniae β-haemolytic: Streptococcus group A, B etc Non-haemolytic: E.g. Enterococcus spp.(Greyer appearance) Gram-positive cocci Negative catalase test	Penicillin Eryhromycin Aminoglycoside
Staphylococcus spp. Eg. S saprophyticus, Staphylococcus aureus	Culture Gram stain and Microscopy Catalase test	Lactose-fermenting (Yellow colonies in CLED); Small, white colonies on BAP Can be hemolytic positive or negative Gram-positive cocci Positive catalase test (effervescence)	Penicillins /with clavulanic acid Cephalosporins Vancomycin
Pseudomonas spp.	Culture Gram stain and Microscopy Oxidase test Biochemical test - TSI	Non-lactose-fermenting; Swarms on BAP; Spready colonies on CLED; Pungent smell Gram-positive bacilli Positive oxidase test Biochemical testTSI - Alkaline slant acidic butt	Penicillin Aminoglycoside
Proteus spp.	Culture Gram stain and Microscopy Oxidase test Biochemical test - TSI, Urease	Non-lactose-fermenting; Swarms on BAP; Glossy colonies; Pungent smell Gram-negative bacilli Negative oxidase test 1) Biochemical test TSI - Alkaline slant acidic butt with H2SUrease – Positive (Purple/pink)	Aminoglycosides Trimethoprim-sulfamethoxazole Ampicillin Cephalosporins
Candida spp.Eg. Candida albicans	Culture Gram stain and Microscopy	Non-lactose-fermenting; Small, flat, white colonies on BAP and CLED; Slightly spready, not glossy Fungus; pseudohyphae	-
Enterococcus faecalis	Culture Gram stain and Microscopy Catalase test Biochemical test – Motility, Indole	Large white colonies; alpha-, beta-, nonhemolytic colonies Gram-positive cocci Negative catalase test Biochemical test Motility – Nonmotile (Growth along stab line)Indole – Negative (No colour change)	Penicillin Vancomycin Gentamicin
Morganella	Culture Gram stain and Microscopy	Large, gray, moist colonies (beta haemolytic) Gram-negative bacili	Aminoglycosides Trimethoprim-sulfamethoxazole
Serratia	Culture Gram stain and Microscopy Catalase test Biochemical test – IMViC (Indole, Methyl red, Voges-Proskauer, citrate), motility	Lactose-fermenting but may produce negative results; Red-pigmented colonies Gram-negative bacilli Positive catalase test (effervescence) 1) Biochemical test Indole – Negative Methy red – Variable Voges-Proskauer - Positive Citrate – PositiveMotility – Motile (Turbidity out of line of inoculation stab)	Cephalosporins Aminoglycosides

Photos of Various Microorganism on Agar Plates:
E.Coli
Klebsiella spp.
Pseudomonas & Klebsiella spp.
Proteus spp.
Candida spp.


Photos are taken by our bacteriology group to "xinjiapo"-GH (Andre,Azhar,Valerie,Boonching)

References:
1. MedlinePlus. (2007) Urinary Tract Infection. Retrieved December 02, 2007, from http://www.nlm.nih.gov/medlineplus/ency/article/000521.htm
http://www.nlm.nih.gov/medlineplus/ency/article/000521.htm > >urinary tract infection
2. MedicineNet, Inc. (2007) Definition of Dysuria. Retrieved December 02, 2007, from http://www.medterms.com/script/main/art.asp?articlekey=3163
http://www.medterms.com/script/main/hp.asp > dysuria
3. Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company

Boon Ching
TG01

MMIC CASE 6

Particulars of Patient
Name: Wong Fei Fei
Sex: Female
Age: 37
Complaints: Fever, pain during urination, vaginal discharge

List of possible organism	Test required	Expected results	treatment
Bacteria Pseudomonas aeruginosa	1) Gram Stain 2) Biochmical Tests: - Catalase test - Oxidase test - TSI agar test 3) Aerobic culture on plates like MacConkey agar plate, blood agar plate or differential media 4) Antibiotic Susceptibility (aminoglycoside + penicillin)	1) Shows gram negative rod 2) Catalase and oxidase positive TSI medium no colour change (K/K/g-/H2S-) 3) Colourless colonies on MacConkey plateColonies grown on blood agar.	Antibiotics that have activity against P. aeruginosa include: - aminoglycosides - quinolones (ciprofloxacin) - cephalosporins (ceftazidime and cefoperazone)
Escherichia coli	1) Gram Stain 2) Biochemical Tests: - TSI agar test - Methyl red test - VP reaction test - Citrate test 3) Aerobic culture on MacConkey agar or EMB agar (or both), blood agar 4) Serology is done using the SSS-Coagglutination test 5) Antibiotic Susceptibility (fluoroquinolone)	1) Gram negative rods 2) • Grows on TSI slant with a (A/A/g+/H2S-) profile • Indole (+) • Methyl red (+) • VP (-) • Citrate (-) 3) On MacConkey agar, deep red colonies are produced. Growth on EMB agar would show black colonies with greenish-black metallic sheen. Hemolysis on blood agar 4) Serology detects O55:K5:H21	Antibiotics which may be used to treat E. coli infection: - nitrofurantoin - fluoroquinolones - meropenem - aminoglycosides - ciprofloxacin
Enterococcus spp.	1) Gram Stain 2) Biochemical tests: - Catalase test 3) Antibiotic Susceptibility (gentamicin + vancomycin)	1) Cocci in tetrad and clusters 2) (-) to weakly (+) catalase	Can treat with vancomycin and gentamicin
Proteus mirabilis	1) Gram stain 2) Biochemical tests: - Indole test - Methyl red test - VP test - Catalase test - Oxidase test - Urease test 3) Aerobic culture on XLD plate, blood agar or differential media 4) Antibiotic Susceptibility (ampicillin)	1) Gram negative rod 2) Indole test (-) Methyl red (+) VP (-) Catalase (+) Oxidase (-) Urease (+) 3) Colonies growns on the agar plates	Antibiotics which may be used to treat: - ampicillin - aminoglycoside - TMP-SMZ - fluoroquinolone - cephalosporin
Chlamydia Trachomatis	1) McCoy cell culture 2) Fluorescent Antibody test/Enzyme-linked Immunoassay 3) DNA probe test 4)Antibiotic Susceptibility (Doxycycline or azithromycin)	1) Cytoplasmic inclusions observed by immunofluore-scence after few days of incubation in cell culture 2) Detect the presence of genus-specific lipopolysaccharide antigen	Can be cured with antibiotics like: - Azithromycin - Doxycycline - Tetracycline
Neisseria Gonorrhoeae	1) Gram stain 2) Biochemical Test - Oxidase test 3) Serology Antibiotic Susceptibility (Penicillin)	1) Gram negative, diplococcus 2) Oxidase (+) IgG and IgA antibodies against gonococcal pili, outer membrane proteins and LPS detected	Recommended treatment: - cephalosporin - Fluoroquinolone plus an antibiotic e.g. ceftriaxone, cefixime, ciprofloxacin
Klebsiella pneumoniae	1) Gram stain 2) Biochemical test: - Citrate test - VP test 3) Aerobic culture on blood agar, MacConkey agar and differential media 4) Antibiotic susceptilibilty (cephalosporin)	1) Gram negative, large and regular capsules 2) Citrate (+) VP (+) 3) Large colonies and very mucoid	Can be cured with antibiotics like: - cephalosporine - aminoglycoside - imipenem
Providencia species	1) Gram stain 2) Biochemical test: - Urease test 3) Aerobic culture on blood agar and differential media 4) Antibiotic susceptilibilty (ceftazidime and cefepime)	1) Gram negative 2) Urease (-) 3) Circular, convex, smooth colonies	Recommended treatment with: - ceftazidime - cefepime - aztreonam - imipenem
Citrobacter	1) Gram stain 2) Aerobic culture on blood agar and differential media 3) Biochemical test:Citrate test	1) Gram negative 2)Circular, convex, smooth colonies 3) Citrate (+)	Antibiotics which may be used to treat: - ampicillin - cephalosporins - fluoroquinolones
Viruses Herpes simplex virus type 2 (HSV-2)	1) Virus isolation using inoculation of tissue culture 2) Serology	1) Viral DNA identified 2) Antibodies of HSV-2 detected	Several antiviral drugs are effective against such infection: - Acyvlovir - Valacyclovir - Vidarabine
Parasites Trichomonas vaginalis	1) Microscopic examination – vaginal discharge examined microscopically in a drop of saline 2) Anaerobic culture	1) Characteristic motile trichomonads observed in wet mount 2) Colonies growth on agar plates	Successful treatment requires destruction of trichomonads by medicine like: - metronidazole (e.g. Flagy) - tinidazole and ornidazole
Fungi Candida albicans	1) Potassium hydroxide (KOH) preparation of the vaginal discharge 2) Culture on fungal or bacteriologic media at room temperature or 37oC	1) Hyphae and pseudo spores of Candida are visible under microscopic examination 2) Culture medium is incubated for several days, during which time colonies of yeast and/or bacteria develop	Usually treated with antimycotics—antifungal drugs e.g. topical clotrimazole, topical nystatin, fluconazole, and topical ketoconazole

Images of possible microorganisms:


Pseudomonas aeruginosa on mac conkey plate (1)

P. mirabilis on an XLD agar plate (2)

C. trachomatis inclusion bodies (brown) in a McCoy cell culture (3)

trichomonas vaginalis wet mount (4)

Candida albicans (4)

K. pneumoniae on a MacConkey agar plate (5)

References:
1. http://www.austincc.edu/microbugz/html/ > macconkey_agar
2. http://en.wikipedia.org/wiki/Main_Page > Proteus_mirabilis
3. http://en.wikipedia.org/wiki/Main_Page > Chlamydia_trachomatis
4. http://www.google.com/ > image search > "microorganism name"
5. http://en.wikipedia.org/wiki/Main_Page > Klebsiella_pneumoniae

Doreen Ng (TG 01)

Case 3 2nd Post

Possible Micro-organism	Microscopy	Culture	Biochemical Tests	Antibiotics Sensitivity Testing	Treatment
Escherichia Coli	Gram negative bacilli	Mac Conkey: Lactose Fermenting(Pink) Sheep Blood Agar: β-hemolytic	TSI: acidic slant/ acidic deep Oxidase Test: Negative IMViC: ++-- Urease Test: Negative	Sensitive to amoxillin, cephalosporins, carbapenems, aztreonam, trimethoprim-sulfamethoxazole, ciprofloxacin, nitrofurantoin, aminoglycosides	No drug is normally needed. Amoxillin used when needed.
Proteus spp.	Gram negative bacilli	Mac Conkey: Non-Lactose Fermenting(colourless) Sheep Blood Agar: Spreading Growth	TSI: alkaline slant with black ppt Oxidase Test: Negative IMViC: ++-+* Urease Test: Positive	Resistance to tetracycline, first generation cephalosporins and ampicillins	Most antibiotics can be used other then the 3 that it is resistant to.
Klebsiella spp.	Gram negative bacilli	Mac Conkey: Lactose Fermenting(Red) Growth in areobic and anareobic condition	TSI: alkaline slant/ acidic deep Oxidase Test: Negative IMViC: --++ Urease Test: Negative	Susceptible to aminoglycosides cephalosporin, gentamicin Resistance to carbenicillin, ampicillin, quinolones, and increasingly to ceftazidime	Genamicin
Serratia	Gram negative bacilli	Mac Conkey: Non-Lactose Fermenting(Colourless)	TSI: acid slant/ acid deep Oxidase Test: Negative IMViC: +-++ Urease Test: Negative	Suceptible to anitbiotics carbapenems, cephalosporin, amikacin Resistant to gentamicin and tobramycin	Carbapenems, cephalosporin, amikancin
Pseudomonas	Gram positive bacilli	Mac Conkey: Non-Lactose Fermenting(colourless)	TSI: alkaline slant/ alkaline deep Oxidase Test: Positive Catalase Test: Positive	Susceptible to piperacillin, imipenem, tobramycin, ciprolorxacin Resistant to penicillin	Tobramycin
Enterococcus	Gram positive cocci	Mac Conkey: Lactose Fermenting(Pink) Sheep Blood Agar: No Haemolysis	Catalase test: Negative	Resistant to Quinupristin, ampicillin and vancomycin	Vancomycin

*Mostly Proteus strain positive except P. mirabilis negative.

Urease Test: Positive - Pink, Negative - Orange

Oxidase Test: Positive - Dark Maroon to Black, Negative - Slight pink colouration or no colouration

IMViC: Indole Test, Methyl Red, Voges-Proskauer, Citrate test. Done when oxidase test is negative.

Indole Test: converts tryptophan into indole, ammonia and pyruvic acid. Bacterial culture grown in tryptophan or peptone for 24 to 48 hours before adding kovac reagent. Positive-Red, Negative- Yellow

Methyl Red Test: If the organism uses the mixed acid fermentation pathway and produces stable acidic end-products, the acids will overcome the buffers in the medium and produce an acidic environment in the medium. When methyl red is added, if acidic end products are present, the methyl red will stay red.

Voges-Proskauer: When the VP reagents are added to MR-VP broth that has been inoculated with an organism that uses the butylene glycol pathway, the acetoin end product is oxidized in the presence of potassium hydroxide (KOH) to diacetyl. Creatine is also present in the reagent as a catalyst. Diacetyl then reacts to produce a red color. Therefore, red is a positive result. If, after the reagents have been added, a copper color is present, the result is negative.

Citrate Test: To see if bacteria is able to use only citrate as carbon and energy source. Positive-Blue, Negative- Green.

Reference:

http://www.wikepedia.com/

Douglas
0503224H
TG01

MMIC CASE 4 (2nd posting)

Particulars of patient
Name: Tong Wei Hong

Sex: Male

Age: 68 years

Clinical diagnosis
Complaints: Fever, chills, excessive phlegm, breathing problems

Diagnosis: Bronchitis

Possible Microorganisms	Description	Microscopy	Culture	Biochemical Tests
Streptococcus pneumoniae	- Gram-positive - Alpha-hemolytic diplococcus bacterium - Fermentative aerotolerant anaerobe - Members of the genus Streptococcus - Found in the nasopharynx - Major cause of pneumonia	Gram stain: Gram positive, lancet-shaped diplococcus (stained purple)	1) Blood agar plate (BAP) with added optochin antibiotic disk: alpha-hemolytic (green) colonies and a clear zone of inhibition around the disk	1) Catalase test: Catalase-negative
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	Gram stain: Gram negative coccobacilli, with no specific arrangement (stained pink)	1) Chocolate agar (with factor X (heme) and factor V (NAD)): Flat, greyish-brown colonies colonies with diameters of 1-2mm	1) Catalase test: Catalase-positive 2) Oxidase test: Oxidase-positive
Moraxella catarrhalis	-Gram-negative, non-motile, aerobic, oxidase-positive diplococcus cocci -Produce beta-lactamase -Major cause of respiratory tract-associated infection, associated with bronchopneumonia	Gram stain: Gram negative kidney bean shaped diplococcus (stained pink)	1) Enriched media (e.g. Mueller-Hinton, modified Thayer-Martin): Non-pigmented or pinkish-grey opaque colonies 2) Chocolate blood agar: Opaque and non-hemolytic colonies being pushed along the surface of the agar like a hockey puck	1) Oxidase test: Oxidase-positive 2) Catalase test: Catalase-positive
Mycoplasma pneumoniae	-Small bacterium lacking a peptidoglycan cell wall -Member of the class Mollicutes -Uniform granular appearance	Nil	Nil	Nil
Chlamydia pneumoniae	-Small bacterium -Produces round, dense, glycogen-negative inclusion -Elementary bodies with pear-shaped appearance	Nil	Nil	Nil
Bordetella pertussis	-One of Bordetella species -Minute gram-negative, aerobic coccobacillus	Gram stain: Gram negative coccobacilli, with no specific arrangement (stained pink)	1) Enriched media- Bordet-Gengou medium (potato-blood-glycerol agar) or BCYE plate that contains penicillin G, using immunofluorescence staining: Mercury-drop colonies Small, faintly staining gram-negative rods	1) Urease test - Urease-negative 2) Nitrase test - Nitrase-negative 3) Citrate test - Citrate-negative 4) Oxidase test - Oxidase-positive

Possible Microorganisms	Serology	Others	Antibiotic Susceptibility Test	Treatment
Streptococcus pneumoniae	Nil	1) Bile solubility: Colonies soluble in bile 2) Capsule swelling test (Quellung reaction): Capsular swelling in the presence of antisera; Quellung reaction-positive	-Resistant to cephalosporins, macrolides (such as erythromycin), tetracycline, clindamycin and the quinolone -Susceptible to Vancomycin	Vancomycin in combination with β-lactam antibiotics
Haemophilus influenzae	Nil	Nil	Susceptible to ampicillin, cephalosporins, cefotaxime	Ampicillin (or amoxicillin) if β-lactamase-negative; cefotaxime (IV) or ceftriaxone
Moraxella catarrhalis	Nil	1) Deoxyribonuclease (DNase) test: Presence of DNase 2) Nitrate reduction test: Nitrate and nitrite levels are reduced.	Susceptible to trimethoprim-sulfamethoxazole, fluoroquinolones, cephalosporins, amoxicillin-clavulanic acid	Trimethoprim-sulfamethoxazole, amoxicillin-clavulanic acid, fluoroquinolones or cephalosporins
Mycoplasma pneumoniae	1) Complement-fixation test: Detects both IgG and IgM specific to M. pneumoniae. A four-fold rise in complement fixation titer during the acute and convalescent phases is indicative of a M. pneumoniae infection. 2) Hemagglutination test: Positive blood test for cold-hemagglutinins A titer of 1:64 or more indicates infection of M. pneumoniae 3) Enzyme immunoassay: Presence of M. pneumoniae specific IgM	Nil	Susceptible to Macrolide antibiotics, doxycyclin, second generation quinolones, tetracyclines, erthromycin	Macrolide antibiotics, doxycyclin, second generation quinolones, tetracyclines, erthromycin
Chlamydia pneumoniae	1) Direct Fluorescent Antibody Test: Detection of IgM or IgG antibodies – a fourfold rise in either the IgM or IgG titers Presence of proteins (antigens) from Chlamydia pneumoniae	Nil	Susceptible to macrolides, tetracycline, fluoroquinolones, erythromycin, clarithromycin	Macrolides, tetracycline, fluoroquinolones, erythromycin, clarithromycin
Bordetella pertussis	1) Direct Fluorescent Antibody (FA) Test: Presence of antibodies Presence of proteins (antigens) from Bordetella pertussis	Nil	Susceptible to erythromycin	Erythromycin

Pictures of Possible Microorganisms:

Streptococcus pneumoniae on blood agar showing alpha hemolysis (green zone surrounding colonies). Note: the zone of inhibition around a optochin disk.

Streptococcus pneumoniae gram stain of a film of sputum

Streptococcus pneumoniae - Quellung (capsular swelling) reaction

Gram stain of Haemophilus influenzae

Haemophilus influenzae on chocolate agar

Moraxella catarrhalis on chocolate agar

Moraxella catarrhalis on BAP

Gram stain of Moraxella catarrhalis

Electron micrograph of Mycoplasma pneumoniae

Chlamydial inclusions containing pear-shaped C. pneumoniae elementary bodies. EB, elementary body; FG, fatty globules; N, nucleus

Photomicrograph of Bordetella pertussis bacteria using gram staining technique

References
1. http://www.google.com/ > image search > "microorganism name"
2. http://en.wikipedia.org/wiki/Main_Page > "microorganism name"
3. http://www.textbookofbacteriology.net/ > S.pneumoniae
4. Geo.F.Brooks, Janet S.Butel, Stephen A. Morse. (2004). Jawetz, Melnick, & Adelberg's Medical Microbiology Twenty-third Edition. Singapore: The McGraw-Hill Companines, Inc

Dorothy Png
TG01
0503239F

MMIC PBL Case 2 (2nd Post)

Name: Kwan Siew Yan (outpatient)
Sex: Female
Age: 29
Complaints: Diarrhea
Diagnosis: Enterocolitis
Antibiotic treatment (if any): Nil
Specimen: Stool

Possible Organisms
1) Salmonella enteritidis
2) Shigella species
3) Campylobacter jejuni
4) Staphylococcus aureus
5) Clostridium difficile
6) Bacillus cereus
7) Enteroinvasive E coli (EIEC)

Investigation required:
1) Culture
2) Microscopy
3) Biochemical tests
4) Serology; ELISA and cytotoxicity tests
5) Antibiotic susceptibility tests

Possible Microorganisms	Culture	Microscopy	Biochemical Tests
Salmonella enteritidis	-MacConkey’s agar: non-lactose fermenting (colourless) colonies -Salmonella-shigella agar: colonies with black centre due to H2S production	-gram-negative rods - motile	-TSI agar test: alkaline slant and acid butt with gas and H2S -IMViC tests: gives test result of - + - + respectively.
Shigella species	-MacConkey’s agar: same as Salmonella enteritidi-Salmonella-shigella agar:colonies without black centre as no H2S is produced	-gram-negative rods - non-motile	-TSI agar test: alkaline slant and acid butt with no gas, no H2S
Campylobacter jejuni	-campylobacter selective media (e.g. Skirrow medium or Campy BAP): grow in scanty amounts at 42 degrees celsius	-curved, gram-negative rods that appear either comma or S-shaped -motile with a single polar flagellum	-positive oxidase test
Staphylococcus aureus	-Blood agar: yellow or gold colonies	-gram-positive cocci in clusters	-catalase-positive -coagulase-positive
Clostridium difficile	- Blood agar	- gram-positive -spore-forming rods	Not done
Bacillus cereus	- Blood agar	- gram-negative -spore-forming rod	Not done
Enteroinvasive E coli (EIEC)	-MacConkey’s agar: lactose-fermenting colonies	-gram-negative rods	-TSI agar test: alkaline slant and acid butt with gas, no H2S - IMViC tests: gives test result of + + - - respectively

Possible Microorganisms	Serology; ELISA and cytotoxicity tests	Antibiotic susceptibility tests	Treatment
Salmonella enteritidis	Not done	-resistant to ampicillin and chloramphenicol	-enterocolitis is self limiting in 2-3 days, thus antibiotic treatment is not required.
Shigella species	Not done	-susceptible to ciprofloxacin, ampicillin, doxycycline and trimethoprim-sulfamethoxazole	-fluid and electrolyte replacement without antibiotics or - one of the following antibiotics: ciprofloxacin, ampicillin, doxycycline and trimethoprim-sulfamethoxazole
Campylobacter jejuni	Not done	-susceptible to erythromycin	- usually symptomatic treatment only; erythromycin for severe disease
Staphylococcus aureus	Not done	- susceptible to penicillin G, nafcillin and vancomycin	-penicillin G for sensitive isolates -nafcillin is used to treat beta-lactamase resistant strains -vancomycin is used to treat isolates resistant to nafcillin
Clostridium difficile	-Exotoxin in stool detected by cytopathic effect on cultured cells. Identified by neutralization of cytopathic effect with antibody -Assessment of the A and B toxins in stool can be detected by using ELISA	-susceptible to metronidazole	-metronidazole. Vancomycin, although effective, should not be used as it may select for vancomycin-resistant enterococci
Bacillus cereus	Not done	-susceptible to vancomycin, chloramphenicol, gentamicin, tetracyclines or sulfonamides	-only symptomatic treatment is given antibiotics such as vancomycin, chloramphenicol, gentamicin, tetracyclines or sulfonamides
Enteroinvasive E coli (EIEC)	Not done	- susceptible to trimethoprim-sulfamethoxazole, aminoglycoside, ampicillin, cephalosporin and sulfonamides	- treatment can be difficult because of multiple drug resistance as mediated by ``R’’ plasmid

MacConkey’s agar is a selective and differential media used to differentiate between Gram negative bacteria while inhibiting the growth of Gram positive bacteria. The addition of bile salts and crystal violet to the agar inhibits the growth of most Gram positive bacteria, making MacConkey agar selective. Lactose and neutral red are added to differentiate the lactose fermenters, which form pink colonies, from lactose nonfermenters that form clear colonies. [2]

Salmonella-shigella agar are designated as moderately selective media based upon the degree of inhibition of gram-positive microorganisms that they inhibit due to their content of bile salts, brilliant green and citrates. [3] This media is used to find out whether the suspected microorganism is either Salmonella enteritidis or Shigella sp.

Skirrow’s agar is peptone and soy protein base agar supplemented with lysed horse blood and vancomycin (inhibits Gram-positives), polymyxin B (antifungal), and trimethoprim (broad spectrum). Campy BAP is brucella agar supplemented with sheep blood and vancomycin, trimethoprim and cephalothin (inhibits streptococci) [4]

Blood agar is an enriched, differential media used to isolate fastidious organisms and detect hemolytic activity. β-hemolytic activity will show complete lysis of red blood cells surrounding colony, while α-hemolysis will only partially lyse hemoglobin and will appear green. γ-hemolysis (or non-hemolytic) is the term referring to a lack of hemolytic activity. [2].

After the microorganisms had been isolated, microscopy is used to observe the morphological characteristics so that to identify the organism. Gram stain is a method of differentiating bacterial species into two large groups (Gram-positive and Gram-negative) based on the chemical and physical properties of their cell walls. Gram-positive bacteria have cell wall made of peptidoglycan which stains purple and Gram-negative bacteria which stain pink. [5]. Spore stain will be required to identify spores. Wet mount microscopy is used initially before isolation of organisms to identify their motility.

Antibiotic Susceptibility Test, also known as Kirby-Bauer test, is performed by swabbing the bacterium on the agar and the antibiotic discs are placed on top. The antibiotic diffuses from the disc into the agar in decreasing amounts the further it is away from the disc. If the organism is killed or inhibited by the concentration of the antibiotic, there will be no growth in the immediate area around the disc. This is called the zone of inhibition. The zone sizes are looked up on a standardized chart to give a result of sensititive, resistant, or intermediate. [6] This can help to provide the necessary treatments for the patient.

Pictures
Salmonella enteritidis on Salmonella-shigella agar
[7]


[8]


Staphylococcus aureus (gram stain)
[9]

Campylobacter jejuni wet mount microscopy[10]


Clostridium difficile (gram stain)[11]


Bacillus cereus on blood agar[12]


Enteroinvasive E coli (EIEC)on MacConkey agar[13]













References
1) Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company
2) http://en.wikipedia.org/ >Agar_plate
3) http://www.bd.com/ >SS_Agar
4) http://www.life.umd.edu/ >Media
5) http://en.wikipedia.org/ >Gram_stain
6) http://www.rlc.dcccd.edu/ >antibiotics
7) http://www.rci.rutgers.edu/ >salmonellashigella
8) http://textbookofbacteriology.net/ >Shigella
9) http://en.wikipedia.org/ >Staphylococcus_aureus
10) http://www.umm.edu/ >fecal culture
11) http://en.wikipedia.org/ >Bacillus_cereus
12) http://www.spiceisle.com/ > Enteric demonstration

Soong Ci Liang
TG01
0503333G

MMIC laboratory diagnosis

Patient particulars:

Name: Wong Fei Hong

Sex: Male

Age: 37 years

Complaints: fever sewlling around operation wound

Diagnosis: wound infection

List of possible organism	Test required	Expected results	treatment
Corynebacterium diphtheriae	1) Gram stain 2) Methylene blue 3) Culture (Loffler medium followed by tellurite plate) 4) PCR assay	1) gram-positive, highly pleomorphic organisms with no particular arrangement 2) Demostrate the presence of metachromatic granules 3) If C. diphtheriae is presence, tellurium in the medium will be reduced to elemental tellurium by the organism, showing a grap-black color of tellurium in the colony 4) Identify the toxin gene presence in the organism isolated form the patient	Antitoxin penicillinG Erythromycin
Pseudomonas aeruginosa	1) Gram stain 2) Culture (EMB or MacConkey’s agar) 3) TSI medium 4) Biochemical test (catalase, oxidase, nitrase, lipase)	1) Gram negative rod with no particular arrangement 2) Culture of patient specimen on MacConkey’s agar produce colourless colonies as the organism does not ferment lactose 3) Alkaline slant and an alkaline butt as the organism does not ferment lactose 4) Catalase, Oxidase, Nitrase and Lipase positive	Antipseudomonal penicillin Aminoglycoside
staphylococcuss aureus	1) Gram stain 2) culture (Mannitol salt agar) 3) coagulase test 4)biochemical test (catalase test)	1) Gram positive cocci in grapelike clusters 2) Golden yellow colonies 3) coagulase positive 4)catalase positive	Mupirocin Beta-lactamose resistant penicillins such as nafcillin, cloxacillin, cephalosporins or vancomycin
Proteus mirabilis	1) Gram stain 2) culture (EMB or MacConkey’s agar)3) TSI agar 4) biochemical test (indole test	1) Gram negative rod 2) Non-lactose fermenting colonies on EMB or MacConkey’s agar 3) alkaline slant and acid butt. 4) indole negative	Ampicillin Tetracycline
group A streptococcus bacterium	1) Gram stain 2) Culture (blood culture) 3) Biochemical test (indole test)	1) Gram positive cocci in chain 2) Beta hemolytic colonies 3) Indole test negative	Penicillin G
Bacteroides fragilis	1) Gram stain 2) Anaerobic Culture blood agar plates containing kanamycin and vaomyycin 3) TSI agar	1) Gram negative rod 2) Colonies should be able to grow on the BAP containing the anitbiotic 3) Red stunt yellow butt	Penicillin G Cephalosporins Aminoglycosises Metronidazole
Pasteurella Multocida	1) Gram stain 2) Culture (blood agr or chocolate agar) 3) oxidase test 4) catalase test	1) Encapsulated gram negative rod 2) positive 3) positive 4)positive	Penicillin G
Aeromonas Hydrophila	1) Gram stain 2) Culture (Sheep blood agar with ampicillin) 3) catalse test 4) oxidase test	1) Gram negative rod 2) Aeromonas Hydrophila is able to grow on the agar plate while the ampicillin will inhibit the grow of other competing organism 3) positive 4) positive	chloramphenicol
Clostridium perfringens	1) Gram stain 2) Culture ( blood agar)	1) Large gram postitive rods 2) Exhibit a double zone of hemolysis	Penicillin G

Types of wound infection and their causative agents:

-Skin infection at the site of preexisting skin leison: Corynebacterium diphtheriae

-Burn wound infection: Pseudomonas aeruginosa

-Wound infection in diabetic patients: Staphylococcuss aureus

-Surgical wound infection: Staphylococcuss aureus

-Abcess infection of brain, lung or abdominal: Bacteroides fragilis

-Wound infection caused by cats or dog bite: Pasteurella multocida

-Traumatic open wound infection: Aeromonas hydrophila

-Gas gangrene wound infection: Clostridium perfringens

Reference:

1) Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company

Wing Fat

Tg01

Compilation of All Potential Microorganisms To Each Case

	Diagnosis	Potential Pathogens	Descriptions
Case 1	Urinary Tract Infection (UTI)	1) Escherichia Coli 2) Klebsiella spp. 3) Streptococcus spp. (GPC) 4) Staphylococcus spp. (GPC) Eg. S saprophyticus, Staphylococcus aureus 5) Pseudomonas spp. 6) Proteus spp. 7) Candida spp. Eg. Candida albicans 8) Enterococcus 9) Morganella 10) Serratia	1) Gram-negative bacilli 2) Gram-negative bacilli 3) Gram-positive cocci 4) Gram-positive cocci 5) Gram-positve bacilli 6) Gram-negative bacilli 7) Gram-negative bacilli 8) Fungus 9) Gram-positive cocci 10) Gram-negative bacilli 11) Gram-negative bacilli
Case 2	Enterocolitis	1) Salmonella enteritidis 2) Shigella species 3) Campylobacter jejuni 4) Staphylococcus aureus 5) Clostridium difficile 6) Bacillus cereus 7) Enteroinvasive Escherichia coli (EIEC)	1) Gram-negative rods 2) Gram-negative rods 3) Gram-negative rods 4) Gram-positive cocci in clusters 5) Gram-positive rods 6) Gram-negative rods 7) Gram-negative rods
Case 3	Urinary Tract Infection (UTI)	1) Escherichia Coli 2) Proteus spp. 3) Klebsiella spp. 4) Serratia 5) Proteus 6) Pseudomonas spp. 7) Streptococcus spp. 8) Enterococcus 9) Candida spp. (Fungus)	1) Gram negative bacilli 2) Gram negative bacilli 3) Gram negative bacilli 4) Gram negative bacilli 5) Gram negative bacilli 6) Gram positive bacilli 7) Gram positive cocci 8) Gram positive cocci 9) -
Case 4	Bronchitis	1) Streptococcus pneumoniae 2) Haemophilus influenzae 3) Moraxella catarrhalis	1) Gram-positive cocci 2) Gram-negative coccobacillus 3) Gram-negative diplococci
Case 5	Wound Infection	1) Corynebacterium diphtheriae 2) Pseudomonas aeruginosa 3) Staphylococcuss aureus 4) Proteus mirabilis	1) Gram positive 2) Gram negative rod 3) Gram positive coccus 4) Gram negative rod
Case 6	Urinary Tract Infection (UTI)	1) Escherichia coli 2) Proteus 3) Chlamydia 4) Gonorrhea 5) Herpes simplex virus type 2 (HSV-2) 6) Candida 7) Parasites

MMic PBL Case 3

MMic PBL



Name: Maisy Hong

Sex: Female

Age: 67

Complaints: Fever, chills, bladder distension; on indwelling catheter
(Bladder distention on indwelling catheter: this refers to implanting a flexible or rigid hollow tube to distend body passages, or to drain fluids from body cavities. [1] )


Diagnosis: Urinary Tract Infection
(A urinary tract infection, or UTI, is an infection that can happen anywhere along the urinary tract. The urinary tract includes the bladder, kidneys, ureters and urethra.[2])

Antibiotic treatment (if any): Nil

Specimen: Urine

Gram	Shape	Possible Microorganism
Negative	bacilli	Escherichia Coli
Negative	bacilli	Proteus spp.
Negative	bacilli	Klebsiella spp.
Negative	bacilli	Serratia
Negative	bacilli	Proteus
Positive	bacilli	Pseudomonas spp.
Positive	cocci	Streptococcus spp.
Positive	cocci	Enterococcus
-	-	Candida spp. (Fungus)

Identification of Microorganism

1. Gram staining and microscopy.
2. Catalase Test.
3. Oxidase Test.
4. Biochemical Test.
5. Aerobic culture on sheep blood agar, Cysteine Lactose Electrolyte Deficient Medium, and Mac Conkey agar.
6. Antibiotics sensitivity testing.

Reference

[1] http://dictionary.reference.com/
[2]MedlinePlus. (2007) Urinary Tract Infection. Retrieved December 02, 2007, from http://www.nlm.nih.gov/medlineplus/ency/article/000521.htm>urinary tract infection

Douglas
TG01

MMIC

Name: Wong Fei Hong
Sex: Male
Age: 37
Complaints: Fever, swelling around operation wound
Specimen: Wound swab
Diagnosis: Wound infection

A wound infection happens when pathogens enter an open wound of the patient. Factors such as disease, surgery and weak immune system increase the patient’s risk of getting wound infection. Signs and symptom of a wound infection includes:
-Redness or swelling around the wound
-Secretion of Pus or watery discharge around the wound area
-foul odour from the wound
-mild fever

Possible pathogens that cause wound infection:
-Corynebacterium diphtheriae
-Pseudomonas aeruginosa
-staphylococcuss aureus
-Proteus mirabilis

Corynebacterium diphtheriae: C. diphtheriae is a facultative anaerobic, gram positive organism that can infect skin lesions in people with poor skin hygiene, causing cutaneous diphtheria. Symptom of cutaneoud diphtheria includes mild fever and swollen wound.

Pseudomonas aeruginosa: Pseudomonas aeruginosa is an aerobic, gram negative rod shape bacterium that can cause burn infection and wound infection. The microorganism is able to produce endotoxin that causes fever.

Staphylococcuss aureus: Staphylococcuss aureus is a facultative, gram positive coccus organism that causes localised infection in forms of abscess and skin infection, which is one of the possible causes to surgical wound infections.

Proteus mirabilis: Proteus mirabilis is A facultative, gram negative rod proteus species that cause uriniry tract infection, but it can also cause wound infection, mostly in hospitalized patients.

Laboratory diagnosis:
-Gram stain
-Culture (aerobic/anaerobic)
-biochemical reactions
-oxidase tests
-catalase tests
-antibiotic susceptibility

Wing Fat
Tg01

MMIC PBL

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

Clinical diagnosis
Complaints: Fever, chills, excessive phlegm, breathing problems
Diagnosis: Bronchitis

Bronchitis is inflammation of the large airways that branch off the trachea (bronchi), usually caused by infection but sometimes caused by irritation from a gas or particle.

In bronchitis, areas of the bronchial wall become inflamed and swollen, and mucus increases. As a result, the air passageway is narrowed. Bacteria and viruses are usually present.

There are two main types of bronchitis:

-Acute bronchitis comes on rapidly, usually after a virus has invaded the upper respiratory tract. Sometimes there is a bacterial infection as well. Viruses most likely to trigger acute bronchitis are those responsible for influenza (the flu) or the common cold.

-Chronic bronchitis is defined as excessive mucus secretion in the bronchi and a chronic or recurrent mucus-producing cough that lasts three or more months and recurs year after year. People who have chronic bronchitis are more susceptible to bacterial infections of the airway and lungs, like pneumonia.

Mycoplasma pneumoniae and Chlamydia pneumoniae often cause bacterial bronchitis in young adults. Among middle-aged and older people, Streptococcus pneumoniae, Haemophilus influenzae, and Moraxella catarrhalis are the most common organisms causing bacterial bronchitis.

Viral bronchitis may be caused by a number of common viruses, including the influenza virus (influenza A and B). A person often has a combination of bacterial and viral bronchitis.

Possible causative agents

The below 3 microorganisms often cause bacterial bronchitis in middle-aged and older people.

Possible Microorganisms	Description
Streptococcus pneumoniae	-Gram-positive -Alpha-hemolytic diplococcus bacterium -Members of the genus Streptococcus -Found in the nasopharynx -Major cause of pneumonia
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
Moraxella catarrhalis	-Gram-negative, aerobic, oxidase-positive diplococcus cocci -Major cause of respiratory tract-associated infection, associated with bronchopneumonia

Investigation required

Specimen: Sputum
*Clear or white sputum suggests a viral infection; yellow or green sputum suggests a bacterial infection.

• Microscopy
  - Gram stain (Gram positive, -negative)


• Culture
  - Aerobic on blood agar plates
  
• Antibiotic Suspectibility
  
• Serology (detection of viruses)
  - Haemagglutinin Inhibition (HI)
  - Enzyme-Linked ImmunoSorbent Assay (ELISA)
  
• Others
  - Oxidase test (Oxidase-positive, -negative)
  - Catalase test (Catalase-positive, -negative)
  

References

1) http://www.merck.com/mmhe/sec04.html > Bronchitis
2) http://en.wikipedia.org/wiki/Main_Page > Moraxella_catarrhalis
3) http://en.wikipedia.org/wiki/Main_Page > Haemophilus_influenzae
4) http://en.wikipedia.org/wiki/Main_Page > Streptococcus_pneumoniae

Dorothy
TG01

MMIC PBL

Name: Kwan Siew Yan (outpatient)
Sex: Female
Age: 29
Complaints: Diarrhea
Diagnosis: Enterocolitis
Antibiotic treatment (if any): Nil
Specimen: Stool

Enterocolitis is the inflammation of the large and small intestines. [1] It is characterized by an invasion of the epithelial and subepithelial tissue of the small and large intestines. Strains that do not invade do not cause this disease. The organisms penetrate both through and between the mucosal cells into the lamina propria, resulting in inflammation and diarrhea. A polmorphonuclear leukocyte will limit the infection to other parts of the body. [2] Symptoms will include:
- diarrhea
- nausea
- vomiting
- fever
- abdominal bleeding
- rectal bleeding
- sluggishness [1]

Possible Organisms Present [2]:
1) Salmonella enteritidis
Characteristics
- facultative anaerobes
- gram-negative rods
- do not ferment lactose
- produce H2S
- motile

Habitat and Transmission
- found in enteric tract of humans and animals
- transmitted mainly by fecal-oral route and foods prepared by chronic carriers

Pathogenesis
- invade the mucosa of the small and large intestines
- infectious dose is at least 10*5 organisms, much greater than infectious dose of Shigella

2) Shigella species
Characteristics
- facultative gram-negative rods
- do not produce H2S
- non-lactose fermenting
- non-motile compared to Salmonella

Habitat and Transmission
- human colon only; human disease
- no animal reservoir for Shigella
- transmitted by the fecal-oral route
- principal factors in transmission include fingers, flies, food and feces

Pathogenesis
- invade the mucosa of the ileum and colon but do not penetrate further; hence sepsis is rare
- most effective pathogens among enteric bacteria as infectious dose is much lower (1-10 organisms) than that of Salmonella

3) Campylobacter jejuni
Characteristics
- curved, gram-negative rods that appear either comma or S-shaped
- motile with a single polar flagellum
- microaerophilic (5% O2 with 10% CO2)
- grow well at 42℃

Habitat and Transmission
- human and animal feces
- transmitted by fecal-oral route
- Food and water contamination with animal feces is the major source of human infection. (e.g. poultry, meat and unpasteurized milk)

Pathogenesis
- invade mucosa of colon but do not penetrate; hence sepsis is rare

4) Staphylococcus aureus
Characteristics
- gram-positive cocci in clusters
- coagulase-positive and catalase-positive

Transmission
- via the hands

Pathogenesis
- produce enterotoxin in foods

5) Clostridium difficile
Characteristics
- anaerobic
- gram-positive
- spore-forming rods

Habitat and Transmission
- human colon and transmitted by fecal-oral route

Pathogenesis
- antibiotics suppress normal flora of colon, allowing them to overgrow and produce large amounts of exotoxins

6) Bacillus cereus
Characteristics
- aerobic
- gram-negative
- spore-forming rod

Habitat and Transmission
- found in grains such as rice (e.g. re-heated rice)

Pathogenesis
- Two enterotoxins are produced

7) Enteroinvasive Escherichia coli (EIEC)
Characteristics
- Facultative gram-negative rods
- Ferment lactose

Transmission
- Fecal-oral route
- Food-borne route

Pathogenesis
- closely resemble Shigella in their pathogenic mechanisms
- penetrate and multiply within epithelial cells of the colon causing widespread cell destruction
- EIEC are invasive organisms which are same as Shigella but they do not produce LT or ST toxin and, unlike Shigella, they do not produce the shiga toxin

References

1) http://www.wrongdiagnosis.com/ >enterocolitis

2) Levinson, W. (2006). Review Of Medical Microbiology and Immunology. (9th ed.). US: The McGraw-Hill Company.

Soong Ci Liang
0503333G
TG01

MMIC PBL

Particulars of Patient

Name: Ong Fei Fei
Sex: Female
Age: 37

Clinical Diagnosis

Complaints: Fever, pain during urination, virginal discharge
Diagnosis: UTI
Antibiotic Treatment: Nil
Particulars of Specimens
Specimen: Vaginal Discharge

Description of Urinary Tract Infection (UTI)

In healthy people, urine in the bladder is sterile—no bacteria or other infectious organisms are present. The channel that carries urine from the bladder out of the body (urethra) contains no bacteria or too few to cause an infection. However, any part of the urinary tract can become infected; an infection anywhere along the urinary tract is called a urinary tract infection.

UTIs are usually classified as upper or lower according to where they occur along the urinary tract. Lower UTIs are infections of the urethra (urethritis) or bladder (cystitis); upper UTIs are infections of the kidneys (pyelonephritis) or ureters (ureteritis).

Causes

UTIs are almost always caused by bacteria, although some viruses, fungi, and parasites can infect the urinary tract as well. Common organisms that cause UTIs include: Escherichia coli and Staphylococcus saprophyticus. Less common organisms include Proteus mirabilis, Klebsiella pneumoniae, Enterobacter spp, Pseudomonas and Enterococcus spp.

Bacteria: Bacterial infections of the lower urinary tract—the bladder and urethra—are very common. Escherichia coli is the most common bacteria. When the person has a kidney stone, Proteus bacteria may be able to grow. These conditions can lead to abnormal vaginal discharge which can be caused by Chlamydia and Gonorrhea.

Viruses: The herpes simplex virus type 2 (HSV-2) may infect the urethra to cause a UTI, making urination painful and emptying of the bladder difficult.

Fungi: Certain fungi or yeasts can infect the urinary tract to cause a UTI. The most common type is Candida, which causes candidiasis.

Parasites: A number of parasites, including certain types of worms, can infect the urinary tract.

Trichomoniasis, caused by a type of microscopic parasite, is a sexually transmitted disease that can produce a copious greenish yellow, frothy discharge from the vagina in women.


Investigation required

Microscopy, culture of specimen and antibiotic susceptibility should be requested.

Doreen Ng (tg 01)

MMIC PBL Case 1

Subject: MMIC
Case: Case 1 - Khong Fay Seah
Complaints: Fever, chills, dysuria,
Diagnosis: Urinary Tract Infection

Urinary Tract Infection: A urinary tract infection, or UTI, is an infection that can happen anywhere along the urinary tract. The urinary tract includes the bladder, kidneys, ureters and u rethra. [1]

Dysuria: Painful or difficult urination. This includes burning on urination. Dysuria is most commonly due to bacterial infection of the urinary tract causing inflammation of the bladder (cystitis) or kidney (pyelonephritis).
In women, dysuria may also reflect inflammation of the vagina (vaginitis) or vulva (vulvitis). And in men, dysuria may be due to inflammation of the prostate (prostatitis) or the urethra (urethritis) from gonorrhoea or chlamydia. [2]

Possible Pathogens

Possible Microorganisms	Descriptions
Escherichia Coli	Gram-negative bacilli
Klebsiella spp.	Gram-negative bacilli
Streptococcus spp. (GPC)	Gram-positive cocci
Staphylococcus spp. (GPC) Eg. S saprophyticus, Staphylococcus aureus	Gram-positive cocci
Pseudomonas spp.	Gram-positve bacilli
Proteus spp.	Gram-negative bacilli
Candida spp. Eg. Candida albicans	Fungus
Enterococcus faecalis	Gram-positive cocci
Morganella	Gram-negative bacilli
Serratia	Gram-negative bacilli

Investigation Required
Microscopy:
1) Gram stain - Differentiate Gram positive and Gram negative bacteria
2) Wet mount and KOH smear - Demonstration of pseudohyphae for identification of Candida spp.

Culture:
Aerobic culture on Blood plate agar(BAP), Cysteine Lactose Electrolyte Deficient medium (CLED), MacConkey (MAC)agar plate.

Other Tests:
1) Catalase Tests
2) Oxidase Tests
3) Biochemical Tests

Antimicrobial Susceptibility Test:
To determine the treatment

References:
1. MedlinePlus. (2007) Urinary Tract Infection. Retrieved December 02, 2007, from http://www.nlm.nih.gov/medlineplus/ency/article/000521.htm
http://www.nlm.nih.gov/medlineplus/ency/article/000521.htm > >urinary tract infection
2. MedicineNet, Inc. (2007) Definition of Dysuria. Retrieved December 02, 2007, from http://www.medterms.com/script/main/art.asp?articlekey=3163
http://www.medterms.com/script/main/hp.asp > dysuria

Boon Ching
Tg01

Treponemal Test – TPPA

Introduction
The Treponema pallidum particle agglutination (TP-PA) test is a treponemal test (confirmation test) for the serologic detection of antibodies to the various species and subspecies of pathogenic Treponema, the causative agents of syphilis, yaws, pinta, bejel, and endemic syphilis. The test is a passive agglutination procedure based on the agglutination of gel particles sensitized with T. pallidum antigens by antibodies found in the patient's serum (1-3). The test is intended as a confirmatory test to replace the microhemagglutination assay for antibodies to T. pallidum (MHA-TP).

Principle
Serum containing antibodies to pathogenic treponemes react with gel particles sensitized with sonicated T. pallidum, Nichols strain (the antigen), to form a smooth mat of agglutinated gel particles in the microtiter tray well.

If antibodies are not present, the particles settle to the bottom of the tray well, forming a characteristic compact button of unagglutinated particles.

The unsensitized gel particle control well for each serum should also show this compact button, or the absence of agglutination.

The TPPA test is used to confirm the reactive results of a nontreponemal screening test for syphilis, such as the VDRL slide test, or as a diagnostic test in patients with a nonreactive nontreponemal test but with signs or symptoms suggestive of late syphilis

Interpretation of results
1) Read the settling patterns of the gelatin particles using an angled mirror (tray viewer) to visualize the patterns from below - Readings are scored on a scale of – to 2+ agglutination
2) Retest serum with samples with a reading of ±

Boon Ching
TG01

special stain

Martius scarlet blue (MSB)

Function: stain fibrin

Principle: the main feature of MSB is the use of a small molecule yellow dye, together with phosphotungstic acid to selectively stain red cells. Phosphotungstic acid blocks the staining of muscle, collagen and most connective tissue fibres. Muscle and fibrin are stained with medium size molecule red dye, while staining of collagen is prevented by phosphotungstic acid. Larger blue dye will then stained the collagen and fibrin.

Control: skeletal or cardiac muscle, soft cartilage, fibrinous appendix

Chemical required: 1% acetic acid
2%phosphotungstic acid
martius blue
martius red
martius yellow
weigert iron haematoxylin

Reagent preparation: martius scarlet blue working solution
martius yellow 1 part
martius blue 2 parts
martius red 3 parts

Procedure:
1) dewax, section to water
2) leave slide overnight in zenker’s solution
3) run in water until colourless
4) stain with weigert iron haematoxylin for 10 minutes
5) blue in water
6) stain in martius scarlet blue working solution for 10 mins
7) wash with acetic acid
8) dehydrate, clear, mount

Result: nuclei: blue/black
Erythrocyes: yellow
Fibrin: red
Connective tissue: blue


Wing Fat
TG01

Biochemistry

For the past two weeks, i am attached to biochemistry laboratory. In this lab, there are quite a number of tests they perform. For example, Urinanalysis, Fungal smear, occult blood, liver function tests, lipid panel and others. Here, in this entry, i will like to discuss about neonatal total bilirubin test and HbA1C.

Neonatal total bilirubin

This test is to screen for and monitor liver disorders, such as jaundice, or liver diseases, such as cirrhosis.

A bilirubinometer which is a direct spectrophotometer is used to measure the total bilirubin in neonatal serum to aid in indicating the risk of bilirubin encephalopathy.

Procedures

1. Centrifuge the capillary tube of neonatal blood for 3 minutes at 3000rpm.
2. By using a pipette, completely fill the cuvette with serum, holding the narrow portion of the cuvette between your thumb and index finger.
   (To avoid erroneous readings, the outside surface of the filled cuvette must clean. The sample seen in the clear rectangular area of the cuvette must be free of bubbles. If bubbles are observed, tap the cuvette lightly to clear them and refill as necessary.)
3. Open the black door on the top of the instrument and insert the cuvette into the sample carrier until the widest portion of the cuvette is seated. Close the door.
4. Press the START button. There will be a 2 second delay to allow the lamp to come to equilibrium, after which the sample carrier will rotate to bring the sample cuvette into the optical path. After a short pause, bilirubin concentration will be displayed.
5. After 15 seconds, the measurement cycle will be complete and the sample carrier will return the sample to the load position. Remove the cuvette.
6. The display will show the bilirubin value for approximately 10 seconds, then clear.

Once the result is read from the display, we will record it down on the patient’s requisition form and send it for verify.

Interpretation of results

• Newborns: When bilirubin levels are high, a condition called jaundice occurs, and further testing is needed to determine the cause. Too much bilirubin may result from the rupture of the red blood cells, or that the liver is incapable of removing bilirubin from the blood. It is not uncommon to see high bilirubin levels (sometimes called neonatal bilirubin) in newborn babies (typically 1–3 days old). Within the first 24 hours of life, up to 50% of full-term newborns, and an even greater percentage of pre-term babies, may have high bilirubin level. At birth, the newborn lacks the intestinal bacteria that help process bilirubin. This is not abnormal and resolves itself within a few days. In other instances, newborns’ red blood cells may have been destroyed because of Rhesus blood typing incompatibility.
• Adults and children: In adults or older children, bilirubin is measured to diagnose and/or monitor liver diseases (such as cirrhosis, hepatitis, or gallstones).

HBA1c

HbA1c is a test that measures the amount of glycosylated hemoglobin in your blood. It is used to measures your blood sugar control over several months and gives a good estimate of how well diabetes is being managed over a period of weeks to months.

Glycosylated hemoglobin (hemoglobin A1c, Hb1c, HbA1c or HgA1c) is a molecule in red blood cells that attaches to glucose (blood sugar). It is a form of hemoglobin used primarily to identify the average plasma glucose concentration over prolonged periods of time. Its name is sometimes abbreviated to A1C. It is formed in a non-enzymatic pathway by hemoglobin's normal exposure to high plasma levels of glucose. Glycation of hemoglobin has been implicated in nephropathy and retinopathy in diabetes mellitus.

Monitoring the HbA1c in diabetic patients may improve treatment.

Normal Results

HbA1c is normal if it is 5% or less. Normal ranges may vary slightly.

Abnormal Results

Abnormal results mean that your blood glucose levels have not been well-regulated over a period of weeks to months. If your HbA1c is above 7%, it means your diabetes is poorly controlled.

That's all for this entry. hope u enjoy reading. =)

Doreen Ng (tg 01)

SIP

Subject: Blood Banking
Topic: Bone Marrow Transplantation (BMT)

Bone marrow contains immature cells (stem cells), which develops into RBCs, WBCs & platelets

Purpose: To replace unhealthy cells with healthy ones in order to survive

Patients: People with diseases of the blood, bone marrow, or certain types of cancer, (e,g., Hodgkin's disease, mulitple myeloma, leukaemia, etc.)

Conditioning/preparative regimen: Treatment with high doses of chemotherapy or total body irradiation, given immediately prior to a transplant.

Purpose: To help remove the patient's disease prior to the BMT & to suppress immune reactions. It also has an immunosuppressive effect which prevents rejection of the BMT by the recipient's immune system.

If donor's bone marrow doesn not match, it'll treat the patient's body as foreign material to be attacked & destroyed, which results in graft vs host disease (GVHD). This is when patient's own immune system destroy the new bone marrow (graft rejection).

During the period when patient waits for the transplanted bone marrow to migrate to the cavities of large bones, setting up 'engraft' to the stage where it begins to produce normal blood cells, the patient is susceptible to infection & excessive bleeding. Platelets are transfused to patients who are bleeding.

Until the donor marrow engrafts, RBCs & platelets will be needed to keep the hemoglobin level above 9g/dl and platelet count > 20,000/ul. Engraftment takes place in 2 to 4 weeks but may be delayed if complications occur.

As a result of the total body irradiation & chemotherapy required for preparation for transplantation, patients are immunosuppressed & susceptible to GVHD, if transfused with blood components that contains live lymphocytes, which are capable of dividing. Tranfusion related GVHD can be prevented by irradiating all blood components.

Why use irradiated & filtered (leucodepleted) blood products?

To prevent patients from febrile non-hemolytic transfusion reaction due to leucocytes & leucocytes fragments in whole blood or packed cells.

Blood cell gamma irradiator is used for irradiating blood components such as red cell or platelet concentrates. The gamma rays effectively stop cells dividing by mitotic division, preventing lymphocyte proliferation.

Since alloimmunisation is caused by leucocytes contained in the blood components, leucodepleted red cells has been used to minimise the likelihood of inducing alloimmunisation.

The immunosuppression also results in great susceptibility to infectious diseases. Most patients will receive anitbiotics, antifungal agents & intravenous immunoglobulin to help prevent fight infection.

ABO Incompatible Transplants:
ABO incompatible transplants may be done when there is patient antibody directed against donor cells (major incompatibility; e.g., patient O, donor A) or donor antibody directed against the patient's red cells (minor incompatibility; e.g., patient A, donor O).

Example 1:
Patient 1's blood group: A+
Donor 1's blood group: O+

Blood product (irradiated & filtered): Packed cell: O+
Platelet/CSP: Group A+ (1st choice)/Group O+

Problem: Continued production of antibody by the new donor lymphocytes while there are original patient's cells continuing to circulate. Thus, the group O donor cells may begin to produce anti-A while the orginal patient A red cells remains, leading to hemolysis.

Solution: This can be lessened by red cell exchange transfusion if necessary.

Group A Recipient with Group O Donor:

Anti-A	Anti-B	Anti-AB	A Cells	B Cells	O Cells	Interpretation
+	-	+	-	+	-	Group A
+/-	-	-	-	+	-	Incomplete Group A
-	-	-	+/-	+	-	Incomplete Group O
-	-	-	+	+	-	Complete Group O

ABO blood grouping of transfused patient:
For incomplete group A, there will be a mixed field reaction when tested with anti-A. Some of the red cells are agglutinated while others are not. The anti-sera will appear to be cloudy due to some free red cells. This occurs when the patient's blood group (A+) is slowly switching to the new donor's blood type (O+).

Example 2:
Patient 2's blood group: O+
Donor 2's blood group: A+

Blood product (irradiated & filtered): Packed cell: O+
Platelet/CSP: Group A+ (1st choice)/Group AB+

Problem: When ABO-incompatible marrow is to be transfused into a patient with circulating antibodies against the donor's ABO type, hemolysis of the red cells in the marrow can be expected.

Prevention: By processing the marrow to remove the red cells.

Following transplant & after engraftment, the patient will become A+ but cannot begin to receive group A red cells until the circulating anti-A has disappeared. Anti-A persists longer than anti-B. Thus, even after transfusion of type A marrow, the patient continues to receive type O red cells containing additonal anti-A that can slow down the appearance of type A red cells.

If delayed red cell engraftment is occurring, the titer of anti-A should be determined & a DAT done to determine if high levels of circulating anti-A are hemolysing newly forming A red cells.

In making the decision when to convert to the new donor ABO type for red cell transfusion, we use the crossmatch to determine the absence of circulating ABO antibody incompatible with the patient's original type. The patient's serum should be tested in the antiglobulin phase to ensure the absence of free ABO-incompatible antibody.

When the ABO antibody to donor type is undetectable & DAT is negative, this is a satisfactory indicator of the safety to begin transfusion the new donor ABO type red cells.

Group O Recipient with Group A Donor:

Anti-A	Anti-B	Anti-AB	A Cells	B Cells	O Cells	Interpretation
-	-	-	+	+	-	Group O
+/-	-	+/-	-	+	-	Incomplete Group A
+	-	+	-	+	-	Complete Group A

dorothy png
tg01

Staining and staining problems

Staining and staining problems

There is 2 main types of stains that is used most commonly. They are the Romanowsky and Papanicolaou(and it's derivatives). Romanowsky-type stain are more rewarding and practical, and readily available in practice situations. Example of such stains are Wright's stain, Giemsa stain, Diff-Quik. Romanowsky-type stain are inexpensive, readily available to practicing veterinarian, easy to prepare, maintain and use. They stain organism and cytoplasm of cells very well.

Smears are first air dried, preserving(fix) the cells, and cause them to adhere to the slide so they do not fall off during the staining procedure. Diff-Quik does not undergo the metachromatic reaction. As a result, granules of some mast cells do not stain, When mast cell granules do not stain, the mast cells may be misclassified as macrophages. This can lead to confusion in examination of some mast-cell tumors. Variation between different Romanowsky-type stains should not cause a problem once the evalutator has become familiar with the stain used routinely.

The procedure recommended for each stain should be followed generally. However, adaptations is to be applied according to the thickness of smear. Thick smear, higher protein concentration, longer stain time. Vice-versa for thin smear.

Poor stain quality often confuse both the novice and the experienced cytologist. Most staining problem can be avoided if the following precaution are taken:

• Use new slides, fresh and well filtered(if needed) stain, and fresh buffer(if needed)


• Stain cytologic preparations immediately after drying


• Take care not to touch the surface of slide or smear at any time.
  
  

The table below shows some common problems and solution.

Problem	Solution
Excessive Blue Staining
Prolonged stain contact	Decrese staining time
Inadequate wash	Wash longer
Specimen too thick	Make thinner smears
Stain, diluent, buffer or wash water too alkaline	Check with pH paper and correct pH
Exposure to formalin vapors	Store and ship cytologic preps separate from formalin containers
Wet fixation in ethanol or formalin	Air dry smears before fixation
Delayed fixation	Fix smears sooner
Surface of the slide was alkaline	Use new slides
Excessive Pink Staining
Insufficient staining time	Increase staining time
Prolonged washing	Decrease duration of wash
Stain or diluent too acidic	Check with pH paper and correct pH; fresh methanol may be needed
Excessive time in red stain solution	Decrease stain time in red solution
Inadequate time in blue stain solution	Increase time in blue stain solution
Mounting coverslip before preparation is dry	Allow preparation to dry completely before mounting coverslip
Weak Staining
Insufficient contact with one or more of the stain solutions	Increse staining time
Fatigued(old) stains	Change stains
Another slide coverd specimen during staining	Keep slides separate
Uneven Staining
Variation of pH in different areas of slide surface	Use new slides and avoid touching their surface before and after preparation
Water allowed to stand on some areas of the slide after staining and washing	Tilt slides close to vertical to drain water from the surface or dry with a fan
Inadequate mixing of stain and buffer	Mix stain and buffer thoroughly
Precipitate on Preparation
Inadequate stain filtration	Filter or change the stains
Inadequate washing of slide after staining	Rinse slides well after staining
Dirty slides used	Use clean new slides
Stain solution dries during staining	Use sufficient stain and do not leave it on slide too long
Miscellaneous
Overstained preparation	Destain with 95% methanol and restain
Refractile artifact on RBC with Diff-Quik stain	Change the fixative

*Reference: Cowell & Tyler (1992), Cytology and Haematology of the Horse.

Cheers
Douglas

SIP

Subject Title: Clinical Chemistry
Name of Test: Total Carotene Test


Carotene is an orange photosynthetic pigment important for photosynthesis. The characteristic orange colour of carrots and red yellow pigments in fruits and vegetables are due to carotenoids. It comes in two primary forms; alpha-carotene and beta-carotene. Beta-carotene is probably the best known of them and is a precursor of Vitamin A. Carotene is stored in the liver and converted to Vitamin A as needed. In addition, carotene is an antioxidant nutrient so it can reduce the risks of cardiovascular diseases and some forms of cancer.


Principle and Procedure of Test
In this test, protein are first removed by precipitation with ethanol. The carotene and other form of carotenoids are extracted with petroleum ether. The characteristics absorbance is measured at 450nm.

Before running the patient sample, two levels of controls are run. Calculations are done to obtain the concentration of carotene. After ensuring that the QC results are acceptable, we will run the patient sample.
1) Pipette 1mL of patient serum sample into the glass test-tube

2) Add 1ml of absolute ethanol into the glass test-tube

3) Vortex the glass test-tube

4) Add 2ml of petroleum ether and vortex them for 10 min to ensure they are well mixed

5) Allow the glass test-tube to stand for 5min

6) After 5 min, extract the top layer of ether and transfer them into the glass microcurvette until it is filled to the top for absorbance readings. This is to enable complete light transmission through the solution

7) Using petroleum ether as blank, place the glass microcurvette into the spectrometer. Check for turbidity in the sample as it can cause false high absorbance readings

8) The absorbance is measured at 450nm

9) Calculations are done to obtain the concentration of carotene. This is as follow:

ODdiff X 14.59 = ____µmol/L

*Note: ODdiff = OD (450nm) – OD blank


Reference Range: 0.93 to 3.73µmol/L


Clinical Interpretation
Concentrations above the reference range shows a high levels of carotene in the body known as carotenemia. Unlike excess Vitamin A, excess carotene is non-toxic. Although carotenemia is not dangerous, it can lead to yellowish/orange discolouration of the skin, mostly occurring in the palms of hands and the soles of the feet. This is due to the consumption of abundance carotene from foods (such as carrot juice) or from supplements. It is nearly impossible to overdose on carotene because the body excretes the excess. This can also be a medical sign of other conditions such as renal diseases. Since carotene itself is harmless, it does not require treatment. Reducing high quantities intake of carotene will gradually remove the colourisation of skin. Sometimes yellowish discolouration of the skin can be misdiagnosed as jaundice. Jaundice affects the colour of the eye while carotenemia does not. A bilirubin serum test can be used to identify whether jaundice is involved.

Concentrations below the reference range shows there is carotene deficiency. Such deficiencies are seldom seen, however, even in people who don't eat fruits or vegetables or take supplements, because so many other foods supply the nutrient. Symptoms of carotene deficiency include dry skin, night blindness and susceptibility to infection.

Total carotene test is a valuable part of diagnosis of Vitamin A deficiency or excessive Vitamin A. This is because carotene is converted to Vitamin A in the body. However, the actual diagnosis requires interpretation of test result in conjunction with other clinical findings.


Soong Ci Liang
0503333G
TG01

CELL-DYN 3700: Principles of Operation

Sample Aspiration

There is 2 modes of aspiration.

• Open Sampler Mode is used to aspirate sample from a collection tube that has been opened and is held under the Open Sample Aspiration Probe.
• Manual Closed Sampler Mode or Automated Sample LoaderMode aspirate blood directly from a capped collection tube by piercing the tube stopper.

Aspiration volume for open mode is 130uL ± 5%, 240uL ± 5% for close mode, and 355uL ± 5% for Sample Loader mode. Sample is aspirated into the Analyzer by the Aspiration Peristaltic Pump, through the Shear Valve.

White Blood Cells(WBC) Analysis

2 measurements: WBC optical count (WOC) and WBC impedance count (WIC).

1. WOC Sheath Syringe dispenses 1.6mL of Sheath Reagent through the Shear Valve, picking up 32uL sample.
2. Sample segment and sheath are then routed to WOC mixing chamber where dilution is bubble-mixed. (1:51)
3. WOC Peristaltic Pump transfers the WOC dilution from the WOC Mixing Chamber to Sample Feed Nozzle in WOC flow cell.
4. A stream of WOC Sheath Reagent is directed through the Flow Cell.
5. WOC Metering Syringe injects 78uL of WOC dilution into the Flow Cellsheath stream.
6. A laser beam is focused on the Flow Cell. As the sample stream intersects the laser beam, the light scattered by the cells is measured at four different angular intervals.

1. WIC/HGB Diluent Syringe dispense 5.25mL of Diluent through Shear Valve, picking up the 20uL WIC/HGB sample segment.
2. Segment and Diluent fouted to Mixing Chamber in the von Beherns WIC Transducer. At the same time, WIC/HGB Lyse Syringe delivers 0.75mL of WIC/HGB Lyse to mixing chamber.
3. The dilution is bubble-mixed. Final dilution is 1:301.
4. Dilution is pulled through the aperture by vacuum, Volumetric metering ensures 200uL used for measurement.
5. Electrical Impedance is used to count WBC as the traverse the aperture.
6. When the count portion of the cycle is completed, the aperture is auto-cleaned by Aperture cleaning circuit.

RBC/PLT Analysis

1. RBC Diluent Syringe dispenses 7.2mL diluent through Shear Valve, picking up 0.74uL RBC/PLT sample segment
2. Sample segment and diluent routed to mixing chamber of von Behrens RBC/PLT Transducer where dilution is bubble-mixed. (1:9760)
3. Dilution is pulled through the aperture by vacuum. Volumetric metering ensures that 100uL of dilution is measured.
4. Electrical Impedance is used to count RBCs and PLTs as they traverse the aperture.

Haemoglobin Analysis

1. After 200uL of WIC /HBG are metered through WIC aperture, remaining dilution is transferred to HGB Flow Cell.
2. HGB concentration is measured spectrophotometrically.

*All information obatined from ABBOTT CELL-DYN 3700 operator's manual.

Cheers
Douglas

Subject: MMIC
Topic: Methicillin Resistant Staphylococcus Aureus (MRSA) Rapid Screening

Introduction
Methicillin-resistant Staphylococcus aureus (MRSA) is a bacterium that causes infections in different parts of the body. It's tougher to treat than most strains of staphylococcus aureus -- or staph -- because it's immune to some commonly used antibiotics.

Recent research suggests that in the identification of MRSA, it is more accurate to either directly detect the gene encoding the methicillin resistance determinant (mecA) or its product, penicillin-binding protein 2' (2a), or PBP2' (PBP2a), which is found in the cell membrane of MRSA. However, as nucleic acid hybridization and DNA amplification techniques such as PCR for detecting the mecA gene are expensive and technically demanding, simple and more inexpensive techniques are required for routine use. MRSA-Screen was developed expressly for this purpose, providing results in 15 minutes with minimal labor and no specialized equipment.

Principle
MRSA Screen is a rapid slide latex agglutination assay which detects the penicillin binding protein 2 (PBP2’) of methicillin-resistant S.aureus. Latex particles are sensitized with a monoclonal antibody against PBP2’ and will specifically react with MRSA to cause agglutination visible to the unaided eye. It will therefore enable the rapid and appropriate antimicrobial therapy for serious MRSA infections.

Equipment & Materials
NMRSA Screen kit
Sensitised Latex 2.5 ml x 1
Control Latex 2.5 ml x 1
Extraction Reagent 1 10 ml x 2
Extraction Reagent 2 5 ml x 1
Test card 55
Mixing stick 220
BAP
Micropipette and tips (50ml)
A beaker, tripod, wire net and a burner for boiling the specimen


Procedures
A. Sample Preparation
1) Colonies believed to be S. aureus taken from BAP or other media, after incubating for 18-24 hours at 35°C are used.

B. PBP2’ Extraction
2) Add 4 drops of Extraction Reagent 1 into a test tube.
3) 4 drops of Extraction Reagent 1 are added into a test tube.
4) A loopful of cells are taken using a platinum loop or other appropriate device and suspended in the test tube.
5) The test tube is placed into a boiling water bath or heating block and heated for 3 minutes.
6) The test tube is removed and allowed to cool to room temperature.
7) A drop of Extraction Reagent 2 is added into the test tube and mixed well.
8) The test tube is centrifuged at 1500 xg for 5 minutes.
9) The supernatant is used as specimen.

C. Latex Agglutination
10) Circles on test card are labeled with Sensitised latex and Control latex for each specimen.
11) 50 ml of specimen is placed onto the test circles
12) Sensitised latex or control latex is added into each respective test circles.
13) They are mixed well using a mixing stick
14) The test card is rotated by hand for 3 minutes and agglutination patterns are observed.

Interpretation
Agglutination is seen with Sensitized but not Control Latex: PBP2’ positive (MRSA)
No agglutination is seen with either latex reagent : PBP2’ negative (MRSA)



Boon Ching

Cytogenetics

For the past 4 weeks, I was attached to Cytogenetics Laboratory. In this entry, I will discuss about peripheral blood culture for chromosome studies.

Peripheral Blood Culture

Specimen Requirement

• 3 - 5ml of peripheral blood in sterile tube wuth sodium heparin as anticoagulant.
• Sterile techniques must be observed when collecting blood.
• The sample is sent to cytogenetics lab as soon as possible.

Clinical Significance

1. T-lymphocytes present in peripheral blood are exposed to a mitogen (e.g. PHA) to stimulate cell divison. They are then arrested at metaphase with the addition of colcemid. Chromosomes for analysis are obtained upon harvesting.
2. Clinical uses of constitutional karyotype obtained from blood cultures include:

• Genetics diagnosis/genetic counselling
• Determine the constitutional karyotype of families of individuals with chromosome abnormalities
• Determine carrier status
• Comparison of lymphocyte karyotypes with those of other tissue e.g. skin, AF or CV
• Confirmation of structural and numerical abnormalities or mosaisim
• Determine the constitutional karyotypes of patient with haematologic malignancies or solid tumors
• Aid in the diagnosis of chromosome breakage syndromes e.g. Fanconi Anaemia
• To provide material for metaphase FISH

Method

1. Thaw 1 tube of complete Rpml medium and 1 tube of complete M199 medium in water bath at 37ºC.
2. Add 0.1ml PHA into each tube.
3. Centrifuge blood specimen at 1200rpm for 10 minutes. Record volume of blood received and note down whether there is haemolysis, jaundice, clot etc. in worksheet.
4. With a sterile transfer pipette, gently pick up »1ml of buffy coat together with some plasma from the blood.
5. Invert transfer pipette and mix the sample thoroughly in the bulb. Distribute the sample into the culture tubes.
6. Tighten caps and mix well by inverting the culture tubes several times.
7. Place the culture tubes in a slant test-tube rack. The slant is at approximately 45º angle and the purpose is to increase the surface area of the culture for gaseous exchange. Leave the rack in a 37ºC incubator for 48 hrs.
8. Invert the tubes every morning to re-suspend pellet.
9. 48 hrs after culture set-up, add 50μl of MTX x 10-5 m working solution into each tube. Mix well by inverting the tubes and re-incubate at 37ºC for another 18 hrs.
10. After 18 hrs, add 50μl of thymidine to each tube.
11. Mix well and incubate at 37ºC for 4 hrs.
12. Proceed to harvesting.

Note: MTX is to block cells in the S (DNA synthesis) phase of cell cycle. Thymidine is to promote longer and better G-banded chromosomes in routine blood cultures.

Harvesting of Peripheral Blood Cultures

Harvest Process

1. Centrifuge tubes at 1200 rpm for 10 minutes. Aspirate supernatant.
2. Loosen pellet gently. Add approximately 8-10ml warmed hypotonic solution into each tube; invert tubes 2-3 times to ensure the suspension is well mixed.
3. At the beginning of handling the first tube, start a timer on 5 minutes counting down for every batch of »12 tubes. Leave the tubes at room temperature for the whole period of 5 minutes.
4. At the end of the 5 minutes, transfer tubes unto a covered centrifuge buckets and spin tubes at 1500 rpm for 6 minutes.
5. Aspirate and discard supernatant.
6. Repeat step 2 and 3.
7. At the end of the 5 minutes, add 1 pipetteful of fixative and invert tubes 2-3 times to mix well.
8. Repeat step 4 and 5.
9. Re-suspend cell pellet by flicking tubes until no pellet clumps are visible.
10. Add another 5 pipetteful of fixative. Invert tubes 2-3 times to mix well.
11. Stand the tubes at room temperature for at least 15 minutes. Aspirate and discard supernatant.
12. Centrifuge the tubes at 1000 rpm for 10 minutes.
13. Re-suspend pellet in 4 pipetteful of fixative. And repeat step 12. check that the suspension is clear after centrifugation, if not repeat this step again especially for clumpy and jelly-like pellets.
14. Re-suspend pellet in 3 pipetteful of fixative and store in refrigerator at 4ºC until ready to make slides. Proceed to slide making.
    

After slide making and staining, the slide is ready for chromosome analysis.
That’s all for this entry. Enjoy reading. Feel free to ask questions. =)

Doreen (tg 01)

p.s. for the interview question, the amniocytes are not lysed. Fixation can strengthen cell membranes, improve chromosome morphology, and rid the sample of debris such as lysed red cells. Hence, after fixation, the cells are ready for slide making and staining.

SIP

Subject: Blood Banking

Topic: Tranfusion Reaction Investigations

Transfusion reaction is any adverse outcome associated with the infusion of blood or blood components. The most common signs & symptoms are chills, fever & urticaria. Patients can show these symptoms during or within several hours after transfusion.

Require:

• 5 ml clotted blood (purple capped tube)
• 5 ml plain blood (yellow capped tube)
• Pre-tranfusion specimen (taken for crossmatching before transfusion reaction)
• Donor pilot tube & segment tube* from blood pack

*pilot tube - blood segment taken from blood pack before transfusion
*segment tube - taken from blood pack after transfusion

1. Exclude clerical errors
The transfused blood pack and patient sample are examined for the following to exclude possible clerical errors in patient or blood identification in the lab:

• Patient identification (name & IC no.)
• Unit ID on both blood bag & label
• Pre-reaction records on patient's buff card (ABO/Rh typing, antibody screening & compatibility results)
• Computer record of both patient & blood unit

2. Visual check
Visual check is done on both the Post I (blood sample) & Post II (blood & urine samples) for hemolysis. (E.g. Haemoglobinaemia, haemoglobulinuria & spherocytosis)

3. Exclude serological errors
a) ABO blood grouping and Rh typing are performed on the following for evidence of a mismatched ABO/Rh or gross incompatibility:

Patient's
- Pre transfusion sample
- Post transfusion sample I (Post I)
- Post transfusion sample II (Post II)

Donor's
- Pilot tube
- Blood pack/segment

b) Direct Antiglobulin Test (DAT) is performed on patient's sample (pre, post I & post II) & donor's sample (pilot tube & segment).
- To demostrate antibodies/complement bound to red cells in vivo.

c) Compatibility testing between patient's samples (Pre, Post I & II serum) & donor's cells are repeated.

d) Antibody screening on the patient's samples (Pre, Post I & II serum) for clinically significant Ab are repeated.

4. Biochemical analysis
Plain blood (yellow cap) sample is sent for biochemical investigations of:

• Serum bilirubin
  (High level of bilirubin -> haemolysis)
• Haptoglobulin
  (Low level of haptoglobin –> increased intravascular haemolysis)

5. Immunohaematological investigation (HLA Ab test)
The test is done when the patient has any 1 of the symptoms (chills, rigors, elevated temperature)and suspected of non-haemolytic febrile transfusion reaction.

6. Bacteriological investigation
The blood pack is sent for sterility testing.
- Perform gram stain & culture the donor's unit to check for bacterial contamination.

Classification

Type of Reaction	Positive Results
Acute Haemolytic Reaction	- Visual changes suggestive of hemolysis - Mismatched ABO/Rh typing - Missed antibody - Missed inappropriate crossmatching - Biochemical hemolysis
Reaction due to Bacterial Contamination	- Visual changes suggestive of bacterial contamination - Positive culture result
Non-Haemolytic Febrile Transfusion Reaction	- Presence of HLA antibodies
Allergic Transfusion Reactions	- Presence of IgA antibody

dorothy
Tg01

SIP

Subject: histopathology
Topic: special stain

Special stains such as PAS for carbohydrate, Gomori’s Methenamine silver nitrate for fungus and Gordon and sweet’s silver impregnation for reticulum fibres can be performed by an automated staining machine. The automated stainer used in the lab is Ventana NexES.(The procedure had been introduced by Sharon and June’s blog so I am not going to repeat) Some special stains can only be performed manually as the kit for that particular stain is not available or temporally out of stock. Perl’s reaction and von kossa’s stain are example of special stains that can be perform manually.

Perl’s reaction

Function: demonstration of iron and haemosiderin

Principle: Dilute hydrochloric acid is used to release haemoglobin or myoglobin in red cells that is tightly complexed with ferric iron. An insouluble blue compound, ferric ferrrocyanide will be formed once the ferric iron reacts with potassium ferrocynade solution.

Chemical required: 20% aqueous hydrochloric acid
20% aqueous potassium ferrocynanide
Nuclear fast red

Procedure:
1) Dewax, and section to water.
2) Prepare perl’s reagent. (equal part of 20% aqueous hydrochloric acid and 20% aqueous potassium ferrocynanide)
3) Treat with perl’s reagent for 20 minutes.
4) Rinse with distil water.
5) Counterstain with nuclear fast red
6) Wash, dehydrate, clear and mount

Results: iron----dark blue
Nuclear----red

Von Kossa’s stain for calcium

Function: demonstrate calcium deposits.

Principle: tissue on the slide is treated with Silver nitrate solution. Under the presence of ultra-violet light, calcium salt is reduced and replaced by silver deposits, and will be appeared as metallic silver.

Chemical required: 10% aqueous silver nitrate
Nuclear fast red

Procedure:
1) Dewax and section to water
2) Soak slides with 10% aqueous silver nitrate and leave under UV light for 3-4 hours
3) Rinse in water
4) Counterstain with nuclear fast red for 1-3 minutes.
5) Wash, dehydrate, clear and mount in DPX.

Results: calcium----black

Wing Fat
Tg01

SIP

Subject Title: Clinical Chemistry

Name of Test: Urine specific gravity

The specific gravity of urine indicates the relative proportions of dissolved solid components to the total volume of the specimen. It measures the concentrating and diluting abilities of the kidney. Water has a specific gravity of 1.000. Since urine is a solution of minerals, salts and organic compounds in water, the specific gravity is greater than 1.000. The more concentrated the urine, the higher the urine specific gravity. Knowledge of the specific gravity is needed in interpreting the results of most tests performed in routine urinalysis. Specific gravity is measured using the refractometer and is determined by the principle of refractometry.

A refractometer

Principle of Test
An indirect method of measuring specific gravity is refractometry. The refractometer does this by measuring the refractive index of the solution. The refractive index of a solution is related to the content of dissolved solids present. It is the ratio of the velocity of light in air to the velocity of light in solution. This ratio varies directly with the number of dissolved particles in solution. The refractometer utilizes a light source, which pass through a chamber containing the specimen and a prism with scale readings. There is a direct relationship between the reading of the degree of refraction of light and the concentration of the specimen. This reading can be converted to specific gravity. Scale readings are in terms of specific gravity, refractive index and serum or plasma total protein. In my lab, we do not use refractometer to measure the total protein of patient sample but use Roche Modular analyzer to measure it. The refractometer is compensated to give correct readings on aqueous solutions at all temperatures ranging from 15°C-37°C. Readings need not be adjusted for either the temperature of the sample or the temperature of the room in which the refractometer is used.

Procedure

Before running any patient samples, we must first do quality control. In my lab, we use KOVA liqua-Trol level 1 (normal) and KOVA liqua-Trol level 2 (abnormal).

1) Label the test request form, urine container and a test-tube

2) Pipette a few ml of random urine into the labeled test-tube

3) Add a few drops of distilled water into the coverslip of the refractometer and clean it using gauze, ensuring the coverslip and prism are clean and dry

4) Add few drops of urine into the coverslip, making sure that no air bubbles are trapped within.

5) Read the specific gravity using the urine specific gravity scale. Avoid lifting the coverslip before the reading is made

6) Record the result on the test request form, which will be later keyed into the LIS

7) Add few drops of distilled water into coverslip again for washing off the urine and dry it using gauze. It is important that the refractometer is always maintained in a clean state

Reference Range

Normal values range from 1.003-1.030 but average is between 1.010 and 1.025. Specific gravity is highest in the first morning specimen, which is usually greater than 1.020 but not exceeding 1.030, due to the highly concentrated urine.

Clinical Interpretation
Low Specific Gravity
Diabetes insipidus, a rare disease caused by impaired functioning of the antidiuretic hormone (ADH), is the most obvious and severe example of the loss of effective concentrating ability of kidneys. This disease is characterized by large volume of urine with low specific gravity. Specific gravity in such case usually ranges between 1.001 and 1.003. Low specific gravity can also occur in patients with glomerulonephristis and pyelonephritis. Pyelonephritis is an ascending urinary tract infection that has reached the pelvis of the kidney. In these diseases, the kidney has lost its ability to reabsorb water due to tubular damage. Thus urine remains dilute. Urine with a fixed low specific gravity is indicative of severe renal damage with disturbance of both the concentrating and diluting abilities of the kidney.

High Specific Gravity
Specific gravity is high in patients when there has been excessive loss of water, as with
sweating, fever, vomiting and diarrhea. Increased secretion of ADH will cause increased tubular water reabsorption and decrease in urine volume. Hence it will have high specific gravity. Trauma, stress, infections, surgery and many drugs cause increased in ADH secretion. Moreover, abnormally high amounts of some of the urinary constituents, in particular glucose and protein, can increase specific gravity.

Urine specific gravity is used for monitoring of diseases and we can use urine osmolality test to further confirm the results. Osmolality is a more exact measurement of urine concentration as specific gravity depends on precise nature of the molecules in the urine and requires correction for the presence of glucose and protein. Urine specific gravity is easy and convenient to test.

Soong Ci Liang
TG01
0503333G

SIP

Subject: MMIC
Topic: Reading of urine cultures

Some of the properties that can be used to identify the organisms
· Colony appearance (colour and shape)
· Ability to ferment lactose
· Ability to haemolyse red blood cells
· Ability to produce cytochrome oxidase

Lactose Fermentor (LF) Vs Non-Lactose Fermentor (NLF)

LF	NLF
Able to utilize lactose on CLED	Unable to utilize lactose on CLED
Forms white to grey colonies on BAP but forms yellow colonies on CLED	Forms white to grey colonies on BAP and CLED
Produces acid causing CLED to turn yellow	May produce alkaline causing CLED to turn blue

Haemolysis

Some organisms are able to cause the lysis of the sheep red blood cells found on BAP. Haemolysis can be detected through holding up the back of plate against a source of light.

Haemolysis on BAP can be classified into two categories:
• α-haemolytic
o greenish-looking colonies
• β-haemolytic
o forms a clearance around the colonies

Oxidase Test
The oxidase test checks for the production of cytochrome oxidase (an enzyme). The oxidase reagent contains a compound that changes color when it becomes oxidized (i.e. chromogenic reducing agent).

Procedure:
A drop of oxidase reagent is dropped onto the test strip. The test strip is then used to lightly touch the colonies.
Positive: Blue (cytochrome oxidase present)
Negative: No colour change

Identification of Microorganism

Microorganism	Colony Appearance	Ferment Lactose	Other Properties
Escherichia Coli	• Flat, grey colonies on BAP • Form yellow colonies on CLED	Yes	-
Klebsiella spp.	• Mucoid, convex colonies • Form white colonies on BAP • Form yellow colonies on CLED	Yes	-
Streptococcus spp. (GPC)	• Small, white colonies on BAP • Form yellow colonies on CLED	Yes	• Catalase negative Haemolysis: • α-haemolytic: eg. Streptococcus pneumoniae • β-haemolytic: Streptococcus group A, B etc • Non-haemolytic: E.g. Enterococcus spp. (Greyer appearance)
Staphylococcus spp. (GPC)	• Small, white, flat colonies on BAP • Form yellow colonies on CLED	Yes	• Catalase positive • Can be hemolytic positive or negative
Pseudomonas spp.	• Swarms on BAP • Spready colonies on CLED	No	• Pungent smell • Oxidase positive • Swarming ability
Proteus spp.	• Swarms on BAP • Glossy	No	• Pungent smell • Swarming ability
Candida spp.	• Small, flat, white colonies on BAP and CLED • Slightly spready • Not glossy	No	-

E. Coli














Klebsiella spp.














Pseudomonas and Klebsiella spp.














Proteus spp.














Candida spp.
















Boon Ching

Haematology

I have been attached to Haematology lab for three weeks. Normally, i performs routine tests like FBC, PBF, ESR and other miscellanous tests like dengue serology test and malaria parasite screening. erm.. so here i will elaborate on the dengue serology test.

Dengue Serology Test

Purpose
It is performed to obtain a qualitative presumptive detection of IgG and IgM antibodies, as well as a presumptive differentiation between primary and secondary infection.

Principle
Serum antibodies of the IgM/IgG class, when present, bind to the anti-human IgM/IgG immobilized in 2 lines across the test strip. The colloidal gold-labeled antiflavivirus monoclonal forms complexes with the dengue antigen that is captured by dengue specific IgM/IgG in the patient’s serum. These complexes are visualized as pink/purple line(s).

Sample requirement
- At least 1ml of capillary/venous blood collected in plain tube
- Centrifuge the sample at 3000 rpm for 10 minutes
- Sample serum should be preferably clear, non-haemolysed, non-lipaemic and non-icteric.

Method:
Use the Denge Duo IgM and IgG Rapid Strip Test Kit (PanBio)
Pipette 10μl of the serum onto the strip kit and the result is to be viewed after 15 minutes. Before conducting this test, the kit is kept in the fridge and so when this test is requested, we have to take it out and keep it in the room temperature for 10-15 minutes.

Dengue results are reported in LIS as follows:

Results Interpretation

Control positive only
If dengue infection is strongly suspected, consider PCR for dengue virus. Within 1st 5 days of illness. If this is an acute specimen, PCR can be performed on the sample, which has been retained in the lab.

IgM and Control positive
Consistent with 1° dengue. Occasional false positive may occur in certain infections such as measles.

IgM, IgG and Control positivie
Consistent with 2° dengue

IgG and Control positive
Consistent with 2° dengue. Cross reactivity may occur with flavivirus.

Primary (1°) infection is characterized by the presence of detectable IgM antibodies 3-5 days after the onset of infection.
Secondary (2°) infection characterized by the elevation of specific IgG antibodies 1-2 days after the onset of infection and in the majority of the cases this generally accompanied by an elevation of IgM.

that's all for this entry. enjoy reading. take care all man. enjoy ur SIP. =)

Doreen (tg o1)

SIP

Subject Title: BLOOD BANK
Topic: Blood banking processes

yep, I was attached to the Blood Bank Laboratory for the whole of 20 weeks. There are 4 stations in the lab, 1) Requisitor 2) Screener 3) Depatching & Crossmatch 4) Issuer. I learnt quite a number of things through observations and some hands-on under supervision. I was not allowed to handle patients' samples yet as results had to be very accurate or else the consequences of wrong results can lead to adverse transfusion reaction.

- Sorting out labels that are returned to the lab after transfusion
- Handling of blood products received from CTM
-> Scan 1) Unit no. 2) Expiry date 3) Product code 4) Blood type into the LIS to document all the stock that are received.
-> Store in refrigerator under pending ABO rechecks for blood units.
-> Store in freezer at -60 degrees celsius for FFP (Fresh Frozen Plasma)
- Retrieval of transfusion records from filing cabinet
- Preparation of ABO tubes for screening and recheck
- ABO recheck of blood units

Purpose: To ensure correct blood type in the blood pack, preventing errors
E.g: Recheck of O+ve blood group
Procedures:
1) Paste the donor blood ID sticker on the "rechecking of donor blood form". Tally the unit no. of the blood pack with the label to prevent errors. (Every blood pack comes with a label which is used to fill in the particulars of the patient, date of transfusion, then issue out together with the blood unit and return back to the lab after transfusion.)
2) Add 1 drop of Anti-AB into a test tube.
3) Add saline into another tube.
4) Remove a segment from the blood pack. Draw the blood sample using a pipette. Dilute the blood in saline.
5) Add 1 drop of blood to the tube containing Anti-AB.
6) Centrifuge for 15 seconds at 3000 rpm.
7) Check for agglutination, record results under "rechecking of donor blood form".

Results: Negative. As O red cells lack of A and B antigens, thus there is no agglutination present with mixed with Anti-AB.

Blood samples are sent to Blood Bank Laboratory for ABO/Rh grouping and cross-match.

Role of a requisitor
When the blood samples are received, tally the doctor's signature/name, patient's NRIC no. and name on the request form with the label on the tube.

- Check with the LIS if the patient requires T1 or T2.
T1- requires ABO/Rh grouping only

T2- requires ABO/Rh grouping & Ab screening (15th day from the last Ab screening or 4th day onwards from the last blood transfusion which is just after last Ab screening.

- Centrifuge the blood tubes to separate the red cells and the serum/plasma.
Serum- top portion of the clotted blood (red tube)
Plasma- top portion of EDTA blood (lavender tube)

- Place the tubes according to the last digit of the patient's NRIC no. in the specimen rack for the screener.

Role of screener
1) Set up controls and calibrations, do daily quality control of working reagents to ensure the accuracy of results for all tests. These reagents are quality controlled daily.

• ABO cells recheck with Anti-A, Anti-B, Anti-AB to ensure that the correct cells are in the respective bottles
  
• Rh-ve & Rh+ve recheck with Anti-D

• Test if the Anti-Human globulin (AHG) reagent is working properly with *check cells & saline
  
  *Check cells are pools of group O RBCs sensitised with IgG
  
  Positive control: 1 drop of check cells with AHG reagent
  Negative control: 1 drop of check cells with 2 drops with of saline
  
  Interpretation of results
  Positive: Agglutination of RBCs
  Negative: No agglutination of RBCs


• Panel (SPO) cells recheck (SI, SII, SIII)
  Using gel card technique
  
  
  - Add 50uL of SI, SII & SIII into each 3 different microtube respectively.
  -Add 25uL of positive control cells (serum diluted with saline which contains the respective Ab) into the first 3 microtubes. Add 25uL of negative control cells (with no Ab) into the last 3 microtubes.
  - Incubate for 15 minutes
  - Centrifuge at 3000rpm for 15 seconds.
  - The results should be like the photo shown below:

*with permission from supervisor

Test: Antibody screening (Microtyping card gel)
Purpose: Screen for clinically significant blood group red cell antibodies in the pretransfusion samples, thus able to select suitabe blood units that will not cause harm to the patient, preventing adverse transfusion reactions. E.g: If the patient has Anti-Mia in the serum/plasma, blood units with the absence of Mia antigens must be issued to prevent transfusion reaction.

We used the ID LISS/Coombs Gel technique instead of the tube method we used in school.
The ID-card "LISS/Coombs" is a antibody screening panel which consists of 6 microtubes containing polyspecific AHG within the gel matrix, Rh-hr, Duffy, Kidd, Lewis, MNS, P, Kell and Mia antigens.


Procedure:
Allow the test cell reagents & samples to reach room temperature before use.
1) Identify the appropriate microtubes of the ID-card "LISS/Coombs" with the patient's name.
2) Remove the aluminium foil as many microtubes as needed.
3) Pipette 50 uL of *SPO cells (SI, SII, SIII) to the appropriate microtubes
*SPO cells - Specific pooled O+ cells with C, e, N antigens, etc
4) Add 25uL of the patient's serum/plasma to each microtube.
5) Incubate the ID-card for 15 minutes in the ID-incubator at 37 degrees celsius.
6) Centrifuge the ID-card for 10 minutes in the ID-centrifuge.
7) Grade & record the results in appropriate worksheet.
8) Leave the gel card & corresponding worksheet for counterchecking by second medical technologist.

Positive results: If the patient's serum/plasma contains abnormal Ab specific to the antigens present in the SPO cells, it forms clumps with the panel cells, resulting in agglutination. Thus, it is unable to pass through the gel and gets trapped at the top portion of the tube. Strong +ve are seen as a line at the gel/fluid interface. Weaker +ve are seen distributed throughout the gel.

Positive samples will be sent to CTM for investigations using the antibody identification panel.

Negative results: If there's absence of abnormal Ab in the patient's serum/plasma, agglutination will not occur. Thus, the non-clump cells are able to pass through the gel and settle at the bottom of the tube. -ve are seen as a red cell button at the bottom of the tube.

*All results are counterchecked by second medical technologist for verification, ensuring accuracy of results & preventing analytical errors. (internal quality control)

Abbreviated crossmatch (Compatibilty testing)

Purpose: To test whether the donor's blood is compatible with the patient's serum, thus able to promote safe transfusion of blood and prevent transfusion reactions.

Procedures:
1) Take the requested blood unit from the refrigerator according to the patient's blood type.
2) Pull out a segment from the blood pack. Draw the blood sample using a pipette. Dilute the blood in saline.
3) Centrifuge at 3000rpm for 15 seconds to wash the cells.
4) Pour away the suspension into the waste bucket and add saline.
5) Add 3 drops of patient's serum into a tube.
6) Add 1 drop of the donor's blood into the same tube.
7) Centrifuge at 3000 rpm for 40 seconds.
8) Read the results under the microscope.

Positive results: Presence of agglutination
Negative results: Donor's RBC is compatible with the patient's (recipient's) serum/plasma, therefore able to proceed with the issuing of blood unit.

Crossmatch specimens are valid for 3 days from the day of receiving after which new specimens are required. (In case of disputes, for investigation & legal cases. The red cells may lyse after that period.) Samples are placed in the specimen racks according to the last digit of the patients' NRIC no. Day 1 samples are placed on the workbench at room temperature for easy retrieval during crossmatching. Day 2 & 3 samples are stored in the refrigerator. At end of day 3, they are bundled in yellow biohazard bags.

Some of the blood products that are issued by Blood Bank:

- Packed cells (without plasma & platelets)
Function: To raise the Hb level up faster as it's more concentrated with no plasma.
Life span: 45 days

- Whole blood
Function: To produce greater expansion of blood volume, especially for patients with massive blood loss
Life span: 36 days

- Platelets (random/apheresis)
Function: To prevent or to treat bleeding due to severely low platelet counts (thrombocytopenia)
Life span: 9 days

- FFP (Fresh Frozen Plasma) & cryoprecipitate
Function: For patients with abnormal or low levels of blood clotting proteins, such as hemophilia.
Life span: when frozen, can be kept for around 1 year. when thawed, had to be used within 24 hours.

- IvIg (Intravenous Gamma Globulin)
Function: To increase patient's immunity against infections

- Human albumin (5%/20%)
Function: For restoration and maintenance of circulating blood volume

- Factor VIII
Function: For treatment of acquired Factor VIII deficiency or hemophilia A

- Autologous blood

- Leco-reduced/ deglycerolysed RBC

Leco-reduced RBC: Where the leukocytes from red cells are filtered off or removed
Deglycerolised RBC: For patients who are sensitised with to IgA protein or to leukocyte or platelet antigens

- Washed cells (freezed with glycerol to preserve the cells)
They're used in cases of emergency when the stock is low. The cells are defrozen & washed to remove the preservatives. The disadvantage of washed cells is that their life span is very short.

yep, that's about it. Free free to ask any questions. I'll try my best to answer them all. Continue enjoy the rest of the SIP period! (: take care

Lastly, wanna share something.
my colleague told me this,
With skills comes good work.
With passion comes artistic work.
With passion & skills will produce a masterpiece.
yep, passion & skills are essential in excelling in your work. =)

Png En Hui Dorothy
0503239F
Tg01

SIP

Subject Title: Histopathology
Topic: Tissue processing

Hi all. I had been attached to a histopathology lab. My SIP consist of 5 components, they are: tissue processing, embedding, microtomy, special staining and cytology. I am posted to the tissue processing station for the first month of my SIP. In this station, my main task is to receive specimen, label specimen and assist the pathologist in recording and labelling of cassettes.

Everyday in the morning, once we received the specimens, we will label the specimens and the request form with a bar coded biopsy number. The sample will then be carried to a room where trimming and examination of specimen is performed. Small specimens, such as appendix, gall bladder, bone marrow core, tonsils, cornea or gastric biopsies will be trimmed or passed into a cassette by a medical technologist. The large and complex specimens such as liver, kidney, intestine, breast, uterus, eye ball or femoral head will be examined and trimmed by a pathologist.

Lab personnel who are in charge of trimming or passing of small specimens into cassettes must make sure that the bar code and the specimen name on the bottle are matched with the request form before commencement of any procedure. Once confirmed, details of the specimen such as number of tissues in the bottle and type of tissue will be recorded. Measurements will also be taken. Specimens will then be trimmed into a smaller pieces using a surgical blade and pass into a cassette. For bone specimen, a bone saw is used to cut the specimen into smaller pieces and is then decalcify before processing. For prostate, whole mount is necessary (a bigger version of tissue block), as the whole entire piece of prostate will be dissected and submitted for investigation. For specimens such as gastric biopsy (about 0.1cm x 0.1cm in size), which are very small, will be coloured with ink and place in a filter paper. The purpose of using ink is to allow the person who is doing embedding to see the tissue clearly so that he/she can transfer the tissue into the mould. The filter paper will prevent lost of tissue during processing. Inks are also used by pathologist to indicate the orientation of the tissue. After trimming, if there is excess tissues, the excess tissues will be set aside for storage and indicate on the requisition form as tissues with reserve.

After passing of tissue into cassettes, the cassettes containing the tissue will be fixed in 10% neutral buffered formalin until all other tissues are ready to be processed. The pH of the formalin will be check daily to ensure that the pH is around 6.8-7.2 as neutral pH will minimize formalin precipitation. New formalin is changed at least twice a week. The principle of fixation is to stop autolysis. Fixatives form cross linkages between protein, thus preserved the tissue's in vivo structure.

The lab used an enclose tissue processor for tissue processing. An enclosed system means that tissues will remains stationary in a closed chamber and processed until the procedure is completed. Tissue processing consists of 3 components:
1) Dehydration: remove water from tissue using graded alcohol
2) Clearing: replace alcohol with an agent that is miscible with wax. Xylene is most commonly used, but some new machine use isopropanol instead of xylene, which make the working environment better.
3) Impregnation: tissue infiltrated with embedding medium.

Different types of tissue will have different processing time. For small fragment of tissues or biopsies, the processing time is usually 2-3 hours. For tissues of normal sizes, the processing time is usually set at 16 hours.

Apart from processing, I had also learnt some safety aspects in a lab, such as how to deal with chemical spillage, and the protocol as follow:
1) Isolate the area of spillage
2) Wear personal protective equipments, such as glove and mask
3) Choose an appropriate spillage kit available in the lab
4) Circulate the spillage with the spillage kit and max them
5) Scoop the spillage, and place the mixture in a bag and tighten it.

I think I will stop here first. Enjoy your SIP!

Wing Fat
TG01

Table

Hi all

Few of you asked about the difference in reference range. I made a table to compare the different parameters.

	Unit	Man	Women	Horse
White Cell Count	10^9/L	3.5-	15.0	6-10
Neutrophil Count	10^9/L	1.9-	9.0	2.5-7.0
Lymphocyte Count	10^9/L	0.9-	4.5	1.2-3.7
Monocyte Count	10^9/L	0.1-	3.1	0.2-0.7
Eosinophil Count	10^9/L	0.0-	0.5	0.0-0.2
Basophil Count	10^9/L	0.0-	0.2	0.0-0.2
Red Cell Count	10^12/L	4.5-5.5	3.8-4.8	7-10
Haemoglobin	g/dL	12.0-18.0	11.0-16.0	12-18
Pack Cell Volume(PCV) or Haematocrit(Hct)	%	40-50	36-46	32-46
Mean Cell Volume(MCV)	fL	80-99	78-97	44-54
Mean Cell Haemoglobin(MCH)	pg	27.0-32.0	25.5-31.5	15-18
Mean Cell Haemoglobin Concentration(MCHC)	g/dL	31.0-	35.9	31-39
RBC Distribution Width(RDW)	%	10.0-	14.5	11.6-14.8
Platelet Count	10^9/L	150-	450	6-210
Fibrinogen	g/L	1.7-	4.0	1-4

Douglas
TG01

SIP

Subject Title: Clinical Chemistry
Topic: Blood Gas Testing

Heys..I have been attached to clinical chemistry lab for the whole of my SIP. In this lab, there are 5 workstations. I had been posted to blood gas station for 2 weeks. I had learnt to use Roche Omni Cobas b 221 system for testing blood gases. This analyzer can measure analytes such as pH, pO2, pCO2, electrolyte ISE(e.g.Ca2+),total haemoglobin, oxyhaemoglobin, carboxyhaemoglobin, methemoglobin and O2 saturation. It consists of reagents such as S1 Rinse Solution, S2 Fluid Pack Solution and waste blood container.

Principle of Measurement
O2: use of clark measurement principle. It is the measurement of current generated by reduction of O2
CO2: measurement of pH change in electrode caused by CO2
pH and Ca2+: potentiometric electrodes based on the measurement of a potential under no current flow. Calculation of these require use of reference electrode
Total haemoglobin: light absorption in whole blood measured at different wavelengths

Calibration, QC, Maintenance
Calibration, quality control and maintenance need to be done before running any patient's samples. Everyday in the morning, calibration results, which include system cal, 1 point cal and 2 point cal, need to be printed out and filed for future audits. Calibrators are not required as the analyzer will automatically calibrate by itself in time intervals. This will plot out a calibration curve and to determine any errors in the measurement of the analyzer so that is adjusted to show true values. After that, quality controls need to be done to ensure the results are within the control limits. QC is performed daily at 9am and 4pm. The commercial control ampoules are stored at 2-8 degree celsius and are temperature sensitive. Hence, it has to "grip warm" in the palm for about 10min before running to achieve successful QC results. The results will also have to be filed. Lastly, maintenance has to be done to make sure it is in good condition. This includes:
- Checking thermal paper supply
- Checking reagents level
- Cleaning the fill port(where the blood is pumped in)with wet cotton swab for removing blood stains
Now, the analyzer will be ready to run patient's samples. These samples are collected in heparinzed syringe which contains anticoagulant to prevent clotting of blood

Procedures
1)Stamp time for the patient's sample
2)Ensure is sent in ice bag
3)Label barcode on both the syringe and request form
4)Order tests requested into LIS
5)Mix the syringe well and check for blood clots before pumping into analyzer
6)Check the analyzer is at ready mode and having green signal before pumping
7)Pump blood into analyzer
8)Scan barcode

Test Results
Analytes-------- Reference Range
pH--------------> 7.350-7.450
pCO2------------> 35.0-45.0 mmHg
pO2-------------> 75.0-100.0 mmHg
O2 saturation---> 95.0-100.0%
Ca2+------------> 1.140-1.320 mmol/L
Total haemoglobin---> 11.5-17.4 g/dL
Oxyhaemoglobin------> 95.0-99.0%
Carboxyhaemoglobin--> 0.5-2.5%
Methemoglobin-------> 0.0-1.5%

Clinical Interpretation
After the results are out, we have to check for any abnormal high or low levels which will be flagged red in the LIS. For example if the pH is less than 7.1 and pCO2 is more than 45 mmHg, we need to call the nurse to inform these panic values. This shows that the patient is having respiratory acidosis. If there is abnormal high pCO2, we have to check if blood sample appear dark and re-run the test. Always ensure pO2 is directly proportionally to O2 saturation. After verifying the results, we can file them into LIS. Therefore, blood gas testing is for diagnostic and therapeutic reasons

That's all for now. Enjoy your SIP!
Soong Ci Liang
0503333G
TG01

SIP

Subject Title: CCHEM/ HAEM/ LMQA/ MMIC
Topic : Bacteriology - Blood Culture

Definition:
A blood culture is a test to determine if microorganisms such as bacteria, mycobacteria, or fungus are present in the blood. A sample of blood is put in a special laboratory preparation and is incubated in a controlled environment for 1 to 7 days.

Principle:
Bacteria growing in a suitable broth causes turbidity of medium or gas production. The BACTEC Fluorescent series blood culture system, detects carbon dioxide (CO2) production by multiplying bacteria.

Materials:
Media contains Soybean-Casein Digest broth with resin and 0.05% w/v sodium polyanethol sulfonate (SPS). All bactec media are dispensed with added CO2. Anaerobic media are prereduced and dispensed with CO2 and N2.

Procedures:
1. Vials are arranged and labeled in racks.
2. If requisition form presents infectious endocarditis in its clinical diagnosis, a red ribbon is tied on the vials for indication.
3. Vials’ barcodes are scanned and incubated aerobically at 35ºC and are agitated throughout the routine 5-day protocol.
4. Negative vials are removed after few days while positive vials are removed from BACTEC machine for flagging, Gram Stain and subculture in appropriate plates.

False Negative Results:
Delay in transportation could lead to false negative results. Inoculated Bactec/F Plus vials can only be left at room temperature up to 48 hours before placing into BACTEC machine.

For photos on BACTEC system and procedures after flagging positive vials, please visit here. (Azhar's entry - linked with permission)


Boon Ching
TG01

Student Internship Programme(SIP)

Subject Title: CCHEM/ HAEM/ LMQA/ MMIC
Topic : SIP
Content:

Hi all

In case you are wondering what am I actually doing at my SIP, no I am not bathing for the horses, not drawing blood or any other body fluid from the horses(done by Vet, Vet nurses, or the horse's traininer themselves. In this hospital for horses, there is a clinical lab of it's own that also runs Haematology(Full Blood Count, Fibrinogen test etc.). I currently work in this laboratory that runs these tests.

There is a seperate lab that test for any performance drugs administered to the horse to prevent cheating.

In the Hospital, there is a X-ray room, Radioactivity room, patient stables(not ward for humans =P) and even a operation theater for the horse! Big operating tables and another big room where horse is given anaesthesia then a crane is used to put the horse onto the operating table. Today(second day of work), there is a surgery for a lame horse and the doctor sent a urgent blood tube to our lab.

My lab is a seperate building from the hospital. This is because there are new instrument boughted by company for clinical test and needed more space to house this equipment. Hence, my supervisor and I have to take a 5 minutes buggy ride to and fro the hospital at least 4 times a day to check if any sample are sent for testing. In my laboratory, the Haemotology is done by the CELL-DYN 3700 from ABBOTT, and clinical tests are done by the OLYMPUS AU400. The Olympus is the new instrument that is bought recently. My supervisor has just finished evaluating the machine and has just switched from the old instrument to this new one. However, there are still several problems and uncertainty that requires trouble-shooting. As the Olympus AU400 is still not stable, and that it is a very sensitive and complicated machine, I will first learn to use the CELL-DYN 3700.

Today, I did the Daily Maintanence. This includes the daily start-up(priming machine and doing background checks), running the Control samples (3-level: Low, High, Normal), Auto-cleaning(cleaning probe and wash block, auto-clean with enzymatic), Daily Shutdown. I also did the FBC for 2 of the samples.

An account in the LIS is created for me so that I can access and order test and record/retrieve results when necessary. I also learn about filling up Laboratory Request Form(LRF) and procedures(who sign who validate results, which copy of LRF to who, lab report out must have who to sign etc.). I also learnt to change reagent for the CELL-DYN3700.

Driving the buggy around the Club needs experience. Some horses are naughty and easily agitated so overtaking them or sudden movement startle them and may cause them to respone vigourously or even violently. Extra care have to be given and observation of the horses' movement and action allows you to tell if the horse is a naughty one or a tame one.

Hope to hear about some of your experience soon too.

Cheers
Yew Long Jie, Douglas
S8819476
0503224H
TG01

OUR FINAL DIAGNOSIS

Murine typhus or rat-bite fever

Murine Typhus

- The patient shows common symptoms like high fever, severe muscle pain (myalgia) and dull red rash, which are also seen in the people infected with murine typhus.

- Since the disease occurs commonly in elderly and the patient is 85 years old, it is highly possible that the patient contracted murine typhus.

- His house is infested with rodents and rats, and he has the habit of feeding stray dogs and cats. Thus these made him widely exposed to the bacteria, as the main transmitters of the disease are fleas, rats and stray cats.

- Murine typhus may result in mild kidney failure which causes excessive urination at night, which explains why his mattress was soiled with urine.

Rat-bite Fever

- The patient shows common symptoms such as moderate fever, chills, a diffuse red rash, which are also seen in the people infected with Rat-bite fever. [1]

- It is caused by a bacterium (S.Moniliformis) which is transmitted from rodent to human via the rodent's urine or mucous secretions. [1]

- It can also be caused by the scratches and bites by rats. This is highly possible as his house is infested with cockroaches and fleas as well as rats.

- It is mostly found in Asia like Singapore and Japan.

References:
http://en.wikipedia.org/wiki/Main_Page > Rat-bite_fever (1)

These are the 5 possible diseases that we deduce the elderly man may be contracted with.

5 possible diseases:

Hantavirus
Leptospirosis
Murine Typhus
Rat-bite fever
Typhoid Fever

REASONS

Hantavirus pulmonary syndrome (HPS)
- a deadly disease transmitted by infected rodents through urine, droppings, or saliva
- symptoms like fever, chills, malaise, fatigue match with those symptoms that the patient had (1)





--> Transmission electron micrograph of the Sin Nombre Hantavirus (1)



Leptospirosis
- Transmitted through contact with water, food, or soil containing urine from infected animals like dogs, rats and mice. This may happen by swallowing contaminated food or water or through skin contact. (2)
- Symptoms like high fever, chills, rash, muscle aches, match with those symptoms that the patient had (2)
- Since he had a habit of feeding stray dogs and cat that might be infected with Leptospira, hence he might be infected through their urine and feces.







--> Scanning electron microscope of a number of Leptospira sp. bacteria atop a 0.1 µm polycarbonate filter(2)

Murine Typhus
- Transmitted through exposure to rat fleas or rat feces, or exposure to other animals such as cats and rats (3)
- Symptoms like extremely high fever, dull red rash, severe muscle pain, chills, match with those symptoms that the patient had (3)
- Might result in mild kidney failure which causes excessive urination at night, which explains why his mattress was soiled with urine.




--> Rickettsia typhi (Bacteria) (6)




Rat-bite fever
- Caused by bacteria, transmitted from rodent to human via the rodent's urine or mucous secretions (4)
- Symptoms like moderate fever, chills, a diffuse red rash, match with those symptoms that the patient had (4)
- Mostly found in Asia (4)

(7)

Typhoid Fever
- Caused by the bacterium Salmonella typhi, transmitted by ingestion of food or water contaminated with feces from an infected person (5)
- Symptoms like high fever, weakness, and rash of flat, rose-colored spots, match with those symptoms that the patient had (5)
- Might result in gastroenteritis, diarrhea, which explains the human excreta present on his mattress

--> Salmonella typhi bacteria (5)

References:
1. http://en.wikipedia.org/wiki/Main_Page > Hantavirus
2. http://en.wikipedia.org/wiki/Main_Page > Leptospirosis
3. http://www.nlm.nih.gov/medlineplus/ency/article/001363.htm > encyclopedia > article001363
4. http://en.wikipedia.org/wiki/Main_Page > Rat-bite_fever
5. http://en.wikipedia.org/wiki/Main_Page > Typhoid_fever
6. http://www.cdc.gov/ > Report > Fatal Rat-Bite Fever
7. http://www.genomenewsnetwork.org/ > Bacteria

List of Possible Diseases

Lyme Disease
Lyme disease is caused by a spirochete bacterium (Borrelia burgdorferi) that is transmitted to humans by hard ticks.
Early symptoms include rash, fatigue, headache, sore throat, muscle and joint aches, heart palpitations, tick paralysis, etc.

Commensal Rodent-borne Diseases
Rats and mice are responsible for the spread, either directly, through contamination of human food with their urine or feces, or indirectly, by way of rodent fleas and mites.

Rat-bite Fever
Rat-bite fever is caused by the bacteria Streptobacillus moniliformis, which is found on the teeth and gums of rats. It is transferred from rats to humans by the bite of the rat.
Symptoms include: difficult red rash, fever, joint pain, chills

Toxoplasmosis
Through contact with cat feces.
Symptoms: Fatigue, Extreme coldness and fever, rashes

Leptospirosis
Reasons: Leptospirosis is a bacterial disease that affects humans and animals. It is caused by bacteria of the genus Leptospira.

Typhoid fever
Reasons are not found yet but most of its symptoms tally with what the patient have.

Salmonella food poisoning
It may caused by the bacteria carried by the rats.

Contact dermatitis
Reason: He may have allergy to the fleas, or even allergy to dog’s and cat’s hair.

Rheumatic fever
It may develop suddenly 2 to 6 weeks after a streptococcal infection. This form of the disease is most common in older people. It generally starts with joint pain and fever. Symptoms vary and include rashes, weakness and joint pain.

Murine or Endemic Typhus
Murine Typhus is another disease that is principally associated with rats and may be occasionally transmitted to humans. Murine Typhus is transmitted when infected flea droppings are scratched into the wounds made by fleas.

Babesiosis
It is a rare infectious disease caused by single-celled microorganisms (protozoa) It is believed that the Babesia protozoa are usually carried and transmitted by ticks (vectors). The sypmtoms are fatigue & fever.

List of websites
1. http://www.crittercontrol.com > diseases > plague
2. http://www.cigna.com/ > healthinfo > nord268
4. http://www.orlandorats.com/ > diseases
5. http://en.wikipedia.org/wiki/Main_Page > Typhoid
6. http://en.wikipedia.org/wiki/Main_Page > Contact_dermatitis
7. http://www.healthopedia.com/ > rheumatic-fever > symptoms
8. http://www.2ndchance.info/ > zoonoses
9. http://www.peteducation.com/ > index > articleid=592
10. http://www.aaapestcontrol.com.au/ > rats

Mmic Tutorials1-4 : A Simulated Case

A 85-year-old-male was found to be staying alone in one-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 neighbours that the elderly man has the habit of feeding stray dogs and cats in the neighbourhood.

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 microorganism[s]?

Expectation

• Good results
• Cooperation
• Team work
• Compromise and Accomodation
• Efficency
• Hard work
• Punctuality
• Initiative