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Medical Microbiology Review

Rita Heuertz, PhD, MT(ASCP)

Saint Louis University

Objectives

1. Review clinical laboratory knowledge base relevant to Microbiology.

2. Link disease conditions with specific microbiologic agents.

3. Discuss specific strategies for identification of different microbiologic

agents.

Major Disease-Producing Microbes Encountered in

Major Organ Systems

Location, Location, Location:

As Important in Microbiology

as it is in Real Estate

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Viral Hepatitis

Serological patterns of viral hepatitis

Hepatitis A, B, C, D, E

Viral Hepatitis

Hepatitis A

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Hepatitis B

CORE WINDOW

REMEMBER: Not everyone spends the same amount of time in each of these stages during disease progression.

Hepatitis B

Positive Diagnosis for Hepatitis B – HBsAg – Positive

– anti-HBc, IgM – Positive

CORE WINDOW: period of time patient may be HBV positive yet appear negative

Follow-Up Indicated: Monitoring Panel – HBsAg – persistence leads to chronic HBV infection

– HBeAg – determines relative infectivity

– Anti-HBe – seroconversion from HBeAg to anti-HBe usually indicates progression towards disease resolution

– Anti-HBs – seroconversion from HBsAg to anti-HBs positivity indicates resolution of disease and establishment of immunity

Progression to Chronic HBV

NOTE: ABSENCE OF ANTI-HBs

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Hepatitis C with Progression to Chronic HCV

Acute HCV with Recovery

• anti-HCV plateau • no HCV RNA

• no ↑ ALT

Hepatitis D (requires HBsAg synthesis)

HDV superinfection of HBV

carrier

HDV coinfection with HBV

HBsAg pos: HDV also pos

HBsAg pos: HDV pos later

Hepatitis E

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Respiratory – Pulmonary

Pharyngitis – Streptococcus pyogenes (GPC)

Otitis media – Streptococcus pneumoniae (GPC)

– Hemophilus influenzae (GNR)

Epiglottitis – Hemophilus influenzae

Pertussis (whooping cough)

– Bordetella pertussis (GNR)

Bronchitis, bronchiolitis – Mycoplasma pneumoniae

– Viruses

Diphtheria – Corynebacterium diphtheriae (GPR)

Thrush – Candida albicans (yeast)

Pneumonia – Hemophilus influenzae, type b (GNR)

– Streptococcus pneumoniae (GPC)

– Mycoplasma pneumoniae

– Klebsiella pneumoniae (GNR)

– Staphylococcus aureus/MRSA (GPC)

Tuberculosis – Mycobacterium tuberculosis

Emerging viral infections – Avian flu (H5N1)

– Novel flu (H1N1)

– SARS

Staphylococcus aureus (MRSA)

S. aureus colonizes anterior nares in 30% of population – pneumonia

Streptococcus pyogenes (Group A)

Beta hemolytic, bacitracin sensitive

Causes

– Pharyngitis

– Noninfectious sequelae (complications)

Post-streptococcal glomerulonephritis

Rheumatic fever

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S. pyogenes (Group A) Sequelae

Pathologic processes of group A strep can extend

to the heart. Scarring and deformation change

capacity of valves to close and shunt blood

properly. A. Normal valves viewed from above. B.

Inset reveals scar tissue on damaged mitral valve.

Post-streptococcal glomerulonephritis Rheumatic fever

Streptococcus pneumoniae

Causes pneumonia (Gram positive cocci)

Optochin sensitive

Klebsiella pneumoniae

Causes pneumonia (Gram negative rod)

Colony morphology Biochemical Testing

IMViC - - + +. Lys -. Nonmotile.

Capsular morphology on BAP

GNR with capsule

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Mycoplasma pneumoniae

Causes primary atypical

pneumonia: “walking

pneumonia” – Nonproductive cough, fever,

malaise, headache, lobar

pneumonia by chest X-ray

Gram negative pleomorphic

bacteria with no cell wall –

hard to stain

Fried egg appearance in

culture

Self-limiting but resolves

with tetracycline or

erythromycin

Hemophilus influenzae

Causes

epiglottitis

flap of cartilage at root of

tongue which is depressed

during swallowing to cover

opening of the windpipe

Hemophilus ID

Hemophilus Growth: X Growth: V Growth: XV Hemolysis

influenzae Neg Neg Pos Neg

parainfluenzae Neg Pos Pos Neg

hemolyticus Neg Neg Pos Pos

parahemolyticus Neg Pos Pos Pos

Growth around

XV only

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Bordetella pertussis

Causes whooping cough

Bordet-Gengou agar

Erythromycin is drug of choice

DTP (diptheria, tetanus,

pertussis)

– Average duration of

protective antibody titers

following vaccination is 12

years.

Corynebacterium diptheriaeae

Elek in vitro toxigenicity test

2. Unknown (toxigenic)

4. Unknown (nontoxigenic)

Arch of

Identity

Positive Immunodiffusion Result

Causes diphtheria

Corynebacterium diptheriaeae

Gram stain Pseudomembrane

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Mycobacterium tuberculosis

LABORATORY DIAGNOSIS staining as AFB

– serpentine cording – is indicator of virulence for M. tuberculosis

culture

– most common patient sample is sputum

– large amounts of bacterial flora, mucus need to be removed to prevent overgrowth, contamination of culture: digestion, decontamination done

digestion: uses liquefying, mucolytic agent (N-acetyl-L-cysteine: NALC)

decontamination: uses NaOH (2-4%)

concentration: specimen concentrated is by centrifugation

concentrate is stained, cultured

– aerobic – requires CO2, 35-37C – slow grower (2-6 weeks)

– media

Lowenstein Jensen (LJ), Middlebrook 7H10 or 7H11

Broth containing casein, albumin, 14C-palmitic acid for radiometric detection (e.g., Bactec machines)

Mycobacterium tuberculosis

AFB stain Colony morphology

Causes

tuberculosis

Mycobacterium tuberculosis

Biochemical ID Tubed Media

Niacin Pos

Nitrate Pos

Catalase Pos

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Candida albicans

Gram stain Colony morphology

Causes thrush

germ tube test: positive

Add yeast colony to

serum (0.5 ml): incubate

2-3h at 37C.

Respiratory Viruses Clinical Signs Virus Lab Test

Common cold * Rhinovirus, coronavirus Virus culture

Pharyngitis * Adenovirus,

parainfluenzavirus 1-4

influenzavirus A & B

Virus culture

Gingivostomatitis,

pharyngitis, tonsillitis

Herpes Simplex Virus HSV IgM/IgG

Virus culture

Infectious mononucleosis Epstein Barr Virus

Cytomegalovirus

Human Herpesvirus 6

EBV serology

CMV IgM/IgG, culture

HHV6 IgM/IgG

Pharyngitis/Koplik’s spots Measles Measles IgM/IgG

Herpangina/hand foot and

mouth disease

Coxsackie A Virus culture

Primary HIV infection HIV HIV serology, culture, PCR

* = self-limiting, lab testing not routinely performed

Epstein Barr Virus – Infectious Mono

Epstein Barr Virus – infectious mononucleosis (in the USA), nasopharyngeal cancer, Burkitt’s

lymphoma, Hodgkin’s lymphoma

ACUTE and CONVALESCENT serum samples show Ab titer increase and decrease with

disease progression, such as for Epstein Barr Virus Viral Capsid Antigen (EBV VCA)

ACUTE CONVALESCENT

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Skin, Tissue

Wounds, Lesions

Skin, Tissue, Wounds, Lesions

Dermatitis, dermaphyte infection – Superficial candidiasis (yeast), ringworm (Tinea corporis), athlete’s foot (Tinea pedis), jock itch

(Tinea cruris)

Rashes – Measles (virus), Scarlet fever (Group A Strep), Toxic shock syndrome (S. aureus), 2ndary syphilis

(treponeme bacteria), Lyme disease (spirochete)

Papules – Warts (virus), scabies (parasitic mite), cellulitis (S. aureus), fasciitis (Group A Strep), myonecrosis

(C. perfringens)

Vesicles – Herpes (virus), chickenpox (varicella virus), scalded skin syndrome (Group A Strep, S. aureus)

Pustules – Impetigo (S. aureus), acne (Propionibacterium), furuncles (S. aureus), carbuncles (S. aureus)

Skin, Tissue, Wounds, Lesions

Burns (P. aeruginosa)

Petechiae, purpura – Rocky Mountain Spotted Fever (rickettsia), meningococcemia (N. meningitidis), plague (Y. pestis),

dengue (virus) (p.826 picture)

Ulcers – Syphilis (treponeme bacteria), herpes (virus), chanchroid (H. ducreyi), impetigo (S. aureus),

histoplasmosis (fungus), anthrax (GPR bacteria), tularemia (GNR bacteria)

Bones – S. aureus osteomyelitis

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Most Common Primary Pyodermas

Tinea infections (fungus)

Ringworm Athlete’s Foot Jock Itch

Tinea corporis Tinea cruris Tinea pedis

Staphylococcus aureus (superficial)

Scalded Skin Syndrome

Impetigo

Furuncle

On Hand

Carbuncle

On Neck

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Staphylococcal Osteomyelitis (with ulna rupture)

Bone aspirate

has WBC and GPC (cl)

Bacteria spread through circulation from other site of infection, enter artery and lodge in small vessels of the bone.

Bacterial growth causes inflammation and damage that manifests as edema (swelling), necrosis (cell death).

Staph aureus and/or Strep pyogenes

Cellulitis

Impetigo

Streptococcus pyogenes

Scarlet fever Strep throat Erysipelas

Scarlet Fever Rash

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Streptococcus pyogenes

Necrotizing fasciitis

Disease begins as innocuous cut in skin

and spreads rapidly to neighboring tissue.

S. pyogenes enzymes digest connective

tissue in skin and toxins poison epidermis

and dermis. As flesh dies, it is sloughed

and Strep spreads into deeper tissue, like

muscle. Potential for disfigurement and

death exists (fatality rate 30 – 50%).

Streptococcus: Disease States

PRIMARY INFECTIONS 1. Scarlet fever (aka scarlatina) – Group A Strep (elevated ASO titer)

2. Pharyngitis (“strep throat”) – Group A Strep

3. Endocarditis

1. Acute bacterial endocarditis – S. aureus or Group A Strep

2. Subacute bacterial endocarditis – Streptococcus viridans group

4. Impetigo (children) – S. aureus or Group A Strep

5. Cellulitis, necrotizing fasciitis – S. aureus or Group A Strep

6. Erysipelas – Group A Strep

7. Group B Streptococcus – β-Streptococcus neonatal infections

1. Early onset: respiratory distress syndrome (RDS), pneumonia

2. Late onset: meningitis

8. Urinary tract infections (UTI) – enterococcus, sometimes Group B Strep

9. Wound infections – Group A or Group D Strep

SECONDARY INFECTIONS 1. Rheumatic fever, rheumatic heart disease – Group A Strep sequelae

2. Acute glomerulonephritis – Group A Strep sequelae

Pseudomonas aeruginosa – burns

Gram stain Colony morphology ID

Oxidase positive Uses glucose

oxidatively rather than fermentatively

(like the enterics)

Pigment producer: lactose neg

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Bacillus anthracis (anthrax)

Gastrointestinal – spore ingestion

Inhalational – woolsorter’s disease

– respiratory distress

Cutaneous – black eschar

– Most commonly encountered

form of anthrax

Bacteroides fragilis (anaerobic wound infections)

Gram stain Colony morphology

Clostridium perfringens (anaerobic infection)

Gram stain Colony morphology

Gas Gangrene

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Clostridium tetani (anaerobic infection)

Gram stain Uncontrollable Muscle Contraction

Tetanus

Gastrointestinal Tract

Gastrointestinal Tract

Stomach Ulcers – Helicobacter pylori

– Perform urease-CLOtest on gastric biopsy

Diarrhea – food/water borne

– Mediated by toxin

Enterotoxigenic E. coli, V. cholerae, C. perfringens, B. cereus, C. difficile,

S. aureus , C. botulinum

– Mediated by infection and mucosal surface invasion

Salmonella spp, Shigella, Campylobacter, Entamoeba histolytica,

Enterohemorrhagic E. coli (e.g., strain O157:H7)

– Mediated by infection and full bowel thickness invasion

Salmonella typhi, Yersinia enteocolitica

– Noninvasive

Giardia lamblia, rotavirus

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Helicobacter pylori

Causes stomach ulcers

Ingestion of urea with labelled carbon labelled carbon in breath H. pylori urease

Food Poisoning: Food Infection

Food Poisoning: ingestion of microbe-produced preformed toxins

Food Infection: ingestion of microbes that grow/replicate, cause infection

Food Poisoning (Toxicity)

Staph aureus Salmonella E. coli O157:H7 Shigella

Infection/Toxin Toxin Infection Toxin Infection

Incubation Period

2-8 hours 18 hours 2-8 hours

2-3 days

Fever Absent Present Absent Present

Symptoms Vomiting, diarrhea, headache

Diarrhea, abdominal pain

Bloody diarrhea Bloody diarrhea

Abdominal cramps

Symptom Duration

24-48 hours 2-5 days 24-48 hours

2-7 days

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Common Pathogens Causing Diarrhea

Clostridium botulinum (anaerobe)

Canned Food Neurological Disease of Toxin

BOTULISM

Clostridium botulinum Toxin Blockade of Neural Transmitter

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Salmonella (GNR)

Typhoid Mary (S. typhi: typhoid fever)

– Identification of carrier status

(asymptomatic yet able to transmit

to others)

Preventatives

– Food handling

Refrigeration, proper cooking

No human vaccine is 100% effective

– Vivotif Berna

– Typhim Vi

Salmonella typhi: IMViC - + - + Lac - H2S + Motile

Shigella (GNR)

Gram stain Colony morphology

Causes

bacillary

dysentery

Shigella: IMViC -/+ + - - Lac - H2S - Nonmotile

Biochemical Rxns of Kligler Iron Agar (KIA) Slants: Primarily Useful for Enterobacteriaceae

NOTE: Triple Sugar Iron Agar (TSI) slants are read in similar manner.

# Slant

(lactose)

Base

(glucose)

Gas?

H2S?

Biochem

Result

1 Alkaline (K) Alkaline (K) Gas? no

H2S? no

All neg:

control

2 Alkaline (K) Alkaline (K) Gas? no

H2S? No

K/K

3 Alkaline (K) Acid (A) Gas? no

H2S? no

K/A

4 Alkaline (K) Acid (A) Gas? no

H2S? yes

K/A H2S

5 Acid (A) Acid (A) Gas? yes

H2S? no

A/A gas

6 Acid (A) Acid (A) Gas? no

H2S? no

A/A H2S

1 3 2 4 5 6

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Carbohydrate Fermentation Tests

TSI agar reactions of

Enterobacteriaceae

(tube slant/base results after

18-24 hours incubation)

Tube 1: A/A gas (acid/acid with gas)

Tube 2: A/A H2S

Tube 3: K/A (alkaline/acid)

Tube 4: K/A, H2S, gas

Tube 5: K/K (alkaline/alkaline)

KIA agar reactions of Enterobacteriaceae (tube slant/base results after 18-24

hours incubation).

Left A: A/A gas (acid/acid with gas) Right A: K/A H2S

Left B: K/A (alkaline slant lactose/acid glucose) Right B: K/A, H2S

Left C: K/K (alkaline/alkaline) Right C: K/K (uninoculated)

Common KIA Results

5% are lac neg & pathogenic

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MOTILITY: the motility test medium has agar concentrations of 0.4% which is

semi-solid thereby allowing free spread of the organism. A single stab into the

medium is made. After overnight incubation, movement away from the stab line

or hazy appearance throughout the medium indicates a motile organism.

Motility Results

IMViC Reactions for Enterobacteriaceae Family

Indole: Negative

Methyl Red: Negative

Voges Proskauer : Positive

Citrate: Positive

K. pneumoniae

Indole: Positive

Methyl Red: Positive

Voges Proskauer: Negative

Citrate: Negative

E. coli

Campylobacter (GNR)

Gram stain Colony morphology

Erythromycin: preferred treatment

Is a food-borne infection (has been associated with undercooked poultry)

Causes fever, cramps, bloody diarrhea (is a self-limiting disease)

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Clostridium difficile (GPR)

Gram stain Colony Morphology Anaerobe

• Causes pseudomembranous colitis

• Is also known as antimicrobic-associated diarrhea

• Treatment: vancomycin

Vibrio cholerae (GNR)

Disease: cholera Symptoms: vomiting, “rice-water stools”, diarrhea (fluid loss up to 20 l/day) Transmission: fecal-oral route – fruits and vegetables grown in sewage-rich soil, ingested uncooked – raw oysters – however: drying, sunlight, acid conditions easily kill Vibrio

Enterobius vermicularis (pinworm)

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Giardia lamblia (parasite)

Microscopy

Cryptosporidium (parasite)

Single cell sporozoa parasite responsible for

contaminated water (e.g., Milwaukee water

outbreak of 1993)

Ascaris lumbricoides (roundworm)

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Central Nervous System

Central Nervous System

Bacterial meningitis

– Group B Streptococcus, Hemophilus influenzae (type b), Streptococcus

pneumoniae, Listeria monocytogenes, Neisseria meningitidis, T. pallidum

(syphilis spirochete)

Mycobacterial meningitis

– M. tuberculosis (HIV is a risk factor)

Fungal meningitis

– Cryptococcus neoformans, Coccidioides immitis

Parasitic meningitis

– Naegleria fowleri

Viral meningitis

– Enteroviruses like poliovirus, coxsackieviruses (A and B), echoviruses

Bacteria that cause CNS infections are highly susceptible to cold

temperatures. Do not put CNS samples in the refrigerator or on ice.

Group B Streptococcus

Gram stain Colony morphology

Risk population for meningitis: newborn infants

birthed through a canal infected with Grp B Strep

Catalase negative

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Listeria monocytogenes

LABORATORY DIAGNOSIS

At risk populations: pregnant women, newborn

infants (sepsis, meningitis), immunocompromised,

elderly

Gram positive rod that resembles lancet shape of

Streptococcus

Growth is with beta hemolysis (resembles Strep)

Catalase positive

Exhibits “umbrella” pattern of motility at room temp

(23-25oC) but not at human body temp (35-37oC)

Capable of growth at 4oC

Grows at high salt concentrations

23oC 37oC Listeriosis is leading

cause of fatal food-

borne infections in USA: heat ready-to-eat foods

to steaming hot before

ingestion

umbrella shape motility at RT

Neisseria meningitidis

Gram stain Culture & oxidase result

Petechial rash commonly develops with meningococcemia (invasion of bloodstream)

Waterhouse-Friderichsen Syndrome – adrenal gland hemorrhage

Risk population for meningitis: newborns-toddlers

ID of Neisseria Pathogens

Lab ID is by cystine trypticase agar (CTA)

sugar utilization method

Organism Glucose Maltose Sucrose Lactose

N. gonorrhoeae Pos Neg Neg Neg

N. meningitidis Pos Pos Neg Neg

N. lactamica Pos Pos Neg Pos

M. catarrhalis Neg Neg Neg Neg

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Cryptococcus neoformans

ID of cryptococcal antigen in CSF

India Ink preparation of CSF for yeast

Bacteremia

Septicemia

Bacteremia, Septicemia

Bacteremia – usually transient periods of microbes in bloodstream (e.g., mouth flora after

vigorous dental flossing)

Septicemia – Common causes in 1960-1970: GNR like E. coli, P. aeruginosa

– Common causes in 1980-1990: GPC like S. aureus, coagulase negative

staphylococci, enterococcus

– 21st century Emerging Resistant Microbes:

Coagulase negative staphylococcus (31%)

S. aureus (20% with 40% of these being MRSA)

enterococcus (9%)

C. albicans (9%)

E. coli (6%)

Klebsiella (5%)

P. aeruginosa (4%) – recent hospital-based survey

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Blood Parasite – Plasmodium

Plasmodium in RBC – Malaria

Urinary Tract

Causes of UTI E. coli is in this family

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E. coli

Leading cause of all urinary track infection (UTI)

IMViC + + - -

E. coli tends to be susceptible to most antimicrobics

Leading Cause of Gram Negative UTI

Enterococcus

Gram stain Colony morphology

Leading Cause of Gram Positive UTI

Genital Tract

Sexually Transmitted Diseases (STD)

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STD and Causative Agents

Neisseria gonorrhoeae (GC)

Gram stain Colony Morphology

JEMBEC system with

Thayer Martin agar for GC

Treponema pallidum (syphilis)

Tissue stain Dark-field microscopy

spirochetes

spirochetes

spirochetes

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Trichomonas vaginalis (parasite)

Parasite – single cell flagellate

Gardernella vaginalis – Clue cells

Describe important

technical procedures

and their application

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Gram Stain for Bacteria

IMViC (and other) Results

Escherichia coli + + - - lac - (usually) lys +

Edwardsiella tarda + + - - lac - H2S + lys +

Klebsiella pneumoniae - - + + lac + capsule lys - Nonmotile

(K. oxytoca is indole +)

Salmonella typhi - + - + lac - H2S + lys + Motile

Shigella -/+ + - - lac - H2S - lys - Nonmotile

Yersinia enterocolitica V + - - lac - H2S - lys –

Motile (25oC), Nonmotile(37oC)

Citrobacter freundii - + - + lac +/V H2S + lys -

Citrobacter diversus + + - + lac V HxS - lys -

Enterobacter aerogenes V - + + lac + orn + lys + Motile

Proteus vulgaris + + - - lac - H2S + lys - Motile

Proteus spp lac - phe + lys -

Antimicrobic Susceptibility by Disk Diffusion

E-test: MIC results with ease

of performance of KB test

Kirby-Bauer Susceptibility Test

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AFB Stain for Mycobacterium

Culture for Mycobacterium

Culture for M. tuberculosis

Cannot culture for M. leprae

KOH Preparation for Fungus/Yeast

KOH destroys mammalian cells: yeast becomes evident

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KOH Prep for Thrush

Candida albicans (direct

KOH prep of patient swab)

Culture for Fungus/Yeast

Candida albicans – pure culture

Saline Wet Prep for Trichomonas

parasite (Trichomonas vaginalis) from vaginal

secretions

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Parasite Testing

Wet direct preparation

from fresh stool – for

trophozoites

Stool for parasite set-up

and concentration – for

cysts

Virus Testing

Cell Culture for Virus Infection and/or Growth

Amplification of Virus-Specific DNA:

Polymerase Chain Reaction (PCR) for Virus

Antibody Testing of Patient Serum for Disease

Presence, Progression, Resolution

Cell Culture for Viruses

Viruses, if present, infect the cells and cause

morphological changes to the cells (e.g., lysis)

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Amplification of Virus DNA (PCR)

EXAMPLE:

HIV gene

PCR for Virus (e.g., H1N1 flu)

Ab Titer for Virus Infection (Acute, Convalescent)

Viremia and antibody kinetics of West Nile virus infection

Detects virus presence, progression, resolution

IgM increases followed by a decrease: IgG then increases and plateaus

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