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Complete Answers - Medical Microbiology (Chapters 1-10)

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CHAPTER 1 - INFECTIVE ENDOCARDITIS

Name Three Bacteria Causing Infective Endocarditis

1. Streptococcus viridans (alpha-hemolytic streptococci) - most common cause of subacute bacterial endocarditis (SBE), especially on previously damaged valves
2. Staphylococcus aureus - most common cause of acute IE; can affect normal valves; common in IV drug users
3. Enterococcus faecalis - common in elderly, associated with GI/GU procedures

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HACEK Agents

• Slow-growing, fastidious organisms requiring enriched media
• Cause subacute endocarditis with large vegetations
• Strong positive predictive value: HACEK bacteremia strongly predicts underlying IE (PPV ~60%)
• Often curable with antibiotics alone without surgery (unlike other gram-negative IE)
• Treatment: Ceftriaxone 2g IV daily for 4-6 weeks (drug of choice)

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Rheumatic Fever - Disease Mechanism / Diagnosis

Etiology

Pathogenesis / Disease Mechanism

1. S. pyogenes M protein contains epitopes that cross-react with human cardiac antigens (myosin, tropomyosin, laminin)
2. Anti-streptococcal antibodies cross-react with cardiac tissue
3. T-cell mediated immune attack on heart valves, joints, brain, skin
4. Repeated attacks cause progressive valve damage (Aschoff bodies - pathognomonic granulomas in myocardium)

Diagnosis - Jones Criteria (Revised 2015)

• Carditis (pancarditis - pericarditis, myocarditis, endocarditis)
• Arthritis (migratory polyarthritis of large joints - most common manifestation)
• Sydenham's chorea (involuntary movements, St. Vitus dance)
• Erythema marginatum (skin rash with clear center, map-like margins)
• Subcutaneous nodules (firm, painless over bony prominences)

• Fever, elevated ESR/CRP, prolonged PR interval, arthralgia (if arthritis not a major criterion), previous RF

• Positive throat culture or rapid strep antigen test
• Elevated/rising streptococcal antibody titers (ASO titer >200 Todd units in adults; >333 in children 5-15 years)

• ASO titre (Antistreptolysin O) - elevated in 80% cases
• Anti-DNase B, anti-hyaluronidase (if ASO negative)
• Throat swab culture - S. pyogenes
• CRP, ESR elevated
• ECG - prolonged PR interval
• Echocardiography for carditis

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CHAPTER 2 - ANEMIA

Blood Culture - Methods and Indications

Indications

• Bacteremia/septicemia
• Fever of unknown origin (FUO)
• Infective endocarditis
• Meningitis
• Pneumonia with bacteremia
• Osteomyelitis
• Urinary tract infection with systemic features
• Post-splenectomy fever
• Immunocompromised patients with fever

Methods

• Collect 10 mL blood (adults) / 1-3 mL (children) per bottle
• Inoculate into aerobic AND anaerobic bottles
• Blood:broth ratio = 1:10 (dilutes inhibitors in blood)
• Incubate at 37°C for 5-7 days
• Subculture if turbidity, gas production, or hemolysis seen
• Blind subculture at 24 hours and 48 hours

• Most widely used currently
• Bottles contain CO2-sensing fluorescent sensor
• Machine detects CO2 production continuously
• Mean detection time: 12-48 hours
• Alerts automatically when positive

• Lyses RBCs and WBCs to release intracellular organisms
• Good for Histoplasma, Cryptococcus, Brucella, Mycobacteria, Bartonella
• Sediment plated directly onto solid media

• Collect BEFORE starting antibiotics
• Collect 2-3 sets from different venipuncture sites, 30-60 min apart (for IE: 3 sets)
• Skin disinfection with 70% alcohol then iodine
• Do not change needles before inoculating bottles (increases contamination)

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Pyrexia of Unknown Origin (PUO) - Etiology

Infections (35-40%) - Most common

• Bacterial: Tuberculosis (most common in India), infective endocarditis, brucellosis, typhoid, abscess (liver, subphrenic, pelvic), osteomyelitis
• Viral: CMV, EBV (infectious mononucleosis), HIV
• Parasitic: Visceral leishmaniasis (kala-azar), malaria
• Fungal: Histoplasmosis, aspergillosis (immunocompromised)

Neoplasms (20-30%)

• Lymphomas (especially Hodgkin's disease) - most common malignancy
• Leukemias
• Renal cell carcinoma (hypernephroma)
• Hepatocellular carcinoma
• Atrial myxoma

Collagen Vascular / Autoimmune (15-20%)

• Adult-onset Still's disease (most common in young adults)
• Systemic lupus erythematosus (SLE)
• Rheumatoid arthritis
• Polyarteritis nodosa
• Temporal arteritis (elderly >50 years)

Miscellaneous (15%)

• Drug fever
• Factitious fever
• Familial Mediterranean fever
• Pulmonary embolism
• Inflammatory bowel disease
• Sarcoidosis

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Clot Culture

1. Collect 5-10 mL of blood WITHOUT anticoagulant into a sterile tube
2. Allow blood to clot at 37°C for 30 min
3. After clot retraction, separate the serum (used for serological tests - Widal)
4. To the clot, add streptokinase (or fibrinolysin) to dissolve the clot
5. Transfer the lysate into peptone water or bile broth
6. Incubate at 37°C for 24-48 hours
7. Subculture onto MacConkey agar and blood agar
8. Identify colonies by biochemical and serological tests

• More sensitive than ordinary blood culture for Salmonella
• Useful when bacteremia is low-grade
• Allows large volume of blood to be processed

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Name Three Blood-Borne Viral Infections

1. HIV (Human Immunodeficiency Virus) - causes AIDS
2. Hepatitis B Virus (HBV)
3. Hepatitis C Virus (HCV)

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Common Microbial Agents (Parasites) Causing Anemia and Type of Anemia

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CHAPTER 3 - ENTERIC FEVER

Typhoid Fever - Pathogenesis

Pathogenesis (Step-by-step)

• Bacteria attach to and invade M cells (microfold cells) overlying Peyer's patches in terminal ileum
• Survive within macrophages (intracellular pathogen)

• Bacteria multiply within mesenteric lymph nodes
• Enter thoracic duct → bloodstream (transient bacteremia)
• Seeded to liver, spleen, bone marrow, gallbladder

• After multiplication in reticuloendothelial system, massive release into bloodstream
• Clinical illness at this stage: sustained high fever (stepladder pattern)
• Relative bradycardia (Faget's sign)

• Gallbladder excretes bacilli into bile → reinfect Peyer's patches
• Hypersensitivity reaction causes necrosis and ulceration of Peyer's patches
• Complications: intestinal perforation, hemorrhage

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Laboratory Diagnosis of Typhoid Fever (Enteric Fever)

1. Bacteriological Methods (Gold Standard = Culture)

• Best in 1st week (highest bacteremia)
• 85-90% positive in first week
• Use bile broth/tryptone soya broth
• Sensitivity: 1st week >80%, 2nd week ~50%, 3rd week ~30%

• Most sensitive (90%) at ALL stages of disease
• Positive even after antibiotic therapy
• Gold standard when blood culture is negative

• Positive in 2nd and 3rd weeks (due to gallbladder excretion)
• Also positive in carriers

• Positive in 3rd week onwards

• Positive in 1st week (early); aspirate and culture rose spots on skin

• Useful in 2nd-3rd week when bacteremia decreases
• More sensitive than routine blood culture

2. Serological Methods

• Detects antibodies against 50-kDa outer membrane protein (OMP) of S. typhi
• IgM positive: acute infection
• IgG positive: past infection or chronic carrier
• Rapid, single serum test

• Detects IgM anti-O9 antibodies (against lipopolysaccharide)
• Semi-quantitative; result in 2 minutes

3. Complete Blood Count

• Leukopenia (characteristic - important clue)
• Relative lymphocytosis
• Anemia, thrombocytopenia in severe cases
• Eosinopenia (absence of eosinophils)

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Widal Test - Uses and Interpretations

• O antigen (somatic lipopolysaccharide) of S. typhi - TO
• H antigen (flagellar) of S. typhi - TH
• H antigens of S. paratyphi A (AH) and S. paratyphi B (BH)

• Serial two-fold dilutions of patient serum (1:10, 1:20, 1:40, 1:80, 1:160, 1:320...)
• Mix with standardized antigen suspension
• Incubate 37°C overnight (18 hours)
• Read agglutination

1. Single titer not diagnostic - endemic areas have background titers
2. False positive: other Salmonella infections, malaria, liver disease, connective tissue disorders
3. False negative: early disease (1st week), antibiotic treatment, immunosuppressed
4. Does not distinguish active from past infection
5. No standard cutoff universally applicable

• Screening test in resource-limited settings
• Retrospective confirmation with paired sera
• Not recommended as sole diagnostic test

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Methods to Detect Carriers of Typhoid Fever

• Chronic fecal carrier: S. typhi in stool >1 year (focus in gallbladder/bile)
• Chronic urinary carrier: S. typhi in urine >1 year (associated with urinary schistosomiasis)
• Convalescent carrier: organism in stool for <3 months post-recovery
• Temporary carrier: asymptomatic excretion

1. Stool culture (repeated) - 3-6 samples on alternate days; use selenite F broth (enrichment medium) then subculture
2. Urine culture - for urinary carriers
3. Duodenal string test (Enterotest): swallow gelatin capsule containing coiled nylon string; capsule dissolves, string absorbs bile; withdraw after 4-6 hours and culture bile
4. Vi antibody test (serological screening): Vi agglutinins >1:10 suggests carrier state; highly sensitive screening test for large populations
5. Bile culture (direct): invasive; only if surgery performed
6. Bone marrow culture: rarely used for carrier detection

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Prophylaxis of Enteric Fever

General (Non-specific) Measures

1. Safe water supply - chlorination, boiling
2. Proper sewage disposal and sanitation
3. Food hygiene - pasteurization of milk, avoid raw vegetables irrigated with sewage water
4. Personal hygiene - handwashing
5. Control of flies
6. Early detection and treatment of cases and carriers
7. Exclusion of carriers from food-handling jobs

Specific (Vaccination)

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Typhoid Vaccines (TAB Vaccine)

1. TAB Vaccine (Killed whole cell vaccine - heat killed, phenol preserved)

• Contains: S. typhi (T), S. paratyphi A (A), S. paratyphi B (B) - killed bacteria
• Route: Subcutaneous
• Schedule: 2 doses, 4-6 weeks apart; booster every 3 years
• Efficacy: 51-67%
• Disadvantages: Severe local reactions, systemic fever/malaise; largely replaced by newer vaccines

2. Vi Polysaccharide Vaccine (ViPS / TYPHIM Vi)

• Contains: Vi capsular polysaccharide antigen of S. typhi (25 μg)
• Route: Single intramuscular injection
• Age: >2 years
• Efficacy: 60-80%, duration 3 years
• Well tolerated; booster every 3 years
• Does NOT protect against paratyphoid

3. Ty21a (Oral Live Attenuated Vaccine - Vivotif)

• Attenuated strain of S. typhi lacking Vi antigen (galE mutant)
• Route: Oral (enteric-coated capsules)
• Schedule: 3 capsules on alternate days (days 1, 3, 5); 4 capsules in USA
• Age: >5 years
• Efficacy: 50-80%, duration 3-7 years
• Stimulates humoral + cell-mediated + secretory IgA
• Booster every 5 years

4. Vi-conjugate Vaccine (Typbar-TCV - Typhoid Conjugate Vaccine)

• Vi polysaccharide conjugated to tetanus toxoid carrier protein
• Route: Single intramuscular dose
• Age: from 6 months
• Efficacy: >80%, long duration
• Recommended by WHO (2018) - preferred for children <2 years
• Included in India's UIP

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CHAPTER 4 - RICKETTSIAL INFECTIONS

Epidemic Typhus

Clinical Features

• Incubation: 1-2 weeks
• Sudden high fever, severe headache, myalgia
• Maculopapular rash appears on day 4-6 - starts on trunk, spreads centrifugally (spares face, palms, soles)
• Altered consciousness, stupor, coma in severe cases
• Complications: myocarditis, pneumonia, gangrene
• Case fatality rate (untreated): 10-40%

Laboratory Diagnosis

1. Weil-Felix reaction: OX19 strongly positive (>1:160); OX2 weakly positive
2. Complement fixation test (CFT): Gold standard serological test
3. Indirect immunofluorescence assay (IFA): Most sensitive and specific
4. ELISA (IgM/IgG)
5. PCR: Most rapid and specific
6. Cell culture: R. prowazekii isolated in Vero cells (BSL-3 facility)

Treatment

• Doxycycline 100 mg twice daily for 7 days (drug of choice)
• Chloramphenicol (alternative)

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Weil-Felix Test (Reaction)

• OX19 (P. vulgaris OX19)
• OX2 (P. vulgaris OX2)
• OXK (P. mirabilis OXK)

1. Non-specific - false positives in Proteus UTI, leptospirosis, liver disease, pregnancy
2. Not sensitive for Q fever and rickettsial pox (negative)
3. Replaced by IFA and ELISA in modern labs
4. Cross-reactions reduce specificity

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Scrub Typhus

Clinical Features

• Incubation: 6-21 days
• Pathognomonic eschar (tache noire): painless black necrotic ulcer at site of mite bite - present in ~50-80%
• Regional lymphadenopathy (tender)
• High fever, severe headache, myalgia
• Rash: maculopapular, starts on trunk day 5-8, spreads to limbs
• Hepatosplenomegaly
• Complications: ARDS, myocarditis, meningoencephalitis, renal failure

Laboratory Diagnosis

1. Weil-Felix OXK positive (OX19 and OX2 negative)
2. IFA (Indirect Immunofluorescence): Gold standard; IgM titer ≥1:200 diagnostic
3. ELISA: IgM detection
4. Immunoperoxidase test
5. PCR: Most sensitive and specific; targets 56-kDa antigen gene
6. Culture: Weil-Felix method in mice; Vero cell culture (BSL-2/3)
7. Buffy coat smear: Intracellular organisms rarely seen
8. Rapid Diagnostic Tests (ICT): Available for field use

Treatment

• Doxycycline 100 mg twice daily for 7-14 days (drug of choice)
• Azithromycin (in pregnancy, children)
• Chloramphenicol (alternative)

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Q Fever

• Inhalation of infected aerosols (most common) from contaminated dried placenta, amniotic fluid, milk, urine, feces during parturition
• No arthropod vector needed (unique among rickettsia-like organisms)
• Consumption of raw milk
• Blood transfusion (rare)

Clinical Features

• Incubation: 2-4 weeks
• Self-limiting febrile illness with severe headache, myalgia
• Atypical pneumonia (20-40% cases)
• Granulomatous hepatitis (fever of unknown origin)
• Rarely: aseptic meningitis, pericarditis

• Culture-negative endocarditis (most common in those with pre-existing valve disease)
• Chronic hepatitis
• Osteomyelitis
• Very high mortality if untreated

Laboratory Diagnosis

1. Weil-Felix: NEGATIVE (all OX antigens negative - unique feature)
2. Phase variation: Phase I antigen (virulent, chronic disease) vs. Phase II antigen (acute disease)
3. CFT/IFA: Anti-Phase II antibodies elevated in acute Q fever; Anti-Phase I antibodies elevated in chronic Q fever/endocarditis
4. ELISA: IgM/IgG
5. PCR: Diagnostic in early disease
6. Culture: BSL-3 facility required (C. burnetii is a bioterrorism agent)

Treatment

• Acute: Doxycycline 100 mg twice daily for 2-3 weeks
• Chronic endocarditis: Doxycycline + hydroxychloroquine for 18 months (minimum)

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CHAPTER 5 - MISCELLANEOUS BACTERIAL BLOODSTREAM INFECTIONS

Spirochaetes of Medical Importance and Diseases Caused

• Gram-negative (but stain poorly with Gram stain)
• Slender, helically coiled, flexible
• Motile by axial filaments (endoflagella) between inner and outer membrane
• Cannot be cultured on ordinary media (Treponema pallidum cannot be cultured in vitro at all)

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Leptospirosis (Weil's Disease) - Pathogenicity and Laboratory Diagnosis

Pathogenicity (Pathogenesis)

1. Entry through skin cuts or mucous membranes
2. Leptospiremic phase (1st week): Organism spreads through blood to all organs (liver, kidneys, meninges, eyes, muscles)
3. Immune phase (2nd week): Antibodies develop; organism cleared from blood but persists in kidney tubules
4. Damage caused by:
   • Direct invasion of endothelial cells (vasculitis)
   • Toxin production (leptospiral lipopolysaccharide)
   • Immune complex deposition

• Mild (anicteric) leptospirosis (90%): Self-limiting febrile illness; headache, myalgia, conjunctival suffusion
• Weil's disease (severe/icteric, 5-10%): Severe jaundice + acute renal failure (acute tubular necrosis) + hemorrhage + hepatitis + myocarditis
• Leptospiral meningitis: Aseptic meningitis
• Pulmonary hemorrhage syndrome (Weil's variant)

Laboratory Diagnosis

• Dark-field microscopy (DFM): During leptospiremic phase (1st week); visualize live leptospires in blood/CSF/urine; S-shaped or question mark-shaped organisms; NOT sensitive
• Silver impregnation staining: Tissues at autopsy
• PCR: Blood (1st week), urine (2nd week onwards); most sensitive early test; 16S rRNA or lipL32 gene target

• Fletcher's semi-solid medium or EMJH medium (Ellinghausen-McCullough-Johnson-Harris)
• Blood: 1st 10 days (aerobic, 28-30°C)
• Urine: 2nd week onwards
• Very slow growing - 4-6 weeks; BSL-2 precautions

• Microscopic Agglutination Test (MAT): Gold standard serological test
  • Detects IgM antibodies; live leptospires used as antigens
  • Titer ≥1:100 (single) or 4-fold rise in paired sera = diagnostic
  • Can identify serovar (epidemiological tool)
• ELISA (IgM): Sensitive from day 4-5 of illness; commercial kits available
• Latex agglutination test: Rapid bedside test
• Macroscopic slide agglutination test (MSAT): Screening test
• Leptodipstick: Rapid IgM detection (field use)

• Liver function tests: elevated bilirubin, transaminases
• Renal function tests: elevated creatinine, BUN
• CPK elevated (myositis)
• CBC: leukocytosis, neutrophilia, thrombocytopenia

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Brucellosis - Clinical Features and Laboratory Diagnosis (Undulant Fever / Human Brucellosis)

• Brucella melitensis (most virulent; goats, sheep) - most common in India
• B. abortus (cattle)
• B. suis (pigs)
• B. canis (dogs)

Clinical Features

• Incubation: 1-8 weeks
• Classic undulant fever: Fever rises and falls in waves (undulations); accompanied by profuse sweating (classically night sweats), chills
• Fatigue, malaise, anorexia, weight loss
• Hepatosplenomegaly
• Arthralgia/arthritis (peripheral and sacroiliac)
• Spondylitis (most common focal complication)
• Orchitis/epididymo-orchitis (males)
• Neurobrucellosis (meningitis, encephalitis - rare)
• Endocarditis (rare but most common cause of death)

Laboratory Diagnosis

• Blood culture in Castaneda's diphasic medium (liquid + agar slope) - most sensitive
• Bone marrow culture: most sensitive (positive even after antibiotics)
• Brucella requires CO2 (5-10%) for growth (B. abortus - capnophilic)
• Very slow growing - minimum 6 weeks incubation
• BSL-3 precautions (aerosol hazard)
• Alert lab of suspicion!

• Serum Agglutination Test (SAT / Standard agglutination test - SAT/Wright's test): Detects antibodies to smooth LPS
  • Titer ≥1:160 single, or 4-fold rise in paired sera = diagnostic
  • False negatives due to blocking antibodies (prozone effect) in chronic brucellosis
• Rose Bengal Plate Test (RBPT): Rapid screening test (colored antigen)
• ELISA: More sensitive; IgM (acute), IgG (chronic/relapse)
• Coombs (anti-human globulin) test: Detects blocking (IgG) antibodies in chronic brucellosis where SAT is negative
• 2-Mercaptoethanol (2-ME) test: Destroys IgM; residual agglutination by IgG indicates past/chronic infection
• CFT
• IFA

• CBC: leukopenia, lymphocytosis
• Liver biopsy: granulomas (non-caseating)

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CHAPTER 6A - HIV/AIDS

Enumerate Sexually Transmitted Diseases (STDs)

• Syphilis (Treponema pallidum)
• Gonorrhea (Neisseria gonorrhoeae)
• Chlamydia (Chlamydia trachomatis - non-gonococcal urethritis, LGV)
• Chancroid (Haemophilus ducreyi)
• Donovanosis/Granuloma inguinale (Klebsiella granulomatis)
• Mycoplasma genitalium

• HIV/AIDS
• Herpes simplex (HSV-1, HSV-2)
• Human Papillomavirus (HPV) - genital warts, cervical cancer
• Hepatitis B, C
• Molluscum contagiosum (pox virus)
• Cytomegalovirus (CMV)

• Trichomonas vaginalis (trichomoniasis)
• Pubic louse (Phthirus pubis)
• Scabies (Sarcoptes scabiei)

• Candida albicans (vulvovaginal candidiasis)

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HIV - Morphology / Structure

Classification

• Family: Retroviridae; Subfamily: Lentivirinae
• Two types: HIV-1 (pandemic, worldwide) and HIV-2 (West Africa, less virulent)

Morphology

• Size: 80-120 nm (spherical)
• Enveloped virus

Structure (Labeled Diagram Components)

OUTER ENVELOPE (lipid bilayer derived from host cell)
├── gp120 (surface glycoprotein - binds CD4 receptor)
└── gp41 (transmembrane glycoprotein - mediates fusion)
    [gp120 + gp41 = gp160 precursor]

MATRIX (p17) - between envelope and core

CORE/CAPSID (cone-shaped - p24)
├── 2 copies of ssRNA (+sense) - genomic RNA
├── Reverse transcriptase (RT) - p51/p66
├── Integrase (p32)
├── Protease (p11)
└── Nucleocapsid protein (p7)

• gag: Encodes core proteins (p24 capsid, p17 matrix, p7 nucleocapsid)
• pol: Encodes enzymes (reverse transcriptase p51/p66, integrase p32, protease p11)
• env: Encodes envelope glycoproteins (gp120, gp41)

• gp120: Major envelope antigen; target for entry inhibitors; high variability
• gp41: Transmembrane; used in antibody detection
• p24: Core antigen; detected early by p24 antigen test (before antibodies appear)
• p17: Matrix protein

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Pathogenesis of HIV Infection

Key Cellular Target

1. gp120 binds CD4 receptor + co-receptor (CCR5 on macrophages in early disease; CXCR4 on T cells in late disease)
2. gp41 mediates membrane fusion
3. Viral core enters cytoplasm

Stages of Infection

• Massive viral replication (high viral load)
• Acute retroviral syndrome: mononucleosis-like illness (fever, lymphadenopathy, pharyngitis, rash)
• CD4 count drops transiently then recovers
• HIV-specific immune response develops (CTLs, antibodies)

• Viral replication continues but controlled by immune system
• Gradual decline of CD4 count (~50-100 cells/μL/year)
• CD4 count >500 cells/μL: clinically normal
• Persistent generalized lymphadenopathy (PGL) may be present

• Constitutional symptoms: fever, weight loss, diarrhea
• Opportunistic infections begin (herpes zoster, oral candidiasis, TB)
• CD4 <200: AIDS-defining

• Severe immunodeficiency
• Serious opportunistic infections and malignancies

1. Direct viral cytopathic effect
2. CTL (CD8+) killing of infected cells
3. Apoptosis (programmed cell death) via gp120-CD4 binding
4. Syncytia formation
5. Immune activation and chronic inflammation
6. Depletion of CD4+ T cell precursors

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Opportunistic Infections in HIV/AIDS

By Category:

• Tuberculosis (most common OI worldwide, especially India) - at any CD4 count
• Mycobacterium avium complex (MAC) - CD4 <50
• Recurrent bacterial pneumonia
• Salmonella septicemia

• Pneumocystis jirovecii pneumonia (PCP) - most common OI in developed world
• Cryptococcal meningitis (Cryptococcus neoformans)
• Esophageal/disseminated candidiasis
• Histoplasmosis, coccidioidomycosis (disseminated)
• Penicilliosis (Talaromyces marneffei) - Southeast Asia

• Cerebral toxoplasmosis (Toxoplasma gondii) - most common focal brain lesion; CD4 <100
• Cryptosporidiosis (Cryptosporidium parvum) - chronic profuse watery diarrhea
• Isosporiasis (Cystoisospora belli)
• Microsporidiosis (Enterocytozoon bieneusi)
• Visceral leishmaniasis (co-infection - atypical presentation)
• Strongyloidosis (hyperinfection syndrome)

• CMV retinitis (CD4 <50) - most common cause of blindness in AIDS
• HSV - chronic ulcers
• Herpes zoster - multidermatomal
• Progressive multifocal leukoencephalopathy (PML) - JC virus
• EBV - oral hairy leukoplakia

• Kaposi's sarcoma (HHV-8)
• CNS lymphoma
• Invasive cervical cancer (HPV)

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Laboratory Diagnosis of HIV (NACO Guidelines)

Testing Strategy (NACO Strategy III - for diagnosis)

• All 3 positive = HIV positive
• If 1 or 2 positive = indeterminate → repeat testing

Rapid Tests / ELISA

• Detect HIV-1 and HIV-2 antibodies (IgG/IgM)
• Window period: 3-12 weeks (time between infection and detectable antibody)
• 4th generation ELISA: Combo test detecting both p24 antigen AND antibodies - reduces window period to ~2-3 weeks

Confirmatory Tests

• Western Blot: Detection of antibodies against specific HIV proteins (gp160, gp120, gp41, p24, p31, p51, p55, p66)
  • Positive: ≥2 bands (WHO criteria): gp120/gp160 + p24 + gp41
  • Limited use in India due to cost
• Line Immunoassay (LIA)

Early Diagnosis / Nucleic Acid Tests (NAT)

• HIV RNA PCR (Viral Load): Quantifies copies/mL; used to monitor treatment, not primary diagnosis
• HIV DNA PCR: Detection of proviral DNA; used for infant diagnosis (<18 months, where maternal antibodies interfere)
• p24 Antigen test: Detects viral protein; positive before antibodies; used in blood banks

CD4 Count

• Normal: 800-1000 cells/μL
• HIV monitoring: measured every 3-6 months
• AIDS definition: CD4 <200 cells/μL
• Measured by flow cytometry

Monitoring Progression of HIV Infection

1. CD4 count: Best predictor of current immune status
2. Viral load (HIV RNA PCR): Best predictor of disease progression rate; guides ART decisions; goal of ART: undetectable (<20-50 copies/mL)
3. Clinical stage (WHO staging 1-4)
4. CBC, LFT, RFT (for ART toxicity monitoring)

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Case Answer: 40-year-old Male with Vesicular Lesions + HIV Reactive

1. Immediate action: Wash wound with soap and water for 5 minutes; do NOT squeeze or suck; irrigate mucous membranes with water/saline
2. Risk assessment of source patient:
   • Test source for HIV (rapid test with consent)
   • If source unknown/unavailable: assess epidemiological risk
3. PEP Regimen (28 days):
   • Tenofovir (TDF 300 mg) + Lamivudine (3TC 300 mg) + Lopinavir/ritonavir (LPV/r 400/100 mg twice daily)
   • Alternative: TDF + 3TC + Efavirenz (EFV)
   • Duration: 28 days
4. Follow-up:
   • Baseline HIV test at time of exposure
   • Repeat at 6 weeks, 12 weeks, 6 months
   • Consider HIV RNA at 4 weeks (if 4th generation test available)
5. Counseling: Abstain from sex/use condoms during follow-up; avoid blood donation, breastfeeding

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Case: 35-year-old Male, IV Drug Abuse, Unexplained Fever, Weight Loss, Diarrhea, Lymphadenopathy 8 months

• Avoid sharing needles/syringes (needle exchange programs)
• Use sterile injection equipment
• Opioid substitution therapy
• HIV testing and counseling
• Condom use
• Treat STIs promptly
• ART for HIV-positive individuals (Treatment as Prevention - TasP)
• PMTCT (Prevention of Mother to Child Transmission)

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Post-Exposure Prophylaxis (PEP) - Detailed NACO Guidelines

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CHAPTER 6B - VIRAL HEMORRHAGIC FEVER

Classification of Arboviruses

Classification by Taxonomy:

Arboviruses in India:

1. Dengue (Aedes aegypti) - DENV 1-4
2. Chikungunya (Aedes aegypti, Ae. albopictus)
3. Japanese Encephalitis (Culex tritaeniorhynchus)
4. West Nile fever (Culex)
5. Yellow fever (historically in imported cases)
6. Zika virus (Aedes aegypti)

1. Kyasanur Forest Disease (KFD) - Haemaphysalis spinigera - Karnataka
2. Crimean-Congo Hemorrhagic Fever (CCHF) - Hyalomma ticks - Gujarat, Rajasthan

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Dengue Virus - Epidemiology, Pathogenesis, Clinical Disease, Complications, Lab Diagnosis

Pathogenesis

1. Mosquito bite → skin: Virus replicates in dendritic cells (Langerhans cells), local lymph nodes
2. Viremia: Spreads to blood; infects monocytes/macrophages, lymphocytes
3. Primary infection (1st infection): Uncomplicated dengue fever
4. Secondary infection (different serotype - heterotypic):
   • Pre-existing antibodies from 1st infection are non-neutralizing
   • Antibody-Dependent Enhancement (ADE): Non-neutralizing antibodies + FcγR receptors facilitate virus entry into MORE monocytes/macrophages → higher viremia
   • Activated T cells release massive cytokines
   • Increased vascular permeability → plasma leakage
   • Thrombocytopenia (platelet consumption, antibody-mediated platelet destruction)
   • Dengue Hemorrhagic Fever / Dengue Shock Syndrome

Clinical Manifestations

• Sudden high fever (biphasic - "saddleback" pattern)
• Severe headache, retroorbital pain
• Myalgia and arthralgia (breakbone fever)
• Flushing of face
• Rash: erythematous → maculopapular ("islands of white in a sea of red")
• Leukopenia, thrombocytopenia

• Fever (2-7 days)
• Hemorrhagic manifestations (positive tourniquet test, petechiae, purpura, epistaxis, gum bleeding)
• Thrombocytopenia (<100,000/mm³)
• Evidence of plasma leakage (rising hematocrit ≥20%, pleural effusion, ascites, hypoalbuminemia)

• All criteria of DHF PLUS
• Narrow pulse pressure (<20 mmHg) or hypotension
• Rapid, weak pulse
• Cold, clammy skin

Complications

• Dengue shock syndrome (hypovolemic shock)
• Acute respiratory distress syndrome (ARDS)
• Liver failure (dengue hepatitis - liver the "shock organ")
• Myocarditis
• Encephalitis/encephalopathy
• Acute kidney injury
• Hemolytic uremic syndrome
• Severe bleeding (internal hemorrhage)

Laboratory Diagnosis

• NS1 antigen detection (ELISA or Rapid test): NS1 protein shed into blood; highly sensitive (>90%); positive from day 1-9
• PCR (RT-PCR): Most sensitive; identifies serotype; positive day 1-5
• Virus isolation: Mosquito cell lines (C6/36 cells), Vero cells - research only

• IgM ELISA: Detectable from day 5-6; peak at 2 weeks; indicates current/recent infection
• IgG ELISA: Rises from day 7-10; persists for years; high IgG from day 1 = secondary infection
• Hemagglutination inhibition (HAI) test: 4-fold rise in titer diagnostic
• Plaque reduction neutralization test (PRNT): Gold standard serological; identifies serotype

• Leukopenia (hallmark)
• Thrombocytopenia (<100,000 - DHF; <20,000 - severe)
• Hematocrit rise (>20%) - indicates plasma leakage
• NS1 positive = dengue confirmed

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Case: 29-year-old Female, High Fever, Joint Pain, Myalgia, Petechial Rash, Jaundice, Hepatosplenomegaly, Platelets 30,000

• Transmitted by the bite of infected Aedes aegypti mosquito (day-biting; urban)
• Positive tourniquet test (≥20 petechiae per inch² over cubital fossa) confirms increased vascular fragility

• NS1 antigen ELISA (if early - within 5 days of fever)
• IgM ELISA (if >5 days)
• RT-PCR for dengue RNA (most specific)
• Combo rapid test (NS1 + IgM/IgG)
• CBC: thrombocytopenia confirmed, rising hematocrit

• Dengue Shock Syndrome (DSS)
• Severe bleeding (GI hemorrhage, intracranial hemorrhage)
• ARDS
• Dengue hepatitis/acute liver failure
• Myocarditis
• Acute kidney injury
• Death

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Kyasanur Forest Disease (KFD)

Clinical Features

• Incubation: 3-8 days
• Biphasic illness:
  • Phase 1: Sudden fever, severe headache, myalgia, GI symptoms (3-10 days), hemorrhagic manifestations (epistaxis, hematemesis)
  • Remission: 1-2 weeks
  • Phase 2 (30-50% of patients): Neurological manifestations (meningitis, encephalitis, tremors, mental disturbances)
• Leukopenia, thrombocytopenia, elevated liver enzymes
• Case fatality: 2-10%

Diagnosis

1. Virus isolation from blood (BSL-3)
2. RT-PCR (from blood - early phase)
3. IgM ELISA (from day 5)
4. Plaque reduction neutralization test (PRNT)

Prevention

• Formalin-inactivated KFD vaccine (bivalent) - available in Karnataka; 2 primary doses + annual booster
• Tick repellents, protective clothing
• Rodent control

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Ebola Virus

Transmission

• Contact with blood, secretions, organs of infected humans or animals
• Funeral practices with direct contact with body
• Nosocomial transmission (through PPE breaches)
• NOT transmitted by air (no aerosol transmission in natural settings)

Clinical Features

• Incubation: 2-21 days
• Sudden fever, fatigue, myalgia, headache
• Sore throat → nausea, vomiting, diarrhea, rash
• Hemorrhagic manifestations: Maculopapular rash, ecchymosis, bleeding from multiple sites
• Multi-organ failure: hepatitis, renal failure, DIC
• CFR: 25-90% (Zaire strain)

Laboratory Diagnosis (HIGH RISK - BSL-4)

1. RT-PCR: Most sensitive; positive 3-10 days after exposure; method of choice
2. ELISA (IgM/IgG): Detectable from day 7-10
3. Virus isolation: BSL-4 facility only
4. Antigen detection ELISA: Rapid diagnosis
5. Electron microscopy: Characteristic "shepherd's crook" or "6 shape" filamentous particles
6. Immunohistochemistry: On biopsy/autopsy samples

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Chikungunya Fever

Clinical Features

• Incubation: 2-4 days
• Acute phase: Sudden fever, severe polyarthritis/arthralgia (small joints - fingers, wrists, ankles), myalgia
• Maculopapular rash over trunk
• Chronic arthritis (months to years) - distinguishing feature from dengue
• Rarely hemorrhagic (unlike dengue)
• Leukopenia, lymphopenia

Lab Diagnosis

• RT-PCR: Positive days 1-5 (viremia)
• IgM ELISA: Positive from day 5
• IgG ELISA: Paired sera (4-fold rise)
• Virus isolation (cell culture - BSL-3)

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Lassa Fever

Clinical Features

• Incubation: 6-21 days
• Fever, weakness, headache, sore throat
• Retrosternal chest pain, vomiting, diarrhea
• Hemorrhage (30%): epistaxis, gum bleeding, melena
• Neurological: encephalopathy, deafness (characteristic - sensorineural deafness common sequel)
• CFR: 1% overall; 15-25% in hospitalized; 80-90% in severe cases

Lab Diagnosis

1. RT-PCR: Gold standard (BSL-4)
2. ELISA (IgM, IgG, antigen)
3. Virus isolation (BSL-4 only)
4. Immunofluorescence

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CHAPTER 7 - MALARIA AND BABESIOSIS

Parasites Found in Blood (Hemoparasites)

• Plasmodium vivax, P. falciparum, P. malariae, P. ovale, P. knowlesi (malaria)
• Babesia spp.
• Trypanosoma brucei (African sleeping sickness - blood/lymph)
• Trypanosoma cruzi (Chagas disease)
• Leishmania donovani (in monocytes)

• Wuchereria bancrofti
• Brugia malayi
• Loa loa
• Mansonella spp.

• Borrelia spp. (spirochaete)

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Plasmodia of Medical Importance

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Life Cycle of Plasmodium

In Humans (Asexual - Schizogony):

• Sporozoites injected by female Anopheles mosquito → reach liver within 30-60 min
• Invade hepatocytes → form liver schizonts (exoerythrocytic schizonts)
• Rupture → release merozoites (10,000-30,000 per schizont)
• Duration: P. falciparum (5.5 days), P. vivax (8 days), P. malariae (13 days)
• Hypnozoites: Dormant liver forms in P. vivax and P. ovale → responsible for relapses (months-years later)

• Merozoites → invade RBCs → form trophozoites (ring stage → ameboid stage)
• Schizogony: Nucleus divides → merozoites within RBC (blood schizont)
• Erythrocytic schizont ruptures → merozoites released → fever (coincides with rupture)
• Cycle: 48h (P. vivax, falciparum, ovale) or 72h (P. malariae)

• Some merozoites → differentiate into gametocytes (male microgametocytes, female macrogametocytes)
• Ingested by mosquito during blood meal

In Mosquito (Sexual - Sporogony):

• Female Anopheles mosquito ingests gametocytes
• Exflagellation: Microgamete fertilizes macrogamete → zygote
• Zygote → ookinete → penetrates stomach wall → oocyst
• Oocyst divides → sporozoites (thousands)
• Sporozoites migrate to salivary glands
• Transmitted to next human host on biting

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P. falciparum - Morphology, Pathogenesis, Lab Diagnosis, Complications

Morphology (Blood smear characteristics)

• Ring forms (early trophozoites): Multiple rings per RBC (multiply infected); delicate; appliqué (accolé) position at cell margin; "headphone" ring (2 chromatin dots)
• No ameboid trophozoites seen in peripheral blood (sequestration)
• Schizonts: Rarely seen in peripheral blood; 16-32 merozoites when seen
• Gametocytes: Crescentic/banana/sickle-shaped - PATHOGNOMONIC of P. falciparum
• RBCs: Normal size, no enlargement; no Schüffner's dots (Maurer's clefts/spots seen)
• No hypnozoites; no relapse

Pathogenesis of Falciparum Malaria

1. Cytoadherence (Rosetting and sequestration):
   • P. falciparum exports PfEMP1 (P. falciparum Erythrocyte Membrane Protein 1) to RBC surface
   • PfEMP1 causes RBCs to adhere to endothelium (CD36, ICAM-1, VCAM-1) → capillary sequestration in brain, lung, kidney
   • Also causes rosetting (infected RBCs bind uninfected RBCs)
   • Sequestration prevents splenic clearance
2. Cytokine release: TNF-α, IL-1 → fever, rigors
3. Anemia: Rapid hemolysis + ineffective erythropoiesis + splenic sequestration
4. Complications from sequestration:
   • Cerebral malaria: brain capillaries blocked → hypoxia, encephalopathy
   • ARDS: lung sequestration
   • Renal failure: acute tubular necrosis
   • Hypoglycemia

Complications of Falciparum Malaria

1. Cerebral malaria (most dreaded): seizures, altered consciousness, coma, death
2. Severe anemia (Hb <7 g/dL)
3. Acute pulmonary edema / ARDS (non-cardiogenic)
4. Acute kidney injury (hemoglobinuria/black water fever)
5. Hypoglycemia (glucose consumption by parasites + quinine stimulating insulin)
6. Circulatory collapse / Algid malaria (septicemic shock)
7. Abnormal bleeding / DIC
8. Hyperparasitemia (>5% parasitized RBCs)
9. Black water fever (see below)

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Black Water Fever

• Massive intravascular hemolysis
• Hemoglobinuria (port-wine/cola-colored/"black" urine)
• Severe anemia
• Jaundice
• Acute renal failure

• Massive immune-mediated hemolysis of parasitized AND non-parasitized RBCs
• Free hemoglobin in plasma → filtered by kidney → precipitates in tubules → acute tubular necrosis
• Associated with repeated quinine treatment and G6PD deficiency

• Urine: Black/dark brown (hemoglobinuria, not hematuria - urine clears on centrifugation in hematuria, not in hemoglobinuria)
• High mortality (25-50% untreated)

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Laboratory Diagnosis of Malaria

1. Microscopy (Gold Standard)

• 20x more sensitive than thin smear
• RBCs lysed → concentrate parasites
• Stain: Giemsa (standard), Leishman's, Field's stain
• Used to DETECT malaria and quantify parasite density

• Preserves RBC morphology
• Used to IDENTIFY species
• Stain: Giemsa or Leishman's

2. Rapid Diagnostic Tests (RDTs)

3. Molecular Tests

• PCR: Most sensitive (detects <5 parasites/μL); gold standard for species identification
• Quantitative PCR: Quantifies parasite load
• LAMP (Loop-mediated isothermal amplification): Rapid, field-applicable

4. Fluorescent Microscopy

• QBC (Quantitative Buffy Coat): Acridine orange staining; centrifuged capillary tube; fluorescent parasites concentrated at buffy coat
• DAPI staining

5. Serology

• Not useful for acute malaria (antibodies persist)
• IFA, ELISA used for blood donor screening and epidemiological surveys

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Case Answer: 23-year-old Male, Intermittent Fever + Chills + Splenomegaly + Parasites in RBCs

• Rupture of RBCs releases merozoites + pyrogenic substances → fever/chills/rigors
• Hypersplenism → splenomegaly
• Anemia (hemolysis)
• Vivax: splenomegaly, anemia, relapse; rarely severe disease
• P. vivax can cause severe disease: ARDS, thrombocytopenia, cerebral malaria (less common than Pf)

• Thick and thin blood smear (Giemsa stain):
  • Vivax: Enlarged RBCs with Schüffner's dots, ameboid trophozoites, 16 merozoites per schizont (Rosette schizont)
  • Ring stage, ameboid trophozoites, schizonts, gametocytes all seen
• RDT (HRP2/pLDH)
• PCR for confirmation

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CHAPTER 8 - VISCERAL LEISHMANIASIS AND TRYPANOSOMIASIS

Case: 31-year-old from Bihar, Splenomegaly + Anemia + Fever + Amastigotes in Macrophages on Bone Marrow

• Macrophages with amastigotes ingested by sandfly during blood meal
• Amastigotes → promastigotes in sandfly gut
• Promastigotes multiply → migrate to proboscis
• Injected as metacyclic promastigotes during next blood meal

• Promastigotes injected → phagocytosed by macrophages
• Inside macrophage phagolysosome → transform to amastigotes (Leishman-Donovan bodies)
• Multiply by binary fission → rupture macrophage → infect more macrophages
• Amastigotes disseminate to liver, spleen, bone marrow (all RES organs)

Parasitological Diagnosis

• Bone marrow aspirate (Giemsa stain): Most preferred; 80-85% sensitivity; amastigotes in macrophages
• Splenic aspirate (fine needle): Highest sensitivity (93-99%); risk of hemorrhage
• Liver biopsy, lymph node aspirate: Less sensitive

• Novy-Nicolle-McNeal medium (diphasic): Blood agar base with defibrinated rabbit blood + balanced salt solution overlay
• Used to culture Leishmania (promastigotes grow in liquid phase at room temperature)

Serological Tests

• rK39 ICT (Rapid test): Most widely used; recombinant K39 antigen strip; 97% sensitivity, 97% specificity; most practical field test
• Direct Agglutination Test (DAT): Titer ≥1:3200 diagnostic; freeze-dried antigen available; field-applicable
• ELISA: High sensitivity; uses various recombinant antigens
• Complement fixation test (CFT) - Napier's aldehyde test (formol gel test): Historical; crude test; non-specific

Formol-Gel (Aldehyde) Test (Napier's test):

• Serum + formol (2 drops of 40% formalin)
• Positive: Serum solidifies (due to massive hypergammaglobulinemia in kala-azar)
• Very non-specific but cheap

• PCR: Blood, bone marrow; identifies species; most sensitive

Treatment of Kala-azar

• Liposomal Amphotericin B (L-AmB): Drug of choice in India (Miltefosine resistance increasing); National Kala-azar Elimination Programme
• Miltefosine: Oral; teratogenic; resistance emerging
• Amphotericin B deoxycholate: Alternative; nephrotoxic
• Antimonials (SSG - Sodium Stibogluconate): High resistance in Bihar; no longer recommended

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Post-Kala-Azar Dermal Leishmaniasis (PKDL)

• Occurs months to years after apparently successful treatment of kala-azar
• L. donovani parasites persist in skin
• Manifestations: Hypopigmented/erythematous macules → papules → nodules on face/trunk
• Important reservoir for transmission (sandfly bites skin lesions)
• Diagnosis: Skin slit smear, skin biopsy, PCR, rK39 (less sensitive in PKDL)

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Chagas Disease

Clinical Features

• Romaña's sign: Painless periorbital edema (unilateral) - entry through conjunctiva
• Chagoma: Indurated skin lesion at entry site
• Fever, lymphadenopathy, hepatosplenomegaly

• Chagasic cardiomyopathy: Dilated cardiomyopathy, arrhythmias, sudden death (most common cause of death)
• Megaesophagus (dysphagia)
• Megacolon (constipation)

Lab Diagnosis

• Acute: Blood smear → trypomastigotes (C-shaped/U-shaped with kinetoplast)
• Xenodiagnosis: Uninfected triatomine bugs fed on patient → examine bug feces for parasites
• Serology: ELISA, IFA (chronic disease)
• PCR: Most sensitive
• Hemoculture: NNN medium

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Classify Leishmaniasis

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CHAPTER 9 - LYMPHATIC FILARIASIS

Classification of Filariasis

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Lymphatic Filariasis (W. bancrofti) - Pathogenesis and Lab Diagnosis

Life Cycle

1. L3 larvae injected by mosquito bite → migrate to lymphatics
2. Develop into adult worms (male + female) in lymphatic vessels
3. Adults mate → females release microfilariae into blood
4. Microfilariae circulate in blood - nocturnal periodicity (peak 10 PM - 2 AM coinciding with Culex biting time)

Pathogenesis

1. Adult worms in lymphatics → lymphatic inflammation (lymphangitis, lymphadenitis) → lymphatic dilation and dysfunction
2. Immunopathology: T-helper immune response → granuloma formation → lymphatic obstruction
3. Chronic lymphatic obstruction → lymphedema → elephantiasis (massive tissue hypertrophy)
4. Bacteria enter through skin (secondary infection) → worsen lymphedema
5. Chyluria: Dilated lymphatics rupture into urinary tract → lymph in urine (milky white urine)
6. Hydrocele (most common manifestation in males)
7. Tropical pulmonary eosinophilia (TPE): Hyperimmune response; eosinophilia >3000/μL, pulmonary symptoms, high IgE, microfilariae absent from blood

Laboratory Diagnosis

• Peripheral blood smear (night blood - between 10 PM and 2 AM): Giemsa-stained thick smear; identify microfilariae
• Concentration methods:
  • Knott's concentration technique (2 mL blood + 10 mL 2% formalin → centrifuge → examine sediment)
  • Millipore filtration (Nuclepore polycarbonate membrane)
• Microfilaria characteristics of W. bancrofti:
  • Sheathed (sheath stains pink with Giemsa - contrast with B. malayi where sheath stains blue-pink)
  • No nuclei in tail tip (important distinguishing feature)

• Problem: Microfilaremia is nocturnal - patient not available at night, OR diurnal periodicity testing needed
• Method: Give DEC (Diethylcarbamazine / Hetrazan) 2-6 mg/kg orally during DAYTIME
• DEC drives microfilariae from lung capillaries into peripheral blood within 30-45 minutes
• Collect blood 30 min after DEC → examine for microfilariae
• Drawback: Can cause acute reactions (Mazzotti reaction) in heavy microfilaremia

• ICT (Immunochromatographic test for W. bancrofti antigen): Detects adult worm antigen; can be done on daytime blood; high sensitivity (96-100%); does not require nocturnal sampling
• Og4C3 ELISA: Quantitative antigen test

• IgG4 antibody detection (less specific due to cross-reactions)

• Ultrasound (scrotal): "Filarial dance sign" - live adult worms moving in scrotal lymphatics
• Lymphoscintigraphy: Assess lymphatic function

• Urine appearance: milky white
• Fat globules on microscopy
• Ether test: clear on addition of ether (confirms fat)
• Sudan III staining of urine: fat globules stain orange-red

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CHAPTER 10 - SYSTEMIC CANDIDIASIS AND SYSTEMIC MYCOSES

Candida albicans - Morphology, Pathogenicity, Lab Diagnosis

Morphology

• Dimorphic fungus: exists as yeast form (at 37°C/in body) and hyphal/pseudohyphal form (in environment/less optimal conditions)
• Yeast form: Oval, budding cells (blastoconidia), 3-5 μm
• Pseudohyphae: Elongated cells that remain attached end-to-end; constrictions at septa
• True hyphae (in tissues)
• Chlamydoconidia (thick-walled resting spores at hyphal tips): characteristic of C. albicans; produced on cornmeal agar at 25°C
• Germ tubes: Short hyphal projections produced in serum at 37°C for 2-3 hours - pathognomonic (Reynolds-Braude phenomenon)

Pathogenicity / Infections Caused

• Opportunistic pathogen - causes disease when normal defenses compromised
• Risk factors: Antibiotics (disrupts normal flora), corticosteroids, immunosuppression, diabetes, HIV/AIDS, extremes of age, catheters/prosthetics, neutropenia

1. Superficial/mucocutaneous:
   • Oral candidiasis (thrush) - white plaques, AIDS marker
   • Esophageal candidiasis (AIDS-defining)
   • Vulvovaginal candidiasis
   • Cutaneous candidiasis (skin folds, napkin rash)
   • Onychomycosis, paronychia
2. Systemic/Deep:
   • Candidemia (fungemia): commonest nosocomial fungal infection
   • Disseminated candidiasis: Kidney (most common target), retina, brain, liver, spleen, heart
   • Candidal meningitis
   • Candidal endocarditis (prosthetic valves)
   • Hepatosplenic candidiasis (in neutropenia recovery)

Laboratory Diagnosis of Candidiasis

• KOH mount (10%): Yeast cells with pseudohyphae/hyphae
• Gram stain: Gram-positive oval budding yeast cells with pseudohyphae
• Periodic Acid-Schiff (PAS) stain: Tissue sections
• Calcofluor white (fluorescence): Most sensitive for direct microscopy
• Tzanck smear (for mucosal lesions)

• Sabouraud Dextrose Agar (SDA): Smooth, creamy, white colonies
• CHROMagar Candida: Color-based species identification
  • C. albicans: Green colonies
  • C. tropicalis: Steel blue/metallic blue
  • C. krusei: Pink/purple, flat
  • C. glabrata: Purple

• Take C. albicans colony → inoculate in human/animal serum
• Incubate at 37°C for 2-3 hours
• Examine under microscope: germ tubes (cylindrical projections without constriction at base = true hyphae, NOT pseudohyphae)
• Positive only in C. albicans and C. dubliniensis
• Quick, cheap, specific test

• Cornmeal agar + Tween 80, at 25°C
• C. albicans forms chlamydoconidia (large, thick-walled terminal/lateral spores)

• Mannan antigen + anti-mannan antibody (combined): Most sensitive (80%)
• Beta-D-glucan (BDG) assay: Pan-fungal; detects all Candida, PCP, Aspergillus (not Cryptococcus or Mucor)
• Candida precipitins (CPA - counter immunoelectrophoresis)

• PCR: High sensitivity for candidemia

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Germ Tube Test (Reynolds-Braude Phenomenon) - Detailed

1. Pick a small amount of Candida colony from culture plate
2. Inoculate into 0.5 mL human/horse serum (must use serum, not broth)
3. Incubate at 37°C for 2-2.5 hours (NOT more than 3 hours - pseudohyphae form later)
4. Place a drop on glass slide, cover with coverslip
5. Examine under 40x objective

• Cylindrical, parallel-sided projection from yeast cell
• No constriction at point of origin (from cell wall) - this distinguishes from pseudohyphae which have constrictions
• Length: approximately equal to width of parent cell
• Arise directly from cell wall

• Positive (germ tubes present): Presumptively identifies C. albicans (and C. dubliniensis)
• Negative: Other Candida species

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Histoplasmosis / Histoplasma capsulatum - Lab Diagnosis

Clinical Forms

1. Asymptomatic (most common)
2. Acute pulmonary histoplasmosis (flu-like)
3. Chronic pulmonary (resembles TB)
4. Disseminated histoplasmosis (immunocompromised/HIV - CD4 <150)

Laboratory Diagnosis

• Giemsa/PAS stain of bone marrow smear, blood smear, BAL
• Yeast form (tissue/37°C): Oval, 2-4 μm, small budding yeast within macrophages - "NARROW-BASED budding"
• Intracellular; resembles LD bodies (kala-azar) but Histoplasma is a fungus (GMS-positive)
• Methenamine Silver Stain (GMS): Stains fungal cell walls black

• SDA at 25°C: Slow-growing white cotton-like mycelial colony (2-4 weeks)
• Microscopy of culture: Macroconidia (tuberculate macroconidia/chestnut burr): large, thick-walled spores with finger-like projections - PATHOGNOMONIC
• Microconidia: Small, oval conidia (infective particles)
• At 37°C: Dimorphic conversion → yeast form
• BSL-3 (highly infectious in mycelial form!)

• Complement fixation test (CFT): Titer ≥1:8 suggestive; ≥1:32 diagnostic
• Immunodiffusion (ID): M and H precipitin bands; H band = active disease
• ELISA

• Urinary Histoplasma antigen (galactomannan-like polysaccharide): Most sensitive (92%) for disseminated disease; also detectable in serum, BAL, CSF
• Cross-reacts with Blastomyces, Coccidioides

• PCR (research settings)

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Coccidioidomycosis

Clinical Forms

1. Asymptomatic (60%)
2. Acute pulmonary (flu-like, "flu-like illness in desert traveler")
3. Chronic pulmonary (cavitary disease)
4. Disseminated (skin, bones, joints, meninges) - higher risk in immunocompromised, dark-skinned races, pregnancy

Laboratory Diagnosis

• Spherules (tissue form, 10-80 μm) containing endospores - PATHOGNOMONIC
• KOH/PAS/GMS staining of sputum, BAL, tissue biopsy
• Spherules mature → rupture → release endospores → form new spherules

• SDA at 25°C: Rapid growth (3-5 days); white-grey cottony colonies
• Microscopy: Arthroconidia (barrel-shaped cells in chains, alternate cells) - extremely infectious
• BSL-3 laboratory required (arthrospores highly infectious)

• IgM (tube precipitin test / latex agglutination): Acute/early infection
• IgG (complement fixation / immunodiffusion): Indicates dissemination; high titers in meningitis
• ELISA

• Positive = past/current infection; remains positive for life
• Negative in disseminated/severe disease (anergy)

• Urinary antigen test (cross-reacts with Histoplasma)

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Regarding the Attached PDF (Micro CNS TB)

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• These answers cover all standard question patterns for MBBS/MD microbiology exams
• For long essays: combine pathogenesis + lab diagnosis + clinical features
• For short notes: focus on unique/distinctive features (e.g., germ tube test for C. albicans, banana gametocytes for P. falciparum, Weil-Felix OXK for scrub typhus)
• For clinical case questions: identify diagnosis → pathogenesis → confirm diagnosis → management