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CHAPTER 1 - INFECTIVE ENDOCARDITIS

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Q: Name three bacteria causing infective endocarditis

1. Staphylococcus aureus - most common overall cause; causes acute IE
2. Viridans streptococci (e.g., S. sanguinis) - most common cause of subacute bacterial endocarditis (SBE)
3. Enterococcus - common in IV drug users and elderly; associated with left-sided IE

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Q: HACEK agents

• H - Haemophilus parainfluenzae
• A - Aggregatibacter species (A. actinomycetemcomitans, A. aphrophilus, A. paraphrophilus)
• C - Cardiobacterium hominis
• E - Eikenella corrodens
• K - Kingella kingae

• Account for ~5% of total IE cases
• Typically has a subacute course
• Occurs in patients with pre-existing valvular defects or those undergoing dental procedures
• Aortic and mitral valves most commonly affected
• They are capnophilic (optimal growth in presence of 5-10% CO2)
• Require prolonged incubation (up to 1 week)

• Blood cultures on automated systems (e.g., BacT/ALERT)
• Identification by automated systems (MALDI-TOF)
• Molecular methods: broad-range bacterial PCR targeting 16S rRNA gene followed by sequencing

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Q: Rheumatic Fever - Disease mechanism / Diagnosis

Pathogenesis (Disease Mechanism):

1. Precipitating infection: ARF follows upper respiratory tract infection with Group A streptococci (M serotypes 1, 3, 5, 6, 14, 18, 24)
2. Genetic predisposition: HLA-DR2 and HLA-DR4 confer susceptibility
3. Autoimmune/Molecular Mimicry theory: Antibodies targeted against streptococcal antigens (M protein) cross-react with human tissue antigens (heart, joints)
4. These cross-reactive antibodies bind to valvular endothelium, leading to damage to heart valves
5. Results in Rheumatic Heart Disease (RHD) with chronic valvular damage

Clinical Manifestations (Major Jones Criteria):

• Carditis (pancarditis affecting endocardium, pericardium, myocardium)
• Migratory polyarthritis - most common manifestation; joints become hot, swollen, red, tender; moves from one joint to another
• Chorea (Sydenham's chorea) - involuntary movement disorder affecting head and limbs
• Erythema marginatum - pink macular rashes that appear and disappear
• Subcutaneous nodules - painless, small mobile lumps beneath skin over bony prominences

Diagnosis - Revised Jones Criteria (2015):

• Carditis (clinical or subclinical)
• Polyarthritis only
• Chorea, Erythema marginatum, Subcutaneous nodules

• Carditis, Monoarthritis or polyarthritis, Polyarthralgia, Chorea, Erythema marginatum, Subcutaneous nodules

• Polyarthralgia (low-risk), Monoarthralgia (high-risk)
• Hyperpyrexia (≥38.5°C)
• ESR ≥60 mm/h and/or CRP ≥3 mg/dL
• Prolonged PR interval on ECG

• Initial ARF: Two major OR one major + two minor criteria
• Evidence of preceding streptococcal infection: rising ASO titre or positive throat culture

• ARF without carditis: 5 years or till age 21 (whichever longer)
• ARF with carditis but no residual valvular disease: 10 years or age 21
• ARF with persistent valvular disease: 10 years or age 40 (sometimes lifelong)

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CHAPTER 2 - ANEMIA (BLOODSTREAM INFECTIONS)

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Q: Blood culture - methods and indications

Indications:

• Bacteremia or fungemia (suspected)
• Infective endocarditis
• Pyrexia of unknown origin (PUO)
• Septicemia/sepsis
• Typhoid and paratyphoid fever
• Brucellosis
• Meningitis
• Immunocompromised patients with fever
• Neonatal sepsis

Methods of Blood Culture:

• Blood collected aseptically by venipuncture
• Two blood culture sets should be collected at an interval of 1 hour between 1st and 2nd set
• Alternatively, 3 or more blood culture sets can be collected over a 24-hour period
• A blood culture set = blood from single venipuncture divided into two bottles (aerobic + anaerobic)
• Volume: 8-10 mL per bottle in adults; 1-3 mL in children
• Blood:broth ratio = 1:5 to 1:10 (dilutes inhibitory substances in blood)
• Incubated at 37°C

• Most widely used modern method
• Continuously monitors CO2 production by bacteria using fluorescent or colorimetric sensors
• Flags positive bottles automatically
• Most sensitive and faster than conventional methods

• A single bottle with both solid and liquid media
• Liquid medium covers the solid agar slope
• Bottle is tilted periodically to spread liquid over solid medium
• Advantage: dual detection, fewer subcultures needed
• Used for: Brucella, leptospira

• Blood is collected without anticoagulant; allowed to clot
• Clot is washed and cultured
• Advantage: concentrates organisms (intracellular) in leukocytes
• Used for: Salmonella typhi (higher yield than broth culture in some studies), Brucella

• Blood is lysed using saponin; centrifuged to concentrate organisms
• Pellet is plated onto solid media
• Best method for fungi and mycobacteria

• Gram stain from flagged positive bottles
• Subculture onto blood agar, chocolate agar, MacConkey agar
• Sensitivity testing of isolates

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Q: Pyrexia of Unknown Origin (PUO) - Etiology

Etiology (classified):

• Bacterial: Tuberculosis (most common infectious cause), infective endocarditis, brucellosis, typhoid, abscess (intra-abdominal, hepatic, pelvic)
• Viral: EBV (infectious mononucleosis), CMV, HIV
• Parasitic: Visceral leishmaniasis (kala-azar), malaria
• Fungal: Histoplasmosis, candidiasis

• Lymphoma (Hodgkin's and Non-Hodgkin's)
• Leukemia
• Renal cell carcinoma (hypernephroma)
• Hepatocellular carcinoma
• Other solid tumors

• Adult Still's disease (most common in young adults)
• Systemic lupus erythematosus (SLE)
• Rheumatoid arthritis
• Polyarteritis nodosa, Giant cell arteritis (in elderly)

• Drug fever
• Inflammatory bowel disease
• Sarcoidosis
• Factitious fever

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Q: Clot Culture

1. Collect 5-10 mL blood without anticoagulant into a sterile tube
2. Allow to clot at room temperature
3. Serum is separated (can be used for serology)
4. Clot is washed with sterile saline to remove RBCs
5. Clot is emulsified/digested with streptokinase or SPS (sodium polyanethol sulfonate)
6. The lysate is inoculated into culture media

• Concentrates organisms (useful for intracellular pathogens)
• Higher yield for Salmonella typhi compared to broth cultures in the second and third weeks of typhoid fever
• The separated serum can simultaneously be used for Widal test

• Typhoid fever (weeks 2-3)
• Brucellosis
• Leptospirosis

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Q: 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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Q: Common microbial agents (parasites) causing anemia and type of anemia

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CHAPTER 3 - ENTERIC FEVER

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Q: Typhoid fever - pathogenesis and laboratory diagnosis

Causative Agent:

Pathogenesis:

• Intestinal hemorrhage
• Intestinal perforation (most serious complication)

• Week 1: Fever (stepladder pattern), headache, abdominal pain, constipation (early), rose spots on abdomen
• Week 2: High fever, splenomegaly, relative bradycardia (Faget sign), hepatomegaly
• Week 3: Complications (perforation, hemorrhage)
• Week 4: Defervescence, recovery

Laboratory Diagnosis:

• Week 1-2: BEST specimen - blood culture (sensitivity ~80% in week 1)
• Positive in 80-90% cases in 1st week
• Clot culture can also be used
• Automated systems (BacT/ALERT, BACTEC) or conventional

• Most sensitive method (90-95% positivity even after antibiotic therapy)
• Positive throughout the disease
• Especially useful in partially treated cases

• Detects agglutinating antibodies against O antigen (somatic) and H antigen (flagellar)
• Significant titre: O antigen ≥1:80 (>1:160 in endemic areas), H antigen ≥1:160
• 4-fold rise in titre between acute and convalescent sera is significant
• Performed from 2nd week onwards (antibodies appear after 1 week)
• Limitation: false positives in endemic areas, cross-reactions, prior vaccination

• Positive from 2nd-3rd week and in carriers
• Best specimen for detecting chronic carriers

• Positive from 3rd-4th week in ~25% cases

• Detects IgM and IgG antibodies against 50 kDa outer membrane protein (OMP) of S. typhi
• IgM positive = current infection
• IgG positive = past infection
• More sensitive and specific than Widal

• Detects IgM antibodies to O9 antigen of S. typhi
• Rapid test, result in 2 minutes

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Q: Widal Test - Uses and Interpretations

• S. typhi O (somatic/LPS antigen)
• S. typhi H (flagellar antigen)
• S. paratyphi A H antigen
• S. paratyphi B H antigen

1. Serodiagnosis of typhoid and paratyphoid fever
2. Helps when blood culture is negative (e.g., partially treated cases)
3. Epidemiological surveys

• Single titre: O antibody ≥1:80 (or >1:160 in endemic areas) is significant; H antibody ≥1:160 is significant
• Most significant: 4-fold or greater rise in antibody titre in paired sera (acute + convalescent, 2 weeks apart)
• O antibodies rise first (appear by end of 1st week), reach peak by 3rd-4th week, and disappear within months
• H antibodies rise later, persist for years, and may indicate past infection or vaccination

• O agglutination: granular, compact clumps (somatic)
• H agglutination: floccular, fluffy, cotton-wool-like clumps (flagellar)

1. Previous typhoid vaccination (raises H antibody)
2. Prior exposure in endemic areas
3. Cross-reactions with other Salmonella serotypes or Enterobacteriaceae (Brucella, Yersinia)
4. Other fevers (malaria, dengue, liver disease)

1. Early infection (antibodies not yet risen)
2. Partially treated patients
3. Immunocompromised hosts
4. Some strains may not stimulate antibody production

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Q: Methods to detect carriers of typhoid fever

• Temporary/convalescent carrier: <3 months after illness
• Chronic carrier: >1 year (usually in gallbladder/bile duct; chronic cholecystitis)
• Contact/healthy carrier: No history of illness

• Most important method
• Multiple stool cultures (at least 3) should be performed
• S. typhi is excreted in stool for prolonged periods

• Less sensitive
• Occasionally positive in chronic carriers

• Direct sampling from duodenum via duodenal string test (Enterotest)
• Higher sensitivity than stool culture for biliary carriers
• The string capsule is swallowed, and after 4-6 hours the string is retrieved and cultured

• Less useful for carrier detection; H antibody may be high in vaccinated persons
• Vi antibody: Detection of antibodies against Vi (virulence) antigen - high Vi antibody titre (≥1:20) suggests chronic carrier state. Vi test is more specific for carrier detection.

• High Vi antibody titre indicates a chronic carrier
• Used as a screening test before stool culture

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Q: Prophylaxis of Enteric Fever

Non-specific / General Measures:

• Safe water supply (chlorination, boiling)
• Proper sewage disposal
• Food safety and hygiene
• Hand washing
• Detection and treatment of carriers (especially food handlers)
• Fly control

Specific (Vaccines):

1. Ty21a (oral live attenuated vaccine)
2. Vi polysaccharide vaccine (Vi CPS)
3. TAB vaccine (killed whole cell vaccine) - now largely replaced
4. Typhoid conjugate vaccine (TCV) - Vi antigen conjugated to tetanus toxoid; recommended by WHO for children

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Q: Typhoid Vaccines (TAB Vaccine)

• Heat-killed, phenol-preserved whole cells of:
  • Salmonella typhi (T)
  • Salmonella paratyphi A (A)
  • Salmonella paratyphi B (B)

• Significant side effects
• Poor immunogenicity for paratyphoid component

Modern Typhoid Vaccines:

• Contains purified Vi capsular polysaccharide
• SC or IM injection, single dose
• Protects for 2-3 years
• Not effective in children <2 years (T-independent antigen)
• Efficacy: ~70%

• Attenuated mutant of S. typhi (lacks Vi antigen, galE mutant)
• 3 oral doses on alternate days
• Efficacy: ~70% for 5-7 years
• Contraindicated in immunocompromised

• Vi polysaccharide conjugated to tetanus toxoid carrier protein
• Single IM injection
• Effective in children >6 months
• Provides T-cell dependent immunity; longer protection (>2 years)
• Recommended by WHO (2018) for endemic countries

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CHAPTER 4 - RICKETTSIAL INFECTIONS

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Q: Epidemic Typhus

• Man is the reservoir; louse becomes infected by feeding on a rickettsiemic patient
• Louse feces containing rickettsiae are deposited on skin; infection occurs by scratching the bite site (rubbing louse feces into the wound)

• Occurs in conditions of overcrowding, poor hygiene, war, famine, disaster situations
• Worldwide but mainly in mountainous regions of Africa, South America, Asia

• Incubation period: 1-2 weeks
• Sudden onset of high fever (up to 40°C), severe headache, myalgia
• Maculopapular rash appears on 4th-6th day, starting on trunk and spreading centrifugally (spares face, palms, soles)
• Progressive obtundation, delirium, coma in severe cases
• Complications: myocarditis, pneumonia, renal failure, DIC

• Weil-Felix test: OX-19 and OX-2 agglutinate; OX-K negative
• Complement fixation test (CFT)
• Indirect fluorescent antibody (IFA) - gold standard
• PCR
• Giemsa or immunofluorescence staining of tissue/skin biopsy

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Q: Weil-Felix Test (Reaction)

• Proteus OX-19 - agglutinated by antibodies against R. rickettsii, R. prowazekii, R. typhi
• Proteus OX-2 - agglutinated by antibodies against R. rickettsii, R. conorii
• Proteus OX-K - agglutinated by antibodies against Orientia tsutsugamushi (scrub typhus)

• Low sensitivity and specificity
• False positives: UTI due to Proteus, Borrelia, leptospirosis, brucellosis, liver disease
• Q fever and Rickettsialpox give NEGATIVE results (no cross-reactivity)
• Currently replaced by more specific tests (IFA, ELISA)
• Considered a screening test only

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Q: Scrub Typhus

• Mites are both vectors AND reservoir (transovarial transmission in mites)
• Humans are accidental dead-end hosts

• Incubation: 6-21 days
• Eschar (tache noire): Painless black eschar at site of chigger bite - pathognomonic
• High fever, headache, myalgia
• Lymphadenopathy (especially regional near eschar)
• Maculopapular rash on trunk (4th-5th day), spreads to limbs
• Hepatosplenomegaly
• Complications: pneumonitis, meningitis, myocarditis, ARDS

• Weil-Felix test: OX-K positive (OX-19 and OX-2 negative)
• IFA (gold standard) - titre ≥1:200 diagnostic
• Rapid diagnostic tests (ICT)
• PCR
• Giemsa staining of peripheral blood smear - intracytoplasmic organisms in monocytes
• Isolation in L929 cell line (BSL-3 required)

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Q: Q Fever

• NOT transmitted by arthropod vector (unlike other rickettsiae)
• Inhalation of infected dust/aerosols from parturient animals (cattle, sheep, goats) - most common route
• Contact with infected birth products (placenta, amniotic fluid)
• Ingestion of unpasteurized milk
• Rarely: tick bite (Dermacentor, Amblyomma)

• Acute Q fever: Fever, headache, myalgia, hepatitis (granulomatous), atypical pneumonia (like a "flu-like illness")
• Chronic Q fever (Endocarditis): Most serious complication; occurs months-years later; mainly involves pre-existing abnormal/prosthetic valves; culture-negative endocarditis

• Weil-Felix test is NEGATIVE (no cross-reactivity with Proteus OX antigens)
• Phase variation: C. burnetii exists in two phases:
  • Phase I: Virulent form (from naturally infected animals); smooth LPS
  • Phase II: Avirulent form (in laboratory passage); rough LPS
• Serology (IFA - gold standard):
  • Acute Q fever: Phase II IgG/IgM antibodies predominate
  • Chronic Q fever/Endocarditis: Phase I IgG ≥1:800 highly suggestive
• PCR (blood, tissue)
• Culture (BSL-3): not routine
• Modified Duke criteria: Single positive blood culture or Phase I IgG antibody titre ≥1:800 = major criterion for IE

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CHAPTER 5 - MISCELLANEOUS BACTERIAL BLOODSTREAM INFECTIONS

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Q: Spirochaetes of medical importance - diseases, pathogenicity and lab diagnosis of Leptospirosis (Weil's disease)

Spirochaetes of Medical Importance:

Leptospirosis (Weil's Disease):

• Tightly coiled, thin, flexible spirochaetes
• 6-20 µm long, 0.1 µm wide
• Hooked ends (characteristically bent at one or both ends like a question mark)
• Motile by axial filaments (periplasmic flagella)
• Cannot be seen by Gram stain; visible by dark-field microscopy

• Reservoir: Wild and domestic animals (rats most important; also cattle, dogs, pigs)
• Transmission: Contact with water/soil contaminated with urine of infected animals (leptospires excreted in urine)
• Entry: Through skin abrasions or mucous membranes (conjunctiva, oral)
• Occupational risk: Farmers, sewer workers, veterinarians, miners, military personnel
• Recreational risk: Swimming in contaminated water

1. Entry through skin/mucous membrane → leptospiraemia (1st week)
2. Dissemination to all organs via blood
3. Vasculitis (endothelial damage) is the key mechanism
4. Target organs: Liver, kidneys, lungs, meninges
5. Immune phase (2nd week): Antibodies appear, organisms cleared from blood but persist in kidneys

• Anicteric leptospirosis (90% cases): Mild; biphasic fever
  • Phase I (Leptospiraemic): High fever, myalgia (severe, especially calf muscles - pathognomonic), headache, conjunctival suffusion (redness without discharge), nausea, vomiting. Lasts 3-7 days.
  • Phase II (Immune/Leptospiruric): Fever recurs; meningismus; leptospiruria begins
• Weil's Disease (5-10% cases - severe icteric leptospirosis):
  • Jaundice (hepatic involvement)
  • Renal failure (acute tubular necrosis) - oliguria/anuria
  • Hemorrhagic manifestations (epistaxis, hemoptysis, thrombocytopenia)
  • Myocarditis
  • Mortalitiy: 5-15% in Weil's disease

• Week 1 (leptospiraemic phase): Blood, CSF
• Week 2 onwards (immune/leptospiruric phase): Urine

• Dark-field microscopy of blood (week 1) or urine (week 2)
• Leptospires appear as bright, coiled, motile organisms against dark background
• Silver staining of tissue sections

• EMJH (Ellinghausen-McCullough-Johnson-Harris) medium or Fletcher's semi-solid medium
• Incubated at 28-30°C for up to 6 weeks
• Blood culture: Week 1; Urine culture: Week 2 onwards
• Very slow-growing; results take weeks

• MAT (Microscopic Agglutination Test) - GOLD STANDARD
  • Live leptospiral antigens mixed with patient serum
  • Agglutination seen under dark-field microscopy
  • Titre ≥1:100 in clinical context, or 4-fold rise in paired sera is diagnostic
  • Can identify the specific serovar
• ELISA: Detects IgM antibodies; used for rapid screening
• Rapid dipstick/ICT tests: Detect leptospiral antibodies

• Most sensitive in early leptospiraemic phase
• Can detect leptospiral DNA in blood, urine, CSF

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Q: Brucellosis - clinical features and laboratory diagnosis (Undulant fever / Human brucellosis)

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

• Ingestion of unpasteurized milk/dairy products from infected animals
• Direct contact with infected animal products (abortus material, carcasses) - occupational risk
• Inhalation of aerosols in abattoir workers
• NOT person-to-person (rare exceptions)

• Brucella is a facultative intracellular organism
• Survives inside macrophages by inhibiting phagolysosomal fusion
• Spreads to lymph nodes → bloodstream → RES (liver, spleen, bone marrow, lymph nodes)
• Granuloma formation in infected tissues

Clinical Features:

• Insidious onset of fever
• Undulant fever: Characteristic relapsing, remitting fever pattern (rises in evening, subsides in morning with drenching sweats)
• Severe malaise, weakness, myalgia, arthralgia
• Headache
• Splenomegaly, hepatomegaly, lymphadenopathy

• Continued fever, weight loss, constitutional symptoms

• Localized complications: Spondylitis (Brucella spondylitis - lumbar spine most common), sacroiliitis, peripheral arthritis, orchitis/epididymitis, endocarditis (severe, culture-negative), neurobrucellosis

Laboratory Diagnosis:

• Isolation of Brucella from blood, bone marrow, or other tissues is gold standard
• Bone marrow culture: Most sensitive (85-90%)
• Blood culture: Positive in 50-80%
• Culture requires prolonged incubation (up to 6 weeks at 37°C)
• Growth in CO2 (especially B. abortus)
• Biphasic (Castaneda) medium preferred
• Automated systems (BACTEC, BacT/ALERT)
• CAUTION: Brucella is a BSL-3 organism; lab must be alerted

• Serum Agglutination Test (SAT)/Wright's agglutination test:
  • Single titre ≥1:160 significant; 4-fold rise in paired sera diagnostic
  • Titre >1:320 with compatible clinical features = probable brucellosis
  • Detects IgM (prozone effect possible at high titres - use dilutions)
• Rose Bengal Plate Test (RBPT):
  • Rapid, simple screening test
  • Uses buffered acid antigen of B. abortus
  • High sensitivity, used for screening
• ELISA: More sensitive; detects IgM, IgG, IgA
• 2-Mercaptoethanol (2-ME) test:
  • 2-ME destroys IgM, leaving only IgG agglutination
  • Persistent IgG (post 2-ME agglutination ≥1:160) = current/chronic infection
  • Differentiates acute (high IgM) from chronic (IgG dominant) infection
• Coombs test: Detects incomplete (non-agglutinating) antibodies in chronic brucellosis

• Highly sensitive and specific
• Can detect Brucella DNA from blood, bone marrow, other specimens

• Gram-negative small coccobacilli (may need 10× objective as they are very faint)
• Modified Ziehl-Neelsen stain: Brucella stains pink against blue background (partially acid-fast)

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CHAPTER 6 - HIV/AIDS

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Q: Enumerate STDs. Morphology, pathogenesis, lab diagnosis of HIV. Opportunistic infections. Prophylaxis.

Sexually Transmitted Diseases (STDs):

1. Syphilis (Treponema pallidum)
2. Gonorrhea (Neisseria gonorrhoeae)
3. Chlamydia (Chlamydia trachomatis)
4. Chancroid (Haemophilus ducreyi)
5. Lymphogranuloma venereum (LGV) (C. trachomatis L1-L3)
6. Granuloma inguinale/Donovanosis (Klebsiella granulomatis)
7. Mycoplasma genitalium

1. HIV/AIDS
2. Herpes simplex virus type 2 (HSV-2) - genital herpes
3. Human Papillomavirus (HPV) - genital warts, cervical cancer
4. Hepatitis B, C
5. Molluscum contagiosum (MCV)

1. Trichomoniasis (Trichomonas vaginalis)
2. Pubic lice (Phthirus pubis)
3. Scabies (Sarcoptes scabiei)

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Structure/Morphology of HIV:

• Spherical, enveloped virus, ~100-120 nm diameter

1. Envelope (lipid bilayer): Derived from host cell membrane during budding
   • Contains gp120 (surface glycoprotein/knobs) - binds to CD4 receptors
   • gp41 (transmembrane glycoprotein) - mediates fusion with host cell
   • Together form gp160 precursor (cleaved by host protease)
2. Matrix protein (p17/MA): Lines inner surface of envelope; stabilizes the virion
3. Capsid (p24/CA): Conical/truncated cone-shaped core; contains:
   • Two copies of positive-sense single-stranded RNA genome (+ssRNA)
   • Reverse transcriptase (RT/p66/p51) - RNA-dependent DNA polymerase
   • Integrase (p32/IN) - integrates proviral DNA into host genome
   • Protease (p10) - cleaves polyprotein precursors
4. Nucleocapsid (p7/NC): Surrounds and protects RNA

• Structural genes: gag (p17, p24, p7), pol (RT, integrase, protease), env (gp120, gp41)
• Regulatory genes: tat, rev (essential)
• Accessory genes: vif, vpr, vpu/vpx, nef

• Core antigens: p24 (most important - detected in ELISA/4th generation tests), p17, p7
• Envelope antigens: gp120, gp41, gp160
• Pol antigens: p66, p51 (RT), p32 (integrase), p10 (protease)

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Pathogenesis of HIV Infection:

• HIV binds via gp120 to CD4 receptor (on CD4+ T lymphocytes, macrophages, dendritic cells)
• Co-receptors required: CCR5 (for macrophage-tropic, R5 strains - early infection) or CXCR4 (for T-cell tropic, X4 strains - late infection)
• gp41 mediates fusion; viral core enters cytoplasm

• HIV reverse transcriptase converts ssRNA → double-stranded DNA (dsDNA)
• Error-prone process → generates genetic diversity/mutations (drug resistance)

• Viral dsDNA is transported to nucleus
• Integrase integrates viral DNA into host chromosome → forms provirus
• Provirus can remain latent indefinitely

• Activated CD4+ cells transcribe proviral DNA → viral RNA
• Tat protein: transactivator, dramatically increases transcription
• Rev protein: facilitates transport of viral RNA from nucleus
• Viral proteins assembled; protease cleaves polyproteins into functional units
• New virions bud from cell membrane

• Direct killing by viral budding
• Apoptosis of infected and uninfected CD4+ cells
• Immune destruction of HIV-infected cells
• Normal CD4 count: 500-1500 cells/µL
• When CD4 <500: Symptomatic HIV
• When CD4 <200: AIDS-defining (opportunistic infections occur)

• Acute HIV syndrome (Primary HIV infection): 2-4 weeks after exposure; flu-like illness (fever, lymphadenopathy, rash, pharyngitis); high viral load, transient drop in CD4+
• Clinical latency (asymptomatic): 8-10 years average; gradual CD4 decline; virus still replicating
• AIDS: CD4 <200/µL or AIDS-defining condition

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Laboratory Diagnosis of HIV:

• Screening test
• 4th generation ELISA (HIV Combo test) detects:
  • Anti-HIV 1 + 2 antibodies (IgM + IgG)
  • p24 antigen (core antigen - detects acute infection before antibody development)
• Reduces window period to ~15 days

• Immunochromatographic tests
• Results in 30 minutes
• NACO strategy: Three rapid tests (Strategy III)

• Test 1 (A1): Most sensitive ELISA/rapid test for screening
• If A1 positive → Test 2 (A2): Different antigen/different principle
• If A2 positive → Test 3 (A3): Third assay with different principle
• All three positive = HIV positive
• If A1+A2 positive but A3 negative = Indeterminate → repeat testing after 14 days
• A1 negative = HIV negative

• Detects antibodies to specific HIV proteins on nitrocellulose strips
• Positive: ≥2 bands including gp41, gp120/gp160 and p24 (WHO criteria)

• Quantifies HIV-1 RNA copies/mL plasma
• Uses RT-PCR or bDNA technique
• Used for: Monitoring treatment response, detecting acute HIV (window period), diagnosis in neonates
• Target for treatment success: Viral load <50 copies/mL (undetectable)

• Flow cytometry to count CD4+ T cells
• Used for: Staging, decision to start ART, monitoring treatment, opportunistic infection prophylaxis
• CD4 <200/µL = AIDS

• Detects HIV p24 core antigen
• Positive during: Acute HIV (before antibodies), late AIDS (when antibodies fall)
• Incorporated in 4th generation combo ELISA

• 1st generation (IgG ELISA): 6-8 weeks
• 3rd generation: 3-4 weeks
• 4th generation (combo): ~15-18 days
• PCR: 10-12 days

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Opportunistic Infections in HIV/AIDS:

• Tuberculosis (most common OI in India)
• Herpes zoster (reactivation)
• Oral candidiasis (thrush)
• Kaposi's sarcoma (HHV-8)

• Pneumocystis jirovecii pneumonia (PCP) - most common OI in western countries
• Toxoplasma gondii encephalitis (CD4 <100)
• Cryptococcal meningitis (Cryptococcus neoformans)
• CMV retinitis/colitis (CD4 <50)
• Mycobacterium avium complex (MAC)

• Toxoplasma gondii - cerebral toxoplasmosis (CD4 <100): ring-enhancing lesions on CT/MRI
• Cryptosporidium parvum - chronic profuse watery diarrhea
• Cyclospora cayetanensis - diarrhea
• Isospora belli (Cystoisospora belli) - diarrhea
• Microsporidium spp. - diarrhea, keratoconjunctivitis
• Leishmania donovani - visceral leishmaniasis (unusual dissemination)
• Giardia lamblia - more severe/prolonged diarrhea

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Monitoring Progression of HIV:

1. CD4+ T cell count (absolute + percentage):
   • Most important clinical marker
   • Monitor every 3-6 months
   • Guides when to start ART and OI prophylaxis
2. HIV Viral Load (Plasma HIV RNA):
   • Best predictor of rate of disease progression
   • High viral load = rapid progression
   • Measured at baseline, 4-8 weeks after starting ART, then every 3-6 months
   • Goal: <50 copies/mL
3. Clinical staging (WHO stages I-IV)
4. Other markers (less used):
   • Beta-2 microglobulin
   • Neopterin levels
   • p24 antigen levels

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Post-Exposure Prophylaxis (PEP) for HIV - NACO Guidelines:

• High risk: Needle-stick with blood, deep injury, hollow-bore needle
• Lower risk: Mucous membrane or non-intact skin exposure

1. Immediately wash wound with soap and water (do not squeeze)
2. Flush mucous membranes with water
3. Do NOT use bleach/caustic agents

• TDF (Tenofovir) + 3TC (Lamivudine) + LPV/r (Lopinavir/ritonavir)
• Alternative: TDF + 3TC (Emtricitabine, FTC) + EFV (Efavirenz)
• Duration: 28 days
• Must be started within 72 hours (ideally within 2 hours; earlier = more effective)
• After 72 hours, PEP is not effective

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Q: Case - 40-year-old male with recurrent herpes zoster + HIV

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Q: Case - 35-year-old male, IV drug abuse, unexplained fever, weight loss, diarrhea, lymphadenopathy for 8 months

• Universal precautions for healthcare workers
• Safe sex practices (condoms)
• Avoid sharing needles (needle exchange programs, harm reduction)
• Screening blood/blood products
• Prevention of mother-to-child transmission (PMTCT)
• Pre-exposure prophylaxis (PrEP): TDF+FTC daily for high-risk individuals
• Voluntary counseling and testing (VCT)
• NACO guidelines for linkage to care and ART

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CHAPTER 6 - VIRAL HEMORRHAGIC FEVER

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Q: Classify arboviruses. General properties. Arboviruses in India. Diagnosis.

Classification of Arboviruses:

• Genus Alphavirus: Chikungunya, Eastern/Western/Venezuelan equine encephalitis

• Genus Flavivirus: Dengue, Yellow fever, Japanese encephalitis, West Nile virus, Zika virus, KFD (Kyasanur Forest Disease)

• Genus Phlebovirus: Sandfly fever
• Genus Nairovirus: Crimean-Congo hemorrhagic fever (CCHF)
• Genus Orthobunyavirus: La Crosse encephalitis

• Genus Orbivirus: Colorado tick fever

General Properties of Arboviruses:

• RNA viruses
• Enveloped (except reoviruses)
• Transmitted by arthropod vectors (mosquitoes, ticks, sandflies)
• Zoonotic - maintained in animal reservoir (birds, rodents) + arthropod cycle
• Humans are incidental (dead-end) hosts
• Can cause fever, encephalitis, hemorrhagic fever

Arboviruses in India:

• Dengue virus (DENV 1-4) - Aedes aegypti mosquito
• Chikungunya virus (CHIKV) - Aedes aegypti, A. albopictus
• Japanese Encephalitis virus (JEV) - Culex tritaeniorhynchus
• Kyasanur Forest Disease virus (KFDV) - Haemaphysalis ticks (hard ticks)
• West Nile virus - Culex mosquitoes
• Zika virus - Aedes mosquitoes

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Q: Dengue Virus / Dengue Fever / Dengue Hemorrhagic Fever

Pathogenesis:

• Mild disease; serotype-specific immunity develops

• Antibody-Dependent Enhancement (ADE): Non-neutralizing antibodies from 1st infection facilitate enhanced uptake of 2nd serotype into macrophages (via Fc receptors)
• Massive viral replication in macrophages → high viremia
• Cytokine storm (TNF-α, IL-1, IL-6) → increased vascular permeability, thrombocytopenia

Clinical Manifestations:

• Incubation: 4-10 days
• High fever (biphasic/"saddle-back"), severe headache, retro-orbital pain
• "Breakbone fever" - severe myalgia and arthralgia
• Maculopapular rash (4th-5th day, "islands of white in sea of red")
• Positive tourniquet test

• Grade I: Fever + positive tourniquet test
• Grade II: Grade I + spontaneous hemorrhage
• Grade III: Grade II + circulatory failure (narrowing pulse pressure, hypotension)
• Grade IV: Profound shock (undetectable pulse/BP) = Dengue Shock Syndrome (DSS)

Complications:

1. Dengue Shock Syndrome (DSS)
2. Hepatitis/hepatic failure
3. Encephalitis/encephalopathy
4. Myocarditis
5. Acute respiratory distress syndrome (ARDS)
6. Secondary bacterial infections

Laboratory Diagnosis:

• NS1 Antigen test (Non-structural protein 1): Rapid immunochromatographic test; very sensitive in early disease (day 1-5); specificity 100%
• RT-PCR: Detects dengue RNA; gold standard for acute phase; serotype identification

• IgM antibody (MAC-ELISA): Detects IgM; positive from day 4-5 after fever onset
  • Primary dengue: IgM appears earlier, higher levels
  • Secondary dengue: IgM lower, IgG higher (IgG rises much faster = "secondary response")
• IgG antibody: IgG >1280 in acute phase suggests secondary dengue
• Dengue IgM/IgG ratio: IgM:IgG <1.2 = secondary dengue
• Haemagglutination inhibition (HI) test: 4-fold rise between acute and convalescent sera

• CBC: Leukopenia (WBC <5000), thrombocytopenia (<100,000 = DHF), rising hematocrit (>20% rise = plasma leakage)
• NS1+IgM combo rapid test

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Q: Case - 29-year-old female with high fever, joint pain, petechial rashes, jaundice, hepatosplenomegaly, low platelets (30,000), positive tourniquet test

• Dengue Hemorrhagic Fever (DHF)
• Transmitted by bite of Aedes aegypti mosquito (day-biting)

• Acute phase (day 1-5): NS1 antigen test + RT-PCR
• After day 5: Dengue IgM ELISA
• CBC: Thrombocytopenia, leukopenia, rising hematocrit

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Q: Kyasanur Forest Disease (KFD)

• Incubation: 3-8 days
• Biphasic illness:
  • Phase 1: Abrupt onset of high fever, severe headache, myalgia, bradycardia, conjunctival congestion; hemorrhagic manifestations (epistaxis, hematemesis)
  • Brief remission (7-21 days)
  • Phase 2 (in ~50%): Neurological manifestations (meningoencephalitis - tremors, mental disturbance, vision disturbances)
• Leukopenia, thrombocytopenia, elevated liver enzymes
• Mortality: 3-5%

• RT-PCR (acute phase)
• IgM and IgG ELISA (serology)
• Virus isolation (BSL-4 required)
• CBC: Leukopenia, thrombocytopenia

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Q: Ebola Virus / Ebola Virus Diagnosis

• Filamentous (pleomorphic) virus; can appear as U-shaped, 6-shaped, branching forms
• Non-segmented, negative-sense single-stranded RNA (-ssRNA)
• Enveloped; ~80 nm diameter, variable length

• Direct contact with blood, secretions, organs, body fluids of infected humans or animals
• Nosocomial transmission (healthcare workers without PPE)
• Preparation of bushmeat ("hunting hypothesis")
• Sexual transmission (survivors can shed virus in semen for months)
• NOT airborne under natural conditions

• Infects monocytes, macrophages, dendritic cells → systemic spread
• Massive cytokine storm → multiorgan failure
• Disseminated intravascular coagulation (DIC)
• Endothelial damage → hemorrhage

• Incubation: 2-21 days (average 8-10 days)
• Sudden onset: Fever, severe headache, myalgia, weakness
• Vomiting, diarrhea
• Hemorrhagic phase: Bleeding from multiple sites, petechiae, ecchymoses
• Impaired kidney and liver function
• Mortality: 50-90%

• RT-PCR (gold standard): Detects Ebola RNA; highly sensitive from day 3 of symptoms
• Antigen detection ELISA
• IgM/IgG ELISA (from day 7)
• Electron microscopy: Characteristic filamentous morphology
• ALL specimens must be handled at BSL-4

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Q: Chikungunya Fever

• Incubation: 1-12 days (average 2-4 days)
• Sudden-onset high fever
• Severe, incapacitating joint pain (arthralgia/arthritis) - "Chikungunya" = "that which bends up" in Makonde language
• Joint swelling, morning stiffness
• Maculopapular rash
• Headache, myalgia
• Chronic arthritis can persist for months-years (distinguishes from dengue)
• Hemorrhage is RARE (unlike dengue)

• Acute phase (day 1-5): RT-PCR (most sensitive); Virus isolation
• Day 5 onwards: IgM ELISA (MAC-ELISA); IgM positive for months
• Rapid ICT tests for IgM

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Q: Lassa Fever

• Contact with food/household items contaminated with rat urine/feces
• Eating infected rats (considered a delicacy)
• Person-to-person: Direct contact with blood/body fluids
• Nosocomial transmission

• Incubation: 6-21 days
• Gradual onset: Fever, malaise, headache
• Pharyngitis (exudative), chest pain, vomiting, diarrhea, abdominal pain
• Facial edema
• Hemorrhagic manifestations in severe cases (<20%)
• Deafness (sensorineural - major complication even after recovery)
• High mortality in pregnancy (~87% in 3rd trimester)
• Overall mortality: ~1%; hospitalized: 15-25%

• RT-PCR (gold standard - acute)
• Antigen detection ELISA
• IgM/IgG ELISA
• BSL-4 handling required

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CHAPTER 7 - MALARIA AND BABESIOSIS

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Q: Parasites found in blood (hemoparasites)

1. Plasmodium spp. (malaria) - inside RBCs
2. Babesia spp. (babesiosis) - inside RBCs
3. Trypanosoma spp. (T. brucei, T. cruzi) - in blood/plasma
4. Leishmania donovani - inside monocytes/macrophages
5. Microfilariae (Wuchereria bancrofti, Brugia malayi, Loa loa) - in blood
6. Toxoplasma gondii - in blood (during acute parasitemia)

1. Plasmodium falciparum - malignant tertian malaria (most dangerous)
2. Plasmodium vivax - benign tertian malaria (most common worldwide)
3. Plasmodium ovale - ovale tertian malaria
4. Plasmodium malariae - quartan malaria
5. Plasmodium knowlesi - zoonotic (from macaque monkeys in Southeast Asia)

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Q: Life cycle, pathogenesis, lab diagnosis of P. falciparum

Life Cycle:

1. Female Anopheles mosquito takes blood meal from infected human → ingests gametocytes
2. In mosquito midgut: Microgametes and macrogametes fuse → zygote → ookinete → oocyst
3. Oocyst ruptures → releases sporozoites
4. Sporozoites migrate to salivary glands

1. Mosquito injects sporozoites into human bloodstream
2. Sporozoites travel to liver within 30 minutes
3. Invade hepatocytes → differentiate into trophozoites → schizonts
4. Each schizont releases merozoites (hepatic/exo-erythrocytic merozoites)
5. P. falciparum and P. malariae: NO dormant liver stage (no hypnozoites)
6. P. vivax and P. ovale: Form hypnozoites (dormant liver stage) → responsible for relapses

1. Hepatic merozoites invade RBCs → form ring trophozoite
2. Ring → growing trophozoite → mature trophozoite (ameboid)
3. Schizogony (nuclear division): Schizont forms containing merozoites
4. RBC ruptures → merozoites released → invade new RBCs (cycle repeats)
5. Cycle period: P. falciparum = 48 hours (malignant tertian fever every 48 hours)

• Invades RBCs of ALL ages (young and old)
• Infected RBCs express knobs (PfEMP1) → cytoadherence to endothelium
• Sequestration in deep organs (brain, placenta, lung) → NOT seen in peripheral smear
• Only ring forms and crescentic/banana-shaped gametocytes seen in peripheral blood
• High parasitemia possible (>10%)

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Pathogenesis of P. falciparum:

1. Fever: Merozoite release + pyrogens (GPI anchors, hemozoin) → stimulate cytokine release (IL-1, IL-6, TNF-α)
2. Anemia (multifactorial):
   • Direct lysis of parasitized RBCs
   • Destruction of non-parasitized RBCs (bystander hemolysis)
   • Dyserythropoiesis (bone marrow suppression)
   • Spleen hyperactivity
3. Cytoadherence and sequestration (P. falciparum SPECIFIC):
   • Infected RBCs express PfEMP1 (P. falciparum Erythrocyte Membrane Protein 1) on surface knobs
   • PfEMP1 binds ICAM-1 (cerebral vessels), CD36, CSA (placenta)
   • Leads to sequestration in deep capillaries → microvascular obstruction → organ damage
4. Cerebral malaria:
   • Sequestration in cerebral vessels → impaired blood flow
   • Cytokine-mediated BBB disruption
   • Coma, seizures
5. Placental malaria:
   • PfEMP1 binds CSA (chondroitin sulfate A) in placenta → low birth weight, maternal anemia
6. Blackwater fever: Massive intravascular hemolysis → hemoglobinemia, hemoglobinuria (dark urine) → acute tubular necrosis, renal failure

Complications of Falciparum Malaria:

1. Cerebral malaria (most dangerous): Coma, seizures, mortality >20%
2. Blackwater fever: Hemolytic crisis → hemoglobinuria (black/dark urine)
3. Severe anemia (<5 g/dL Hb)
4. Acute renal failure
5. ARDS (pulmonary edema)
6. Hypoglycemia (especially in pregnant women on quinine)
7. Thrombocytopenia
8. Algid malaria (septic shock-like picture)
9. Hyperpyrexia (>41°C)
10. Splenic rupture

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Life Cycle of P. vivax

• P. vivax invades only young RBCs (reticulocytes)
• Schüffner's dots visible in infected RBCs (not in P. falciparum)
• Infected RBCs are enlarged
• Forms hypnozoites in liver → responsible for relapses (not recrudescence)
• Fever cycle: 48 hours (benign tertian)
• Gametocytes are round (not crescent-shaped)
• Less severe disease; cerebral malaria and blackwater fever are rare

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Laboratory Diagnosis of Malaria:

• Thick smear: Screening; more sensitive; concentrates parasites; cells lysed
• Thin smear: Species identification; morphological features better preserved; stained with Giemsa or Leishman stain

• Immunochromatographic tests (ICT)
• Detect parasite antigens:
  • HRP-2 (Histidine-Rich Protein 2): P. falciparum specific
  • pLDH (Parasite Lactate Dehydrogenase): All species
  • Aldolase: Pan-malarial
• Results in 15-20 minutes; no microscope needed
• Cannot determine parasitemia level

• Blood tube with acridine orange (fluorescent dye) + hematocrit centrifugation
• Parasites fluoresce bright orange/green
• More sensitive than thick smear
• Cannot easily identify species

• Most sensitive; species identification, drug resistance detection
• Not routine; used for research/reference labs

• Detects antibodies; not useful for acute diagnosis (past exposure)
• Used for epidemiological surveys, blood donor screening

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Q: Case - 23-year-old male, endemic area, intermittent fever with chills/rigors, intense sweating, splenomegaly, parasites inside RBCs on peripheral smear

• Causative agent: Plasmodium vivax (most likely given "endemic area" + typical tertian pattern)
• Vector: Female Anopheles mosquito (night-biting)

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CHAPTER 8 - VISCERAL LEISHMANIASIS AND TRYPANOSOMIASIS

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Q: Case - 31-year-old man from Bihar, splenomegaly, anemia, fever, bone marrow aspirate showing amastigotes in macrophages

• Parasite: Leishmania donovani
• Clinical diagnosis: Kala-azar (Visceral Leishmaniasis)

1. Infected sandfly (Phlebotomus argentipes in India) bites human → injects promastigotes
2. Promastigotes engulfed by macrophages in skin/RES
3. Within macrophages, promastigotes transform into amastigotes (Leishman-Donovan bodies/LD bodies)
4. Amastigotes multiply intracellularly by binary fission
5. Macrophage ruptures → amastigotes infect new macrophages, spread to spleen, liver, bone marrow

1. Sandfly bites infected human → ingests amastigotes with blood meal
2. In sandfly midgut: Amastigotes → promastigotes (flagellated, extracellular)
3. Promastigotes multiply, migrate to proboscis
4. Ready for injection into next host during blood meal

• Bone marrow aspirate (BMA): Most common clinical specimen; sensitivity 70-90%
• Splenic aspirate: Most sensitive (95-99%) but risk of hemorrhage (done only by specialists)
• Lymph node aspirate
• Liver biopsy
• Peripheral blood (rarely positive)
• Stain: Giemsa or Leishman stain
• LD bodies (amastigotes): 2-3 µm oval organisms inside macrophages; nucleus + kinetoplast (rod-shaped) visible (two distinct dots - "double dot" appearance)

• NNN (Novy-MacNeal-Nicolle) medium - biphasic medium (agar base overlaid with Locke's/Defibrinated rabbit blood saline)
• Aspirate inoculated → promastigotes appear (motile flagellates)
• Incubated at 22-25°C

• Hamster is the most susceptible experimental animal
• Intradermal/intracardiac inoculation → develop kala-azar features

• DAT (Direct Agglutination Test): Detects IgG antibodies against whole promastigotes; titre ≥1:3200 diagnostic; used in field settings
• rK39 ICT rapid test (rK39 strip test): Most commonly used; detects IgG against rK39 antigen (conserved kinesin-related protein); sensitivity 90-100%; highly specific; results in minutes
• ELISA: Using soluble Leishmania antigen or rK39
• CFT (Complement Fixation Test): Classical Chopra's antimony test, largely replaced
• Leishmania formol-gel test (aldehyde test): Non-specific; 1 drop of 40% formaldehyde added to patient serum → coagulation/gelation in positive case; highly non-specific, historical interest only

• PCR on blood, bone marrow, or buffy coat
• More sensitive than microscopy, especially for immunocompromised patients

• First-line: Miltefosine (oral, 28 days) - first oral drug for kala-azar; used in India for VL
• Second-line/severe cases: Liposomal Amphotericin B (IV, 3-5 mg/kg/day for 3-5 doses) - highly effective, low toxicity
• Amphotericin B deoxycholate (conventional): Effective but more toxic
• Sodium stibogluconate (SSG/Pentostam): First-line in Africa and other regions; resistance in India limits use
• Miltefosine + Amphotericin B: Combination therapy in drug-resistant cases

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Q: LD bodies (Draw and label)

• Small (2-3 µm), oval/round organisms
• Found inside macrophages (intracellular)
• On Giemsa stain:
  • Nucleus: Large, round, stains red-purple
  • Kinetoplast: Small, rod-shaped, stains deeply purple; located adjacent to nucleus - gives characteristic "double dot" or "shepherd's crook" appearance
  • Cytoplasm: pale blue/lavender
• Groups of LD bodies pack the cytoplasm of macrophages

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Q: Post-Kala-Azar Dermal Leishmaniasis (PKDL)

• India: 5-10% of kala-azar patients develop PKDL, typically 6 months to 3 years after apparent cure of VL
• Sudan: 50% of kala-azar patients; occurs during or shortly after treatment

• Starts as hypopigmented or erythematous macules on the face (especially around nose and mouth)
• Progresses to papules, nodules (resembling lepromatous leprosy)
• Skin lesions contain L. donovani amastigotes
• Patients are NOT systemically ill (no fever, no splenomegaly)

• PKDL patients act as a reservoir of infection → continue transmission of kala-azar
• Important source during inter-epidemic periods

• Skin slit-smear for LD bodies
• Skin biopsy → Giemsa stain
• PCR on skin tissue
• rK39 serology

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Q: Chagas Disease

• Bug bites → defecates on skin while feeding → feces with trypomastigotes are rubbed into bite wound or conjunctiva by scratching
• NOT through the bite itself

• Amastigotes in host cells (heart, skeletal muscle) → trypomastigotes in blood → ingested by bug → multiply → metacyclic trypomastigotes in bug hindgut

• Acute Chagas disease: Romaña's sign (painless unilateral periorbital edema when entry is conjunctiva), chagoma (skin lesion), fever, lymphadenopathy
• Chronic Chagas disease (years later):
  • Chagas cardiomyopathy (most serious): Dilated cardiomyopathy, arrhythmias, sudden death
  • Chagasic megacolon and megaesophagus (destruction of enteric neurons)

• Acute: Wet blood film → motile trypomastigotes; Blood smear (Giemsa) → trypomastigotes with C-shape/U-shape
• Xenodiagnosis: Uninfected lab-reared bugs fed on patient → bugs examined for trypanosomes
• Chronic: Serology (ELISA, IFA, indirect hemagglutination)

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Q: Classify Leishmaniasis

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Q: NNN Medium

• Solid base: Agar with 2N NaCl (saline agar)
• Liquid overlay: Defibrinated rabbit blood (or blood saline - Locke's solution) added after autoclaving and cooling the agar

• Aspirate (bone marrow/spleen) inoculated into the liquid phase
• Incubated at 22-24°C (optimal for promastigote stage)
• Examined weekly for motile promastigotes (flagellated forms)

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Q: Serological Tests for Kala-Azar

1. rK39 ICT (Rapid test): Most widely used; strip test detecting IgG to rK39; sensitivity/specificity >90%; point-of-care
2. DAT (Direct Agglutination Test): Titre ≥1:3200; sensitive, field-friendly but requires cold chain
3. ELISA: Using rK39, rKE16 or whole antigen; quantitative
4. Formol-gel (aldehyde) test: Historical; non-specific; coagulation of serum with formaldehyde - replaced by specific tests
5. CFT (Complement Fixation Test): Chopra's reaction - historical interest

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CHAPTER 9 - LYMPHATIC FILARIASIS

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Q: Classify filariasis. Pathogenesis and lab diagnosis of lymphatic filariasis.

Classification of Filariasis:

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Wuchereria bancrofti - Pathogenesis and Lab Diagnosis:

1. Mosquito bites → deposits L3 (infective/filariform larvae) on skin → larvae enter through puncture wound
2. L3 → L4 → adult worms in lymphatic vessels/lymph nodes (mature in ~12 months)
3. Adult worms (male + female): Male 4 cm, female 8-10 cm
4. Female produces microfilariae (L1 larvae)
5. Microfilariae enter bloodstream - nocturnal periodicity (in peripheral blood maximally between 10 PM - 2 AM in Indian strain)
6. Mosquito ingests microfilariae during night blood meal → develop to L3 in flight muscles → migrate to proboscis

Pathogenesis:

• Living worms in lymphatics → lymphangitis (retrograde - from distal to proximal; characteristic of filariasis; unlike bacterial lymphangitis which is proximal to distal)
• Lymphadenitis
• Fever, scrotal pain, epididymo-orchitis

• Lymphatic obstruction → dilatation of lymphatics → lymphedema
• Chronic lymphedema of legs → elephantiasis (thickened, fibrotic skin)
• Lymphedema of scrotum → hydrocele (most common manifestation in males)
• Lymphedema of breast, arm
• Chyluria: Rupture of dilated lymphatics into urinary tract → milky white urine (lymph + urine)
• Tropical Pulmonary Eosinophilia (TPE): Microfilariae trapped in lungs → eosinophilic response; cough, wheeze, high eosinophilia

Laboratory Diagnosis:

• Nocturnal blood collection (10 PM - 2 AM) - essential due to nocturnal periodicity
• Thick blood smear: Most commonly used; Giemsa/Leishman stain; look for sheathed microfilariae
• Knott's concentration technique: 1 mL blood + 9 mL of 2% formalin → centrifuge → examine sediment; increases sensitivity 6-7 fold
• Membrane filtration (Millipore filter): Most sensitive

• Administered during daytime when MF are not in peripheral blood
• DEC 50-100 mg orally → microfilariae appear in peripheral blood within 30-60 minutes (provocation)
• Daytime blood sample collected 20-60 minutes after DEC
• Used for: Detecting occult infections, confirming periodicity in atypical cases

• Og4C3 ELISA / ICT (Immunochromatographic card test): Detects circulating filarial antigen (CFA) of W. bancrofti
• ICT filariasis test: Positive even in daytime (not affected by periodicity)
• Very sensitive and specific; no need for night blood collection

• Ultrasonography (scrotal USG): "Filarial dance sign" - live adult worms visible as moving hyperechoic spots in dilated lymphatics

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Q: Microfilaria

• Microfilaria = L1 larval stage produced by adult female filarial worms
• Circulate in peripheral blood (for W. bancrofti, B. malayi) or in skin (Onchocerca)
• W. bancrofti microfilaria:
  • Sheathed (covered in egg membrane)
  • Nocturnal periodic (maximal 10PM-2AM)
  • No nuclei in tail tip (useful for species ID)
  • 244-296 µm long
• B. malayi microfilaria:
  • Sheathed
  • Has nuclei in tail tip (two discrete subterminal nuclei)
  • Nocturnal periodic

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Q: Chyluria

• Adult worms cause lymphatic obstruction → lymphatics dilate and rupture into renal pelvis/calyx or urinary tract
• Lymph (chyle) enters urine → milky white urine

• Urinalysis: Milky/turbid urine
• Sudan III/IV staining of urine: Stains fat droplets
• Urine becomes clear on adding ether (fat dissolves)
• Microfilariae may be seen in urine centrifuge sediment
• Lymphangiography

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Q: DEC (Hetrazan) Provocation Test

1. Patient is daytime (9 AM-5 PM)
2. Baseline daytime blood smear taken (usually negative)
3. DEC 50-100 mg given orally (or 2-6 mg/kg)
4. Blood collected after 20-60 minutes
5. Thick blood smear prepared and examined for microfilariae

1. Diagnosis of occult filariasis (microfilaremia too low for routine detection)
2. Diagnosing filariasis when only daytime blood samples are possible
3. Confirmation of nocturnal periodicity

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CHAPTER 10 - SYSTEMIC CANDIDIASIS AND SYSTEMIC MYCOSES

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Q: Candida albicans / Candidiasis - morphology, pathogenicity, lab diagnosis

Morphology:

• Dimorphic fungus: Exists as yeast AND hyphae/pseudohyphae
• Yeast form: Oval, thin-walled, 3-6 µm; reproduce by budding (blastoconidia/blastospores)
• Pseudohyphae: Elongated yeast cells that remain attached, forming chains with constrictions at septa (not true hyphae)
• True hyphae: Also produced (especially in tissue invasion)
• Chlamydoconidia (chlamydospores): Large, thick-walled, round spores at terminal or intercalary positions in pseudohyphae; unique to C. albicans; formed in nutrient-deficient conditions at 25°C; seen on corn meal agar with Tween 80

Pathogenicity (Infections caused):

1. Oral candidiasis (Thrush): White curd-like plaques on oral mucosa; bleeds on scraping; common in immunocompromised, neonates, elderly
2. Vulvovaginal candidiasis: Thick white "cottage-cheese" discharge; itching; common in diabetics, pregnant, antibiotics
3. Cutaneous candidiasis: Intertrigo (in skin folds), diaper rash, paronychia
4. Esophageal candidiasis: AIDS-defining illness; painful swallowing (odynophagia)

1. Candidemia (bloodstream infection): Most common life-threatening; intravascular catheters major risk
2. Hepatosplenic candidiasis: In neutropenic patients
3. Renal candidiasis: Hematogenous
4. Candida endocarditis: Especially in IV drug users, prosthetic valves
5. Candida meningitis
6. Ophthalmic candidiasis: Endophthalmitis

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Laboratory Diagnosis:

• Specimen: Swab, scrapings, sputum, CSF, blood, BAL
• KOH mount (10-20% KOH): Reveals yeast cells, pseudohyphae, budding - rapid, easy
• Gram stain: Candida stains gram-positive (yeast forms appear as oval budding cells)
• Calcofluor white stain (fluorescence): Highly sensitive for fungal elements

• Sabouraud Dextrose Agar (SDA): Growth at 37°C (and 25°C) within 24-48 hours
• Cream-colored, smooth, pasty colonies (characteristic smell - yeasty)
• Blood culture (automated - BacT/ALERT): For candidemia

• Most important presumptive identification test for C. albicans
• Principle: C. albicans produces germ tubes (short filamentous extensions from yeast cells WITHOUT a constriction at the point of origin) when incubated in human/horse serum at 37°C for 2-3 hours
• Procedure:
  • Yeast colony suspended in 0.5 mL human serum
  • Incubated at 37°C for 2-3 hours
  • Wet mount examined under microscope
• Positive: Germ tubes (slender, cylindrical filaments about 3× length of yeast cell, no constriction at base)
• Positive species: C. albicans (and C. dublinensis)
• Negative: Other Candida species (C. tropicalis, C. parapsilosis, etc.)

• On Corn meal agar with Tween 80 at 25°C after 48-72 hours
• C. albicans produces chlamydospores (large, round, thick-walled terminal conidia)
• Unique to C. albicans; used for definitive identification

• Differential chromogenic medium
• C. albicans → green colonies
• C. tropicalis → dark blue/metallic blue
• C. krusei → purple with white edge
• Used for rapid speciation

• Beta-D-Glucan (1,3-β-D-glucan) assay: Detects pan-fungal cell wall antigen; elevated in invasive candidiasis; sensitive but not Candida-specific
• Mannan antigen and anti-mannan antibody (ELISA): For invasive candidiasis

• PCR from blood; highly sensitive

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Q: Histoplasmosis / Histoplasma capsulatum - lab diagnosis

• Mold form (25°C): White fluffy colonies; produces macroconidia (thick-walled, tuberculate - "knobby") and microconidia
• Yeast form (37°C, in tissue): Small oval yeast cells (2-4 µm), found inside macrophages (similar to LD bodies but slightly larger and no kinetoplast)

1. Asymptomatic
2. Acute pulmonary histoplasmosis (flu-like)
3. Chronic pulmonary histoplasmosis (mimics TB)
4. Disseminated/progressive histoplasmosis (in immunocompromised/HIV)

• Bone marrow, BAL, blood, lymph node biopsy
• Wright/Giemsa stain: Small oval yeast cells inside macrophages
• GMS (Gomori Methenamine Silver) stain: Stains fungal cell walls black

• SDA at 25°C (mold) and 37°C (yeast)
• Mold: White → brown colonies; tuberculate macroconidia (pathognomonic)
• BSL-3 organism - biohazard precautions required
• Growth takes 2-6 weeks

• Complement Fixation Test (CFT): Anti-histoplasmin and anti-yeast antibodies; titre ≥1:32 significant
• Immunodiffusion (ID): Detects H and M precipitin bands; H band = active infection
• ELISA

• Histoplasma urinary antigen (MVista® ELISA): Most sensitive test; detects polysaccharide antigen in urine/serum; best for disseminated disease; monitor treatment response
• Sensitivity >90% in disseminated histoplasmosis

• PAS or GMS stain on tissue sections
• Oval yeast within macrophages

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Q: Coccidioidomycosis

• Mold form (environmental/25°C): Produces arthroconidia (barrel-shaped; barrel and ghost cells alternate)
• Spherule (37°C in tissue): Large, thick-walled spherule (20-60 µm) filled with endospores (2-5 µm) - pathognomonic

1. Primary pulmonary coccidioidomycosis (60% asymptomatic; 40% flu-like = "Valley fever")
2. Disseminated coccidioidomycosis: Skin, bone, meninges; most serious; more common in dark-skinned individuals, immunocompromised

• Sputum, BAL, pus, CSF
• 10% KOH mount or calcofluor white: Reveals large spherules containing endospores - pathognomonic
• Papanicolaou (PAP) stain or H&E on tissue

• SDA at 25°C → white cottony colonies (arthroconidia)
• EXTREME BIOHAZARD - BSL-3: Arthroconidia are easily aerosolized and highly infectious
• Growth in 1-2 weeks

• IgM by immunodiffusion (ID) or tube precipitation: Positive early (1st 3 weeks); indicates primary/early infection
• IgG by CF (Complement Fixation Test): Indicates chronicity/dissemination; titre ≥1:32 suggests disseminated disease; used to monitor treatment
• Latex agglutination (LA): Rapid screening for IgM

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Q: Germ Tube Test (Reynolds-Braude Phenomenon) - Describe

• Human serum or horse serum (0.5 mL)
• Candida colony
• Microscope

1. A small inoculum (1-2 colonies) of Candida is emulsified in 0.5 mL of human serum in a test tube
2. Incubated at 37°C for 2-3 hours (not more than 3 hours - pseudohyphae formation can cause false positive)
3. A drop of suspension placed on glass slide with coverslip
4. Examined under light microscope (×400)

• Germ tube = slender, elongated tube-like extension from yeast cell
• No constriction at origin (this differentiates from pseudohyphae which have constrictions)
• Length ~3× the diameter of yeast cell
• Represents early hyphal formation

• No germ tube; yeast cells remain as oval buds

• Rapid and cost-effective presumptive identification of C. albicans
• 97% sensitivity for C. albicans
• If positive, further tests (chlamydospore production, CHROMAGAR) confirm identification

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1. Use headings and numbered/bulleted lists
2. Write the bold key terms (organisms, test names, media names)
3. For long essays (HIV, malaria, typhoid), follow the standard format: Definition → Causative agent → Pathogenesis → Clinical features → Lab diagnosis → Treatment
4. For case-based questions (a, b, c, d), answer each sub-part clearly

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MICRO GIT TB 2 - Complete Exam-Ready Answers (Chapters 1-7)

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CHAPTER 5: VIRAL GASTROENTERITIS

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Q. Write a short note on Rotavirus Gastroenteritis / Describe Rotavirus in detail.

• Family: Reoviridae (the only virus family with ds RNA)
• Measures 70-75 nm, icosahedral symmetry
• Triple-layered, wheel-shaped capsid (Latin: rota = wheel)
• Contains 11 segments of ds RNA
• 6 structural proteins (VP1-VP6, except VP5) and 6 non-structural proteins (NSP)
• VP6 = group-specific antigen (determines group A-G)
• VP7 = outer capsid, G-type antigen (serotyping) - induces neutralizing antibodies
• VP4 = spikes on outer capsid, P-type antigen (genotyping) - also induces neutralizing antibodies

• Group A-G based on VP6
• G-types (glycoprotein VP7): G1, G2, G3, G4, G9 etc.
• P-types (protease-sensitive VP4): P[4], P[8] etc.
• Most common worldwide and in India: G1P[8] type (~74% of all isolates)

• Single most important cause of gastroenteritis in young children worldwide
• 1-3 billion diarrheal episodes annually; ~500,000 deaths/year (mainly sub-Saharan Africa, South Asia)
• In India: ~100,000 hospitalizations, ~500,000 OPD visits, ~98,000 deaths annually
• Temperate climate: Group A causes outbreaks in cooler months
• Developing countries (e.g., India): occurs at younger age, less seasonal, more diverse serotypes

• Transmitted by fecal-oral route
• Infects and destroys enterocytes of the small intestine (gastric and colonic mucosa spared)
• Replicates in the cytoplasm of enterocytes → damages transport mechanisms → secretory diarrhea
• NSP4 acts as an enterotoxin; alters epithelial cell function and permeability

• Incubation: 1-2 days; abrupt onset
• Vomiting followed by watery diarrhea, fever, abdominal pain
• Recovery in majority; severe dehydration can occur in young children
• Adults: usually asymptomatic but show seroconversion
• Group B rotavirus: large outbreaks of severe gastroenteritis in adults (China)

1. Direct detection - feces collected early in illness

• Immunoelectron microscopy (IEM): characteristic triple-shelled, wheel-like capsid
• Isolation: difficult; roller culture may enhance replication

1. Antigen detection in stool - ELISA and latex agglutination methods
2. RT-PCR: most sensitive detection method (stool)
3. Typing: G serotypes (VP7) by neutralization; P genotypes (VP4) by RNA sequence typing
4. Serology (ELISA) for antibody rise - used for seroprevalence only

MCQ tip: Rotavac is given orally (NOT intramuscular). No vaccine-induced intussusception has been reported with Rotavac.

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Q. List the viral agents causing gastroenteritis. Describe their pathogenesis and clinical manifestations.

• Calicivirus, 27-35 nm, icosahedral, cup-like depressions on capsid surface (calyx = cup), ss RNA, single major structural protein
• NOT cultivable in cell culture
• Symptoms begin 12-48 hours after exposure: diarrhea, nausea, vomiting, abdominal cramps
• Common food sources: contaminated salad, fresh fruits, shellfish (oysters), water
• Lab diagnosis: EM, RT-PCR (most sensitive), antigen detection (similar to rotavirus)

Key MCQ answers: Most common cause of viral GE = Rotavirus (children); most important cause of epidemic viral GE in adults = Norovirus; NSP4 (rotavirus enterotoxin) acts as the responsible enterotoxin in 6-month-old child with diarrhea; organism causing diarrhea associated with shellfish ingestion = Norovirus; Rotavirus is wheel-shaped.

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CHAPTER 6: INTESTINAL PROTOZOA

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Q. Describe Entamoeba histolytica infection in detail (Pathogenesis, Clinical Features, Lab Diagnosis, Treatment).

• E. histolytica is worldwide; more common in tropics (China, Central/South America, India)
• India: prevalence ~3-10%; 3rd most common parasitic cause of death after malaria and schistosomiasis
• Three morphologically similar, non-pathogenic species: E. dispar, E. moshkovskii, E. bangladeshi

• Single host: man
• Infective form: mature quadrinucleate cyst
• Mode of transmission: fecal-oral (contaminated food/water)
• Excystation in small intestine → 4 trophozoites released
• Trophozoites reach ileocecal region → multiply, colonize → either asymptomatic cyst passers OR amoebic dysentery OR invasive amoebiasis (liver abscess via portal vein)
• Encystation occurs in large intestine only; trophozoites disintegrate in environment but cysts survive

1. Amoebic lectin antigen (Gal/GalNAc lectin): 170 kDa surface protein - main virulence factor for adhesion to intestinal mucosa
2. Amoebapore: forms pores in host cell membranes
3. Cysteine proteases and hydrolytic enzymes: facilitate invasion into GI mucosa

1. Colonization of large intestine
2. Adhesion via amoebic lectin
3. Formation of characteristic flask-shaped ulcers (broad base, narrow neck) - most common site: cecum
4. Invasion → extraintestinal spread via portal vein → most common extraintestinal site: liver (amoebic liver abscess)

• Incubation: 1-4 weeks
• Majority (90%): asymptomatic cyst passers
• ~10% develop intestinal amoebiasis:
• Amoebic dysentery: bloody diarrhea (up to 10 times/day) with mucus and pus, colicky abdominal pain, fever
• Intestinal complications (rare): fulminant amoebic colitis, appendicitis, perforation, toxic megacolon, amoeboma (pseudotumor-like mass in rectosigmoid)

• Direct examination (saline + iodine mount)
• Trophozoites (gold standard for active infection): 20-30 µm, actively motile with finger-like pseudopodia, single nucleus. Presence of ingested RBCs differentiates E. histolytica from E. dispar. Saline mount is better than iodine mount (iodine kills trophozoites). Do NOT report on iodine mount alone.
• Quadrinucleate cysts (diagnostic + infective form): Round, 10-20 µm, 4 nuclei (also 1- or 2-nuclei immature forms). Internal structure appreciated better in iodine mount. Report as "cyst/trophozoite of E. histolytica/E. dispar/E. moshkovskii/E. bangladeshi" (as they are morphologically indistinguishable)
• Stool concentration by formalin-ether sedimentation improves sensitivity
• Permanent staining: trichrome stain or haematoxylin stain (Fig B - trichrome stained trophozoite)

• ELISA detecting 170 kDa lectin antigen: 85% sensitivity, 95% specificity; differentiates E. histolytica (lectin Ag-positive) from E. dispar (lectin Ag-negative)
• ICT (e.g., Triage Parasite Panel): detects 3 antigens simultaneously (Giardia, E. histolytica/dispar, Cryptosporidium parvum) in 15 min; 95% sensitivity and specificity

• Amoebic antigen ELISA (170 kDa lectin Ag in serum): indicates active infection only; disappears after cure
• Amoebic antibody ELISA (IgG vs lectin Ag): antibodies appear late in intestinal amoebiasis, so less useful; used for seroepidemiological studies
• Isoenzyme (zymodeme) analysis: differentiates E. histolytica from E. dispar by distinct zymodeme patterns on electrophoresis; technically demanding

• Nested multiplex PCR targeting 18S rRNA gene: 95% sensitivity, 98-100% specificity; differentiates E. histolytica from E. dispar
• Real-time PCR (18S rRNA gene): more sensitive, quantitates parasite load
• BioFire FilmArray GI Panel: fully automated PCR system detecting Cryptosporidium, Cyclospora, E. histolytica, Giardia, and other diarrheal pathogens

• Colonoscopy: detects flask-shaped amoebic ulcers
• Charcot-Leyden crystals in stool (diamond-shaped, eosinophilic breakdown products)
• Moderate leukocytosis in blood

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Q. Describe Giardia lamblia (Giardiasis) - Life Cycle, Pathogenesis, Lab Diagnosis.

• Also known as G. intestinalis or G. duodenalis
• Named after Dr F. Lambl and Professor A. Giard
• One of the most common parasites causing both endemic and epidemic intestinal disease
• More common in warm climates; prevalence in Indian children: 5-15%

• Pear-shaped (front view) / spoon/sickle-shaped (lateral view) - also described as "tennis racket" or "tear drop" shape
• Size: 10-20 µm length, 5-15 µm width
• "Falling leaf" motility (bilaterally symmetrical, flagellated)
• Bears: 1 pair nuclei, bilobed adhesive ventral disk (for attachment), 4 pairs of flagella, pair of axonemes

• Oval, 8-12 µm length, 7-10 µm width
• Contains 4 nuclei and axonemes
• Cannot differentiate active disease from carrier

• Single host: man
• Infective form: tetranucleated cyst (as few as 10-25 cysts can initiate infection)
• Mode of transmission: fecal-oral (contaminated food/water)
• Excystation in duodenum → trophozoites → multiply by longitudinal binary fission in duodenum
• Trophozoites adhere to duodenal mucosa by adhesive disk
• In active disease, trophozoites are excreted directly in diarrhoeic stool
• Encystation in jejunum → nuclei divide to become 4 → cysts in feces → survive in environment → infective to man

• Trophozoites adhere to duodenal mucosa → disruption of intestinal epithelium → increased permeability → malabsorption:
• Malabsorption of fat (steatorrhoea)
• Disaccharidase deficiencies → lactose intolerance
• Malabsorption of vitamins A, B12, iron
• Protein-losing enteropathy
• Antigenic variation: Giardia undergoes frequent antigenic variation via variant surface protein (VSP) - a cysteine-rich surface protein - helps evade host immune system → persistent/recurrent infection

• Most infected: asymptomatic carriers
• Incubation: 1-2 weeks
• Fatty diarrhoea (steatorrhoea): foul-smelling with foul flatus, mixed with fat/cellular exudate/mucus, NO blood - key feature
• Abdominal pain, bloating, belching, flatulence, vomiting
• Profound weight loss → growth retardation
• Extraintestinal (rare): urticaria, arthritis, anterior uveitis, "salt and pepper" retinal changes
• NO pus cells or RBCs in stool microscopy in giardiasis

• Minimum 3 samples, alternate days (intermittent shedding)
• Trophozoites: better in saline mount and permanent staining (not iodine - iodine kills them)
• Cysts: better in iodine mount than saline mount
• Concentration techniques: zinc sulphate flotation or formalin-ether sedimentation
• Sensitivity: 50-70% with one stool; >90% after three stools

• If stool negative → Enterotest (String test): gelatin capsule attached to thread, swallowed → thread enters duodenum → 4 hours later, thread withdrawn, shaken in saline to release trophozoites → examined by wet mount or permanent stain
• Also useful for: Strongyloides, Cryptosporidium, Clonorchis

• ELISA and direct fluorescent antibody (DFA) tests - detect cyst wall protein antigens; 85-98% sensitivity and 90-100% specificity
• Rapid ICT (e.g., Triage Parasite Panel): simultaneous detection of Giardia, E. histolytica/dispar, and Cryptosporidium

• IFA and ELISA - cannot differentiate recent from past infection; used for epidemiological purposes only

• Tinidazole: 2 g single dose (per oral) = drug of choice (DOC)
• Metronidazole (7-10 days) or Nitazoxanide (3 days) = alternatives

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Q. Write short notes on Opportunistic Intestinal Coccidian Parasitic Infections (Cryptosporidiosis, Cyclosporiasis, Cystoisosporosis).

• Highly refractile, double-walled, acid-fast in nature
• Resistant to routine chlorination, heat, and other disinfectants

• Infective: sporulated oocysts (thick-walled = infective to others; thin-walled = autoinfection)
• Man ingests thick-walled oocysts → excystation → 4 sporozoites → invade brush border epithelium of small intestine (lie inside parasitophorous vacuole near microvilli surface) → trophozoites → schizonts → merozoites → gametocytes → gametes → fertilization → zygote → unsporulated oocysts → sporulation → sporulated oocysts
• Thin-walled oocysts cause autoinfection → responsible for chronic persistent diarrhoea
• Key difference from Cyclospora/Cystoisospora: Cryptosporidium sporulates INSIDE the host enterocytes; the other two sporulate in the environment

• Immunocompetent: mostly asymptomatic; some develop self-limiting diarrhea, anorexia, nausea, abdominal pain, fever; can cause 3-15% of traveller's diarrhoea
• Immunocompromised (AIDS): chronic, persistent, profuse diarrhoea (5-10 L/day), significant fluid/electrolyte loss and weight loss ("cholera-like")
• Extraintestinal: biliary tract infection, sclerosing cholangitis, respiratory, pancreatic
• More severe when CD4 T-cell count <50/µL
• More severe in Cryptosporidium than Cyclospora/Cystoisospora (due to autoinfection)

1. Direct microscopy - stool examination:

• 3 stool samples collected
• Methods: direct wet mount, acid-fast staining, direct fluorescent antibody (DFA), epifluorescence microscopy (autofluorescence of Cyclospora/Cystoisospora), phase contrast microscopy
• Concentration: Sheather's sugar flotation or saturated salt flotation; or formalin-ether sedimentation

1. Antigen detection (Cryptosporidiosis only):

• ELISA for C. parvum-specific coproantigen (oocyst antigen)
• ICT (e.g., Triage Parasite Panel) - simultaneous detection of Cryptosporidium, Giardia, E. histolytica

1. Antibody detection: ELISA for antibodies against oocyst antigens - used for epidemiological purposes, not routine
2. Molecular diagnosis: PCR or real-time PCR targeting 18S rRNA or small-subunit rRNA; can differentiate Cryptosporidium, Cyclospora, and Cystoisospora; also BioFire FilmArray GI Panel
3. Histopathology: Small intestinal biopsy - demonstrates various developmental stages

• Mild cases: self-limiting, ORS
• Cryptosporidiosis: Nitazoxanide (adults); NOT effective in HIV-infected; Paromomycin as alternative
• Cyclosporiasis and Cystoisosporosis: Cotrimoxazole; HIV patients may need long-term suppressive therapy

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Q. Write a short note on Balantidiasis.

• Largest protozoan and the only ciliated parasite of humans
• Produces intestinal disease similar to amoebic dysentery but less severe
• Habitat: large intestine (similar to E. histolytica)
• Reservoir/host: Pig is the main reservoir; man is an accidental host

• Two forms: trophozoite (oval, 40-70 µm, covered with cilia - rotatory motility) and cyst (round, 50-70 µm)
• Both forms are binucleated with a large kidney-shaped macronucleus and a small micronucleus

• Infection by ingestion of contaminated food/water containing cysts (infective form)
• Cysts → trophozoites → multiply in large intestine (asexual: binary fission; sexual: conjugation)
• Both trophozoites and cysts excreted; trophozoites disintegrate but cysts are resistant/infective

• Majority: asymptomatic carriers
• Small number: acute dysenteric disease (similar to amoebic dysentery but less severe; extraintestinal involvement very rare)

• Repeated stool examination (intermittent shedding)
• Trophozoite: oval, 40-70 µm, ciliated, found in dysenteric stool, rotatory motility
• Cyst: round, 50-70 µm, surrounded by cyst wall, found in carriers and chronic cases

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Q. Case Study - Intestinal Amoebiasis (Case vignette: 10-year-old boy, bloody diarrhea, mucus, pus cells, trophozoites 20-30 µm, actively motile with finger-like pseudopodia)

• Evidence: 10-year-old (susceptible age), bloody diarrhea with mucus and pus cells, trophozoites 20-30 µm with finger-like pseudopodia (characteristic of E. histolytica)

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Q. Case Study - Giardiasis (vignette: 8-year-old, recurrent foul-smelling diarrhea, foul flatus, sulphurous belching, weight loss, pear-shaped trophozoites with falling leaf motility)

• Evidence: pear-shaped trophozoites with "falling leaf" motility in stool = pathognomonic of Giardia
• "Sulphurous belching" (foul flatus), fatty/foul-smelling diarrhea, weight loss - classic giardiasis presentation

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Q. Case Study - Cryptosporidiosis (vignette: 30-year-old HIV-positive female, profuse diarrhea 10x/day, 3 weeks, weight loss, stool shows round sporulated oocysts 4-6 µm with 4 sporozoites by modified acid-fast stain)

• Round sporulated oocysts 4-6 µm containing 4 sporozoites, uniformly acid-fast = classic Cryptosporidium parvum

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CHAPTER 7: INTESTINAL HELMINTHS

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Q. Describe Intestinal Taeniasis - Taenia saginata vs Taenia solium (Differences, Life Cycle, Lab Diagnosis)

1. Adult worm: Long, segmented, dorsoventrally flattened tapeworm. Comprises scolex (head), neck, and body (strobila) of several proglottids

• Scolex bears 4 cup-like muscular suckers for attachment
• T. solium: scolex has rostellum with 2 rows of hooklets (armed tapeworm)
• T. saginata: NO rostellum, NO hooklets (unarmed tapeworm)

1. Egg: Round, 31-43 µm, contains embryo (oncosphere) with 3 pairs of hooklets (hexacanth embryo), surrounded by radially striated embryophore
2. Larva (Cysticercus): A muscular organ with bladder-like sac

• Cysticercus bovis (in cattle muscles) = larva of T. saginata
• Cysticercus cellulosae (in pig muscles, brain, eye) = larva of T. solium

• 2 hosts: definitive (man) + intermediate (cattle for T. saginata, pig for T. solium)
• Transmission: Man ingests contaminated undercooked beef/pork containing cysticercus
• In human GIT: larvae → adult worms → self-fertilization → eggs released in feces (diagnostic form)
• In cattle/pigs: Eggs ingested → penetrate intestinal wall → migrate via blood to skeletal muscles → transform into cysticercus (infective to man)

• Majority: asymptomatic; patient becomes aware by noticing proglottids in feces
• Common: mild abdominal pain, nausea, loss of appetite, change in bowel habit
• Perianal discomfort or pruritus when proglottids are discharged

1. Stool examination (saline/iodine wet mount): Eggs (morphologically similar in both species) - round, 31-43 µm, embryophore present, bile-stained (brownish), do NOT float in saturated salt solution

• Proglottids can be differentiated (lateral branches of uterus, accessory lobe in ovary, vaginal sphincter etc.)

1. Antigen detection in stool (ELISA): Taenia-specific coproantigen; cannot differentiate between T. saginata and T. solium
2. Molecular methods: PCR targeting mitochondrial DNA + sequencing can distinguish between Taenia species

• Adequate cooking of beef/pork viscera (at 60°C for 5 minutes OR refrigeration at -10°C for 10 days)
• Salting and pickling are NOT effective
• Effective fecal disposal to prevent infection in cattle/pigs

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Q. Write a short note on Hymenolepis nana (Dwarf Tapeworm).

• Smallest cestode infecting man; most common tapeworm infection worldwide (affects ~75 million people)
• Higher prevalence in children

• Man is the only host (single host)
• Infective form: eggs
• Modes: (i) Ingestion of food/water contaminated with eggs; (ii) Autoinfection (eggs released in their small intestine)
• Eggs → larvae (called cysticercoid larva) → adult worms in small intestine → eggs released in feces → cycle continues

• Stool microscopy: characteristic eggs confirm diagnosis
• Egg characteristics:
• Round to oval, 6-8 µm in size
• Non-bile-stained, colorless in saline mount
• Has two membranes surrounding embryo with 6 hooklets
• Polar filaments at both poles of embryophore (distinguishing feature from H. diminuta which lacks polar filaments)
• Eggs = both infective form AND diagnostic form

H. diminuta (rat tapeworm): Definitive host = rodents; intermediate host = insects; human infection rare. Eggs larger (60-80 × 52-60 µm), NO polar filaments, bile-stained

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Q. Describe Ascaris lumbricoides (Ascariasis) - Life Cycle, Pathogenesis, Lab Diagnosis.

• Largest nematode parasitizing the human intestine
• Soil-transmitted helminth (STH); roundworm; common in tropical countries including India
• Adult worm: cylindrical, 15-35 cm long

1. Adult worm (male and female)
2. Larvae (4 stages: L1-L4)
3. Two types of eggs: fertilized eggs and unfertilized eggs

• Single host: man
• Infective form: embryonated eggs containing L2 larvae
• Mode of transmission: Ingestion of embryonated eggs from contaminated soil, food, water

A gravid female can lay up to 200,000 eggs/day

• Migrating larvae in lungs → immune-mediated hypersensitivity reaction → eosinophilic pneumonia
• Non-productive cough, chest discomfort, fever, dyspnoea
• Transient patchy infiltrates on chest X-ray + transient peripheral eosinophilia

• Malnutrition and growth retardation (adult worms rob nutrition)
• Impairment of educational performance, language learning, motor skills in children
• Intestinal complications: obstruction by bolus of entangled worms → perforation, intussusception, volvulus
• Extraintestinal complications: worms enter and occlude biliary tree → cholecystitis, pancreatitis
• Allergic manifestations: fever, urticaria from toxic fluid released by adult worms

1. Stool examination (saline and iodine wet mount): detecting fertilized and unfertilized eggs

• Concentration by formalin-ether sedimentation (flotation method NOT preferred - unfertilized eggs don't float)
• Decorticated eggs: fertilized eggs that have lost their thick mammillated albuminous coat

1. Adult worms: occasionally seen in stool/sputum by naked eye; or on barium meal X-ray of GIT
2. Larvae: in sputum or gastric aspirates during early pulmonary migratory phase
3. Antibody detection (ELISA): sensitive but cannot differentiate recent from past infection
4. Molecular test (PCR): Ascaris-specific genes in stool; multiplex PCR can differentiate Ascaris, Trichuris, and hookworm simultaneously
5. Eosinophilia: prominent in early lung stage; disappears later
6. Charcot-Leyden crystals in sputum and stool (non-specific)

• Albendazole (400 mg once) or Mebendazole (500 mg twice daily for 3 days) = DOC
• In pregnancy: Pyrantel pamoate (safe)

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Q. Describe Hookworm Infection (Ancylostoma duodenale) - Life Cycle, Pathogenesis, Lab Diagnosis.

• Ancylostoma duodenale = Old World hookworm
• Necator americanus = New World/American hookworm

• Adult worm: small (5-13 mm), has a bent anterior end (hence "hookworm")
• L1 = rhabditiform larva; L3 = filariform larva (infective)
• Eggs and first-stage larvae of A. duodenale and N. americanus are morphologically indistinguishable

• Single host: man
• Infective form: L3 (filariform) larva
• Mode of transmission: skin penetration by L3 larva while walking barefoot on damp, contaminated soil

• Adult worms suck blood from intestinal vessels by:
• Attaching and making cuts in the intestinal wall with buccal capsule and teeth
• Secreting hydrolytic enzymes and anticoagulants (maintain continuous oozing from attachment site)
• Blood loss: A. duodenale: 0.1-0.5 mL/worm/day; Necator: 0.03 mL/worm/day

• Cutaneous lesions: (i) "Ground itch" - pruritic maculopapular rashes at site of skin penetration; (ii) serpentine tracks (subcutaneous larval migration - see Chapter on cutaneous larva migrans)
• Mild transient pneumonitis (larvae passing through lungs), asthma, bronchitis

• Most infections: asymptomatic
• Early intestinal phase (light worm load): epigastric pain, inflammatory diarrhoea, eosinophilia
• Late intestinal phase (chronic, heavy worm load): Iron-deficiency anaemia + protein-energy malnutrition (major manifestation)
• Weakness, shortness of breath
• Rarely: impaired intellectual power and behavioural changes

• Characteristic eggs in feces: oval, 56-75 × 35-40 µm, thin egg shell
• Non-bile-stained, colorless in saline mount
• Ovum segmented with 4 blastomeres (segmented)
• Clear space between egg shell and embryo
• Float in saturated salt solution
• A. duodenale and N. americanus eggs morphologically indistinguishable
• Rhabditiform larvae of hookworm occasionally seen in stool specimens

1. Egg counting: Kato-Katz technique to count eggs per gram of stool (estimate disease burden)
2. Stool Culture: Freshly passed stool → eggs hatch → develop into L3 filariform larvae → L3 stage can differentiate A. duodenale from N. americanus

• Harada-Mori filter paper tube method
• Agar plate technique (more sensitive)

1. Molecular Diagnosis: PCR targeting mitochondrial cytochrome oxidase genes; can differentiate Ancylostoma and Necator
2. Other findings:

• Hypochromic microcytic anaemia
• Eosinophilia
• Hypoalbuminaemia

• Albendazole (400 mg once) or Mebendazole (500 mg once) = DOC
• Symptomatic treatment: oral iron + folic acid for iron-deficiency anaemia; nutritional support for malabsorption

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Q. Describe Enterobius vermicularis (Pinworm/Threadworm Infection, Enterobiasis) - Life Cycle, Clinical Features, Lab Diagnosis (NIH Swab).

• Caused by Enterobius vermicularis - also called pinworm or threadworm (adult worm is small, white, thread-like)
• Common parasitic infection in children

• Only host: man; Infective form: embryonated eggs (carrying L3 larvae)

1. Ingestion of eggs from environment (surfaces, clothes, bedlinen) through contaminated fingers
2. Autoinfection: (i) Exogenous - transferring eggs to mouth with hands that have scratched the perianal area; (ii) Endogenous - retrograde migration of larvae hatched from eggs in the perianal skin

• Eggs usually contain fully developed larvae → hatch in cecum → develop into adult worms
• After fertilization, gravid female worms migrate to large intestine (rectum, colon) → start laying eggs on perianal skin at night
• Adult female worms usually lay ~10,000 eggs/day

• Perianal pruritus (most cardinal symptom) - often worse at night due to nocturnal migration of female worm
• Worms found in undergarments/buttock area of infected children
• Repeated scratching → fingers contaminated → autoinfection
• Abdominal pain and weight loss in heavy infections
• Migration of worm (rare): pinworms may invade female genital tract → vulvovaginitis and pelvic/peritoneal granulomas

• Microscopy of perianal skin samples = test of choice - detects characteristic eggs
• Two methods of specimen collection:

• Cellophane tape applied to perianal region → tape mounted with a drop of saline on a clear glass slide

• Consists of a glass rod attached to a cellophane tape by a rubber band
• The cellophane part of the glass rod is rolled over the perianal and perineal skin → sample collected
• Better than cellophane tape as it collects sample more thoroughly

• A series of 4-6 consecutive tapes may be necessary (female worms migrate intermittently)
• Timing: collect late evening or early morning (when chance of egg deposition is more)

• Plano-convex (one side convex, other flat), 50-60 µm long
• Embryonated egg (freshly passed) contains a tadpole-shaped larva inside
• Non-bile-stained, colorless in saline mount
• Float in saturated salt solution

• Mebendazole OR Albendazole OR Pyrantel pamoate
• Same treatment repeated after 2 weeks
• Treatment of household members is advocated (to eliminate asymptomatic reservoirs)

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Q. Describe Trichuris trichiura (Whipworm - Trichuriasis) - Life Cycle, Clinical Features, Lab Diagnosis.

• Caused by Trichuris trichiura (also called whipworm as the adult worm resembles a whip handle)
• Worldwide; warm/moist climates; commonly infects children; soil-transmitted helminth

• Only host: man
• Infective form: embryonated eggs
• Transmission: ingestion of contaminated food/water carrying embryonated eggs

• Eggs hatch in small intestine → L1 larvae → migrate to large intestine, moult twice (L2 → L3) → develop into adult worms → adult female worms get buried in large intestinal mucosa → fertilize → eggs excreted in feces

• Adult female worms buried in large intestinal mucosa → abdominal pain, anorexia etc.
• Trichuris dysentery syndrome: bloody/mucoid diarrhoea resembling inflammatory bowel disease
• Recurrent rectal prolapse (due to heavy worm load in rectum)
• Malnutrition → growth retardation

• Stool microscopy (direct saline/iodine mount or after concentration): characteristic eggs confirm diagnosis
• Egg characteristics:
• Barrel-shaped (barrel-shaped with mucus plugs at both ends)
• Size: 50 × 23 µm
• Bile-stained (yellowish-brown)
• Float in saturated salt solution
• Whip-shaped adult worms (3-5 cm long) occasionally seen on proctoscopy

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Q. Describe Strongyloides stercoralis (Strongyloidiasis) - Life Cycle, Pathogenesis, Lab Diagnosis. What is Larva Currens?

• Caused by Strongyloides stercoralis
• Common in Southeast Asia including India

• Only female worms seen in small intestine of man; male worms present as free-living in environment
• Ovoviviparous (eggs hatch immediately → larvae; no egg stage in diagnosis)

• Single host: man
• Infective form: L3 (filariform) larva
• Mode of transmission: (1) Skin penetration by L3; (2) Autoinfection (internal autoinfection)

• L1 larvae → moult twice → L3 larvae (infective form): Direct cycle (usually in temperate climate)
• Indirect development: L1 → adult worms (male + female) → fertilization → eggs → L1 → L3 (infective): usually in tropical climate

• 30% of chronically infected are asymptomatic

• Cutaneous larva migrans / Larva currens: Migrating larvae → pathognomonic serpentine/urticarial rash, commonly on the thigh, called larva currens (advancing at 2 cm/hour - very fast, distinguishes from hookworm larva migrans)
• Pulmonary symptoms uncommon compared to Ascaris and hookworm

• Mild to moderate worm load: epigastric pain, nausea, diarrhoea, constipation, blood loss
• Heavy larva load: Hyperinfection syndrome and Disseminated strongyloidiasis

1. Stool Microscopy: reveals characteristic rhabditiform larvae (diagnostic form)

• Repeated examinations and concentration techniques to increase yield
• Hookworm eggs may hatch in stool → releasing rhabditiform larvae → must be differentiated from Strongyloides

1. Microscopy of other specimens: In disseminated strongyloidiasis: stool, sputum, other body fluids, and tissue biopsies (contain high numbers of filariform larvae)
2. Enterotest: Duodenal aspirate can be collected and examined for larvae
3. Stool Culture: Harada-Mori filter paper tube method or agar plate technique (more sensitive) - develops rhabditiform larvae into filariform → distinguishes Strongyloides from hookworm
4. Serology: ELISA using crude larval antigens - greater sensitivity but less specific; cannot differentiate recent from past infection
5. Coproantigen detection: Antigen capture ELISA for excretory-secretory antigen in stool; does NOT cross-react with other helminthic infections
6. Molecular diagnosis: Real-time PCR targeting Strongyloides-specific genes in fecal samples

• Ivermectin (DOC - more effective than albendazole) - even in asymptomatic stage (due to risk of fatal hyperinfection)
• Even asymptomatic cases must be treated

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Q. What is Larva Currens?

• It is a serpentine, urticarial rash produced by the migrating filariform larvae in the subcutaneous tissue
• Characteristically seen on the thigh or perianal area
• It advances at a very rapid rate of ~2 cm/hour (much faster than cutaneous larva migrans of hookworm)
• The rapid movement is the key distinguishing feature that differentiates it from hookworm's cutaneous larva migrans (which advances slowly)

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Q. Describe Soil-Transmitted Helminths (STH).

• High-risk groups: school-age children and women of childbearing age
• Presentation: infected children are nutritionally, intellectually, and physically impaired; malabsorption of nutrients, anaemia, diarrhoea, dysentery (depending on type of helminth)

• Periodic prophylactic measure to prevent high-risk people from worm infestation
• Done once or twice a year based on STH infection prevalence
• Single dose albendazole (400 mg) given to all children aged 1-19 years on deworming day

• WHO aims to eliminate morbidity due to STH infections in children by 2030
• Global target: <2% of children having STH infections of moderate and heavy intensity by 2030

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Q. Compare Taenia saginata and Taenia solium (Differences Table) [see above]

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Q. Write a short note on Hymenolepis nana [see above]

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Summary of Key Drugs (Exam Favorite MCQ Topics):

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Key MCQ Answers (Chapters 5-7):

1. Most common viral cause of gastroenteritis = Rotavirus (a)
2. Most important cause of epidemic viral GE in adults = Norwalk virus (b)
3. 6-month child with diarrhea/vomiting - enterotoxin responsible = NSP4 (a)
4. Organism causing diarrhea associated with shellfish ingestion = Norwalk virus (c)
5. Rotavirus vaccine given by = Oral route (c)
6. Shape of Rotavirus = Wheel-shaped (a)

1. Infective form + invasive form of E. histolytica = Quadrinucleate cyst (infective) + Trophozoite (invasive) = (b)
2. Sporulated oocyst of Cystoisospora belli contains = Two sporocysts and 8 sporozoites (2×4) = (d)
3. Sporulated oocyst of Cyclospora cayetanensis contains = Two sporocysts and 4 sporozoites (2×2) = (c)
4. False statement about Cryptosporidium = High infective dose (b) - actually Cryptosporidium has a LOW infective dose
5. Intermediate host for Sarcocystis suihominis = Pig (a)
6. Microsporida spores better stained by = Modified trichrome stain (MTS) (c)
7. Which bears two nuclei named macronucleus and micronucleus = Balantidium coli (a)
8. DOC for cystoisosporosis = Cotrimoxazole (b)
9. HIV patient with profuse diarrhea, autofluorescing round structures under fluorescence microscopy = Cyclospora cayetanensis (c)
10. Triage Parasite Panel detects all EXCEPT = Cystoisospora belli (d)

1. Cestode without rostellum and hooks = Taenia saginata (c)
2. Cestode eggs NOT bile-stained = Hymenolepis nana (a)
3. Larval form of H. nana = Cysticercoid (d)
4. Intestinal nematode that is NOT oviparous = Strongyloides (b) (it is ovoviviparous)
5. Common name of Trichuris trichiura = Whipworm (d)

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