Week 9: Serological and Molecular Detection of Bacterial and Viral infections

Host-microbe relationships

• symbiotic: host and microbes live tgt for a long time, indigenous microbiota

• commensalistic: no benefit or harm to either organism

• Mutualistic: both host and microbes benefit

• parasitic

Infectivity

Organism’s ability to establish an infection

pathogenicity

ability or an organism to cause disease

virulence

extent of pathology caused by an organism when it infects a host

Bacterial virulence factors

• endotoxin

  • lipid A portion of LPS in gram-negative cell walls

  • powerful stimulator of cytokine release

• Pili

  • adherence to host cells; R to phagocytosis

• Flagella: adherence to host cells; motility

• capsule: blocks phagocytosis, antibody attachment, complement

• exotoxins

  • potent toxic proteins released from living bacteria

  • neurotoxins, cytotoxins, enterotoxins

Lab detection of bacterial infections

• Culture of causative agent

• microscopic: Gram stain or special stains

• detection of bacterial antigens: ELISA, LFA, LA

• Molecular detection of bacterial DNA/RNA

• Serology: detection of anitbodies generated against bacteria

Serology advantages

• to detect and confirm infections for which other lab methods not avail

• to diagnose infections clin symptoms nonspecific

• current infection indicated by presence of IgM, high IgG titer, or 4x rise in antibody titer between acute and convalescent samples

• determine past exposure to an organism (IgM-, IgG+)

• assess reactivation or re-exposure

Clinical manifestations of GAS

GAS sequelae

• acute rheumatic fever

• Poststreptococcal glomerulonephritis

lab diagnosis of acute GAS infections

• culture on sheep blood agar

  • small translucent colonies surrounded by clear zone beta hemolysis

• rapid assays to detect GAS antigens

  • lateral flow immunochromatographic assay (LFA)

Antistreptolysin O (ASO)

• nephelometric methods currently used that measure light scatter produced by immune complexes containing streptolysin antigen

• Titer elevated in 85%of pts with acute rheumatic fever

• doesn’t increase in pts with skin infection

Anti-DNAse B

• produced by both rheumatic fever and impetigo patients

• tested by EIA and nephelometric methods

Streptozyme test

• detects antibodies to 5 streptococcal products

  • ASO

  • anti-hyaluronidase (AHase)

  • anti-streptokinase (ASKase)

  • Anti-nicotinamide-adenine dinucleotide (anti-NAD)

  • Anti-DNAse B

Helicobacter pylori

• GN microaerophilic spiral bacterium

• transmission likely by fecal-oral route

• major cause of gastric and duodenal ulcers

• can survive in acid environment bc of production of urease, which provides buffering zone around bacteria

• treated with antibiotics and anti-ulcer meds

• if untreated, can lead to gastric carcinoma or mucosa-associated lymphoid tumors

Detection of H. pylori infection

• detect urease in stomach biopsy (CLOtest)

• urea breath test

• antigens/antibodies

  • ELISA is method of choice

  • IgG in serum indicates active infection

  • titers decrease after successful treatment

Mycoplasma pneumoniae

• tiny bacteria that lack cell wall

• leading cause of resp infections

  • fever, headache, malaise, cough

  • walking pneunomia

  • Raynaud syndrome

  • causes Steven-John syndrome in minority of cases

• spread by resp droplets

Lab diagnosis of M. pneumoniae infection

• culture: produces mulberry colonies with ‘fried egg’ appearance on specialized media

  • gold standard

• ABs to M. pneumoniae

  • most useful diagnostic assay

  • IgM antibodies = recent infection

  • IgG antibodies = possible reinfection

• cold agglutinins

  • present in about 50% of pts but not specific for infection

• molecular methods: film array resp panel

Rickettsial infections

• obligate intracellular GN bacteria

• spotted fever group (rocky mountain spotted fever)

• typhus group (epidemic typhus)

• organisms transmitted by arthropods through biting on an infection animal

Rocky mountain spotted fever (RMSF)

• caused by R. rickettsii

• transmitted by 3 species of ticks

• headache, nausea, vomiting, diarrhea, skin rash; death

• diagnosis by clinical presentation, serology by IFA

Syphilis

• sexually transmitted diseases caused by spirochete Treponema pallidum

• rapidly destroyed by heat, cold, and drying

• direct contact with open lesion needed

• transmission to fetus during pregnancy

• bloodborne transmission rare

Clinical manifestations of syphilis

• primary stage: development of chancre

• secondary: generalized lymphadenopathy, malaise, fever, pharyngitis, rash

• latent stage: asymptomatic

• tertiary stage: Gummatous, cardiovascular, neurosyphilis

• treated with penicillin when detected in early stages

Congential syphilis

• transmission of treponemes to fetus occurs when pregnant woman has early-stage or latent syphilis

• causes death in 10% of cases

• live-born infants may be asymptomatic at birth but develop symptoms later

Lab diagnosis of syphilis

• direct detection

  • demonstration of treponemes in active lesions

  • dark-field microscopy

  • fluorescent antibody staining

• serological tests: nontreponemal/treponemal

Nontreponemal tests

• detect antibody against cardiolipin (reagin), a lipid released from membranes of cells damages as a result of infection

• VDRL test

• RPR test

• look for flocculation

• screen: test undiluted patient serum

• titer: test twofold dilutions of patient serum

VDRL test

• patient serum mixed on a slide with cardiolipin-lecithin-cholesterol antigen suspension

• viewed under light microscope for flocculation

RPR test

• patient serum mixed on a card with charcoal particles with cardiolipin antigen

• observe for macroscopic flocculation

Treponemal tests

• detect antibody to T. pallidum

• fluorescent treponemal absorption (FTA-ABS)

• T. pallidum particle agglutination (TP-PA)

• automated immunoassays

  • ELISA

  • CLIA

  • MFI

FTA-ABS test

• an indirect immunoflorescence test for antibody to T. pallidum

• patient serum incubated with sorbent to remove cross-reacting anitbodies

• absorbed patient serum incubated with microscope slide fixed with T. pallidum

• wash → AhG conjugated with fluorescein is added

• after 2nd incubation and wash → slide examined under fluorescent microscope

TP-PA test

• pt serum and controls diluted and incubated wit unsensitized gel particles or gel particles sensitized with T. pallidum antien

• (+) = agglutination

• (-) = no agglutination (button)

Reverse sequence algorithm for syphilis

• screen with an automated immunoassay for T. pallidum antibody

• confirm pos results with an RPR

• perform TP-PA on samples with discrepant results

Lyme disease

• caused by spirochete bacterium Borrelia burgdorferi

• transmitted by Ixodes ticks

• main resevoir: white-footed mouse

Clinical manifestations of lyme disease

• Stage 1: localized rash

• Stage 2: early dissemination

• Stage 3: late dissemination with arthritis

Two-tiered testing for lyme

Western blot results for B. burgdorferi antibodies

• Patient serum incubated with nitrocellulose membrane containing electrophoresed B. burgdorgeri antibodies

• + IgM: 2-3 characteristic bands

• +IgG: 5-10 bands

Leptospirosis

• zoonotic infection associated with occupational and recreational activities

• humans are exposed by mucous membrane contact with urine-contaminated water

• causes febrile episode that can progress to severe disease involving renal, liver, pulmonary, CNS

• lab testing:

  • IgM screening with ELISA, ImmunoDOT, LFA

  • MAT is gold standard for confirmation

Virus structure

• submicroscopic particles (nm)

• core of DNA or RNA

• protein coat (capsid)

• some have outer envelope

Virus life cycle

Immune defenses against viruses

• innate

  • skin and mucous membrane barriers

  • recognition of PAMPs on virus-infected host cells

  • interferons a and B

• humoral antibody response

  • antibodies attack free virus particles

  • viral neutralization, opsonization, C’ fixation, ADCC

• cell-mediated immunity

  • interferon y and IL-2 produced by Th1 cells

  • host cells containing intracellular virus destroyed by CTLs

Viral escape mechanisms

• mutations result in production of new viral antigens (influenza)

• Viruses block action of immune system components (HCV binding C3b)

• Suppression of immune response (CMV reducing MHC1 expression)

• Immune function altered (EBV stimulating polyclonal B-cell activation)

• latent state established

Lab testing for viral infections

• serologic tests

  • distinguishes between current and past infection

    • IgM (+), IgG(±): current or recent infection; congenital infection

    • IgM (-), IgG (+): past infection

  • antibody titers used to monitor course of infection

  • assess immune status

    • virus-specific IgG indicates immunity

• molecular tests

  • detect active infection

  • quantitative tests - guide antiviral therapy

Hepatitis viruses

• transmitted fecal-oral route: Hep A/E

• transmitted via parenteral route: Hep B/D/C

hepatitis clin and lab findings

• general flu-like symptoms early in infection

• pain in upper right quadrant of abdomen

• hepatomegaly and liver tenderness with progression

• jaundice, dark urine, light feces

• elevated bilirubin and liver enzymes (ALT)

HAV

• RNA virus transmitted by

  • fecal-oral route

  • close person-to-person contact

  • ingestion of contaminated food or water

• produces acute hepatitis in majority of adults

• infections in children usually asymptomatic

• Formalin-inactivated HAV vaccine

• HAV immune globulin recommended if unimmunized persons exposed

• acute infection indicated by (+) IgM and anti-HAV

• immunity indicated by (+) total anti-HAV along with (-) IgM anti-HAV

HEV

• RNA virus with 4 genotypes

• HEV-1/2 transmitted primarily through ingestion of feces-contaminated drinking water

• HEV-3/4 transmitted mainly by consumption of infected pork

• mostly asymptomatic or self-limiting infections

• severe infections possible (in immunocompromised or pregnant)

• acute infection indicated by IgM anti-HEV

• IgG anti-HEV indicates past exposure

• can detect HEV RNA in blood or stool samples during acute infection

HBV

• DNA virus transmitted through parenteral or perinatal routes

  • sexual contact

  • IV drug use

  • occupational needlestick injury

  • during birth process

• acute infection: symptoms increase with age

• chronic infection

  • persists for 6 months or more

  • occur sin 90% of infected infants, 10% of infected adults

  • increases in risk of liver cirrhosis or hepatocellular carcinoma

• infection preventable by immunization

• HBIG recommended for unimmunized persons exposed to HBV

Hepatitis B surface antigen (HBsAg)

• protein on outer envelope of virus

• excess circulates in virus-like particles in blood

• marker for active HBV infection

• component of hepatitis B vaccine

Hepatitis Be antigen (HBeg)

• protein in core of HBV

• marker of active viral replication

• indicates high degree of infectivity

Anti-HBc

• directed against hepatitis B core antigen

• IgM anti-HBc indicates current or recent acute infection; detects “core window”

• total anti-HBc consists mainly of IgG and can indicate a current or past infection

Anti-HBe

• directed against HBeAg

• indicates recovery from hep B

Anti-HBs

• directed against HBsAg

• indicates immunity to hep B$

Typical markers in Acute HBV infection

Typical markers in chronic HBV infection

HDV

• RNA virus that requires presence of HBV

• transmitted through parenteral or perinatal routes

• coinfection with HBV

  • usually results in acute, self-limited hepatitis

  • (+) for anti-HDV, IgM anti-HBc

• Superinfection of chronic HBV carriers

  • chronic liver disease with accelerated progression to cirrhosis and liver failure

  • (+) for anti-HDV, IG anti-HBc

• HDV RNA= marker of active viral replication

HCV

• RNA virus with 7 genotypes

• transmitted by exposure to contaminated blood, sexual contact, perinatal

• most infections asymptomatic at first but develop into chronic liver disease

• anti-HCV IgG used for screening and diagnosis

• Qualitative HCV RNA used for confirmation

• quantitative molecular test used to monitor viral load during antiviral therapy

• genotyping used to determine best therapy

EBV

• DNA herpes virus most commonly transmitted by intimate contact with saliva secretions

• begins in oropharynx in B cells and epithelial cells and spreads through lymphoreticular system

  • infectious mononucleosis

  • lymphoproliferative disorders

  • certain malignancies (Burkitt lymphoma)

Infectious mononucleosis (IM)

• absolute lymphocytosis

• 20% or more atypical lymphocytes

• heterophile antibody

  • reacts with antigens from 2 or more species

  • monospot

  • paul-bunnet test

Antibodies to EBV antigens

• early antigens: EA-D {acute IM}, EA-R

• late antigens: viral capsid antigens, IgM anti-VCA (acute) IgG anti-VCA (acute or past IM)

• latent phase antigens: EBV nuclear antigens (EBNA), anti-EBNA appear during convalescent IM

CMV

• DNA herpes virus transmitted through oral secretions, genital secretions, congenitally, transfusion/transplantation

• may be asymptomatic or induce mononucleosis-like syndrome in health individuals

• in sick people, can disseminate to lungs, liver, GI tract, CNS, and eyes and cause life-threatening infections

• may cause congenital defects an decreased survival in infants

Lab diagnosis of CMV

• direct virus detection

  • viral culture

  • ID of CMV antigens

  • molecular tests for CMV DNA

• serology

  • used to screen blood and organ donors; pregnant women

  • presence of IgG anti-CMV indicates infection

  • low avidity antibodies indicative of recent infection

VZV

• DNA herpes virus

• transmitted by inhalation of infected resp secretions or aerosols from skin lesion

• cause of: varicella, Herpes zoster (shingles)

Lab diagnosis of VZV

• diagnosis is usually based on characteristic clin findings

• real-time PCR

• serology is most useful in determining immunity

Rubella

• RNA virus transmitted through resp droplets or across placenta

• cause of German measles

• can cause:

  • congenital abnormalities

  • miscarriage

  • stillbirth in infants born to infected mothers

Lab testing for rubella

• serology

  • presence of IgG used to screen for immunity

  • congenital infection indicated by IgM antibodies in fetal blood, cord blood, or neonatal serum

  • current infection indicated by rubella-specific IgM or fourfold rise IGG

  • low avidity antibodies indicate recent infection

• viral culture

• molecular methods

Rubeola virus - measles

• RNA virus transmitted through resp droplets

• cause of: measures, subacute sclerosing panencephalitis (SSPE)

• diagnosis based on clin presentation and confirmed by serology

Rubula virus- mumps

• RNA virus transmitted through resp droplets, saliva, fomites

• most common clin manifestation: parotitis

• diagnosis based on clin presentation

• confirmation by culture or RT-PCR

HTLV-I/II

• closely related to retroviruses that preferentially infect T lymphs

• transmission mainly bloodborne, sexual contact, perinatal (breastfeeding)

• HTLV-I cause of adult T-cell leukemia/lymphoma (ATL_ and HAM/TSP

• serologic tests for antibodies to HTLV I/II are used to diagnosis infections and screen blood donors

• ELISA or CLIA used to screen; Western blot or LIA used for confirmation of + results

Human Immunodeficiency virus (HIV)

• cause of AIDS

• HIV-1

  • cause of most HIV infections worldwide

  • 4 groups (M, O, N, P)

  • 9 subtypes in group M (ABCDFGHJK)

• HIV-2

  • originated in West Africa

Modes of HIV transmission

• sexual contact involving exchange of body fluids

• contact with blood or other body fluids

• perinatal: mother to child

Characteristics of HIV

• retrovirus

• contains 2 copies of ssRNA

• reverse transcriptase transcribes viral RNA into DNA

• surrounded by protein coat (capsid)

• outer envelope of glycoproteins embedded in lipid bilayer

Replication of HIV

• attachment of HIV into host cell

• main target: CD4 Th

• coreceptor required

• fusion and undercoating

• RT converts viral RNA into complementary DNA

Immune responses to HIV

• innate defenses

  • NK cells mediate cytolysis of HIV-infected cells

  • dendritic cells stimulate release of cytokines that have antiviral effects

• humoral antibody production

  • antibodies are detected by 6 weeks after infection

  • antibodies produced later may prevent HIV from infecting host cells and participate in ADCC

• cell-mediated immunity

  • T cells produce cytokines with antiviral activity

  • CTLs destroy HIV-infected host cells

HIV escape from immune responses

• genetic mutations rapidly occur, generating new viral mutants with altered antigens

• HIV can down regulate expression of MHC-I molecules on infected host cells

• HIV can survive as a latent provirus for prolonged periods

• CD4 T cells are prime targets of destruction

HIV primary infection

• acute, early infection

• may be asymptomatic or have flu-like syndrome that resolves

• high level of viremia and decrease in CD4 T-cell #

HIV clinical latency

• absence of clinical symptoms

• decrease in viremia increase in CD4 T-cell #

AIDS

• resurgence of viremia and decrease in CD4 T-cell #

• profound immunosuppression, with appearance of life-threatening opportunistic infections and malignancies

Antiretroviral therapy (ART)

• drugs that block various steps of HIV replication cycle

• most effective when used in combination (CART)

• have significantly improved morbidity and mortality of HIV-infected persons and have reduced the rate of perinatal transmission

Previous testing algorithm

• screen for HIV1/2 antibodies by ELISA or rapid EIA

• confirm + test results by repeating ELISA, followed by Western Blot

CDC algorithm

• labs should begin antigen/antibody HIV screening immunoassay, followed (when reactive) by an HIV-1/2 antibody differentiation immunoassay

• when differentiation assay interpretation is negative or indeterminate for HIV-1, perform HIV nucleic acid test (NAT)

Disease monitoring

• peripheral blood CD4 T-cell counts

• quantitative viral load assays

• drug resistance testing

CD4 T-cell Enumeration

• these counts are best indicator of immune function in HIV-infected individuals

• incubate peripheral blood with fluorescent-labeled anti-CD4; analyze results by flow cytometry

• in untreated patients, CD4 T-cell # declines progressively, and CD4 T: CD8 T-cell ratio is less than 1:1

• CD4 T-cell count of less than 200 cells/uL indicates stage 3 infection (AIDS)

quantitative viral load assays

• measure amount of HIV RNA circulating in patient plasma

• methods: qPCR, bDNA

• HIV RNA detectable about 11 days after infection

Phenotype resistance testing

• determine ability of HIV from clinical samples to grow in presence of antiretroviral drugs

• involve sophisticated technologies only performed by specialized reference labs

Testing of infants younger than 18 months

• maternal antibodies in infant serum can complicate serologic test results

• qualitative HIV-1 DNA PCR using infant’s peripheral blood mononuclear cells is preferred method

• serologic testing at 12-18 months of age may be used to confirm diagnosis