LABORATORY DIAGNOSIS OF INFECTIOUS DISEASES

OBJECTIVES

Know available diagnostic technologies for ID

Understand specific specimen for specific diagnostic test

Understand procedure for specimen collection

DIAGNOSTIC TECHNOLOGIES

Gram stain/Microscopy

Cultures.

Three major culture media

Enrichment: chocolate and sheep blood

Selective: Thayer-Martin

Differential: MacConkey-ability to ferment lactose

DIAGNOSTIC TECHNOLOGIES

Antigen Detection Assays

Most has poor sensitivity and specificity

57% sensitivity and 98% specificity for pneumococcal pneumonia

Conc. Urine EIA for Legionella pneumophila serogroup 1 has 89% sensitivity and 100% specificity

Immunochromatographic assay has better sensitivity and are faster

DIAGNOSTIC TECHNOLOGIES

ImmunoserologyHemagglutination

EIA

Latex agglutination

Compliment fixation

Immunoflorecent

LIMITATIONS OF CONVENTIONAL CLINICAL MICROBIOLOGY

CultureLabor intensiveNeed for special mediaProlonged period of time to cultureSome organisms are uncultivable on artificial mediaPotential health hazards

Antigen DetectionNegative tests require confirmationEffected by poor specimen collectionLow microbe burden

SerologyUnhelpful during early stage of infectionNot quite useful in immunocompromised patients

MOLECULAR DIAGNOSTICS

Most widely used is PCRHigh sensitivity

High specificity

Diversity

Nucleic acid probesDo not amplify DNA

MOLECULAR DIAGNOSTICS

Polymerase Chain Reaction

Specific PCR: Uses primers to known DNA targets. So far 31 clinical bacterial gene sequence are known and 38 in progress

• Use when conventional diagnostics are inadequate, time consuming, difficult and hazardous

Broad range PCR: uses complementary primers to conserved regions shared by a given taxonomic group

• Used in cases of B. henselae and Mycobacterium spp

MOLECULAR DIAGNOSTICS

Multiplex PCRUses single clinical specimen to investigate several potential pathogens simultaneously

• Encephalitis/meningitis panel: HSV,VZV, CMV HHV-6, EBV, Enteroviruses

Real-time PCRUtilizes a fluorescent labeled probeRequires small volumes thus takes 30-60 minutes to complete

Leading uses for nucleic acid based tests

Nonculturable agentsHuman papilloma virus Hepatitis B virus

Fastidious, slow-growing agentsMycobacterium tuberculosisLegionella pneumophilia

Highly infectious agents that are dangerous to cultureFrancisella tularensisBrucella speciesCoccidioidis immitis

Leading uses for nucleic acid based tests

In situ detection of infectious agentsHelicobacter pyloriToxoplasma gondii

Agents present in low numbersHIV in antibody negative patientsCMV in transplanted organs

Organisms present in small volume specimensIntra-ocular fluidForensic samples

Leading uses for nucleic acid based tests

Differentiation of antigenically similar agentsMay be important for detecting specific virus genotypes associated with human cancers (Papilloma viruses)

Antiviral drug susceptibility testingMay be important in helping to decide anti-viral therapy to use in HIV infections

Non-viable organismsOrganisms tied up in immune complexes

Leading uses for nucleic acid based tests

Molecular epidemiologyTo identify point sources for hospital and community-based outbreaks

To predict virulence

Culture confirmation

OTHER USES OF MOLECULAR DIAGNOSTICS

Viral load monitoring

Viral genotyping

Bacterial resistance detection

Bacterial genotyping

LIMITATION OF PCR TECHNOLOGIES

Cost

False positives caused by amplification of contaminants

Only sample from normally sterile sites should be considered for broad-range PCR

Specimen is required to be refrigerated or stored in alcohol before processing

LIMITATION OF PCR TECHNOLOGIES

Specimen should be frozen until amplification

No antimicrobial sensitivity is available

Needs the clinician to name the suspect

RAPID DIAGNOSTIC TESTS

High sensitivity and specificity

High negative and positive predictive values

High accuracy compared to gold standard

Simple to perform

Rapid turn around time

Cost effective

Pathogens Mechanism Sensitivity(%)

Specificity(%)

PositivePredictiveValue (%)

Negative PredictiveValue (%)

Time to perform test

Group A Streptococcus

Detects group A staphylococcal carbohydrate antigen by immunoassay

89-94(Compared to culture)

95-99 55-89 90-97 5 minutes

Helicobacter pylori Detects immunoglobulin G antibodies specific to H. pylori

85-90(Compared to biopsy)

80-89 85 79 5-10 minutes

Borrelia burgdorferi Detects antibodies to B. burgdorferi using recombinant antigen

72(Compared to ELISA)

97 Not available Not available 20 minutes

Collection and Processing of Clinical Specimen

Diagnostic TechnologiesCultureAntigen detectionSerologyMolecular diagnostics

Rapid Diagnostic TestCLIA-Waived testsOther rapid non-CLIA waived tests

COLLECTION AND PROCESSING OF CLINICAL SPECIMEN

Most important aspect of laboratory medicine

Insufficient quantity

Contamination

Improper transport media

Delay in transportation

Inappropriate storage

COLLECTION AND PROCESSING OF CLINICAL SPECIMEN

Collecting Blood

Clean with 70% ethyl alcohol

Disinfect with 10% povidone-iodine

Allow to dry for at least 1 minute

No wiping!

Clean the rubber stopper of the bottle

Use alcohol for Bactec bottle to prevent cracking

COLLECTION AND PROCESSING OF CLINICAL SPECIMEN

Collect enough blood1-2ml in neonate2-3ml in infants3-5ml in children10-20ml in adolescent

Rapid inoculationA 3 hour delay result in 25% reduction in recovery of S. pneumoniae

Paisley JW, Lauer BA. Pediatric blood cultures. Clin Lab Med 1994; 14: 17

Roback MG, Tsai AK, Hanson KL. Delayed incubation of blood culture bottles: Effect on recovery rate of S. pneumoniae. Pediatr Emerg Care 1994; 10: 268

COLLECTION AND PROCESSING OF CLINICAL SPECIMEN

Collecting urineClean-voided midstream urineUse of urine bagCatheterized specimen/ Suprapubic aspiration

Collecting CSFCSF is hypotonicRefrigeration can render fastidious bacteria non-viableCell count decreases by 32% after 1 hour and 50% after 2 hours

Steele RW, Mormer DJ, O’Brien MD, et al. Leukocyte survival in cerebrospinal fluid. J Clin Microbiol 1986; 23: 965

COLLECTION AND PROCESSING OF CLINICAL SPECIMEN

Insufficient quantity/qualitySmall quantity for optimal analysis

Poor specimen e.g. eye cultures for chlamydiae should have enough cellular element

ContaminationDuring collection

During transport

Contamination in the lab

COLLECTION AND PROCESSING OF CLINICAL SPECIMEN

Improper transport mediaPrevent dryingMaintain optimal physiochemical environmentPrevent oxidation and destruction of enzymesProvide adequate nutrients

Three major culture mediaEnrichment: chocolate and sheep bloodSelective: Thayer-MartinDifferential: MacConkey-ability to ferment lactose

COLLECTION AND PROCESSING OF CLINICAL SPECIMEN

Delay in transportationHolding conditions are specimen or pathogen specific

Urine: 2˚ C to 8˚C

Inoculated blood: 35˚ C to 37˚C

SPECIFIC EXAMPLESSpecimen for isolation of N. gonorrhoeae should be inoculated into a specific media, transported within 30 minutes of collection, incubated at 35˚-37˚C in 5-10% co2

Stool for ova & parasite should be placed in preservatives

CSF is held in room temperature and never refrigerated

Stool for C. difficle must be refrigerated or frozen

SUMMARY

If you are not sure of how to collect a specimen call microbiology before collection

If you are unsure what to send, call ID before collection

If you are not sure of specific clinical syndrome, save a sample

You are asked to attend the delivery of a term infant because the baby is small for gestational age, and prenatal ultrasonography revealed periventricular cerebral calcifications. The infant’s birth weight is 2,000 g. On physical examination, you note hepatosplenomegaly and a petechial rash on the face and trunk.

Of the following, the BEST laboratory test for diagnosing the cause of these findings is

A. Nasopharyngeal culture for herpes simplex

B. Rapid plasma reagin for syphilis

C. Serum immunoglobulin (Ig) G titer for rubella

D. Serum IgM titer for toxoplasmosis

E. Urine culture for cytomegalovirus

A 12-year-old girl comes to your office complaining of headache, malaise, fever to 101 F (38.3 C), rhinorrhea, and a sore throat for the past 3 to 4 days. Most recently, she developed hoarseness and cough. On physical examination, you note crackles and wheezes throughout the lung fields. Chest radiography demonstrates interstitial

infiltrates in the lower lung fields bilaterally.

Of the following, the BEST test to order to confirm the patient’s diagnosis is:

A. Direct fluorescent antibody for Bordetella pertussis

B. Enzyme immunoassay for respiratory syncytial virus

C.Serology for Mycoplasma pneumoniae

D.Throat culture for group A Streptococcus

E. Viral culture for parainfluenza

A 16-year-old boy presents with a 5-day history of low-grade fever, headache, mild nasal congestion, and a persistent cough associated with posttussive syncope, he reports that it is hard for him to catch his breath after one of his coughing episodes. His immunizations are up to date. Several of his classmates are ill with similar symptoms. Chest radiography results are normal.

Of the following, the test that is MOST likely to aid in the diagnosis of this patient is:

A. Cold agglutinin test

B. Mantoux skin test with purified protein derivative

C.Monospot

D.Pertussis direct fluorescent antibody

E. Sputum Gram stain

A 5-year-old girl develops fever, swelling of the parotid gland, and headache.

Of the following, the BEST diagnostic test for this child is:

A. Bacterial culture of parotid duct secretions

B. Epstein-Barr virus serology

C.Mumps serology

D.Serum amylase

E. Viral culture of respiratory secretions

A 3-year-old boy presents with a large, non-tender, rubbery anterior cervical lymph node. You prescribe a course of dicloxacillin, but there is no change in the node. Results of Mantoux purified protein derivative skin test reveal 8 mm in duration.

Of the following, the BEST diagnostic procedure to undertake in this patient now is:

A. Biopsy of the node

B. Chest radiography

C.Excision of the node

D.Gastric aspirates for culture

E. Needle aspiration of the node

A newborn has evidence of symmetric intrauterine growth restriction. Evaluation reveals microcephaly with intracranial calcifications, “salt and pepper” retinopathy, hearing deficit, enlarged liver and spleen, and

purpura. Laboratory evaluation documents thrombocytopenia.

Of the following, the test MOST likely to confirm the diagnosis in this infant is:

A. Cytology of a conjunctival swab

B. Rapid plasma reagin test

C. Serology of blood

D. Urine assay for interferon

E. Viral culture of urine

Of the following, the BEST direct stain to detect Mycobacterium tuberculosis is the

A. Calcofluor white stain

B. Giemsa stain

C. Gram stain

D. Kinyoun stain

E. Periodic acid-Schiff stain

A 9-year-old boy has had a nonproductive cough for the past 3 weeks. He has been afebrile and otherwise feeling well. On physical examination, you note widespread rales. Chest radiography reveals bilateral, diffuse infiltrates. You

diagnose pneumonia, most likely due to Mycoplasma pneumoniae.

Of the following, the test that would BEST confirm the diagnosis is:

A. Bacterial culture of sputum

B. Blood culture

C.Gram stain of sputum

D.Mycoplasma-specific immunoglobulin M

E. Serum cold agglutinins

A 5-year-old boy is hospitalized in January with fever and seizures. Lumbar puncture reveals clear cerebrospinal fluid that has a white blood cell count of 47/ cu mm, all of which are lymphocytes. On physical examination, he appears obtunded but arouses with painful stimuli. Neurologic examination reveals no focal findings.

Of the following, the diagnostic test that is MOST likely to reveal the etiology of this child’s illness is:

A. Bacterial culture of cerebrospinal fluid of herpes simplex

B. Polymerase chain reaction test of cerebrospinal fluid for herpes simplex

C. Streptococcus pneumoniae bacterial antigen test of cerebrospinal fluid

D. Viral culture of cerebrospinal fluid

E. Viral culture of nasopharyngeal and rectal swabs

A newborn has hepatosplenomegaly, purpuric rash, jaundice, thrombocytopenia, and microcephaly. Computed tomography of the head demonstrates cerebral calcifications

Of the following, the MOST appropriate diagnostic testing for this infant includes:

A. Maternal human immunodeficiency virus serology

B. Serologic testing of mother and infant for cytomegalovirus

C.Serologic testing of mother and infant for Toxoplasma

D.VDRL on infant and maternal sera

E. Viral culture of swabs of infant’s throat and conjunctivae

A 12-year-old girl is brought to your office of evaluation following 4 weeks of diarrhea, abdominal pain, and weight loss. You suspect giardiasis.

Of the following, the MOST reliable next step to establish the diagnosis is to examine a single stool sample for:

A. Giardia antigen

B. Leukocytes

C.Ova and parasites

D.pH

E. Reducing substances

A 4-month-old infant develops severe paroxysmal coughing 10 days after the onset of nasal congestion and rhinorrhea. His mother reports that often 15 to 20 coughs occur in rapid succession.

Of the following, the BEST test to establish the diagnosis is:

A. Bronchoscopy that demonstrates the presence of a foreign body

B. Culture of a nasal swab that grows a small gram-negative coccobacillus

C.Culture of a nasal swab that shows viral growth

D.pH probe that demonstrates gastroesophageal reflux

E. Skin testing with demonstration of allergies