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 Immunoserology Hemagglutination EIA Latex agglutination Compliment fixation Immunoflorecent

LIMITATIONS OF CONVENTIONAL CLINICAL MICROBIOLOGY Culture Labor intensive Need for special media Prolonged period of time to culture Some organisms are uncultivable on artificial media Potential health hazards Antigen Detection Negative tests require confirmation Effected by poor specimen collection Low microbe burden Serology Unhelpful during early stage of infection Not quite useful in immunocompromised patients

MOLECULAR DIAGNOSTICS Most widely used is PCR High sensitivity High specificity Diversity Nucleic acid probes Do 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 PCR Uses single clinical specimen to investigate several potential pathogens simultaneously Encephalitis/meningitis panel: HSV,VZV, CMV HHV-6, EBV, Enteroviruses Real-time PCR Utilizes a fluorescent labeled probe Requires small volumes thus takes minutes to complete

Leading uses for nucleic acid based tests Nonculturable agents Human papilloma virus Hepatitis B virus Fastidious, slow-growing agents Mycobacterium tuberculosis Legionella pneumophilia Highly infectious agents that are dangerous to culture Francisella tularensis Brucella species Coccidioidis immitis

Leading uses for nucleic acid based tests In situ detection of infectious agents Helicobacter pylori Toxoplasma gondii Agents present in low numbers HIV in antibody negative patients CMV in transplanted organs Organisms present in small volume specimens Intra-ocular fluid Forensic samples

Leading uses for nucleic acid based tests Differentiation of antigenically similar agents May be important for detecting specific virus genotypes associated with human cancers (Papilloma viruses) Antiviral drug susceptibility testing May be important in helping to decide anti-viral therapy to use in HIV infections Non-viable organisms Organisms tied up in immune complexes

Leading uses for nucleic acid based tests Molecular epidemiology To 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

PathogensMechanismSensitivity (%) Specificity (%) Positive Predictive Value (%) Negative Predictive Value (%) Time to perform test Group A Streptococcus Detects group A staphylococcal carbohydrate antigen by immunoassay (Compared to culture) minutes Helicobacter pyloriDetects immunoglobulin G antibodies specific to H. pylori (Compared to biopsy) minutes Borrelia burgdorferiDetects antibodies to B. burgdorferi using recombinant antigen 72 (Compared to ELISA) 97Not available 20 minutes

Collection and Processing of Clinical Specimen Diagnostic Technologies Culture Antigen detection Serology Molecular diagnostics Rapid Diagnostic Test CLIA-Waived tests Other 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 blood 1-2ml in neonate 2-3ml in infants 3-5ml in children 10-20ml in adolescent Rapid inoculation A 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 urine Clean-voided midstream urine Use of urine bag Catheterized specimen/ Suprapubic aspiration Collecting CSF CSF is hypotonic Refrigeration can render fastidious bacteria non-viable Cell 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/quality Small quantity for optimal analysis Poor specimen e.g. eye cultures for chlamydiae should have enough cellular element Contamination During collection During transport Contamination in the lab

COLLECTION AND PROCESSING OF CLINICAL SPECIMEN Improper transport media Prevent drying Maintain optimal physiochemical environment Prevent oxidation and destruction of enzymes Provide adequate nutrients Three major culture media Enrichment: chocolate and sheep blood Selective: Thayer-Martin Differential: MacConkey-ability to ferment lactose

COLLECTION AND PROCESSING OF CLINICAL SPECIMEN Delay in transportation Holding conditions are specimen or pathogen specific Urine: 2˚ C to 8˚C Inoculated blood: 35˚ C to 37˚C

SPECIFIC EXAMPLES Specimen 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% co 2 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