Edward W. Taggart

Evaluation of an In Vitro Assay for Gamma Interferon Production in Response to Mycobacterium tuberculosis Infections

ABSTRACT The tuberculin skin test (TST) is the “gold standard” for detecting infection with Mycobacterium tuberculosis. We compared the TST using purified protein derivative to the QuantiFERON-TB test (QFT). Two groups were examined. Group 1 individuals (n = 66) (low risk) were at low risk for exposure to M. tuberculosis and were not Mycobacterium bovis BCG vaccinated. Group 2 (n = 29) include individuals who were likely to have been exposed to a high prevalence of M. tuberculosis infections and were BCG vaccinated. Group 1 individuals were given a TST. Group 2 individuals were not given a TST because of possible adverse reactions. A 10- to 15-mm indurated area 48 h after TST was considered positive. A positive QFT result was defined as a significant gamma interferon response to M. tuberculosis antigen, Mycobacterium avium antigen, and a nonspecific mitogen stimulus and no response in the negative control. In group 1, 60 of 66 individuals (90.9%) were negative by both methods, and 1 person was positive by both methods. There was one QFT-negative, TST-positive case, one QFT-positive, TST-negative case, and three conditional QFT-positive, TST-negative cases. In group 2, 12 of 29 (41.4%) were positive by QFT and considered likely to be TST positive because of prior BCG vaccination. QFT testing in our low-risk group resulted in an agreement of 96.8%, a sensitivity of 50%, and a specificity of 98.4% compared with TST results. QFT testing with TST in low-risk groups can aid in the detection of latent M. tuberculosis infections.

Performance of diagnostic tests to detect respiratory viruses in older adults

Abstract The performance of 4 laboratory methods for diagnosis of viral respiratory tract infections (RTI) in older adults was evaluated. Seventy-four nasopharyngeal (NP) swab specimens were obtained from 60 patients with RTI at a long-term care facility over 2 respiratory seasons. Sixteen specimens were positive for a respiratory virus by at least 1 method. Multiplex reverse transcriptase polymerase chain reaction (RT-PCR) by the Luminex xTAG® Respiratory Viral Panel (RVP) detected 16 (100%) of the positive specimens, RVP of 24-h culture supernatant detected 8 (50%), direct fluorescent antibody testing detected 4 (25%), rapid culture detected 2 (12.5%), and rapid antigen testing detected none. For a comparison group, RVP was performed on NP swabs from 20 outpatient children with RTI. The mean fluorescence intensity by RVP was significantly lower for positive adult patients than pediatric patients (P = 0.0373). Our data suggest that older adult patients shed lower titers of viruses, necessitating a highly sensitive assay such as RT-PCR to reliably detect respiratory viral pathogens.

Comparison of Complement Fixation With Two Enzyme-Linked Immunosorbent Assays for the Detection of Antibodies to Respiratory Viral Antigens

We compared complement fixation (CF) for the measurement of antibodies against influenza A, influenza B, respiratory syncytial virus (RSV), human adenovirus, and parainfluenza viruses 1, 2, and 3 (para-1, para-2, and para-3) with 2 enzyme-linked immunosorbent assays (ELISA kits, A and B). The IgG ELISA kits compared very well with each other except for the influenza A and B IgG ELISAs. The IgG ELISAs, in general, did not agree with CF In contrast, the IgM ELISAs compared well with CF and each other except for the consensus parainfluenza panel from ELISA B. The poor agreement of the IgG ELISAs with the CF test can be explained by the increased sensitivity of the ELISAs and differences between CF antigens and the ELISA antigens. The influenza A and influenza B ELISA antigens differed between both kits, which may explain their poor agreement. The ELISA is a suitable replacement for CF, providing greater sensitivity, isotype specificity, and ease of use.

Evaluation of a PCR Probe Capture Assay for the Detection of Toxoplasma gondii Incorporation of Uracil N-Glycosylase for Contamination Control

Toxoplasma gondii is a cyst-forming parasite of clinical relevance in humans primarily because of the neurologic abnormalities it can cause. In some clinical circumstances, it is desirable to detect the pathogen directly. We modified a commercially available Toxoplasma polymerase chain reaction (PCR) probe capture assay by incorporating uracil N-glycosylase (UNG) to prevent carryover amplicon contamination. In addition, UNG inactivation and DNA denaturation were accomplished chemically to simplify the DNA hybridization to the capture probe. The incorporation of UNG effectively eliminated carryover contamination; the probe capture assay showed a log increase in detection sensitivity compared with standard agarose gel electrophoresis. To assess sensitivity and possible inhibition of amplification, different sample types were spiked with Toxoplasma organisms. After DNA extraction and PCR amplification, a sensitivity of 2 tachyzoites for the assay was determined in buffered saline, cerebrospinal fluid (CSF), serum, and amniotic fluid; 20 tachyzoites for whole blood; and 200 tachyzoites for brain tissue. An additional 20 human serum and CSF samples submitted for Toxoplasma serologic testing were run by the PCR method. Of these, only an IgM-positive CSF sample was PCR positive. The Toxoplasma PCR probe capture assay showed good sensitivity and was not substantially inhibited by several different clinically relevant samples.

Comparison of 10 indirect fluorescent antibodies to detect and type influenza A specimens.

CONTEXT Management of influenza infections relies on rapid, accurate, and sensitive diagnostic techniques. Influenza A (IA) strain typing has become more important since the emergence of highly pathogenic avian and novel influenza strains and the high frequency of oseltamivir resistance in circulating H1N1 isolates. OBJECTIVE To analyze the performance of indirect fluorescent antibody testing for subtyping a broad range of IA strains. DESIGN Ten indirect fluorescent antibody reagents were used to detect and type 100 archived IA respiratory specimens from 1986 through 1995 and 2006 through 2007 and a reassortant, nonpathogenic H5N1 sample. Both direct specimen and cultured isolates were tested. Reverse transcription-polymerase chain reaction was used to confirm indirect fluorescent antibody results. Three H1N1-, 2 H3N2-, and 1 H1-H2-H3-H5-specific antibodies (Chemicon Diagnostics), an IA pool reagent (Trinity Biotech), and H1, H3, and H1-H3-specific antibodies (Centers for Disease Control and Prevention) were used. RESULTS Reverse transcription-polymerase chain reaction confirmed all 100 isolates as IA and identified 71 as H1, 22 as H3, and 7 as non-H1-H3. Sensitivity of direct specimen testing ranged was 18.3% to 57.7% for the H1 reagents, 36.4% to 50.0% for the H3 reagents, and 40.0% to 53.8% for the pool reagents. Subtyping was more sensitive on cultured isolates than direct specimens. Specificity for all antibodies was 89.7% to 100%. The H5N1 sample was positive by direct testing and culture (reverse transcription-polymerase chain reaction, Centers for Disease Control and Prevention H5N1 pool, Chemicon H1-H2-H3-H5). No cross-reactivity was observed when the 10 antibodies were tested against other common respiratory viruses. CONCLUSIONS When positive, IA subtyping antibodies can serve as a useful diagnostic tool when multiple influenza virus subtypes are cocirculating with different susceptibility patterns.

Flow cytometric detection and serotyping of enterovirus for the clinical laboratory.

Abstract Culture and serotyping of human enteroviruses by fluorescence microscopy are time-consuming and labor-intensive. Flow cytometry has the potential of being more rapid, sensitive, and objective but has not been used for these purposes in a clinical laboratory. Primary rhesus monkey kidney (PMK) cells were inoculated with several enterovirus serotypes and stained with enterovirus-specific antibodies for flow cytometry and indirect fluorescence antibody testing (IFA). Kinetic studies of coxsackievirus B1 and echovirus 30 infection of PMK cells were performed on days 1–4 after inoculation. Flow cytometry results for echovirus 6, 9, 11, and 30 and coxsackievirus B1 correlated with IFA in all cases. Coxsackievirus B1 and echovirus 30 infections were detected 1 day earlier by flow cytometry than IFA. Flow cytometry can be effectively used for detecting enterovirus-infected cells in a clinical laboratory with the advantages of better quantitation of low levels of infection and earlier detection of virally infected cells in culture systems.

Verification of performance specifications for a US Food and Drug Administration-approved molecular microbiology test: Clostridium difficile cytotoxin B using the Becton, Dickinson and Company GeneOhm Cdiff assay.

CONTEXT US Food and Drug Administration (FDA)-approved diagnostic tests based on molecular genetic technologies are becoming available for an increasing number of microbial pathogens. Advances in technology and lower costs have moved molecular diagnostic tests formerly performed for research purposes only into much wider use in clinical microbiology laboratories. OBJECTIVE To provide an example of laboratory studies performed to verify the performance of an FDA-approved assay for the detection of Clostridium difficile cytotoxin B compared with the manufacturers performance standards. DESIGN We describe the process and protocols used by a laboratory for verification of an FDA-approved assay, assess data from the verification studies, and implement the assay after verification. RESULTS Performance data from the verification studies conducted by the laboratory were consistent with the manufacturers performance standards and the assay was implemented into the laboratorys test menu. CONCLUSION Verification studies are required for FDA-approved diagnostic assays prior to use in patient care. Laboratories should develop a standardized approach to verification studies that can be adapted and applied to different types of assays. We describe the verification of an FDA-approved real-time polymerase chain reaction assay for the detection of a toxin gene in a bacterial pathogen.

Identifying respiratory viruses in nasal mucus from children.

Large amounts of respiratory viruses are shed in nasal secretions by children. Nasal mucus was compared with nasopharyngeal swabs as a source for respiratory virus testing. Multiplex reverse transcription-polymerase chain reaction detected virus in nasal mucus specimens in 73% (11/15) of positive cases, demonstrating the potential utility of less invasive specimens when a highly sensitive method is used for respiratory virus detection.