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 an in vitro assay for interferon gamma production in response to the Mycobacterium tuberculosis-synthesized peptide antigens ESAT-6 and CFP-10 and the PPD skin test.

We compared the tuberculin skin test (TST) to QuantiFERON-TB (QFT) and QuantiFERON-TB Gold (QFT-G) for the detection of latent tuberculosis. The QFT-G uses synthesized early secretory antigenic target 6 and culture filtrate protein 10 peptide antigens instead of purified protein derivative (PPD) antigens. The study included 137 adults in 3 groups: 1 (n = 81), at low risk for Mycobacterium tuberculosis (TB) and not vaccinated for Mycobacterium bovis bacillus Calmette-Guérin (BCG); 2 (n = 30), probably had TB exposure and were BCG vaccinated; and 3 (n = 26), at low risk for TB, not BCG vaccinated, but previously had a positive TST result. Positive results were as follows: group 1: TST 3 (3.7%); QFT 9 (11.1%); and QFT-G, 0 (0.0%); group 2: TST 26 (86.7%); QFT, 15 (50.0%); and QFT-G, 5 (16. 7%); and group 3: TST, 26 (100.0%); QFT, 13 (50.0%); and QFT-G, 9 (34.6%). The QFT-G demonstrated less cross-reactivity with BCG antigen and was more specific than QFT and TST in low-risk individuals.

A comparison of Thermo Electron RSV OIA to viral culture and direct fluorescent assay testing for respiratory syncytial virus.

BACKGROUND: Rapid diagnostic methods for respiratory syncytial virus are useful tools available for the clinician. OBJECTIVES: The Thermo Electron RSV OIA (optical immunoassay kit) was prospectively compared with direct immunofluorescent assay and viral culture at Primary Children's Medical Center, Salt Lake City, Utah. STUDY DESIGN: Specimens from three hundred and thirty patients exhibiting respiratory symptoms were collected for testing by the three methods above. Several specimens were positive by both OIA and DFA with a negative culture result. These culture results were verified by RT-PCR analysis. RESULTS: Overall, 107 specimens were positive for RSV by the reference tests (culture or RT-PCR). DFA analysis identified an additional 40 patient specimens positive for other respiratory viruses. Compared to the reference tests the sensitivity, specificity, positive, and negative predictive values of the OIA for detection of RSV were 87.9%, 99.6%, 98.9% and 94.5%, respectively. CONCLUSIONS: The rapid OIA assay format proved to be cost effective, and simple to use in comparison to DFA and viral culture. Negative rapid test results should still be confirmed with a secondary test.

A comparison of Binax NOW to viral culture and direct fluorescent assay testing for respiratory syncytial virus.

The Binax NOW immunochromatographic assay for respiratory syncytial virus was prospectively compared with direct fluorescent assay and viral culture at Primary Children's Medical Center, Salt Lake City, Utah during February 2003. Three hundred ten patient specimens were collected for testing, of which 102 specimens were positive for respiratory syncytial virus by the reference tests, direct immunofluorescence assay (DFA), and culture or molecular analysis. DFA analysis identified an additional 40 patient specimens positive for other respiratory viruses. Compared to the reference tests, the sensitivity, specificity, and positive and negative predictive values of the rapid immunochromatographic assay for detection of respiratory syncytial virus were 89.2%, 100.0%, 100.0%, and 94.9%, respectively. This rapid assay format proved to be cost-effective and simple to use in comparison to DFA and viral culture. Negative rapid test results should still be confirmed with a secondary test.

Use of heat labile UNG in an RT-PCR assay for enterovirus detection.

A reverse transcription-polymerase chain reaction (RT-PCR) assay was developed to replace the Roche AMPLICOR Enterovirus Test used in our laboratory from 1996 to 1999. The new assay design was optimized to match or exceed the performance of the Roche AMPLICOR Enterovirus test kit with respect to analytical sensitivity and specificity, contamination control, ease of use and availability of reagents. This new assay uses a heat labile form of the enzyme uracil DNA glycosylase (UNG) for amplicon contamination control and an RT-PCR enzyme mixture, enabling a one tube/one step amplification. RNA preparation was undertaken using a commercial extraction kit. End detection was accomplished using a probe-capture enzyme immuno assay (EIA) plate format. This EV RT-PCR assay exceeds the performance of the Roche AMPLICOR Enterovirus assay in a direct comparison. The combined enzymological approach has potential application to a wide variety of assays requiring sensitive RNA detection and stringent contamination control, including those utilizing real time detection methods.

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 manufacturer's 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 manufacturer's performance standards and the assay was implemented into the laboratory's 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.

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.

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.

Performance of enterovirus genotyping targeting the VP1 and VP2 regions on non-typeable isolates and patient specimens.

Culture and serotyping of human enteroviruses are time-consuming and labor-intensive. Targeted nucleic acid sequencing has emerged as a powerful alternative to conventional methods. Many published genotyping assays use two-step reverse transcription and polymerase chain reaction (RT-PCR), nested PCR protocols, and/or reflexive testing algorithms. The performance of a one-step RT-PCR protocol, a more clinically practical approach, was evaluated. The VP1 and/or VP2 region of archived enterovirus isolates (n=36, representing 32 serotypes), patient enterovirus isolates not typeable by immunofluorescence antibodies (n=50), and enterovirus from direct patient specimens (48 cerebrospinal fluid, 2 plasma/serum, 1 blood) were amplified and sequenced for genotype identification. The analytical sensitivity of the genotyping assays was 100-fold less than detection by RT-PCR of the 5'-untranslated region. Thirty-four of 36 archived isolates could be genotyped by combining results of VP1 and VP2 target sequencing. Non-typeable isolates included 17 echovirus 18, 6 enterovirus 68, 6 rhinovirus, and 7 which could not be classified further. From clinical specimens, 23 of 51 (45%) could be identified using VP2 typing and the most common types were coxsackievirus B1, echovirus 30, and echovirus 6. Using a one-step RT-PCR without nesting, most enterovirus isolates and a subset of clinical samples with high viral titer could be genotyped.

Performance of diagnostic tests to detect respiratory viruses in older adults.

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.

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

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.

Description and validation of a novel real-time RT-PCR enterovirus assay.

BACKGROUND: Enteroviruses are a leading cause of aseptic meningitis in adult and pediatric populations. We describe the development of a real-time RT-PCR assay that amplifies a small target in the 5' nontranslated region upstream of the classical Rotbart enterovirus amplicon. The assay includes an RNA internal control and incorporates modified nucleotide chemistry. METHODS: We evaluated the performance characteristics of this design and performed blinded parallel testing on clinical samples, comparing the results with a commercially available RT-PCR assay (Pan-Enterovirus OligoDetect kit) that uses an enzyme immunoassay-like plate end detection. RESULTS: We tested 778 samples and found 14 discrepant samples between the 2 assays. Of these, the real-time assay detected 6 samples that were negative by the OligoDetect kit, 5 of which were confirmed as positive by sequence analysis using an alternative primer set. Eight discrepant samples were positive by the OligoDetect kit and real-time negative, with 6 confirmed by sequencing. Overall, detection rates of 97% and 96% were obtained for the OligoDetect kit and real-time assays, respectively. Sequence analysis revealed the presence of a number of single nucleotide polymorphisms in the targeted region. The comparative sensitivities of the 2 assays were equivalent, with the limit of detection for the real-time assay determined to be approximately 430 copies per milliliter in cerebrospinal fluid. CONCLUSIONS: This novel real-time enterovirus assay is a sensitive and suitable assay for routine clinical testing. The presence of single nucleotide polymorphisms can affect real-time PCR assays.

Evaluation of an in vitro assay for gamma interferon production in response to Mycobacterium tuberculosis infections.

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.