Kathleen A. Stellrecht

Comparison of Multiplex PCR Assay with Culture for Detection of Genital Mycoplasmas

ABSTRACT Ureaplasma, spp. Mycoplasma genitalium, and Mycoplasma hominis are associated with infection of the genitourinary tract, reproductive failure, and neonatal morbidity and mortality. We have developed a multiplex PCR for the detection of these Mycoplasmatales in a single amplification reaction. The analytical sensitivities of this assay were 10.8, 10.8, and 8.8 CFU for each organism, respectively. This multiplex PCR was compared to culture on 26 cervical swabs, 2 vaginal swabs, 4 female urine specimens, 49 semen samples, 2 male urine specimens, and 1 nonspecified sample. A total of 21 specimens were culture positive (25%); 17 of these were PCR positive. An additional 11 specimens were PCR positive but culture negative. Of the 21 culture-positive specimens, 17 (81%) grew Ureaplasma spp. and 4 (19%) grew Mycoplasma spp. Of the 28 PCR-positive specimens, Ureaplasma spp. was detected in 23 (82%), M. hominis was detected in 3 (11%), and both were detected in 2 (7%). In a confirmatory analysis, all samples were tested by amplification of a second target of the ureaplasma genome. True-positive cases were defined as a positive result by culture or by both amplification assays. The multiplex PCR detected organisms in 26 of the 30 true-positive specimens, as well as in 2 other specimens. Based on a 36% prevalence of infection, the sensitivity, specificity, and positive and negative predictive values of multiplex PCR analyses were 87, 96, 94, and 93%, respectively. Multiplex PCR offers a rapid, sensitive, and easy method to detect genital mycoplasmas.

Deep Sequencing Reveals Mixed Infection with 2009 Pandemic Influenza A (H1N1) Virus Strains and the Emergence of Oseltamivir Resistance

Mixed infections with seasonal influenza A virus strains are a common occurrence and an important source of genetic diversity. Prolonged viral shedding, as observed in immunocompromised individuals, can lead to mutational accumulation over extended periods. Recently, drug resistance was reported in immunosuppressed patients infected with the 2009 pandemic influenza A (H1N1) virus within a few days after oseltamivir treatment was initiated. To better understand the evolution and emergence of drug resistance in these circumstances, we used a deep sequencing approach to survey the viral population from an immunosuppressed patient infected with H1N1/2009 influenza and treated with neuraminidase inhibitors. This patient harbored 3 genetic variants from 2 phylogenetically distinct viral clades of pandemic H1N1/2009, strongly suggestive of mixed infection. Strikingly, one of these variants also developed drug resistance de novo in response to oseltamivir treatment. Immunocompromised individuals may, therefore, constitute an important source of genetic and phenotypic diversity, both through mixed infection and de novo mutation.

The impact of an enteroviral RT-PCR assay on the diagnosis of aseptic meningitis and patient management

BACKGROUND Enterovirus (EV) is a major cause of aseptic meningitis and non-specific febrile illness in children. Since the majority of patients are hospitalized for possible bacterial infection, a rapid test for the diagnosis of enteroviral meningitis (EVM) may reduce hospitalizations and unnecessary treatments. OBJECTIVE To review the impact of an EV reverse transcriptase-polymerase chain reaction (RT-PCR) assay for the diagnosis of EVM on patient management. STUDY DESIGN CSF from 1056 patients admitted to the hospital between 1998 and 2001 was tested using EV RT-PCR. The results were correlated with CSF counts, diagnosis, test turnaround time (TAT) and length of hospital stay (LOS). RESULTS EV RT-PCR was positive for 113 patients (11%). Of these cases, 92% occurred during the summer months and 77% were in children <19 years of age. Children <3 years old with EVM frequently had non-specific clinical findings and lacked pleocytosis. There was a significant correlation between decreasing LOS and TAT (r(2)=0.97, P<0.001). CONCLUSION RT-PCR testing for EVM is an important tool to aid in the diagnosis of children with non-specific febrile illness. This test impacted patient management as measured by shortened patient stays, which should translate into significant health care savings.

Multi-Site PCR-based CMV viral load assessment-assays demonstrate linearity and precision, but lack numeric standardization: a report of the association for molecular pathology.

Viralload (VL) assessment of cytomegalovirus (CMV) by real-time PCR is an important tool for diagnosing and monitoring CMV viremia in patients with compromised immune systems. We report results from a sample exchange organized by members of the Association for Molecular Pathology that compared PCR results from 23 laboratories; 22 such laboratories used a laboratory-developed real-time PCR assay and one laboratory used a competitive PCR assay. The samples sent to each laboratory were comprised of a dilution panel of CMV virion-derived reference materials that ranged from 0 to 500,000 copies/ml. Accuracy, linearity, and intralaboratory precision were established for the different laboratory-developed assays. Overall, PCR results were linear for each laboratory (R(2) > 0.97 in all but two). While 13 laboratories showed no significant quantitative assay bias, 10 laboratories reported VLs that were significantly different compared with expected values (bias range, -0.82 to 1.4 logs). The intralaboratory precision [mean coefficient of variance of 2% to 5% (log-scale)] suggested that changes in VLs of less than 3- to fivefold may not be significantly different. There was no significant association between laboratory-specific technical variables (PCR platform, calibrator, extraction method) and assay linearity or accuracy. These data suggested that, within each laboratory, relative VL values were linear, but additional method standardization and a CMV DNA reference standard are needed to allow laboratories to achieve comparable numeric results.

Comparison of three commercial RT-PCR systems for the detection of respiratory viruses

Abstract Background Due to the insensitivity of rapid tests for respiratory viruses, nucleic acid amplification tests are quickly becoming the standard of care. Objectives and study design The performance of the FilmArray Respiratory Panel (RP) and Verigene RV+ (RV+) were compared in a retrospective analysis of 89 clinical specimens previously determined to be positive for the following viruses by our test of record, Prodesse (Pro): influenza A (29, FluA), influenza B (13, FluB), respiratory syncytial virus (12, RSV), human metapneumovirus (10, hMPV), parainfluenza (14, PIV), and adenovirus (10, AdV). Samples positive for influenza A, B or RSV were tested by both methods, while the remainder were tested by RP only. True positives were defined as positive by two or more assays. Results Limit of detection (LOD) analyses demonstrated Pro had the lowest LOD for all FluA strains tested, PIV1, PIV2 and AdV; RV+ had the lowest LOD for FluB; and RP had the lowest LOD for RSV, PIV3 and hMPV. Of the 55 samples tested by RV+, all 54 true positive samples were positive by RV+. Of the 89 samples tested by RP, 85 of the 88 true positive samples were positive by RP. From these results, the overall sensitivities for influenza A, B and RSV were 100% and 98% for RV+ and RP, respectively. The overall sensitivity of RP for all viruses was 97%. Conclusions In summary, these systems demonstrated excellent performance. Furthermore, each system has benefits which will ensure they will all have a niche in a clinical laboratory.

Multicenter Evaluation of the Performance Characteristics of the NucliSens HIV-1 QT Assay Used for Quantitation of Human Immunodeficiency Virus Type 1 RNA

ABSTRACT The analytical performance of the NucliSens HIV-1 QT assay, a highly sensitive test based on nucleic acid sequence-based amplification technology, was evaluated in a multicenter trial. Assay specificity was evaluated with 502 plasma (EDTA) specimens from human immunodeficiency virus type 1 (HIV-1)-seronegative volunteer donors. No HIV-1 RNA was reported in any of the donor specimens. Analytical sensitivity and reproducibility were estimated with panels prepared from a high-titer well-characterized HIV-1 RNA stock (5.84 × 108 RNA copies/ml). The assays dynamic range was linear from 106 to 101 HIV-1 RNA copies, with a lower detectable limit of 25 copies/ml and a 95% detection rate of 176 copies/ml. Sensitivity of the assay to detect HIV-1 RNA in clinical specimens from patients (n = 101) and in commercially available or prepared panels (n = 24) was compared with NASBA HIV-1 RNA QT (an earlier version of NucliSens HIV-1 QT) and with the Food and Drug Administration-approved standard and ultrasensitive AMPLICOR HIV-1 MONITOR, version 1.0, assays. Detection of HIV-1 RNA was reproducible over a 5-log range (mean standard deviation = 0.15 log). The NucliSens and the standard AMPLICOR assays were equivalent in detection of HIV-1 RNA (concentration, 103 to 105 copies/ml) in 57 clinical specimens. The NucliSens assay was more sensitive in detecting HIV-1 RNA at lower concentrations (≤102 copies/ml) (44 of 44) than either the standard AMPLICOR test (12 of 19) or the NASBA assay (10 of 25). A 25% increase in HIV-1 RNA detection frequency with panels was observed with the NucliSens assay (23 of 24) compared with the standard AMPLICOR test (17 of 24). The new assay was highly specific and demonstrated good sensitivity with a broad linear dynamic range.

A one-step RT-PCR assay using an enzyme-linked detection system for the diagnosis of enterovirus meningitis

BACKGROUND Enteroviruses are the most common cause of meningitis in the United States, with an estimated 50000-75000 cases each year. Enteroviral meningitis (EVM) is frequently a diagnosis of exclusion, as viral cultures lack sensitivity and require prolonged incubation periods. OBJECTIVE To develop a sensitive and rapid test for the diagnosis of EVM. STUDY DESIGN A rapid, one-step, reverse transcriptase-polymerase chain reaction (RT-PCR) was used in a prospective analysis of 160 patients who had cerebrospinal fluid (CSF) tested for enterovirus. RESULTS Of the 160 patients, 14 were excluded due to missing CSF viral culture data. A total of 14 were CSF culture positive (10 with pleocytosis) and 19 were PCR positive (15 with pleocytosis). The ability to detect enterovirus by either culture or PCR correlated significantly with the white blood cell count in the CSF (P=0.001). Based on a clinical definition of enterovirus culture positive and pleocylosis: ten had definite EVM and 12 had probable EVM (pleocytosis without any other cause). Four had possible EVM (CSF culture positive without pleocytosis) and 18 had pleocytosis due to other causes. PCR was positive in all ten patients with definite EVM. Five out of 12 patients with probable EVM and three out of four patients with possible EVM. No patients with pleocytosis due to other causes were PCR positive and one patient that was defined as EVM negative (culture negative and no pleocytosis) was PCR positive. Overall, PCR was positive in 18 out of the 26 patients with a likelihood of EVM, while CSF culture was positive in only 14 cases. Our results demonstrated that RT-PCR enhances the sensitivity of enterovirus detection in CSF (69 vs. 54% for culture). CONCLUSION The diagnosis of EVM is difficult to make clinically: the enhanced sensitivity and rapid turn around time of PCR will be of great clinical benefit.

Low Prevalence of Listeria monocytogenes in Human Stool

Listeria monocytogenes is a foodborne pathogen that is found widely in the environment and in a variety of ready-to-eat foods, yet human invasive infection is relatively rare (five cases per million people annually in the United States). Despite wide exposure to this organism, little is known about the prevalence of L. monocytogenes in human stool, and it is not known whether human fecal dispersal contributes to human foodborne transmission. We cultured 827 stool specimens (well formed and loose-watery) from individuals from four large metropolitan areas of New York state for L. monocytogenes and found only 1 (0.12%) positive specimen. L. monocytogenes was also isolated from the blood of the person with the single positive specimen, and the two isolates were indistinguishable by molecular subtyping (both were ribotype DUP-1042B). This provides further evidence that human L. monocytogenes fecal carriage among persons with and without diarrheal disease is remarkably low. Unlike the case for other foodborne pathogens (e.g., Salmonella), human shedders probably do not contribute significantly to L. monocytogenes contamination of foods. However, we observed a single individual with invasive listeriosis that shed the pathogen in feces, indicating the potential for fecal dispersal of L. monocytogenes from persons with listeriosis.

Premarket Evaluations of the IMDx C. difficile for Abbott m2000 Assay and the BD Max Cdiff Assay

ABSTRACT Clostridium difficile-associated diarrhea is a well-recognized complication of antibiotic use. Historically, diagnosing C. difficile has been difficult, as antigen assays are insensitive and culture-based methods require several days to yield results. Nucleic acid amplification tests (NAATs) are quickly becoming the standard of care. We compared the performance of two automated investigational/research use only (IUO/RUO) NAATs for the detection of C. difficile toxin genes, the IMDx C. difficile for Abbott m2000 Assay (IMDx) and the BD Max Cdiff Assay (Max). A prospective analysis of 111 stool specimens received in the laboratory for C. difficile testing by the laboratorys test of record (TOR), the BD GeneOhm Cdiff Assay, and a retrospective analysis of 88 specimens previously determined to be positive for C. difficile were included in the study. One prospective specimen was excluded due to loss to follow-up discrepancy analysis. Of the remaining 198 specimens, 90 were positive by all three methods, 9 were positive by TOR and Max, and 3 were positive by TOR only. One negative specimen was initially inhibitory by Max. The remaining 95 specimens were negative by all methods. Toxigenic C. difficile culture was performed on the 12 discrepant samples. True C. difficile-positive status was defined as either positive by all three amplification assays or positive by toxigenic culture. Based on this definition, the sensitivity and specificity were 96.9% and 95% for Max and 92.8% and 100% for IMDx. In summary, both highly automated systems demonstrated excellent performance, and each has individual benefits, which will ensure that they will both have a niche in clinical laboratories.

The Use of Intravenous Palivizumab for Treatment of Persistent RSV Infection in Children With Leukemia

Palivizumab is a humanized monoclonal antibody used to decrease the threat of respiratory syncytial virus (RSV) infection among children at high risk. There are no standard guidelines due to conflicting data on palivizumab’s use in the treatment of RSV lower respiratory tract infections. Intravenous (IV) palivizumab was shown to be well tolerated and associated with decreased mortality in high-risk children who have RSV disease. However, it did not prevent lower respiratory tract infections and did not affect the survival rate of allogeneic stem cell transplant recipients who had RSV infection. We present 2 children with acute lymphocytic leukemia (ALL) and persistent RSV infection while receiving chemotherapy. Patient A is a 4-year-old male with Down syndrome, ALL, and persistent RSV infection for at least 3 months. Patient B is a 3-year-old female with pre–B cell ALL whose chemotherapy intensification phase was delayed due to a month-long RSV infection. RSV infections were determined by using real-time polymerase chain reaction assays from nasopharyngeal swabs before IV palivizumab therapy; patient A was positive for RSV at 36 cycles and patient B was positive for RSV at 29 cycles. RSV infection was cleared in both patients within 72 hours after receiving IV palivizumab (patient A: 16 mg/kg; patient B: 15 mg/kg). IV palivizumab may be a treatment option for persistent RSV infection among immunocompromised patients.