Frederick S. Nolte

Risk Factors for Perinatal Transmission of Hepatitis C Virus (HCV) and the Natural History of HCV Infection Acquired in Infancy

BACKGROUND The goal of the present study was to assess risk factors for perinatal hepatitis C virus (HCV) transmission and the natural history of infection among HCV-infected infants. METHODS In a cohort study, 244 infants born to HCV-positive mothers were followed from birth until age > or =12 months. Maternal serum was collected at enrollment and delivery; infant serum was collected at birth and at 8 well-child visits. Testing included detection of antibody to HCV, detection of HCV RNA (qualitative and quantitative), and genotyping. HCV-infected infants were followed annually until age 5 years. RESULTS Overall, 9 of 190 (4.7% [95% confidence interval (CI), 2.3%-9.1%]) infants born to mothers who were HCV RNA positive at delivery became infected, compared with 0 of 54 infants born to HCV RNA-negative mothers (P=.10). Among HCV RNA-positive mothers, the rate of transmission was 3.8% (95% CI, 1.7%-8.1%) from the 182 who were human immunodeficiency virus (HIV) negative, compared with 25.0% (95% CI, 4.5%-64.4%) from the 8 who were HIV positive (P<.05). Three infected infants resolved their infection (i.e., became HCV RNA negative). In multivariate analysis restricted to HCV RNA-positive mothers, membrane rupture > or =6 h (odds ratio [OR], 9.3 [95% CI, 1.5-179.7]) and internal fetal monitoring (OR, 6.7 [95% CI, 1.1-35.9]) were associated with transmission of HCV to infants. CONCLUSION If duration of membrane rupture and internal fetal monitoring are confirmed to be associated with transmission, interventions may be possible to decrease the risk of transmission.

MultiCode-PLx System for Multiplexed Detection of Seventeen Respiratory Viruses

ABSTRACT The MultiCode-PLx system (EraGen Biosciences, Inc., Madison, WI) for the detection of respiratory viruses uses an expanded genetic alphabet, multiplex PCR chemistry, and microsphere flow cytometry to rapidly detect and specifically identify 17 different respiratory viruses directly in clinical specimens. The MultiCode-PLx system was tested in parallel with direct fluorescent-antibody (DFA) staining and rapid shell vial culture (R-mix cells; Diagnostic Hybrids, Inc. Athens, OH) with 354 respiratory specimens from adult patients that were submitted to the clinical virology laboratory at the Emory University Hospital. Single-target PCRs were performed with retained samples to confirm the positive results obtained with the MultiCode-PLx system for viruses not covered by DFA and R-mix culture (metapneumovirus, coronaviruses [CoV], parainfluenza viruses 4a and 4b, and rhinoviruses) and to resolve any discrepancies between the DFA and R-mix culture and the MultiCode-PLx results for viruses common to both systems. Respiratory viruses were detected in 77 (21.8%) and 116 (32.7%) specimens by DFA and R-mix culture and with the MultiCode-PLx system, respectively. Among the viruses common to both systems, the MultiCode-PLx system detected significantly more influenza A viruses (P = 0.0026). An additional increased diagnostic yield with the MultiCode-PLx system resulted from the detection of human metapneumovirus (HMPV) in 9 specimens, human CoV (HCoV) in 3 specimens, and human rhinovirus (HRV) in 16 specimens. Also, two mixed viral infections were detected by the MultiCode-PLx system (HCoV OC43 and HRV infections and HMPV and HRV infections), but none were detected by DFA and R-mix culture. Single-target PCRs verified the results obtained with the MultiCode-PLx system for 73 of 81 (90.1%) specimens that had discordant results or that were not covered by DFA and R-mix culture. The MultiCode-PLx system provides clinical laboratories with a practical, rapid, and sensitive means for the massively multiplexed molecular detection of common respiratory viruses.

Clinical Evaluation of Two Methods for Genotyping Hepatitis C Virus Based on Analysis of the 5′ Noncoding Region

ABSTRACT We compared the performance characteristics of a standardized direct sequencing method (TRUGENE HCV 5′NC; Visible Genetics Inc., Toronto, Ontario, Canada) and a reverse hybridization line probe assay (INNO-LiPA HCV II; Bayer Corp., Tarrytown, N.Y.) for genotyping of hepatitis C virus (HCV). Both methods are based on detection of sequence heterogeneity in the 5′ noncoding (5′NC) region. Concordance between the genotyping methods was assessed by testing 172 samples representing the six major genotypes. Sequence analysis of the more phylogenetically informative nonstructural 5B (NS5B) region was also done with 148 (86%) samples to confirm the accuracy of and resolve discrepancies between the 5′NC genotyping results. The sensitivities of the methods were assessed by using the 5′NC amplicon from both the qualitative and quantitative AMPLICOR HCV tests (Roche Diagnostics Corp., Indianapolis, Ind.). The ability of the methods to detect mixed-genotype infections was determined with mixtures of two different genotypes at relative concentrations ranging from 1 to 50%. Both 5′NC methods were able to genotype 99.4% of the samples with type agreement for 99.5% and subtype agreement for 68.2% of the samples. No or ambiguous subtype results were found by the line probe assay for 16.5% and by the TRUGENE 5′NC test for 17.1% of the samples. Discrepancies occurred between the line probe assay and NS5B results at the type level for 1.4% of the samples and at the subtype level for 14.2% of the samples. Discrepancies also occurred between the TRUGENE 5′NC and NS5B results at the type level for 2% of the samples and at the subtype level for 8.1% of the samples. We also found two distinct strains of HCV classified as type 2 by analysis of the 5′NC region that were type 1 by analysis of the NS5B region. The sensitivities of the two 5′NC genotyping methods were comparable and dependent on the amplification test used (∼103 IU/ml with the qualitative HCV RNA tests and ∼105 IU/ml with the quantitative HCV RNA tests). Genotype mixtures were successfully identified at a relative concentration of 5% by the line probe assay and 10% by the TRUGENE 5′NC test. In conclusion, the performance characteristics of the 5′NC methods were similar and both methods produced accurate results at the genotype level but neither method should be used for subtyping.

Multicenter beta trial of the GeneXpert enterovirus assay.

ABSTRACT The GeneXpert Dx system (Cepheid, Sunnyvale, CA) is a fully integrated and automated nucleic acid sample preparation, amplification, and real-time detection system. It consists of an instrument, a personal computer, and disposable fluidic cartridges. The analytical sensitivity and specificity of the GeneXpert enterovirus assay (GXEA) were determined with a panel of 63 different enterovirus serotypes and 24 other microorganisms, respectively. The potential for blood, hemoglobin, white blood cells, and excess protein to interfere with the assay was also assessed. The performance parameters of the GXEA were determined at three sites with 102 cerebrospinal fluid (CSF) samples obtained from patients with suspected meningitis. All samples were tested for enterovirus RNA with locally developed reverse transcription-PCR (RT-PCR) assays at the trial sites and with a seminested RT-PCR and an analyte-specific reagent (Cepheid) at a reference laboratory. The 5′ nontranslated region was the target for all of the PCR assays except the seminested RT-PCR, which amplified a VP1 sequence. The VP1 amplicon was sequenced to identify the enterovirus types. Consensus reference laboratory RT-PCR results were used to classify cases of enteroviral meningitis. The GXEA detected all of the enterovirus serotypes and none of the other microorganisms tested except rhinovirus 16. The assay was unaffected by moderate amounts of blood or blood components. Thirty-six (35%) of the CSF samples tested had at least one positive PCR result. Eleven different enterovirus serotypes were identified in the positive samples. The GXEA had a sensitivity of 97.1% (95% confidence interval [CI], 84.7 to 99.9%) and a specificity of 100% (95% CI, 94.6 to 100%) for the diagnosis of enteroviral meningitis.

Branched DNA Signal Amplification for Direct Quantitation of Nucleic Acid Sequences in Clinical Specimens

In this chapter I have reviewed the development of bDNA as a method for quantitation of nucleic acid targets and the application of this technology to the study of infectious diseases and cell biology. The ability to quantify viral nucleic acids in clinical specimens has led to a better understanding of the pathogenesis of chronic viral infections such as HIV-1, HCV, and HBV. The information provided by these methods can also be important in the management of patients with these infections. The prognostic value of a single baseline HIV-1 RNA level rivals that surgical staging procedures for cancer, which are among the most powerfully predictive tests in medicine (Mellors et al., 1996). These methods have been used to assess rapidly the effects of antiviral therapy, which has both expedited the development of antiviral drugs and improved the management of patients with HIV-1 and HCV infections. bDNA has several characteristics that distinguish it from the quantitative target amplification systems, including better tolerance of target sequence variability, more direct measurement of target, simpler sample preparation, and less sample-to-sample variation. However, the first- and second-generation bDNA assays lacked sensitivity compared with the target amplifications systems. The changes incorporated into the third-generation assays have effectively increased the signal-to-noise ratio to such a high level that the analytical sensitivity of system 8 bDNA approaches that of PCR. In theory, bDNA can be made even more sensitive by increasing both the sample volume and the signal-to-noise ratio. Nonspecific hybridization can be further reduced by finding more effective blockers for the solid phase or by redesigning the amplifier molecule or the solid phase itself. The increased sensitivity may create new applications for the technology in filter and in situ hybridization assays.

Development and Evaluation of a Real-Time PCR Assay for Detection of Klebsiella pneumoniae Carbapenemase Genes

ABSTRACT We developed a novel real-time PCR assay to detect Klebsiella pneumoniae carbapenemases (KPCs) and used this assay to screen clinical isolates of K. pneumoniae and Klebsiella oxytoca for the presence of blaKPC genes. The TaqMan real-time PCR assay amplified a 399-bp product from the blaKPC gene. The amplicon was designed so that the genes for isoenzymes KPC-1, -2, and -3 could be easily distinguished by subsequent restriction digestion of the amplicon with the enzymes BstNI and RsaI. The assay was validated with reference strains obtained from the Centers for Disease Control and Prevention that contained each of the three described isoenzymes and 69 extended-spectrum β-lactamase-producing clinical isolates (39 K. pneumoniae and 30 K. oxytoca isolates). Subsequently, the blaKPC PCR assay was used to confirm the presence of blaKPC genes in any meropenem-resistant Klebsiella spp. The PCR assay detected blaKPC in all of the reference strains, in 6 of 7 meropenem-resistant isolates, and in 0 of 62 meropenem-susceptible clinical isolates. The PCR assay was then used to confirm the presence of blaKPC in an additional 20 meropenem-resistant isolates from 16 patients. Restriction digestion of the PCR amplicons identified two blaKPC gene variants in our patient population: 9 isolates with C and 17 with T at nucleotide 944, consistent with blaKPC-2 and blaKPC-3, respectively. The real-time PCR assay is a rapid and accurate method to detect all KPC isoenzymes and was useful in documenting the presence and dissemination of KPC-producing strains in our patient population.

Molecular Diagnostics for Detection of Bacterial and Viral Pathogens in Community-Acquired Pneumonia

Abstract Traditional microbiological methods for detection of respiratory tract pathogens can be slow, are often not sensitive, may not distinguish infection from colonization, and are influenced by previous antibiotic therapy. Molecular diagnostic tests for common and atypical causative pathogens of community-acquired pneumonia have the potential to dramatically increase the diagnostic yield and decrease the time required to render results. Unfortunately, these tests often lack standardization and are not widely available. Consideration should be given to the development and evaluation of companion molecular diagnostic tests for detection of respiratory pathogens in future clinical trials of antimicrobials intended to treat community-acquired pneumonia.

Furunculosis Due to Mycobacterium mageritense Associated with Footbaths at a Nail Salon

ABSTRACT We report two cases of lower-extremity furunculosis caused by Mycobacterium mageritense. Both patients were patrons of the same nail salon, where they received footbaths prior to pedicures. M. mageritense bacteria isolated from two whirlpool footbaths were determined to be closely related to the patient isolates by pulsed-field gel electrophoresis.

Evaluation of a Multiplexed PCR Assay for Detection of Respiratory Viral Pathogens in a Public Health Laboratory Setting

ABSTRACT There are numerous viral and bacterial causes of respiratory disease. To enable rapid and sensitive detection of even the most prevalent causes, there is a need for more-simplified testing systems that enable researchers and clinicians to perform multiplexed molecular diagnostics quickly and easily. To this end, a new multiplexed molecular test called the MultiCode-PLx respiratory virus panel (PLx-RVP) was developed and then implemented in a public health laboratory setting. A total of 687 respiratory samples were analyzed for the presence of 17 viruses that commonly cause respiratory disease. As a comparator, the samples were also tested using a standard testing algorithm that included the use of a real-time influenza virus A and B reverse transcription-PCR test and routine viral culture identification. The standard testing algorithm identified 503 (73%) samples as positive and 184 as negative. Analyzing the same 687 samples, the PLx-RVP assay detected one or more targets in 528 (77%) samples and found 159 samples negative for all targets. There were 25 discordant results between the two systems; 14 samples were positive for viruses not routinely tested for by the Wisconsin State Laboratory of Hygiene, and 13 of these were confirmed by real-time PCR. When the results of the standard testing algorithm were considered “true positives,” the PLx-RVP assay showed an overall sensitivity of 99% and an overall specificity of 87%. In total, the PLx-RVP assay detected an additional 40 viral infections, of which 11 were mixed infections.

A multicenter, randomized trial of daily high-dose interferon-alfa 2b for the treatment of chronic hepatitis C : Pretreatment stratification by viral burden and genotype

A multicenter, randomized trial of daily high-dose interferon-alfa 2b for the treatment of chronic hepatitis C: pretreatment stratification by viral burden and genotype