Persing, David H.

ABSTRACTBackground:The cause of fulminant hepatic failure in children remains unknown, but a viral origin has been suspected in most cases. The recently discovered blood‐borne virus, hepatitis G, has been suggested as a possible causative agent.Method:Six consecutive children who underwent liver transplantation for fulminant hepatic failure were studied. The children were tested for hepatitis G virus antibodies and hepatitis G virus RNA by polymerase chain reaction after excluding other causes of fulminant hepatic failure.Results:No evidence of hepatitis G virus infection was found in these patients.Conclusion:Hepatitis G virus is unlikely to be a common cause of fulminant hepatic failure in pediatric patients from the upper midwestern United States.

ABSTRACT Human babesiosis in the United States is caused predominantly by Babesia microti , a tick-transmitted blood parasite. Improved testing methods for the detection of infection with this parasite are needed, since asymptomatic B. microti infection represents a potential threat to the blood supply in areas where B. microti is endemic. We performed immunoscreening of an expression library of genomic DNA from a human isolate of B. microti (strain MN1). Among 17 unique immunoreactive clones, we identified 9 which represent a related family of genes with little sequence homology to other known sequences but with an architecture resembling that of several surface proteins of Plasmodium . Within this family, a tandem array of a degenerate six-amino-acid repeat (SEAGGP, SEAGWP, SGTGWP, SGTVGP) was found in various lengths between relatively well conserved segments at the N and C termini. In order to examine within-clone variation, we developed a PCR protocol for direct recovery of a specific bmn1-6 homologue directly from 30 human blood isolates, 4 corresponding hamster isolates, and 5 geographically corresponding Peromyscus leucopus (white-footed mouse) isolates. Isolates from the hamsters had the same sequences as those found in the corresponding human blood, suggesting that genetic variation of bmn1-6 does not occur during passage. However, clones from different patients were often substantially different from each other with regard to the number and location of the degenerate repeats within the bmn1-6 homologue. Moreover, we found that strains that were closely related geographically were also closely related at the sequence level; nine patients, all from Nantucket Island, Mass., harbored clones that were indistinguishable from each other but that were distinct from those found in other northeastern or upper midwestern strains. We conclude that considerable genetic and antigenic diversity exists among isolates of B. microti from the United States and that geographic clustering of subtypes may exist. The nature of the bmn1-6 gene family suggests a mechanism of antigenic variation in B. microti that may occur by recombination, differential expression, or a combination of both mechanisms.

Abstract Over the past several years, the development and application of molecular diagnostic techniques has initiated a revolution in the diagnosis and monitoring of infectious diseases. Microbial phenotypic characteristics, such as protein, bacteriophage, and chromatographic profiles, as well as biotyping and susceptibility testing, are used in most routine laboratories for identification and differentiation. Nucleic acid techniques, such as plasmid profiling, various methods for generating restriction fragment length polymorphisms, and the polymerase chain reaction (PCR), are making increasing inroads into clinical laboratories. PCR-based systems to detect the etiologic agents of disease directly from clinical samples, without the need for culture, have been useful in rapid detection of unculturable or fastidious microorganisms. Additionally, sequence analysis of amplified microbial DNA allows for identification and better characterization of the pathogen. Subspecies variation, identified by various techniques, has been shown to be important in the prognosis of certain diseases. Other important advances include the determination of viral load and the direct detection of genes or gene mutations responsible for drug resistance. Increased use of automation and user-friendly software makes these technologies more widely available. In all, the detection of infectious agents at the nucleic acid level represents a true synthesis of clinical chemistry and clinical microbiology techniques.

Until recently, the laboratory diagnosis of central nervous system (CNS) infections with herpes simplex virus (HSV) has been limited by poor sensitivity and/or specificity. We assessed the diagnostic utility of PCR for detection of HSV in over 2,100 specimens referred to the Mayo Clinic from August 1993 to May 1996. DNA extracted from cerebrospinal fluid (CSF) samples with IsoQuick was amplified by PCR with oligonucleotide primers directed to the DNA polymerase gene of HSV, yielding a 290-bp amplicon. HSV DNA was detected in 150 (135 by gel electrophoresis, 15 by Southern blotting only) of 2,106 (7.1%) specimens. PCR-positive CNS disease occurred in patients ranging in age from 13 days to 89 years; 59% of the cases occurred in patients between the ages of 30 and 69, and 21 (14%) of the patients were infants. Genotype analysis was not routinely performed; however, amplification of a 335-bp product within the thymidine kinase gene of HSV revealed 13 positions within a span of 80 nucleotides that accurately identified the two serotypes of the virus according to 14 reference strains. We conclude that PCR detection of HSV DNA in CSF specimens should be considered an emerging "gold standard" for the laboratory diagnosis of CNS infections with this virus.

A second-generation recombinant immunoblot assay (RIBA 2.0) is used in the United States to confirm infection with hepatitis C virus (HCV) in samples that are anti-HCV (enzyme immunoassay) positive. In some cases, indeterminate results of RIBA 2.0, which are defined as reactivity to a single antigen species or reactivity limited to two proteins derived from the same coding region of the HCV genome, are encountered. This study was performed to establish the significance of indeterminate RIBA 2.0 results in relation to HCV RNA detection, high positivity for the c22-3 band, and the HCV genotype as determined by direct DNA sequencing. Ninety-six samples with indeterminate RIBA 2.0 results were studied. HCV RNA was detected in 21 of 34 (62%) samples with high reactivity to c22-3 and in 8 of 62 (13%) samples with low reactivity to c22-3. The HCV genotype distribution in samples that were RIBA 2.0 indeterminate and HCV RNA positive was significantly different from that in samples of a control group with positive results for both the RIBA 2.0 and HCV PCR. These results suggest that highly positive c22-3 samples are likely to be associated with HCV viremia and that infection with less common HCV genotypes is more commonly associated with indeterminate RIBA 2.0 results.

Recombinant Lyme disease vaccines based on purified preparations of outer surface protein A (OspA) have been shown to be effective in preventing transmission of Borrelia burgdorferi in experimental animal models and are now being tested in humans. Since the most widely used screening tests for Lyme disease are based on a whole-cell sonicate of B. burgdorferi, serologic false positivity in vaccinated persons could result from reactivity to OspA within the antigen preparation. In order to avoid serologic false positivity in vaccinated subjects, we developed an immunoassay based on a low-passage-number, naturally occurring variant of B. burgdorferi which lacks the plasmid encoding OspA and OspB. The use of an antigen preparation derived from this organism provided sensitive and specific detection of B. burgdorferi seropositivity in experimental animals and in human Lyme disease cases. The OspA-B-negative enzyme-linked immunosorbent assay (ELISA) also appeared to be capable of discriminating the vaccinated state from vaccine failure and natural infection in experimental animals. Sera from human subjects participating in a vaccine trial gave false-positive results with an ELISA based on an OspA-containing strain, but no such reactivity was observed when the OspA-negative ELISA was used. We conclude that low-passage-number OspA-B-negative isolates in immunoassays may become useful for the immunologic discrimination of the vaccinated state, natural infection, and vaccine failure.

A new solution-phase DNA hybridization capture assay for the rapid detection of the mecA gene in clinical isolates of Staphylococcus was compared with multiplex PCR and disk diffusion methods. The assay uses a DNA capture probe immobilized on paramagnetic particles and a second DNA probe labeled with an acridinium ester. Bacteria from 24-h cultures are lysed, and the lysates are hybridized with the DNA probes. After magnetic separation to remove unhybridized labeled probe, the mecA gene is detected by the chemiluminescence of the hybridized probe. Four hundred consecutive staphylococcal isolates were assayed, including 147 S. aureus and 253 coagulase-negative Staphylococcus isolates. Among the S. aureus isolates, 14 of 147 were MecA+ by both the hybridization capture assay and PCR; 133 of 147 were MecA negative by both assays (positive and negative predictive values, 100%). Comparison of disk diffusion results with those obtained by genotypic methods indicated that 14 of 147 S. aureus isolates judged to be resistant were positive by both methods; 119 of 147 were Oxs and negative by both genotypic methods (positive and negative predictive values, 50 and 100%, respectively). The remaining 14 S. aureus isolates were MecA- Oxr; among these, 13 were beta-lactamase hyperproducers. For coagulase-negative staphylococci, 130 of 253 were MecA+ by the hybridization capture assay; 129 of 130 of these isolates were positive by PCR (positive and negative predictive values, 99.2 and 100%, respectively). Comparison with the disk diffusion assay showed that 128 of the coagulase-negative MecA+ isolates were Oxr; 111 of 253 were MecA- and Oxs (positive and negative predictive values, 90.8 and 99.1%, respectively). Thirteen coagulase-negative isolates were MecA-Oxr; among these, three were beta-lactamase hyperproducers. Comparison of the hybridization capture assay results with PCR indicates that the DNA hybridization assay is a sensitive and specific test for the detection of the mecA gene in clinical isolates of Staphylococcus.

Detection of mutations in the rpoB gene of Mycobacterium tuberculosis can be used as an accurate predictor of rifampin resistance in the majority of strains tested. Simple but highly accurate screening methods must be developed for the detection of these mutations. Either DNA sequence analysis or single-strand conformation polymorphism (SSCP) screening can be used to detect rpoB mutations, but these techniques either are expensive or yield results that may prove difficult to interpret when used in a clinical setting. This report describes the use of dideoxy fingerprinting (ddF) as a postamplification screening method to identify rifampin-resistant genotypes. The ddF protocol was performed on the amplified rpoB fragment with no preparatory steps, thus making ddF practical for laboratories equipped for polyacrylamide gel electrophoresis. When compared with the results of SSCP analysis, ddF results were more easily interpreted and contained more sequence-dependent information that facilitated differentiation of functionally significant and silent mutations. The ddF method was used for genotypic determination of rifampin susceptibility of 20 multidrug-resistant strains of M. tuberculosis. The results of this analysis were concordant with DNA sequence analysis and conventional clinical laboratory methods.