Hepatitis C virus (HCV) genotyping of samples from 184 patients with chronic HCV infection by the Trugene 5NC genotyping kit, based on sequence analysis of the 5 noncoding region (5 NCR), and the InnoLiPA assay was evaluated. In addition to these methods, the 184 samples were also analyzed by sequencing of part of the NS5B of the HCV genome after in-house PCR amplification, as a means of validating results obtained with the 5 NCR. The distribution of the genotypes typed by NS5B sequence analysis was as follows: 1a, 41 samples; 1b, 58 samples; 1d, 1 sample; 2a, 5 samples; 2b, 2 samples; 2c, 7 samples; 3a, 46 samples; 4a, 7 samples; 4c, 1 samples; 4e, 9 samples; 5a, 6 samples; 6a, 1 sample. The Trugene and InnoLiPA assays gave concordant results within HCV types in 100% of cases. The ability to discriminate at the subtype level was 76 and 74% for the Trugene and the InnoLiPA assays, respectively.Hepatitis C virus (HCV) is considered the major cause of posttransfusion non-A, non-B hepatitis. The viral genome, a positive-sense single-stranded RNA of about 9,400 nucleotides (5), is characterized by a high genetic heterogeneity like other RNA viruses. HCV isolates show four levels of genetic variability: types, subtypes, isolates, and quasispecies (3). An HCV genotype is therefore used with the histological results from liver biopsy and viral load for counseling individual patients about the risk-benefit ratio of therapy (17,21,22). HCV genotypes are distributed differently depending on geography and the etiology of infection (15,25). For the purpose of nomenclature, it has been proposed that HCV be classified into types, corresponding to the main branches in the phylogenetic tree, and subtypes, corresponding to the more related sequences within the major groups (23,24). HCV genotypes can be established by methods based on PCR typing and/or serological typing (2, 4, 16). The high degree of conservation in the 5Ј noncoding regions (5Ј NCR) has made it the target of choice for reverse transcriptase PCR-based detection assays. Moreover, several PCR typing methods, such as reverse dot blot (26), restriction fragment length polymorphism (10), cleavase fragment length polymorphism, dideoxy fingerprinting, heteroduplex mobility analysis (28), and hybridization to genotype-specific probes (18), exploit sequence-based differences and/or differences in secondary structure of the 5Ј NCR for HCV genotyping (9). Nucleotide sequence analysis is the reference method for identifying different genotypes of HCV (9, 13). However, because this method is expensive and timeconsuming and requires special equipment for sequencing, it has been restricted to the research setting and considered impractical for large clinical studies. A standardized sequencing assay has recently been developed for routine determination of HCV genotypes. In order to determine whether direct sequencing could be a routine tool for the determination of hepatitis C virus genotype, we assessed this first commercial type of HCV genotyping method (Trugene 5ЈNC HCV genot...

Cystic lymphoepithelial lesions of salivary glands (CLLSG) are nodular or diffuse salivary gland enlargements that are observed in patients who tested positive for human immunodeficiency virus type 1 (HIV-1). Two cases of CLLSG are reported. Particular emphasis is placed on the presence of HIV-1 major-core protein (P24), HIV-1 RNA sequences, Epstein-Barr virus (EBV) DNA sequences, and lymphocyte receptor gene rearrangement. Lymphoid alterations consisted of explosive hyperplasia with a prominent follicular reticular dendritic cell (DRC) network and numerous intrafollicular CD8+ lymphocytes. Intrafollicular DRC strongly expressed HIV-1 major-core protein and HIV-1 RNA, indicating that most DRCs actively replicated the HIV-1 virus. The presence of active HIV-1 replication within DRC and the absence of clonal EBV infected lymphoid population strongly suggest that CLLSG pathogenesis is primarily induced by HIV-1. The presence of oligoclonal immunoglobulin gene rearrangements in our cases, however, suggest the need of long-term follow-up of such patients to determine whether CLLSG could be a benign prelymphomatous disease.

Groups of 3, 17, and 28-day-old Swiss mice were inoculated intracerebrally with JHM virus, the neurotropic strain of mouse hepatitis virus (MHV), and studied serially by virologic and morphologic techniques. Beginning 2--5 days post-inoculation, all groups of infected mice developed CNS lesions which were destructive in the 3-day-old group and demyelinative in the 17 and 28-day-old animals. Infectious virus could be isolated from the brain, spinal cord, and liver. Electron microscopy demonstrated the virus to be pantropic in the CNS with virions occurring within ependymal cells, astrocytes, neurons, oligodendrocytes, endothelial cells, and cell of haematogenous origin. Giant cell formation was a constant feature. In regions of demyelination, oligodendrocytes exhibited a propensity to proliferative aberrant membrane. Myelin degradation was accompanied by membrane vesiculation and by the stripping action of macrophages. The lesions were not due to CNS elements in the inoculum since in animals inoculated with normal CNS suspensions from appropriate age groups failed to show lesions. The morphogenesis of JHM virions was followed ultrastructurally as was the formation of syncytia in the different cell types. In addition to delineating virus morphogenesis and myelin pathology, the results underscore the pantropic nature of JHM virus in the CNS, the synstemic nature of the infection, and that oligodendrocytes were the principal targets.

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Background-The extent to which the prognosis for AIDS and death of patients initiating highly active antiretroviral therapy (HAART) continues to be affected by their characteristics at the time of initiation (baseline) is unclear.Methods-We analyzed data on 20,379 treatment-naive HIV-1-infected adults who started HAART in 1 of 12 cohort studies in Europe and North America (61,798 person-years of followup, 1844 AIDS events, and 1005 deaths).Results-Although baseline CD4 cell count became less prognostic with time, individuals with a baseline CD4 count <25 cells/µL had persistently higher progression rates than individuals with a baseline CD4 count >350 cells/µL (hazard ratio for AIDS = 2.3, 95% confidence interval [CI]: 1.0 to 2.3; mortality hazard ratio = 2.5, 95% CI: 1.2 to 5.5, 4 to 6 years after starting HAART). Rates of AIDS were persistently higher in individuals who had experienced an AIDS event before starting HAART. Individuals with presumed transmission by means of injection drug use experienced substantially higher rates of AIDS and death than other individuals throughout follow-up (AIDS hazard ratio = 1.6, 95% CI: 0.8 to 3.0; mortality hazard ratio = 3.5, 95% CI: 2.2 to 5.5, 4 to 6 years after starting HAART).Conclusions-Compared with other patient groups, injection drug users and patients with advanced immunodeficiency at baseline experience substantially increased rates of AIDS and death up to 6 years after starting HAART.

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Our objective was to study possible etiologic factors of asymmetric periflexural exanthem of childhood (APEC) among a large panel of microbiologic agents not yet investigated. To do so, we designed a prospective case-control study using throat, stool, blood, and skin samples, and enlisted 37 children with APEC and 37 age-matched controls without eruption seen consecutively from February 1995 to April 1996 from a mixed referral center and community-based population. No interventions were done. Used as the main outcome measure was the differences in the two groups for microbiologic investigations. No significant statistical differences between cases and controls for virus and bacteria investigated were found. No microorganism was identified as a possible etiologic agent in any of the APEC patients. APEC is not a nonspecific cutaneous eruptive pattern to several common microbiologic agents. More sophisticated molecular approaches are needed to address its etiology.

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