Transmission of Hepatitis C Virus From Organ Donors Despite Nucleic Acid Test Screening.

Nucleic acid testing (NAT) for hepatitis C virus (HCV) is recommended for screening of organ donors, yet not all donor infections may be detected. We describe three US clusters of HCV transmission from donors at increased risk for HCV infection. Donor's and recipients' medical records were reviewed. Newly infected recipients were interviewed. Donor-derived HCV infection was considered when infection was newly detected after transplantation in recipients of organs from increased risk donors. Stored donor sera and tissue samples were tested for HCV RNA with high-sensitivity quantitative PCR. Posttransplant and pretransplant recipient sera were tested for HCV RNA. Quasispecies analysis of hypervariable region-1 was used to establish genetic relatedness of recipient HCV variants. Each donor had evidence of injection drug use preceding death. Of 12 recipients, 8 were HCV-infected-6 were newly diagnosed posttransplant. HCV RNA was retrospectively detected in stored samples from donor immunologic tissue collected at organ procurement. Phylogenetic analysis showed two clusters of closely related HCV variants from recipients. These investigations identified the first known HCV transmissions from increased risk organ donors with negative NAT screening, indicating very recent donor infection. Recipient informed consent and posttransplant screening for blood-borne pathogens are essential when considering increased risk donors.

CD4- and CD3-T lymphocyte reference values of immunocompetent urban and rural subjects in an African nation.

Studies on the reference values of CD4 and CD3 T cells in healthy individuals have continued to gain significance because of the importance of these immunological markers in the initiation of combination antiretroviral therapy (cART). The aim of the present study was to determine and compare the reference values of CD4 and CD3 T cells in urban and rural Nigerians who were human immunodeficiency virus (HIV), hepatitis B virus (HBV) and hepatitis C virus (HCV) negative. After ethical clearance and informed consent, 1123 subjects who met the inclusion criteria [mean age = 24.4 (± 11.2) years] were recruited in this study. Blood samples were analysed using the BD FACScount cytometer according to the manufacturer's instructions. Of the overall 1123 subjects, reference means of CD4, CD3 and CD4/CD3 ratio were 1030 ± 367, 1757 ± 609 cells/µl and 0.59 ± 0.08, respectively. Five hundred and fifty-one (49.1%) were an urban population with the mean CD4, CD3 and CD4/CD3 T cell ratio of 1032 ± 369, 1761 ± 612 cells/µl and 0.59 (±0.08), respectively. The remaining 572 (50.9%) were of a rural population with the mean CD4, CD3 and CD4/CD3 T cell ratio of 1028 ± 459, 1753 ± 958 cells/µl and 0.59 ± 0.13, respectively. Subjects with higher CD4 and CD3 T cells were more likely to be female than male (P < 0.05). There was no significant difference between the T cell values of the two populations (P > 0.05). Our findings provide new insight in the CD4 and CD3 T cell reference values of Nigerians.

Coordinated evolution of the hepatitis B virus polymerase.

The detection of compensatory mutations that abrogate negative fitness effects of drug-resistance and vaccine-escape mutations indicates the important role of epistatic connectivity in evolution of viruses, especially under the strong selection pressures. Mapping of epistatic connectivity in the form of coordinated substitutions should help to characterize molecular mechanisms shaping viral evolution and provides a tool for the development of novel anti-viral drugs and vaccines. We analyzed coordinated variation among amino acid sites in 370 the hepatitis B virus (HBV) polymerase sequences using Bayesian networks. Among the HBV polymerase domains the spacer domain separating terminal protein from the reverse-transcriptase domain, showed the highest network centrality. Coordinated substitutions preserve the hydrophobicity and charge of Spacer. Maximum likelihood estimates of codon selection showed that Spacer contains the highest number of positively selected sites. Identification of 67% of the domain lacking an ordered structure suggests that Spacer belongs to the class of intrinsically disordered domains and proteins whose crucial functional role in the regulation of transcription, translation and cellular signal transduction has only recently been recognized. Spacer plays a central role in the epistatic network associating substitutions across the HBV genome, including those conferring viral virulence, drug resistance and vaccine escape. The data suggest that Spacer is extensively involved in coordination of HBV evolution.

Evaluation of viral heterogeneity using next-generation sequencing, end-point limiting-dilution and mass spectrometry.

Hepatitis C Virus sequence studies mainly focus on the viral amplicon containing the Hypervariable region 1 (HVR1) to obtain a sample of sequences from which several population genetics parameters can be calculated. Recent advances in sequencing methods allow for analyzing an unprecedented number of viral variants from infected patients and present a novel opportunity for understanding viral evolution, drug resistance and immune escape. In the present paper, we compared three recent technologies for amplicon analysis: (i) Next-Generation Sequencing; (ii) Clonal sequencing using End-point Limiting-dilution for isolation of individual sequence variants followed by Real-Time PCR and sequencing; and (iii) Mass spectrometry of base-specific cleavage reactions of a target sequence. These three technologies were used to assess intra-host diversity and inter-host genetic relatedness in HVR1 amplicons obtained from 38 patients (subgenotypes 1a and 1b). Assessments of intra-host diversity varied greatly between sequence-based and mass-spectrometry-based data. However, assessments of inter-host variability by all three technologies were equally accurate in identification of genetic relatedness among viral strains. These results support the application of all three technologies for molecular epidemiology and population genetics studies. Mass spectrometry is especially promising given its high throughput, low cost and comparable results with sequence-based methods.

What factors determine the severity of hepatitis A-related acute liver failure?

The reason(s) that hepatitis A virus (HAV) infection may progress infrequently to acute liver failure are poorly understood. We examined host and viral factors in 29 consecutive adult patients with HAV-associated acute liver failure enrolled at 10 sites participating in the US ALF Study Group. Eighteen of twenty-four acute liver failure sera were PCR positive while six had no detectable virus. HAV genotype was determined using phylogenetic analysis and the full-length genome sequences of the HAV from a cute liver failure sera were compared to those from self-limited acute HAV cases selected from the CDC database. We found that rates of nucleotide substitution did not vary significantly between the liver failure and non-liver failure cases and there was no significant variation in amino acid sequences between the two groups. Four of 18 HAV isolates were sub-genotype IB, acquired from the same study site over a 3.5-year period. Sub-genotype IB was found more frequently among acute liver failure cases compared to the non-liver failure cases (chi-square test, P < 0.01). At another centre, a mother and her son presented with HAV and liver failure within 1 month of each other. Predictors of spontaneous survival included detectable serum HAV RNA, while age, gender, HAV genotype and nucleotide substitutions were not associated with outcome. The more frequent appearance of rapid viral clearance and its association with poor outcomes in acute liver failure as well as the finding of familial cases imply a possible host genetic predisposition that contributes to a fulminant course. Recurrent cases of the rare sub-genotype IB over several years at a single centre imply a community reservoir of infection and possible increased pathogenicity of certain infrequent viral genotypes.

Robust hepatitis B virus genotyping by mass spectrometry.

Genotyping of hepatitis B virus (HBV) is important for tracking HBV infections, prognosticating the development of severe liver disease, and predicting outcomes of therapy. Current genotyping methods can be laborious and costly and rely on subjective data interpretation. To identify less expensive but equally reliable alternatives, we compared "gold standard" sequencing to a novel mass spectrometry approach. Sera from individuals with acute or chronic HBV infection (n = 756), representing all genotypes, were used to PCR amplify the HBV S gene. All amplicons were subjected to base-specific cleavage and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). The resulting mass peak patterns were used to identify HBV genotype by automated comparison to peak patterns simulated from reference sets of HBV sequences of known genotypes. The MALDI-TOF MS data and phylogenetic analysis of HBV sequences produced completely concordant results. Several parameters such as genetic relatedness of tested HBV variants to the reference set, chronic infections, and the quality of PCR products can lower the MS score but never affected the accuracy of the genotype call. This new streamlined MS-based method provides for rapid and accurate HBV genotyping, produces automated data reports, and is therefore suitable for routine use in diagnostic settings.

Quantification of human immunodeficiency virus-1 viral load using nucleic acid sequence-based amplification (NASBA) in north central Nigeria.

BACKGROUND: Viral load (VL) quantification is considered an integral part of the standard care in human immunodeficiency virus (HIV) infected individuals but in Nigeria as in most of sub-Saharan Africa, this has not reached the majority ofpatients. METHODS: We report the first field application of the NucliSens EasyQ HIV-1 platform for the real time quantification of HIV-1 VL combining NASBA amplification and real time detection with molecular beacons among HIV-1 infected individuals in north central Nigeria where the predominant HIV-1 subtypes are CRF02_AG and G. CD4+ counts were enumerated using a fluorescence-activated cell sorter system. RESULTS: Of one hundred and forty nine (n=149) plasma sample from patients with mean age of 32 years and made up of 77 males and 72 females, fifty {n=50 (37.9%); 28 males and 22 females} had VLs below the lower detection limit (LDL=25 IU/ml) set by the assay while eighty-two {n=82 (62.1%); 39 males and 43 females} had VL levels above the LDL. Furthermore, 13 of 82 (15.9%) patients with viral loads above the LDL had VLs between 26-1000 IU/ml while 69 (84.1%) had VLs of 1001-2,400,000 IU/ml. 17 (11.4%) of the samples could not be analyzed due to poor viral amplification. Among individuals with both CD4+ and VL results (n=56), those with CD4+ of 1-418 cell/microl presented with higher VL usually above 45,000 IU/ml when compared with those with CD4+ of over 500 cell/microl. CONCLUSION: Our findings highlight the pattern, usefulness and feasibility ofVL quantification by NucliSens EasyQ in monitoring HIV-1 patients in Nigeria.

Phylogenetic analysis of human parvovirus b19 sequences from eleven different countries confirms the predominance of genotype 1 and suggests the spread of genotype 3b.

Phylogenetic analysis of 166 human parvovirus B19 sequences from 11 different countries attributed 91.57% to genotype 1, 5.42% to genotype 3b, and 3.01% to genotype 3a. Very similar viruses of genotype 1 circulated widely in Europe and Israel. Genotype 3b seems to show an increasing spread outside of Africa.

High prevalence of hepatitis B virus among female sex workers in Nigeria.

Hepatitis B virus (HBV) infection is endemic in Nigeria and constitutes a public health menace. The prevalence of HBV infection in many professional groups has been described in Nigeria. However, literature on HBV infection among female sex workers (FSW) in Nigeria is scanty. FSW in Nigeria are not subjected to a preventive control of HBV infection. This study assesses the extent of spread of HBV among FSW in Nigeria. Seven hundred and twenty (n = 720) FSW (mean age = 26.7 years) were tested for hepatitis B surface antigen (HBsAg) by a double antibody sandwich ELISA method. The overall HBV prevalence among the FSW was 17.1%. FSWs between the ages of 31-35 year (20.5%) and those with 'age-at-first-sex' below 10 years of age (28%) were most affected. This high prevalence of a vaccine preventable disease is unacceptable, therefore, vaccination of this high risk HBV reservoir group should be considered worthwhile.

Herd immunity to measles virus in a population of student nurses and medical students following a widespread outbreak of clinical measles in Ibadan, Nigeria.

Following an outbreak of serologically--and virologically--confirmed measles requiring large-scale hospitalisation of children in Ibadan, Nigeria, the herd immunity to measles virus among medical students and student nurses was determined. Of the 200 students tested, none lacked haemagglutination--inhibiting antibody to measles virus. The titre of HI--antibody ranged from 2(5) to 2(10). Describing a titre of 2(9) as very high, a significantly higher proportion of student nurses than medical students (P < 0.05) had very high antibody titres to measles virus. There was however no statistical difference between the sexes (P > 0.05). Using a commercial Enzyme Immuno Assay kit (EIA), anti measles IgM could not be detected from any of the students. Thus a clear evidence of recent infection with measles virus during the outbreak could not be detected among the students, a probable indication that student nurses and medical students may not participate in the maintenance of wild measles virus within the hospital environment in developing countries like Nigeria.