High rate of hepatitis C virus and human immunodeficiency virus false-positive results in serologic screening in sub-Saharan Africa: adverse impact on the blood supply.

BACKGROUND: False positivity in blood screening may cause unnecessary deferral of healthy donors and exacerbate blood shortages. An international multicenter study was conducted to estimate the frequency of HCV and HIV false seropositivity in seven African countries (Burundi, Cameroon, Democratic Republic of Congo, Madagascar, Mali, Mauritania, and Niger). STUDY DESIGN AND METHODS: Blood donations were tested for hepatitis C virus (HCV) and human immunodeficiency virus (HIV) with rapid detection tests (RDTs), third-generation enzyme immunoassays (EIAs), or fourth-generation EIAs. HCV (456/16,613 [2.74%]) and HIV (249/16,675 [1.49%]) reactive samples were then confirmed with antigen/antibody assays, immunoblots, and nucleic acid testing. Partial viral sequences were analyzed when possible. RESULTS: The HCV reactivity rate with RDTs was significantly lower than with EIAs (0.55% vs. 3.52%; p < 0.0001). The HIV reactivity rate with RDTs was lower than with third-generation EIAs (1.02% vs. 2.38%; p < 0.0001) but similar to a fourth-generation assay (1.09%). Only 16.0% (57/357) and 21.5% (38/177) of HCV and HIV initial reactive samples, respectively, were repeatedly reactive. HCV and HIV infections were confirmed in 13.2% and 13.7%, respectively, of repeated reactive donations. The predominant HCV genotype 2 and 4 strains in West and Central Africa showed high genetic variability. HIV-1 subtype CRF02_AG was most prevalent. CONCLUSION: High rates (>80%) of unconfirmed anti-HCV and anti-HIV reactivity observed in several sub-Saharan countries highlights the need for better testing and confirmatory strategies for donors screening in Africa. Without confirmatory testing, HCV and HIV prevalence in African blood donors has probably been overestimated.

Parvovirus B19 seroprevalence, viral load, and genotype characterization in volunteer blood donors from southern Brazil.

Usually transmitted via respiratory droplets, parvovirus B19 (B19V) can also be acquired by blood transfusion especially because of viral persistence, resistance to blood treatment procedures, and high viral load during the early infection phase. This is particularly problematic in immunocompromised or anemic patients where the infection can have a severe outcome. As B19V DNA was detected in blood donations from South Brazil during a viral metagenomic survey performed by Next-Generation Sequencing, the objective of this retrospective study was to evaluate the seroprevalence, B19V DNA presence and circulating genotypes in a Hospital Blood Transfusion Service in Santa Maria city in South Brazil (Rio Grande do Sul state). Among 480 volunteer blood donors, 53.9% (n = 258 of 479) were anti-B19V IgG-positive, and 9 (1.9%) plasma samples presented B19V DNA. In almost all cases (n = 7 of 9, 77.8%), B19V DNA load was accompanied by the presence of anti-B19V IgG suggesting a persistent infection. The sequencing of the strains demonstrated that all belong to genotype 1 which is the most prevalent worldwide. The analysis of the recipient information of the positive for B19V DNA units revealed no related posttransfusion adverse effects. Our results demonstrate for the first time, B19V seroprevalence, viral load, and genotypes among blood donors from South Brazil and give a light for the circulation and impact of this B19V in this part of the country.

A member of a new Picornaviridae genus is shed in pig feces.

During a study of the fecal microbiomes from two healthy piglets using high-throughput sequencing (HTS), we identified a viral genome containing an open reading frame encoding a predicted polyprotein of 2,133 amino acids. This novel viral genome displayed the typical organization of picornaviruses, containing three structural proteins (VP0, VP3, and VP1), followed by seven nonstructural proteins (2A, 2B, 2C, 3A, 3B, 3C(pro), and 3D(pol)). Given its particular relationship with Parechovirus, we propose to name it "Pasivirus" for Parecho sister clade virus, with "Swine pasivirus 1" (SPaV1) as the type species. Fecal samples collected at an industrial farm from healthy sows and piglets from the same herd (25 and 75, respectively) with ages ranging from 4 to 28 weeks were analyzed for the presence of SPaV1 by one-step reverse transcription (RT)-PCR targeting a 3D region of 151 bp. SPaV1 was detected in fecal samples from 51/75 healthy piglets (68% of the animals) and in none of the 25 fecal samples from healthy sows, indicating that SPaV1 circulates through enteric infection of healthy piglets. We propose that SPaV1 represents the first member of a novel Picornaviridae genus related to parechoviruses.

Identification of the first human gyrovirus, a virus related to chicken anemia virus.

We have identified in a skin swab sample from a healthy donor a new virus that we have named human gyrovirus (HGyV) because of its similarity to the chicken anemia virus (CAV), the only previously known member of the Gyrovirus genus. In particular, this virus encodes a homolog of the CAV apoptin, a protein that selectively induces apoptosis in cancer cells. By PCR screening, HGyV was found in 5 of 115 other nonlesional skin specimens but in 0 of 92 bronchoalveolar lavages or nasopharyngeal aspirates and in 0 of 92 fecal samples.

Residual risk of mother-to-child transmission of hepatitis B virus infection despite timely birth-dose vaccination in Cameroon (ANRS 12303): a single-centre, longitudinal observational study.

BACKGROUND: In sub-Saharan Africa, administration of hepatitis B virus (HBV) birth-dose vaccines remains suboptimal. Evidence is scarce on whether African countries should focus on increasing vaccine coverage or developing strategies incorporating additional measures, such as peripartum antiviral prophylaxis to pregnant women at high risk. To better inform decision makers, we estimated the residual risk of mother-to-child transmission despite HBV birth-dose vaccine in Cameroon. METHODS: We did a single-centre, longitudinal observational study. Pregnant women were systematically screened for HBV surface antigen (HBsAg) at Tokombéré District Hospital (Tokombéré district, Cameroon). Children born to HBsAg-positive mothers in 2009-16 who received the HBV birth-dose vaccine and three subsequent doses of pentavalent vaccine at 6, 10, and 14 weeks were followed up prospectively in 2015-17. In children, capillary blood was obtained for HBsAg rapid test and dried blood spots to quantify HBV DNA concentrations. Venous blood was also collected from HBsAg-positive children. Mother-to-child transmission was confirmed by whole-genome sequencing. FINDINGS: Between Jan 31, 2009, and Dec 31, 2016, 22 243 (66·8%) of 33 309 pregnant women accepted antenatal HBV screening, of whom 3901 (17·5%) were HBsAg positive. 2004 (51·4%) of 3901 children who were born to HBsAg-positive mothers received the HBV birth-dose vaccine, of whom 1800 (89·8%) also completed the three-dose pentavalent vaccine. In total, the current analysis included 607 children who had a follow-up serosurvey. The prevalence of HBsAg was 5·6% in children who received the birth-dose vaccine in less than 24 h, 7·0% in those who received it 24-47 h after birth, and 16·7% in those who received it 48-96 h after birth (ptrend=0·083). 35 (89·7%) of 39 infected children were born to mothers positive for HBV e antigen with high HBV DNA of 5·3 log10 IU/mL or more. Whole-genome sequencing of HBV in infected mother-child pairs confirmed high identity proportions of 99·97-100%. INTERPRETATION: We documented a substantial risk of mother-to-child transmission despite timely administration of the HBV birth-dose vaccine within 24 h after birth. To reach WHO's elimination targets, peripartum antiviral prophylaxis might be required in parts of Africa, in addition to increasing coverage of the HBV birth-dose vaccine. FUNDING: Agence nationale de recherches sur le sida et les hépatites virales (ANRS).

Human polyomavirus related to African green monkey lymphotropic polyomavirus.

While studying the virome of the skin surface of a patient with a Merkel cell carcinoma (MCC) by using unbiased, high-throughput sequencing, we identified a human polyomavirus nearly identical to human polyomavirus 9, a virus recently reported in blood and urine of renal transplantion patients and closely related to the African green monkey lymphotropic polyomavirus. Specific PCR analysis further identified this virus in 2/8 patients with MCC but in only 1/111 controls without MCC. This virus was shed for ≥20 months by the MCC index patient and was on the skin of the spouse of the index patient. These results provide information on the viral ecology of human skin and raise new questions regarding the pathology of virus-associated skin disorders.

Evaluation of high-throughput sequencing for identifying known and unknown viruses in biological samples.

High-throughput sequencing furnishes a large number of short sequence reads from uncloned DNA and has rapidly become a major tool for identifying viruses in biological samples, and in particular when the target sequence is undefined. In this study, we assessed the analytical sensitivity of a pipeline for detection of viruses in biological samples based on either the Roche-454 genome sequencer or Illumina genome analyzer platforms. We sequenced biological samples artificially spiked with a wide range of viruses with genomes composed of single or double-stranded DNA or RNA, including linear or circular single-stranded DNA. Viruses were added at a very low concentration most often corresponding to 3 or 0.8 times the validated level of detection of quantitative reverse transcriptase PCRs (RT-PCRs). For the viruses represented, or resembling those represented, in public nucleotide sequence databases, we show that the higher output of Illumina is associated with a much greater sensitivity, approaching that of optimized quantitative (RT-)PCRs. In this blind study, identification of viruses was achieved without incorrect identification. Nevertheless, at these low concentrations, the number of reads generated by the Illumina platform was too small to facilitate assembly of contigs without the use of a reference sequence, thus precluding detection of unknown viruses. When the virus load was sufficiently high, de novo assembly permitted the generation of long contigs corresponding to nearly full-length genomes and thus should facilitate the identification of novel viruses.

Development and clinical validation of loop-mediated isothermal amplification (LAMP) assay to diagnose high HBV DNA levels in resource-limited settings.

OBJECTIVE: A massive scale-up of testing and treatment is indicated to globally eliminate hepatitis B virus (HBV) infection. However, access to a polymerase chain reaction (PCR), a key test to quantify HBV DNA levels and determine treatment eligibility, is limited in resource-limited countries. We have developed and evaluated the loop-mediated isothermal amplification (LAMP) assay to diagnose clinically important HBV DNA thresholds defined by the WHO (≥20 000 and ≥ 200 000 IU/mL). METHODS: Pan-genotypic primer sets were designed on conserved HBV gene regions. Accuracy of LAMP to identify highly viraemic patients was evaluated in 400 and 550 HBV-infected people in France and Senegal, respectively. RESULTS: Our primers successfully detected eight major HBV genotypes/sub-genotypes (A1/2/3/B/C/D/E/F) with a detection limit ranging between 40 and 400 IU/mL. In France, the area under the receiver operating characteristic curve (AUROC), sensitivity and specificity of bead-based extraction and real-time turbidimetric LAMP were 0.95 (95% CI 0.93-0.97), 91.1% and 86.0%, respectively, to diagnose HBV DNA ≥20 000 IU/mL; and 0.98 (0.97-0.99), 98.0% and 94.6% for ≥200 000 IU/mL. The performance did not vary by viral genotypes. In Senegal, using a field-adapted method (reagent-free boil-and-spin extraction and inexpensive end-point fluorescence detection), the AUROC, sensitivity and specificity were 0.95 (0.93-0.97), 98.7% and 91.5%, respectively, to diagnose HBV DNA ≥200 000 IU/mL. The assay was not adapted to discriminate low-level viraemia. DISCUSSION: We have developed a simple, rapid (60 min), and inexpensive (US$8/assay) alternative to PCR to diagnose high viraemia ≥200 000 IU/mL. HBV-LAMP may contribute to eliminating HBV mother-to-child transmission by identifying high-risk pregnant women eligible for antiviral prophylaxis in resource-limited countries.

New insights into Human Pegivirus-1 (HPgV-1) genotypes diversity in sub-Saharan Africa.

In the framework of a viral discovery research program using metagenomics, Human Pegivirus-1 reads (HPgV-1, formerly known as GBV-C) were detected in plasma pools of healthy blood donors from seven sub-Saharan African countries. For five of these countries, Mauritania, Mali, Niger, Burundi and Madagascar, no data about HPgV-1 genotypes was reported to date. To confirm our metagenomic findings and further investigate the genotype diversity and distribution of HPgV-1 in Africa, 400 blood donations from these five localities as well as from Cameroon, the Democratic Republic of Congo (DRC) and the Burkina Faso were screened with a RT-nested PCR targeting the viral 5'NCR region. Amplified products were sequenced, and the virus was genotyped by phylogenetic analysis. Out of the 400 plasma samples tested, 65 were positive for HPgV-1 RNA and 61 were successfully genotyped. Among these, 54 strains (88.5%) clustered with genotype 1, six (9.8%) with genotype 2 and one (1.6%) with genotype 5. Genotype 1 was observed in all countries studied, except in Madagascar, genotype 2 was detected in Mauritania and Madagascar, and genotype 5 in DRC. Overall, our results extend the geographic distribution of HPgV-1 in Africa and provide six additional nearly complete genomes. Considering that some HPgV-1 genotypes have been reported as potential predictive indicators of lower disease progression in HIV-1 infected subjects, further investigations should be conducted to better understand the positive impact, if any, of this virus.

Deep sequencing applied to the analysis of viromes in patients with beta-thalassemia.

To date, blood banks apply routine diagnosis to a specific spectrum of transfusion-transmitted viruses. Even though this measure is considered highly efficient to control their transmission, the threat imposed by emerging viruses is increasing globally, which can impact transfusion safety, especially in the light of the accelerated viral discovery by novel sequencing technologies. One of the most important groups of patients, who may indicate the presence of emerging viruses in the field of blood transfusion, is the group of individuals who receive multiple transfusions due to hereditary hemoglobinopathies. It is possible that they harbor unknown or unsuspected parenterally-transmitted viruses. In order to elucidate this, nucleic acids from 30 patients with beta-thalassemia were analyzed by Illumina next-generation sequencing and bioinformatics analysis. Three major viral families: Anelloviridae, Flaviviridae and Hepadnaviridae were identified. Among them, anelloviruses were the most representative, being detected with high number of reads in all tested samples. Human Pegivirus 1 (HPgV-1, or GBV-C), Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV) were also identified. HBV and HCV detection was expected due to the high seroprevalence in patients with beta thalassemia. Our results do not confirm the presence of emerging or unsuspected viruses threatening the transfusion safety at present, but can be used to actively search for viruses that threaten blood transfusion safety. We believe that the application of viral metagenomics in multiple-transfused patients is highly useful to monitor possible viral transfusion threats and for the annotation of their virome composition.

Molecular characterization and expression analysis of a P-glycoprotein homologue in Toxoplasma gondii.

ATP-binding cassette (ABC) transporters represent an important family of membrane proteins involved in drug resistance and other biological activities. The present study reports on the characterization of a P-glycoprotein (Pgp), TgABC.B1, in the protozoan parasite Toxoplasma gondii. The protein encoded by the TgABC.B1 gene displays the typical (TMD-NBD)2 structural organization of the "full" ABC transporter and shows significant identity and similarity with two apicomplexan Pgps; Pgh1 in Plasmodium falciparum and CpABC3 in Cryptosporidium parvum. The TgABC.B1 gene is a single copy gene transcribed into a full-length mRNA of 4.3kb and expressed as a protein of approximately 150kDa, which cellular localization revealed a membrane-associated labelling in tachyzoites. The TgABC.B1 gene is constitutively expressed in the three major T. gondii genotypes but demonstrated a higher expression in virulent type I, at both transcriptional and translational levels. Further characterization of this Pgp-like protein will increase our knowledge of the membrane transport system in this parasite and could result in the identification of a new therapeutic target in Toxoplasma.

Early MinION™ nanopore single-molecule sequencing technology enables the characterization of hepatitis B virus genetic complexity in clinical samples.

Until recently, the method of choice to characterize viral diversity consisted in cloning PCR amplicons of full-length viral genomes and Sanger-sequencing of multiple clones. However, this is extremely laborious, time-consuming, and low-throughput. Next generation short-read sequencing appears also limited by its inability to directly sequence full-length viral genomes. The MinION™ device recently developed by Oxford Nanopore Technologies can be a promising alternative by applying long-read single-molecule sequencing directly to the overall amplified products generated in a PCR reaction. This new technology was evaluated by using hepatitis B virus (HBV) as a model. Several previously characterized HBV-infected clinical samples were investigated including recombinant virus, variants that harbored deletions and mixed population. Original MinION device was able to generate individual complete 3,200-nt HBV genome sequences and to identify recombinant variants. MinION was particularly efficient in detecting HBV genomes with multiple large in-frame deletions and spliced variants concomitantly with non-deleted parental genomes. However, an average-12% sequencing error rate per individual reads associated to a low throughput challenged single-nucleotide resolution, polymorphism calling and phasing mutations directly from the sequencing reads. Despite this high error rate, the pairwise identity of MinION HBV consensus genome was consistent with Sanger sequencing method. MinION being under continuous development, further studies are needed to evaluate its potential use for viral infection characterization.

High prevalence of cyclovirus Vietnam (CyCV-VN) in plasma samples from Madagascan healthy blood donors.

Cycloviruses, small ssDNA viruses belonging to the Circoviridae family, have been suggested as possible causes of enteric, respiratory and neurological disorders in human patients. One of these species, cyclovirus-Vietnam (CyCV-VN), initially isolated from cerebrospinal fluid samples of patients with unexplained neurological disorders, has since been reported in serum samples from chronically patients infected with HBV, HCV or HIV, in Italy. On the other hand, CyCV-VN was not detected in serum samples from healthy individuals. Here, we report on a high prevalence of 43.4% (40/92) of CyCV-VN in plasma samples from asymptomatic blood donors from Madagascar. Interestingly, this virus was not detected by metagenomics and PCR in six other African countries, suggesting regional differences in CyCV-VN prevalence across Africa. Phylogenetic analysis based on the complete genomes showed that CyCV-VN sequences isolated from blood were most closely related to sequences previously reported from human stool in Madagascar. Further investigations using larger cohorts are required to determine the global epidemiology, the natural history and the pathological significance, if any, of CyCV-VN infection in humans.

Identification and expression analysis of ABC protein-encoding genes in Toxoplasma gondii. Toxoplasma gondii ATP-binding cassette superfamily.

The ATP-binding cassette (ABC) transporters are one of the largest evolutionarily conserved families of proteins. They are characterized by the presence of nucleotide-binding domains (NBDs), which are highly conserved among organisms. In the present study, we used human and protozoan ABC sequences, and ATP-binding consensus motifs to screen the Toxoplasma gondii TwinScan2 predicted proteins database. We identified 24 ABC open reading frames (ORFs), whose deduced amino acid sequences exhibited all the typical biochemical features of the ABC family members. Fifteen of them clustered into five of the seven families of human ABC proteins: six ABCBs (drug, peptides and lipid export), two ABCCs (organic anion conjugates and drug export), one ABCE (Rnase L inhibitor, RLI, antibiotic resistance and translation regulation), one ABCF (drug resistance and regulation of gene expression) and five ABCGs (drug export and resistance). The nine other ORFs were represented by four ABCHs (energy-generating subunits), four SMCs (structural maintenance of chromosomes) and one member of unclear origin, whose closest homologue was the yeast Elf1 protein (mRNA export factor). A notable feature of the Toxoplasma ABC superfamily seems to be the absence of genes encoding ABCA and ABCD members. Expression analysis of ABC genes in tachyzoite and bradyzoite stages revealed the presence of ABC transcripts for all genes studied. Further research on the implication of these ABC proteins will increase our knowledge of the basic biology of Toxoplasma and provide the opportunity to identify novel therapeutic targets. To our knowledge, this is the first report of ABC transporters in T. gondii.

Viral metagenomics applied to blood donors and recipients at high risk for blood-borne infections.

BACKGROUND: Characterisation of human-associated viral communities is essential for epidemiological surveillance and to be able to anticipate new potential threats for blood transfusion safety. In high-resource countries, the risk of blood-borne agent transmission of well-known viruses (HBV, HCV, HIV and HTLV) is currently considered to be under control. However, other unknown or unsuspected viruses may be transmitted to recipients by blood-derived products. To investigate this, the virome of plasma from individuals at high risk for parenterally and sexually transmitted infections was analysed by high throughput sequencing (HTS). MATERIALS AND METHODS: Purified nucleic acids from two pools of 50 samples from recipients of multiple transfusions, and three pools containing seven plasma samples from either HBV-, HCV- or HIV-infected blood donors, were submitted to HTS. RESULTS: Sequences from resident anelloviruses and HPgV were evidenced in all pools. HBV and HCV sequences were detected in pools containing 3.8×10(3) IU/mL of HBV-DNA and 1.7×10(5) IU/mL of HCV-RNA, respectively, whereas no HIV sequence was found in a pool of 150 copies/mL of HIV-RNA. This suggests a lack of sensitivity in HTS performance in detecting low levels of virus. In addition, this study identified other issues, including laboratory contaminants and the uncertainty of taxonomic assignment of short sequence. No sequence suggestive of a new viral species was identified. DISCUSSION: This study did not identify any new blood-borne virus in high-risk individuals. However, rare and/or viruses present at very low titre could have escaped our protocol. Our results demonstrate the positive contribution of HTS in the detection of viral sequences in blood donations.

P-glycoprotein inhibitors modulate accumulation and efflux of xenobiotics in extra and intracellular Toxoplasma gondii.

We examined xenobiotic transport and the effects of P-glycoprotein (Pgp) inhibitors on efflux function in Toxoplasma gondii tachyzoites. The fluorescence emission of JC-1 and daunorubicin (Pgp substrates) was determined in both extracellular tachyzoites and T. gondii-infected human KB cells. Dye accumulation and efflux were modulated by verapamil (Vp) and cyclosporin A (CsA), both of which are Pgp inhibitors. Red JC-1 emission was measured from 10(6) extracellular tachyzoites, using spectrofluorometry. The increase in red emission was significant from 1 microM concentration of both drugs and was higher with CsA than with Vp. Compared with untreated tachyzoites, JC-1 efflux was inhibited by 3 microM CsA and 3 microM Vp. With intracellular tachyzoites, the fluorescence distribution of daunorubicin (DNR) between the parasitophorous vacuole and the host cell was modulated by Vp and CsA. In media free of CsA and Vp, DNR emission inside intracellular tachyzoites was very weak, as observed by confocal microscopy. In the presence of CsA or Vp, DNR emission was markedly enhanced in tachyzoites but not in the whole vacuole. The modulation of DNR uptake seems to involve the tachyzoite membrane rather than the parasitophorous vacuole or host cell membranes. It suggests that Vp would inhibit the DNR efflux from intracellular tachyzoites through a transitory effect. In conclusion, these two Pgp inhibitors increase both extracellular and intracellular dye accumulation in living T. gondii, pointing to the existence of a transmembrane transport mediated by a Pgp homologue located on the parasite membrane complex.

Sulfadiazine resistance in Toxoplasma gondii: no involvement of overexpression or polymorphisms in genes of therapeutic targets and ABC transporters.

Several treatment failures have been reported for the treatment of toxoplasmic encephalitis, chorioretinitis, and congenital toxoplasmosis. Recently we found three Toxoplasma gondii strains naturally resistant to sulfadiazine and we developed in vitro two sulfadiazine resistant strains, RH-R(SDZ) and ME-49-R(SDZ), by gradual pressure. In Plasmodium, common mechanisms of drug resistance involve, among others, mutations and/or amplification within genes encoding the therapeutic targets dhps and dhfr and/or the ABC transporter genes family. To identify genotypic and/or phenotypic markers of resistance in T. gondii, we sequenced and analyzed the expression levels of therapeutic targets dhps and dhfr, three ABC genes, two Pgp, TgABC.B1 and TgABC.B2, and one MRP, TgABC.C1, on sensitive strains compared to sulfadiazine resistant strains. Neither polymorphism nor overexpression was identified. Contrary to Plasmodium, in which mutations and/or overexpression within gene targets and ABC transporters are involved in antimalarial resistance, T. gondii sulfadiazine resistance is not related to these toxoplasmic genes studied.