High prevalence of GB virus C/hepatitis G virus RNA among Brazilian blood donors

The aim of this study was to investigate the presence of the Hepatitis G Virus on a population of blood donors from S o Paulo, Brazil and to evaluate its association to sociodemographic variables. Two RT-PCR systems targeting the putative 5'NCR and NS3 regions were employed and the former has shown a higher sensitivity. The observed prevalence of HGV-RNA on 545 blood donors was 9.7% (CI 95% 7.4;12.5). Statistical analysis depicted an association with race/ethnicity, black and mulatto donors being more frequently infected; and also with years of education, less educated donors presenting higher prevalences. No association was observed with other sociodemographic parameters as age, gender, place of birth and of residence. DNA sequencing of nine randomly chosen isolates demonstrated the presence of genotypes 1, 2 and 3 among our population but clustering of these Brazilian isolates was not detected upon phylogenetic analysis.     

HIV-1 subtype H near-full length genome reference strains and analysis of subtype-H-containing inter-subtype recombinants

OBJECTIVE: To characterize near-full-length genomes of two HIV-1 subtype H strains. To extend sequence data to include full env and gag, and analyse and redefine, previously documented subtype H strains. DESIGN: Near-full-length genomes of HIV-1 env subtype H strains VI991 and VI997 were amplified, cloned, sequenced, phylogenetically analysed and compared with a panel of 23 HIV-1 group M reference isolates. The mosaic nature of previously published subtype H strains VI557 and CA13 was reanalysed. MATERIALS AND METHODS: Peripheral blood mononuclear cells (PBMC) from individuals harbouring strains VI991 and VI997 were co-cultivated with PHA stimulated donor PBMC. Near-full-length genomes of VI991 and VI997, and gag and env genes of CA13 and VI557, were amplified by polymerase chain reaction, cloned and sequenced. Intersubtype recombination analyses were performed by similarity plot, bootscanning and phylogenetic analysis. RESULTS: Near-full-length clones of HIV-1 VI991 and VI997 are representative of subtype H. They form a phylogenetic cluster with the only previously described subtype H representative HIV-1 90CF056.1, regardless of the genome region analysed. VI557 is redefined as a gag and env subtype H mosaic virus containing unclassified fragments. CA13 is a complex intersubtype recombinant between subtypes A, H and unclassified strains CONCLUSION: Near-full-length genome analysis identified HIV-1 VI991 and VI997 as two new subtype H representatives. These reagents will allow defining and classifying non-recombinant as well as recombinant HIV-1, eventually helping to solve the puzzle of HIV-1 subtypes.     

HTLV-1/2 seroprevalence and coinfection rate in Brazilian first-time blood donors: an 11-year follow-up

The seroprevalence and geographic distribution of HTLV-1/2 among blood donors are extremely important to transfusion services. We evaluated the seroprevalence of HTLV-1/2 infection among first-time blood donor candidates in Ribeir?o Preto city and region. From January 2000 to December 2010, 1,038,489 blood donations were obtained and 301,470 were first-time blood donations. All samples were screened with serological tests for HTLV-1/2 using enzyme immunoassay (EIA). In addition, the frequency of coinfection with hepatitis B virus (HBV), hepatitis C virus (HCV), human immunodeficiency virus (HIV), Chagas disease (CD) and syphilis was also determined. In-house PCR was used as confirmatory test for HTLV-1/2. A total of 296 (0.1%) first-time donors were serologically reactive for HTLV-1/2. Confirmatory PCR of 63 samples showed that 28 were HTLV-1 positive, 13 HTLV-2 positive, 19 negative and three indeterminate. Regarding HTLV coinfection rates, the most prevalent was with HBV (51.3%) and HCV (35.9%), but coinfection with HIV, CD and syphilis was also detected. The real number of HTLV-infected individual and coinfection rate in the population is underestimated and epidemiological studies like ours are very informative.     

High prevalence of diverse forms of HIV-1 intersubtype recombinants in Central Myanmar: geographical hot spot of extensive recombination

OBJECTIVES: To investigate the molecular epidemiology and genetic structure of HIV-1s causing the epidemic in Central Myanmar and to explore the genesis of HIV epidemic in this area. DESIGN: A molecular epidemiological investigation was conducted in 1999-2000 in the city of Mandalay among high-risk populations and the structural features of circulating HIV-1s were analyzed. METHODS: HIV-1 genotypes of 59 specimens were screened based on gag (p17) and env (C2/V3) regions. Near full-length nucleotide sequences of HIV-1 isolates with subtype discordance were determined and their recombinant structures were characterized. RESULTS: Three lineages of HIV-1 strains, including CRF01_AE (27, 45.8%), subtype B' (Thailand variant of subtype B) (15, 25.4%) and subtype C (8, 13.6%), were distributed in Mandalay, while substantial portions (9, 15.3%) of specimens showed various patterns of subtype discordance in different regions of HIV-1 genomes. The study on six HIV-1 isolates with subtype discordance revealed that they were highly diverse types of unique recombinant forms (URFs) comprised of various combinations of three circulating subtypes. One URF was a particularly complex mosaic that contained 13 recombination breakpoints between three HIV-1 subtypes. Approximately half of recombinants showed 'pseudotype' virion structures, in which the external portions of envelope glycoproteins were exchanged with different lineages of HIV-1 strains, suggesting the potential selective advantage of 'pseudotype' viruses over parental strains. CONCLUSION: The study revealed the unique geographical hot spot in Central Myanmar where extensive recombination events appeared to be taking place continually. This reflects the presence of highly exposed individuals and social networks of HIV-1 transmission.     

HIV-2 antibody testing of blood donors with doubtful immunoblot results for HIV-1

Summary Antibodies against human immunodeficiency virus type-1 (HIV-1) in samples from blood donors are commonly detected by various enzyme-linked immunosorbent assays (ELISA) and by confirmatory tests, e.g., Western blot or immunofluorescence tests. Immunoblot reactivity, which is directed only towards the HIV-1 core proteins p 18, p 24 and p 55, may represent false-positive reactions. Out of 125,000 blood donations, 140 were repeatably HIV-1 antibody reactive by ELISA; of these, 20 were doubtful positive sera with isolated p 18 and/or p24 bands in the HIV-1 confirmatory assay. Antibodies to HIV-2 are known to cross-react with these HIV-1 core proteins. We therefore assayed the 20 sera by immunofluorescence and immunoblotting for the presence of antibodies to HIV-2. None of these doubtful HIV-1 antibody positive blood donor sera was found to have antibodies to HIV-2.     

HIV-1 CRF_BC recombinants infection in China: molecular epidemic and characterizations

CRF_BC recombinant strains were first identified in China and are one of the most prevalent and characteristically unique HIV-1 subtypes across China. Here we aim to review the published data about HIV-1 CRF_BC recombinant strains epidemic in China and to characterize the genetics, biology and drug resistance of this virus. This study may help to better understand the current situation of HIV-1 CRF_BC prevalence and facilitate the development of vaccines and more efficient anti-HIV-1 regimens in China.     

Screening for haemochromatosis: prevalence among Danish blood donors

Hereditary haemochromatosis is an autosomal recessive disease that is genetically expressed by excessive accumulation of iron in the tissues, resulting in cirrhosis, diabetes mellitus, cardiomyopathy and hypogonadism. As the disease may be diagnosed before the appearance of symptoms, and prevented by repeated phlebotomies, there are strong implications for adoption of a screening procedure. Determinations of transferrin saturation (TS) and serum ferritin concentration (SF) were used to screen 4302 blood donors, who were selected for follow-up studies if they fulfilled any of the following three criteria: (i) TS greater than or equal to 0.7; (ii) TS greater than or equal to 0.5 together with SF greater than or equal to 150 micrograms l-1; (iii) SF greater than or equal to 300 micrograms l-1. A total of 58 subjects who fulfilled at least one of these criteria were reinvestigated, after which 18 individuals still fulfilled at least one criterion. Fifteen subjects having SF greater than or equal to 300 micrograms l-1 were offered liver biopsy and thirteen of these accepted. In one individual, no stainable iron was detected, and two subjects did not fulfil the previously established diagnostic criteria for the diagnosis of hereditary haemochromatosis. Ten subjects who had a high TS and liver iron grade 2-4 according to Bassett were classified accordingly as homozygotes. On the basis of these results, the prevalence of haemochromatosis in Denmark was estimated to be 0.0037-0.0046.     

Hepatitis E virus prevalence in Flemish blood donors

Transmission of hepatitis E virus (HEV) through transfusion of blood components has already been reported in several European countries. Here, we assessed the HEV prevalence in Flemish blood donors. This study is of importance in order to assess the risk of HEV transmission through blood transfusion. We analysed 38 137 blood donation samples that were collected by the Red Cross Flanders during the period May‐June 2015. All samples were screened for the presence of HEV RNA and a selection for HEV‐specific IgM/IgG. After pooling per 6, 11 pools reacted positive during RNA screening. Reactive pools were deconstructed, and individual samples were retested. After deconstruction, seven samples were confirmed as HEV RNA positive. Serological screening of the confirmed RNA‐positive samples showed that six out of these seven samples were HEV IgM positive, of which three donors were also IgG positive. Serological screening was also performed on the samples that constituted the four initially HEV RNA reactive pools where RNA positivity was not confirmed on the individual level. In three pools, we found indirect evidence of recent HEV exposure. Within 356 randomly selected samples, 31 donations were HEV IgG positive. Here we show that at least 1:5448 of blood donations in Flanders may originate from donors that are actively infected with HEV. Upon transfusion, these donations may pose a major threat towards patients at risk. Finally, a serological analysis showed that the anti‐HEV IgG prevalence in Flemish blood donors is 8.71%.     

Hereditary hemochromatosis: population screening based on phenotype in Brazilian blood donors

GOALS: A population of blood donors was screened for hereditary hemochromatosis (HH) based on the phenotype strategy in accordance with the European consensus. STUDY: Nonfasting serum samples were obtained from 1,050 donors. Transferrin saturation (TS) was measured using a threshold of 45%. Donors with a TS > or = 45% were retested in a fasting sample. If TS was elevated, the participants were tested for iron overload by ferritin measurement followed by genetic testing. All donors underwent clinical and laboratory workup for expression of the disease. RESULTS: A total of 775 (74.6%) of the donors were men, 749 (72.1%) white, and had a mean age of 30 years (range, 8-60 years). Mean +/- SD TS was 25.9% +/- 13.1% (range, 2.1%-85.8%), and there were 58 (5.6%) donors with a TS > or = 45%. Fifty-four subjects had a repeat TS in a fasting serum sample with a mean +/- SD TS of 32.1% +/-16.1% (range, 15.4%-63.0%), and 12 donors had a TS > or = 45%. Ten complied with genetic testing and ferritin measurement. The study found four donors with HH-related mutations (C282Y and/or H63D); therefore, a gene allele frequency of 0.4%. Only the C282Y homozygote had diagnostic criteria for HH, defining a disease frequency of 0.1%. None of the donors who were mutations carriers had clinical or laboratory manifestations of organic injury. CONCLUSION: We conclude that this is a feasible screening strategy that, by timely diagnosing HH, allows patients not only to benefit from effective treatment but also to have disease progression halted.     

Evolutionary history of HIV-1 subtype B and F infections in Brazil

OBJECTIVE: To reconstruct the onset date of the HIV-1 B and F epidemics in Brazil based on virus diversification over time. DESIGN: We studied HIV-1 env V3 sequences (210 nt) with a known sampling year isolated from HIV-1 positive patients from Brazil between 1989 and 1997: 101 subtype B sequences and 41 subtype F sequences. METHODS: HIV-1 V3 env sequences were grouped by year of collection and the relationship between the sampling years of HIV-1 sequences and their genetic distance to the reconstructed common ancestor (intra-population divergence) or to other sequences from the same year (intra-population diversity) was examined by using linear regression analysis. RESULTS: Regression analysis of nucleotide distances, revealed a highly significant positive correlation between sampling years of subtype B and F V3 sequences and their intra-population divergence (P < 0.001) or diversity (P < 0.0001). In both subtype populations, the divergence and diversity increased at a rate of 0.5 and 0.9% per year, respectively. Considering these evolutionary rates, we estimate the onset of the subtype B and F HIV-1 epidemics in Brazil during early 1970s and early 1980s, respectively. CONCLUSIONS: The consistent correlation between divergence and diversity of the V3 sequences with their sampling years indicates that the molecular clock is operational in the evolution of the HIV-1 in Brazil's epidemic, and show that subtypes B and F are evolving at a similar rate over time. The dating results suggest a discontinuous introduction of these subtypes in the Brazilian population.     

Short report: occurrence of Leishmania donovani DNA in donated blood from seroreactive Brazilian blood donors

Human visceral leishmaniasis (kala-azar) transmitted by blood transfusion has been described in previous reports. Seroprevalence of antibodies to Leishmania donovani was shown to be related to prior blood transfusions in multiply transfused hemodialysis patients in Natal, Rio Grande do Norte, Brazil. In this study, a possible correlation between seroreactivity and the presence of L. donovani DNA was investigated in asymptomatic healthy blood donors. Sera were tested using the fucose mannose ligand (FML) ELISA, which was shown to have a sensitivity of 100%, a specificity of 96-100%, reliability, and diagnostic and prognostic potential for the detection of human and canine kala-azar, respectively. Leishmanial DNA was assessed by the polymerase chain reaction (PCR) and dot-blot hybridization techniques in blood and bone marrow samples. Among 21 FML-seroreactive asymptomatic blood donors, 5 (24%) were positive by the PCR and 9 (43%) were positive in a dot-blot assay of blood samples, showing a significant correlation (chi2 = 14.24, P < 0.01). No Leishmania DNA was detected in 20 FML non-reactive blood donors. Our results point to the need for control of transmission of kala-azar by blood transfusion in areas endemic for this disease.     

Imbalance in cytokine production by whole blood related to presence of cytopathogenic HIV-1 strains in HIV-1-infected patients

Summary The possible association between the emergence of cytopathogenic HIV-1 variants and disturbance of the cytokine production in the course of HIV-1 infection was studied in 18 infected patients. The cytopathogenicity of the isolates was studied in a microassay based on the use of HIV-1-infectible Hela-CD4 cells carrying the bacterial LacZ gene under the control of the HIV-LTR (P4 cells). In addition, the production of cytokines by heparinized whole blood (HWB) obtained the same day from HIV-1(+) patients was measured. TNF-α was determined in a one-step procedure combining HWB culture in the presence of LPS+PHA for 24 h and detection of cytokines in the same wells. In separate experiments HWB was cultured in the presence of LPS+PHA for 48 h, then the supernatants were collected and stored until assayed by ELISA for IFN-γ and IL-4. Higher TNF-α levels were found in activated HWB of patients with cytopathic strains (n=9) than in patients with non-cytopathic strains (n=9, p=0.02) as assed with P4 cells. A defective production of type 1 cytokine (IFN-γ) and no increased secretion of type 2 cytokines (IL-4) was observed in patients with cytopathic strains. IFN-γ/IL-4 ratios were significantly lower in patients with cytopathic strains (n=9) than in other patients (n=9, p=0.009). The results show that the disarray of cytokine production, as assessed with whole blood culture, is associated with the cytopathogenicity of HIV-1 isolates in HIV-1-infected individuals.     

The prevalence of transfusion transmitted virus infection in blood donors

INTRODUCTION A newly discovered DNA virus,transfusion transmitted virus (TTV), was reported as a cause of post-transfusion hepatitis of unknown etiology in Japan[1]. In order to investigate TTV prevalence in southern China, a study was carried out among blood donors, patients with liver diseases and hemodialysis to determine the epidemiological charateristics.     

既往有偿献血HIV-1感染者中合并感染的HCV对telaprevir和boceprevir天然耐药分析

目的分析既往有偿献血人群HIV-1感染者中合并感染的HCV对telaprevir和boceprevir的天然耐药,为HCV的耐药监测及临床抗病毒治疗提供基础数据。方法收集既往有偿献血人群HIV阳性患者的血浆样本,检测其HCV抗体,应用巢式PCR扩增HCV NS3/4A区,对测序结果进行耐药分析,计算耐药率,评价耐药毒株的流行趋势。结果从150份符合要求的样本里共获得70份(46.67%)HCV NS3/4A基因序列。检测出耐药相关突变4例(5.71%),其中对boceprevir耐药2例(2.86%),耐药位点为R117H和A156V;对telaprevir耐药2例(2.86%),耐药位点为R117H和A156V;对telaprevir可能耐药2例(2.86%),耐药位点为T54S。尚未发现V36A/M/L/C、Q41H/R、F43C/S、T54A、V55A、Q80R、R109K、S138C、R155G/I/K/M/T/Q/S、A156S/T、D168A/E/G/H、V170A/T/I、N174G/S、L175M等已报道的耐药位点。其他突变中,氨基酸替换率超过90%的变异位点有I35V、A40T、R62K、I64L、V71I、S66G、S91A和T42S;在系统进化树中,耐药序列呈点状散在分布,未发现聚集性。结论既往有偿献血HIV-1感染者中合并感染的HCV对telaprevir和boceprevir存在天然耐药,耐药相关突变率分别为5.71%和2.86%。其他氨基酸位点出现突变率较高,但是未发现与耐药相关。各耐药菌株之间未发现传播关联性。