Distinct resistance mutation and polymorphism acquisition in HIV-1 protease of subtypes B and F1 from children and adult patients under virological failure.

The goal of this work was to compare the differences between human immunodeficiency virus type 1 (HIV-1) of B and F1 subtypes in the acquisition of major and minor protease inhibitor (PI)-associated resistance mutations and of other polymorphisms at the protease (PR) gene, through a cross sectional study. PR sequences from subtypes B and F1 isolates matched according to PI exposure time from Brazilian patients were included in this study. Sequences were separated in four groups: 24 and 90 from children and 141 and 99 from adults infected with isolates of subtypes F1 and B, respectively. For comparison, 211 subtype B and 79 subtype F1 PR sequences from drug-naïve individuals were included. Demographic and clinical data were similar among B- and F1-infected patients. In untreated patients, mutations L10V, K20R, and M36I were more frequent in subtype F1, while L63P, A71T, and V77I were more prevalent in subtype B. In treated patients, K20M, D30N, G73S, I84V, and L90M, were more prevalent in subtype B, and K20T and N88S were more prevalent in subtype F1. A higher proportion of subtype F1 than of subtype B strains containing other polymorphisms was observed. V82L mutation was present with increased frequency in isolates from children compared to isolates from adults infected with both subtypes. We could observe a faster resistance emergence in children than in adults, during treatment with protease inhibitors. This data provided evidence that, although rates of overall drug resistance do not differ between subtypes B and F1, the former accumulates resistance at higher proportion in specific amino acid positions of protease when compared to the latter.

Near full-length genome analysis of low prevalent human immunodeficiency virus type 1 subclade F1 in São Paulo, Brazil.

BACKGROUND: The genetic diversity of the human immunodeficiency virus type 1 (HIV-1) is critical to lay the groundwork for the design of successful drugs or vaccine. In this study we aimed to characterize and define the molecular prevalence of HIV-1 subclade F1 currently circulating in São Paulo, Brazil. METHODS: A total of 36 samples were selected from 888 adult patients residing in São Paulo who had previously been diagnosed in two independent studies in our laboratory as being infected with subclade F1 based on pol subgenomic fragment sequencing. Proviral DNA was amplified from the purified genomic DNA of all 36 blood samples by 5 fragments overlapping PCR followed by direct sequencing. Sequence data were obtained from the 5 fragments of pure subclade F1 and phylogenetic trees were constructed and compared with previously published sequences. Subclades F1 that exhibited mosaic structure with other subtypes were omitted from any further analysis RESULTS: Our methods of fragment amplification and sequencing confirmed that only 5 sequences inferred from pol region as subclade F1 also holds true for the genome as a whole and, thus, estimated the true prevalence at 0.56%. The results also showed a single phylogenetic cluster of the Brazilian subclade F1 along with non-Brazilian South American isolates in both subgenomic and the full-length genomes analysis with an overall intrasubtype nucleotide divergence of 6.9%. The nucleotide differences within the South American and Central African F1 strains, in the C2-C3 env, were 8.5% and 12.3%, respectively. CONCLUSION: All together, our findings showed a surprisingly low prevalence rate of subclade F1 in Brazil and suggest that these isolates originated in Central Africa and subsequently introduced to South America.

Drug resistance among chronic HIV-1-infected patients naïve for use of anti-retroviral therapy in Sao Paulo city.

Primary infection with drug-resistant HIV appears to be increasing in the regions where HAART is widely available, which may reduce efficacy of first-line antiretroviral therapy. To determine prevalence of antiretroviral drug-resistant mutations in newly diagnosed subjects in a clinical setting where HAART has been widely used since 1997. One hundred and thirty-six HIV-1-infected adult patients were diagnosed with HIV infection between January 2000 and December 2006 in the HIV out-clinic at the HC/FMUSP, Sao Paulo city. These antiretroviral naïve patients were mainly referred from the blood bank, situated in the same building or elsewhere in the city. The samples were genotyped to provide HIV protease and reverse transcriptase sequence data. Major antiretroviral drug resistance mutations were classified according to Shafer et al. [Shafer, R.W., Rhee, S.Y., Pillay, D., et al., 2007. HIV-1 protease and reverse transcriptase mutations for drug resistance surveillance. AIDS 21, 215-223]. Thirteen cases had no DNA amplification, and 123 patients were successfully analyzed, with a mean age of 37 years and 89 (72%) were males. Antiretroviral drug resistance mutations were detected in 8/123 patients (6.5%), all eight were heterosexuals and HIV asymptomatic, the mean of the CD4 cells count was 323 cells/mm(3), and the RNA plasma viral load was 4.7 log(10)/mL. We found NRTI (n=2, 1.6%), NNRTI-resistant (n=2, 1.6%) mutations, and one cases with PI mutation (0.8%). Three cases (2.4%) showed mutations for NRTI, NNRTI or PI, simultaneously. Eighty-two percent were HIV-1 B subtype, and HIV-1 F (6.5%), HIV-1 C (5.7%) and recombinant viruses (5.8%) were observed. In an unselected cohort, primary drug resistance was seen in 6.5% of the naïve for drug ART use. These results indicate that HIV drug resistance testing should be a practical approach in monitoring first-line ART. In addition, HIV-1 C seems to be emerging in Sao Paulo city.

Trends in antiretroviral drug resistance and clade distributions among HIV-1--infected blood donors in Sao Paulo, Brazil.

BACKGROUND: We analyzed rates of drug resistance mutations in antiretroviral-naive São Paulo blood donors with recently acquired or established HIV-1 infections and characterized clade diversity in this population. METHODS: Six hundred forty-eight seropositive blood donor specimens were identified at the Blood Center of São Paulo between July 1998 and March 2002. To discriminate recent infections, samples were subjected to the standardized testing algorithm for recent HIV seroconversion (less-sensitive enzyme immunoassay) testing algorithm. There were 531 samples with a sufficient volume of serum to attempt polymerase chain reaction (PCR) and viral sequencing; 341 (64%) samples yielded a PCR product that could be sequenced for the reverse transcriptase and protease genes. Mutations were analyzed using the 2005 International AIDS Society mutation list. RESULTS: Of 341 specimens successfully analyzed, 21 (6.3%; 95% confidence interval [CI]: 3.9% to 9.3%) had drug-resistant mutations. The proportion of resistant strains was 12.7% (95% CI: 5.2% to 24.5%) among recently infected individuals compared with 5.0% (95% CI: 2.8% to 8.2%) among those with long-standing infections (P = 0.03). No change in the proportion of drug-resistant strains was observed among recently infected donor samples from the first half of the study period (4 of 32 samples) as compared with the second half (3 of 23 samples; P = 0.95). Of the 341 samples, 277 (81.2%) were classified as subtype B, 25 (7.3%) as subtype F1, 13 (3.8%) as subtype C, and 26 (7.6%) as recombinant strains. The distribution of HIV-1 subtypes was similar among recent and long-standing infected individuals and over time. CONCLUSIONS: The prevalence of drug-resistant mutations among newly diagnosed persons in São Paulo city is low and similar to what has been described in Europe and the United States. Although HIV-1 subtype B remains predominant, subtypes F and C and recombinant forms are present in substantial proportions in infected donors.

Prevalence, incidence, and residual risk of human immunodeficiency virus among community and replacement first-time blood donors in São Paulo, Brazil.

BACKGROUND: Concerted efforts have been directed toward recruitment of community rather than replacement donors in Brazil. Time trends and demographic correlates of human immunodeficiency (HIV) prevalence and incidence among first-time (FT) donors in Brazil were examined by donation type. HIV residual risk from FT-donor transfusions, and projected yield of p24 antigen and nucleic acid test (NAT) screening were estimated. STUDY DESIGN AND METHODS: HIV prevalence data and seroreactive specimens were obtained at Fundação Pró-Sangue/Hemocentro-de-São Paulo from 1995 to 2001. To estimate incidence, confirmed-positive samples from July 1998 through December 2001 were tested with a less-sensitive (detuned) enzyme immunoassay to detect recent seroconversions. Incidence data were used to estimate residual risk and p24 and NAT yield based on published window periods (WPs). RESULTS: HIV prevalence was 22 percent higher among the FT community donors than replacement donors (19.6 vs. 16.1 per 10,000; p < 0.01) and 48 percent higher among men than women (19.1 vs. 12.9; p < 0.01). In the multivariable logistic regression, both variables remained significant predictors of HIV prevalence. HIV prevalence decreased from 20.4 (1995) to 13.1 per 10,000 FT donations (2001). HIV incidence was 2.7 per 10,000 person-years. The estimated rate of infected antibody-negative donations was 14.9 per 1,000,000 units (95% confidence interval, 9.8-20.0). It was estimated that addition of p24 antigen, minipool NAT, and individual-donation NAT assays would detect 3.9 (2.0-5.8), 8.3 (5.3-11.3), and 10.8 (7.1-14.5) WP units per 1,000,000 FT donations, respectively. CONCLUSION: HIV incidence and residual transfusion risk estimates are approximately 10 times higher in Brazil FT donors compared to US and European FT donors. Community FT donors had higher HIV prevalence than replacement FT donors. The yield of p24 antigen or RNA screening will be low in Brazilian donors, but substantially higher than in US donors.

[The discarding of blood units and the prevalence of infectious diseases in donors at the Pro-Blood Foundation/Blood Center of São Paulo, São Paulo, Brazil]. / Descarte de bolsas de sangue e prevalência de doenças infecciosas em doadores de sangue da Fundação Pró-Sangue/Hemocentro de São Paulo.

OBJECTIVES: To analyze the changes in the proportion of blood units discarded from 1991 through 2001 at the Pro-Blood Foundation/Blood Center of São Paulo (Fundação Pró-Sangue/Hemocentro de São Paulo), which is the largest blood bank in Latin America, and to determine the prevalence of infectious diseases among donors at the Blood Center in November 2001. METHODS: We compiled data concerning the discarding of blood units due to the presence of serological markers for communicable diseases at the Blood Center during the period from 1991 through 2001. To determine the prevalence of infectious diseases, 9 942 screened samples were analyzed in November 2001; all reactive samples underwent confirmatory tests. RESULTS: Over the study period there was a significant decrease in the percentage of units discarded, from 20% in 1991 to 9% in 2001. In November 2001 the prevalence of infectious diseases among donors was: 0.04% for human immunodeficiency virus (HIV), 0.21% for hepatitis C virus (HCV), 0.06% for human T-lymphotropic virus (HTLV), 0.14% for Chagas' disease, and 1.10% for syphilis. For hepatitis B virus, the prevalences found were: 0.14% for anti-HBc and HBsAg, 1.68% for anti-HBc and anti-HBs, and 1.67% for isolated anti-HBc. CONCLUSIONS: The decrease in the discarding of blood units and in infectious diseases among donors at the Blood Center of São Paulo reflects the increase in the Center's percentage of repeat donors.

Evaluation of the concomitant use of two different EIA tests for HIV screening in blood banks.

OBJECTIVE: In 1998, the Brazilian Ministry of Health made it mandatory for all blood banks in the country to screen donated blood for human immunodeficiency virus (HIV) concomitantly using two different enzyme immunoassay (EIA) tests. Concerned with the best use of available resources, our objective with this study was to evaluate the usefulness of conducting two EIA screening tests instead of just one. METHODS: We analyzed data from 1999 through 2001 obtained by testing 698 191 units of donated blood using two EIA HIV screening tests concomitantly at the Pro-Blood Foundation/Blood Center of São Paulo (Fundação Pró-Sangue/Hemocentro de São Paulo), which is a major blood center in the city of São Paulo, Brazil. All samples reactive in at least one of the two EIA tests were submitted for confirmation by a Western blot (WB) test, and the persons who had donated those samples were also asked to return and provide a follow-up sample. RESULTS: Out of the 698 191 blood units that were donated, 2 718 of them (0.4%) had to be discarded because they were reactive to at least one of the EIA tests. There were two WB-positive donation samples that were reactive in only one HIV EIA screening test. On their follow-up samples, both donors tested WB-negative. These cases were considered false positive results at screening. Of the 2 718 donors who were asked to return and provide a follow-up sample, 1 576 of them (58%) did so. From these 1 576 persons, we found that there were two individuals who had been reactive to only one of the two EIA screening tests and who had also been negative on the WB at screening but who were fully seroconverted on the follow-up sample. We thus estimated that, in comparison to the use of a single EIA screening test, the use of two EIA screening tests would detect only one extra sample out of 410 700 units of blood. CONCLUSIONS: Our data do not support the use of two different, concomitant EIA screening tests for HIV. The great majority of HIV-positive donors have already seroconverted and will be detected using only a single EIA screening test. Only persons who are going through the process of seroconversion may be missed by using a single EIA test and detected using two EIAs for screening. To screen out these individuals and decrease the residual risk of HIV transmission from the blood donated in our center, the use of other techniques, such as nucleic acid testing (NAT) or a p24 antigen assay, would be more effective.

Descarte de bolsas de sangue e prevalência de doenças infecciosas em doadores de sangue da Fundação Pró-Sangue/Hemocentro de São Paulo / The discarding of blood units and the prevalence of infectious diseases in donors at the Pro-Blood Foundation/Blood Center of Säo Paulo, Säo Paulo, Brazil

OBJETIVO: Analisar a evoluçäo, de 1991 a 2001, do descarte sorológico na Fundaçäo Pró-Sangue/Hemocentro de Säo Paulo, o maior banco de sangue da América Latina, e verificar a prevalência de doenças infecciosas entre doadores dessa instituiçäo no ano de 2001. MÉTODOS: Foram compilados os dados de descarte sorológico relativos aos anos de 1991 a 2001. Para determinar a prevalência de doenças infecciosas, foram analisadas 9 942 amostras triadas em novembro de 2001, sendo as amostras reativas submetidas a testes confirmatórios. RESULTADOS: Foi encontrada uma diminuiçäo percentual significativa de descarte, de 20 por cento em 1991 para 9 por cento em 2001. A prevalência de doenças infecciosas entre doadores em 2001 foi de 0,04 por cento para vírus da imunodeficiência humana (VIH); 0,21 por cento para vírus da hepatite C (VHC); 0,06 por cento para vírus T-linfotrópico humano (HTLV); para vírus da hepatite B (VHB), as prevalências foram de 0,14 por cento para anti-HBc + HBsAg, 1,68 por cento para anti-HBc + anti-HBs e 1,67 por cento para anti-HBc isolado; 1,10 por cento para sífilis; e 0,14 por cento para doença de Chagas. CONCLUSÄO: A diminuiçäo no descarte e a prevalência de doenças infecciosas entre doadores da Fundaçäo Pró-Sangue/Hemocentro de Säo Paulo em 2001 refletem o aumento na porcentagem de doadores de repetiçäo nesse banco de sangue

Evaluation of the concomitant use of two different EIA tests for HIV screening in blood banks

OBJECTIVE: In 1998, the Brazilian Ministry of Health made it mandatory for all blood banks in the country to screen donated blood for human immunodeficiency virus (HIV) concomitantly using two different enzyme immunoassay (EIA) tests. Concerned with the best use of available resources, our objective with this study was to evaluate the usefulness of conducting two EIA screening tests instead of just one. METHODS: We analyzed data from 1999 through 2001 obtained by testing 698 191 units of donated blood using two EIA HIV screening tests concomitantly at the Pro-Blood Foundation/Blood Center of Säo Paulo (Fundaçäo Pró-Sangue/Hemocentro de Säo Paulo), which is a major blood center in the city of Säo Paulo, Brazil. All samples reactive in at least one of the two EIA tests were submitted for confirmation by a Western blot (WB) test, and the persons who had donated those samples were also asked to return and provide a follow-up sample. RESULTS: Out of the 698 191 blood units that were donated, 2 718 of them (0.4 percent) had to be discarded because they were reactive to at least one of the EIA tests. There were two WB-positive donation samples that were reactive in only one HIV EIA screening test. On their follow-up samples, both donors tested WB-negative. These cases were considered false positive results at screening. Of the 2 718 donors who were asked to return and provide a follow-up sample, 1 576 of them (58 percent) did so. From these 1 576 persons, we found that there were two individuals who had been reactive to only one of the two EIA screening tests and who had also been negative on the WB at screening but who were fully seroconverted on the follow-up sample. We thus estimated that, in comparison to the use of a single EIA screening test, the use of two EIA screening tests would detect only one extra sample out of 410 700 units of blood. CONCLUSIONS: Our data do not support the use of two different, concomitant EIA screening tests for HIV. The great majority of HIV-positive donors have already seroconverted and will be detected using only a single EIA screening test. Only persons who are going through the process of seroconversion may be missed by using a single EIA test and detected using two EIAs for screening. To screen out these individuals and decrease the residual risk of HIV transmission from the blood donated in our center, the use of other techniques, such as nucleic acid testing (NAT) or a p24 antigen assay, would be more effective