Dulce Bila

Risk of extended viral resistance in human immunodeficiency virus-1-infected Mozambican children after first-line treatment failure.

Background: Resistant virus may be selected by sub-optimal control of HIV-1 replication during antiretroviral treatment. The incidence and profile of resistance in children receiving World Health Organization-recommended treatment remains to be evaluated on a large scale. Goals: Assessment of the frequency and profile of resistant virus in HIV-1-infected children, treated for at least 6 months with stavudine/zidovudine + lamivudine + nevirapine and presenting virological failure in a large access program in Maputo, Mozambique. Results: Cross-sectional evaluation of plasma HIV-1 viral load (VL) in 495 evaluable children among 512 treated for at least 6 months showed that 360 (72.7%) had a VL of <50 copies/mL of HIV-1 RNA. Genotypic resistance tests were performed in the 84 available samples from the 135 treated children with VL ≥50 copies/mL: 92% of the viruses were resistant to lamivudine and/or nevirapine, and 15% were resistant to stavudine. Twenty children (24%) harbored virus with a extended spectrum of cross-resistance defined as resistance to the 3 drugs of the combination received by the child and/or at least 1 resistance to a drug to which the child had never been exposed (abacavir: 5%, tenofovir: 6%, didanosine: 3.5% and the new generation non nucleoside inhibitor, etravirine: 6%). The only factor identified by multivariate analysis as being associated with this extended resistance profile was the duration of treatment (aOR: 6.67 [95% CI: 1.24–35.93], P = 0.015 for treatment >24 months) with a per month increase of 1.09 (1.02–1.16) P = 0.007. Conclusions: Residual viral replication in children receiving stavudine/zidovudine + lamivudine + nevirapine treatment is associated with a time-dependent risk of acquiring cross-resistance, including resistance to drugs currently used for second-line treatment and also to the new generation of non nucleoside reverse transcritpase inhibitors.

Evolution of Primary HIV Drug Resistance in a Subtype C Dominated Epidemic in Mozambique

Objective In Mozambique, highly active antiretroviral treatment (HAART) was introduced in 2004 followed by decentralization and expansion, resulting in a more than 20-fold increase in coverage by 2009. Implementation of HIV drug resistance threshold surveys (HIVDR-TS) is crucial in order to monitor the emergence of transmitted viral resistance, and to produce evidence-based recommendations to support antiretroviral (ARV) policy in Mozambique. Methods World Health Organization (WHO) methodology was used to evaluate transmitted drug resistance (TDR) in newly diagnosed HIV-1 infected pregnant women attending ante-natal clinics in Maputo and Beira to non-nucleoside reverse transcriptase inhibitors (NNRTI), nucleoside reverse transcriptase inhibitors (NRTI) and protease inhibitors (PI). Subtypes were assigned using REGA HIV-1 subtyping tool and phylogenetic trees constructed using MEGA version 5. Results Although mutations associated with resistance to all three drug were detected in these surveys, transmitted resistance was analyzed and classified as <5% in Maputo in both surveys for all three drug classes. Transmitted resistance to NNRTI in Beira in 2009 was classified between 5–15%, an increase from 2007 when no NNRTI mutations were found. All sequences clustered with subtype C. Conclusions Our results show that the epidemic is dominated by subtype C, where the first-line option based on two NRTI and one NNRTI is still effective for treatment of HIV infection, but intermediate levels of TDR found in Beira reinforce the need for constant evaluation with continuing treatment expansion in Mozambique.

Evaluation of a High-Throughput Diagnostic System for Detection of HIV-1 in Dried Blood Spot Samples from Infants in Mozambique

ABSTRACT We performed a comparative analysis between Roche Amplicor HIV-1 DNA test and CAPTAQ assay for the detection of HIV in 830 dried blood spot (DBS) pediatric samples collected in Mozambique. Our results demonstrated no statistical difference between these assays. The CAPTAQ assay approached nearly 100% repeatability/accuracy. The increased throughput of testing with minimal operator interference in performing the CAPTAQ assay clearly demonstrated that this method is an improvement over the Roche Amplicor HIV-1 DNA test, version 1.5.

Trends in Prevalence of HIV-1 Drug Resistance in a Public Clinic in Maputo, Mozambique

Background An observational study was conducted in Maputo, Mozambique, to investigate trends in prevalence of HIV drug resistance (HIVDR) in antiretroviral (ART) naïve subjects initiating highly active antiretroviral treatment (HAART). Methodology/Principal Findings To evaluate the pattern of drug resistance mutations (DRMs) found in adults on ART failing first-line HAART [patients with detectable viral load (VL)]. Untreated subjects [Group 1 (G1; n=99)] and 274 treated subjects with variable length of exposure to ARV´s [6–12 months, Group 2 (G2;n=93); 12-24 months, Group 3 (G3;n=81); >24 months (G4;n=100)] were enrolled. Virological and immunological failure (VF and IF) were measured based on viral load (VL) and T lymphocyte CD4+ cells (TCD4+) count and genotypic resistance was also performed. Major subtype found was C (untreated: n=66, 97,06%; treated: n=36, 91.7%). Maximum virological suppression was observed in G3, and significant differences intragroup were observed between VF and IF in G4 (p=0.022). Intergroup differences were observed between G3 and G4 for VF (p=0.023) and IF between G2 and G4 (p=0.0018). Viral suppression (<50 copies/ml) ranged from 84.9% to 90.1%, and concordant VL and DRM ranged from 25% to 57%. WHO cut-off for determining VF as given by 2010 guidelines (>5000 copies/ml) identified 50% of subjects carrying DRM compared to 100% when lower VL cut-off was used (<50 copies/ml). Length of exposure to ARVs was directly proportional to the complexity of DRM patterns. In Mozambique, VL suppression was achieved in 76% of individuals after 24 months on HAART. This is in agreement with WHO target for HIVDR prevention target (70%). Conclusions We demonstrated that the best way to determine therapeutic failure is VL compared to CD4 counts. The rationalized use of VL testing is needed to ensure timely detection of treatment failures preventing the occurrence of TDR and new infections.

Genetic Characterization of Human T-Cell Lymphotropic Virus Type 1 in Mozambique: Transcontinental Lineages Drive the HTLV-1 Endemic

Background Human T-Cell Lymphotropic Virus Type 1 (HTLV-1) is the etiological agent of adult T-cell leukemia (ATL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP). It has been estimated that 10–20 million people are infected worldwide, but no successful treatment is available. Recently, the epidemiology of this virus was addressed in blood donors from Maputo, showing rates from 0.9 to 1.2%. However, the origin and impact of HTLV endemic in this population is unknown. Objective To assess the HTLV-1 molecular epidemiology in Mozambique and to investigate their relationship with HTLV-1 lineages circulating worldwide. Methods Blood donors and HIV patients were screened for HTLV antibodies by using enzyme immunoassay, followed by Western Blot. PCR and sequencing of HTLV-1 LTR region were applied and genetic HTLV-1 subtypes were assigned by the neighbor-joining method. The mean genetic distance of Mozambican HTLV-1 lineages among the genetic clusters were determined. Human mitochondrial (mt) DNA analysis was performed and individuals classified in mtDNA haplogroups. Results LTR HTLV-1 analysis demonstrated that all isolates belong to the Transcontinental subgroup of the Cosmopolitan subtype. Mozambican HTLV-1 sequences had a high inter-strain genetic distance, reflecting in three major clusters. One cluster is associated with the South Africa sequences, one is related with Middle East and India strains and the third is a specific Mozambican cluster. Interestingly, 83.3% of HIV/HTLV-1 co-infection was observed in the Mozambican cluster. The human mtDNA haplotypes revealed that all belong to the African macrohaplogroup L with frequencies representatives of the country. Conclusions The Mozambican HTLV-1 genetic diversity detected in this study reveals that although the strains belong to the most prevalent and worldwide distributed Transcontinental subgroup of the Cosmopolitan subtype, there is a high HTLV diversity that could be correlated with at least 3 different HTLV-1 introductions in the country. The significant rate of HTLV-1a/HIV-1C co-infection, particularly in the Mozambican cluster, has important implications for the controls programs of both viruses.

Genetic Diversity in HIV-1 Subtype C LTR from Brazil and Mozambique Generates New Transcription Factor-Binding Sites

The HIV-1 subtype C has been substituting the subtype B population in southern Brazil. This phenomenon has been previously described in other countries, suggesting that subtype C may possess greater fitness than other subtypes. The HIV-1 long-terminal repeat (LTR) is an important regulatory region critical for the viral life cycle. Sequence insertions immediately upstream of the viral enhancer are known as the most frequent naturally occurring length polimorphisms (MFNLP). Previous reports demonstrated that the MFNLP could lead to the duplication of transcription factor binding sites (TFBS) enhancing the activity of the HIV-1 subtype C LTR. Here, we amplified and sequenced the LTR obtained from proviral DNA samples collected from patients infected with subtype C from the Southern Region of Brazil (naïve or treatment failure) and Mozambique (only naïve). We confirm the presence of different types of insertions in the LTR sequences of both the countries leading to the creation of additional TFBS. In the Brazilian clinical samples, the frequency of the sequence insertion was significantly higher in subjects experiencing treatment failure than in antiretroviral naïve patients.

Performance characteristics of finger-stick dried blood spots (DBS) on the determination of human immunodeficiency virus (HIV) treatment failure in a pediatric population in Mozambique

Quantitative plasma viral load (VL) at 1000 copies /mL was recommended as the threshold to confirm antiretroviral therapy (ART) failure by the World Health Organization (WHO). Because of ongoing challenges of using plasma for VL testing in resource-limited settings (RLS), especially for children, this study collected 717 DBS and paired plasma samples from children receiving ART ≥1 year in Mozambique and compared the performance of DBS using Abbott’s VL test with a paired plasma sample using Roche’s VL test. At a cut-off of 1000 copies/mL, sensitivity of DBS using Abbott DBS VL test was 79.9%, better than 71.0% and 63.9% at 3000 and 5000 copies/mL, respectively. Specificities were 97.6%, 98.8%, 99.3% at 1000, 3000, and 5000 copies/mL, respectively. The Kappa value at 1000 copies/mL, 0.80 (95% CI: 0.73, 0.87), was higher than 0.73 (95% CI: 0.66, 0.80) and 0.66 (95% CI: 0.59, 0.73) at 3000, 5000 copies/mL, respectively, also indicating better agreement. The mean difference between the DBS and plasma VL tests with 95% limits of agreement by Bland-Altman was 0.311 (-0.908, 1.530). Among 73 children with plasma VL between 1000 to 5000 copies/mL, the DBS results were undetectable in 53 at the 1000 copies/mL threshold. While one DBS sample in the Abbott DBS VL test may be an alternative method to confirm ART failure at 1000 copies/mL threshold when a plasma sample is not an option for treatment monitoring, because of sensitivity concerns between 1,000 and 5,000 copies/ml, two DBS samples may be preferred accompanied by careful patient monitoring and repeat testing.

A16 Primary drug resistance among children infected with HIV-1 in Mozambique: Impact of maternal and neonatal prophylaxis

A14 Ultra-wide and ultra-deep sequencing increases the detection rate of dual HIV-1 infections and recombinants K. Van Laethem, A. Thielen, Y. Schrooten, F. Ferreira, L. Vinken, and M. Daümer KU Leuven, Department Microbiology and Immunology, Rega Institute for Medical Research, Leuven, Belgium, AIDS Reference Laboratory, University Hospitals Leuven, Leuven, Belgium and Institute of Immunology and Genetics, Kaiserslautern, Germany

Compromise of Second-Line Antiretroviral Therapy Due to High Rates of Human Immunodeficiency Virus Drug Resistance in Mozambican Treatment-Experienced Children With Virologic Failure

Background Virologic failure (VF) is highly prevalent in sub-Saharan African children on antiretroviral therapy (ART) and is often associated with human immunodeficiency virus drug resistance (DR). Most children still lack access to routine viral load (VL) monitoring for early identification of treatment failure, with implications for the efficacy of second-line ART. Methods Children aged 1 to 14 years on ART for ≥12 months at 6 public facilities in Maputo, Mozambique were consecutively enrolled after informed consent. Chart review and caregiver interviews were conducted. VL testing was performed, and specimens with ≥1000 copies/mL were genotyped. Results Of the 715 children included, the mean age was 103 months, 85.8% had no immunosuppression, 73.1% were taking stavudine/lamivudine/nevirapine, and 20.1% had a history prevention of mother-to-child transmission exposure. The mean time on ART was 60.0 months. VF was present in 259 patients (36.3%); 248 (95.8%) specimens were genotyped, and DR mutations were found in 238 (96.0%). Severe immunosuppression and nutritional decline were associated with DR. M184V and Y181C were the most common mutations. In the 238 patients with DR, standard second-line ART would have 0, 1, 2, and 3 effective antiretrovirals in 1 (0.4%), 74 (31.1%), 150 (63.0%), and 13 (5.5%) patients, respectively. Conclusion This cohort had high rates of VF and DR with frequent compromise of second-line ART. There is urgent need to scale-up VL monitoring and heat-stable protease inhibitor formulations or integrase inhibitorsfor a more a durable first-line regimen that can feasibly be implemented in developing settings.

A3 Genetic analysis and natural polymorphisms in HIV-1 gp41 isolates from Maputo, Mozambique

Twenty patients with hemophilia (HP) were diagnosed with HIV-1, one to two years after exposure to domestic clotting factor IX (DCF), in 1989–90. Previous analysis of pol and vif genes confirmed that HIV-1 transmission to 20 HPs occurred through infusion of DCF. In this study, we determined full-length env gene sequences (about 2,550 bp) in 21 HPs, three plasma donors whose plasma were used in DCF production, and 45 local controls. The env gene from frozen stored sera obtained 1–3 years after diagnosis as well as from samples collected years after infection, was amplified by RT-PCR, and subjected to direct sequencing. Phylogenetic analysis revealed that all sequences were subtype B, with 109 sequences from 64 patients (20 HPs, 3 serum donors, 41 local controls, LCs) belonging to the Korean subclade (KSB) and 13 sequences from 5 patients (1 HP infected outside Korea, 4 LCs) not in the KSB. Sequences of the env gene from donors O and P plus the 20 HPs comprised two subclusters within KSB. In addition, signature pattern analysis revealed signature nucleotides at 45 positions between the HPs and LCs (P< 0.05). Surprisingly, specific signature nucleotides positions were conserved 100% in clusters O and P only (at 4 and 1 positions, respectively) with none in LCs. Within both clusters, sequence identity was high. These results are consistent with our previous conclusion that these 20 HPs were infected with viruses in the DCF used for treatment. In addition, we found that there are 25 signature pattern residues in amino acids in env gene of KSB distinct from subtype B.