HIV-1 molecular diversity in Brazil unveiled by 10 years of sampling by the national genotyping network.

HIV-1 has diversified into several subtypes and recombinant forms that are heterogeneously spread around the world. Understanding the distribution of viral variants and their temporal dynamics can help to design vaccines and monitor changes in viral transmission patterns. Brazil has one of the largest HIV-1 epidemics in the western-world and the molecular features of the virus circulating in the country are still not completely known. Over 50,000 partial HIV-1 genomes sampled between 2008 and 2017 by the Brazilian genotyping network (RENAGENO) were analyzed. Sequences were filtered by quality, duplicate sequences per patient were removed and subtyping was performed with online tools and molecular phylogeny. Association between patients' demographic data and subtypes were performed by calculating the relative risk in a multinomial analysis and trends in subtype prevalence were tested by Pearson correlation. HIV-1B was found to be the most prevalent subtype throughout the country except in the south, where HIV-1C prevails. An increasing trend in the proportion of HIV-1C and F1 was observed in several regions of the country, while HIV-1B tended to decrease. Men and highly educated individuals were more frequently infected by HIV-1B and non-B variants were more prevalent among women with lower education. Our results suggest that socio-demographic factors partially segregate HIV-1 diversity in Brazil while shaping viral transmission networks. Historical events could explain a preferential circulation of HIV-1B among men who have sex with men (MSM) and non-B variants among heterosexual individuals. In view of an increasing male/female ratio of AIDS cases in Brazil in the last 10-15 years, the decrease of HIV-1B prevalence is surprising and suggests a greater penetrance of non-B subtypes in MSM transmission chains.

Pregnant women co-infected with HIV and Zika: Outcomes and birth defects in infants according to maternal symptomatology.

BACKGROUND: Zika virus (ZIKV) was first isolated in Uganda in 1947. In Brazil, the first reported case of ZIKV infection was in May 2015. Additionally, dengue (DENV) is endemic and there has been a recent outbreak of chikungunya (CHIKV). Since the clinical manifestations of different arboviral infections (AI) can be similar, definitive diagnosis requires laboratory testing. OBJECTIVES: To determine the prevalence of ZIKV, DENV, and CHIKV infections in a Brazilian cohort of HIV-infected pregnant women, to assess clinical/immunological characteristics and pregnancy outcomes of women with evidence of recent AI. STUDY DESIGN: Laboratory diagnosis of ZIKV, DENV and CHIKV infections utilized serological assays, RT-PCR and PRNT. The tests were performed at the first visit, 34-36 weeks of gestation and at any time if a woman had symptoms suggestive of AI. Mann-Whitney tests were used for comparison of medians, Chi-square or Fisher's to compare proportions; p< 0.05 was considered statistically significant. Poisson regression was used to analyze risk factors for central nervous system (CNS) malformations in the infant according to maternal symptomatology. RESULTS: Of 219 HIV-infected pregnant women enrolled, 92% were DENV IgG+; 47(22%) had laboratory evidence of recent AI. Of these, 34 (72%) were ZIKV+, nine (19%) CHIKV+, and two (4%) DENV+. Symptoms consistent with AI were observed in 23 (10%) women, of whom 10 (43%) were ZIKV+, eight (35%) CHIKV+. No CNS abnormalities were observed among infants of DENV+ or CHIKV+ women; four infants with CNS abnormalities were born to ZIKV+ women (three symptomatic). Infants born to ZIKV+ women had a higher risk of CNS malformations if the mother was symptomatic (RR = 7.20), albeit not statistically significant (p = 0.066). CONCLUSIONS: Among HIV-infected pregnant women with laboratory evidence of a recent AI, 72% were ZIKV-infected. In this cohort, CNS malformations occurred among infants born to both symptomatic and asymptomatic pregnant women with Zika infection.

Brazilian network for HIV Drug Resistance Surveillance (HIV-BresNet): a survey of treatment-naive individuals.

INTRODUCTION: In Brazil, more than 487,450 individuals are currently undergoing antiretroviral treatment. In order to monitor the transmission of drug-resistant strains and HIV subtype distribution in the country, this work aimed to estimate its prevalence and to characterize the nationwide pretreatment drug resistance in individuals recently diagnosed with HIV between 2013 and 2015. METHODS: The HIV threshold survey methodology (HIV-THS, WHO) targeting antiretroviral-naive individuals with recent HIV diagnosis was utilized, and subjects were selected from 51 highly populated cities in all five Brazilian macroregions. The HIV pol genotypic test was performed by genomic sequencing. RESULTS: We analysed samples from 1568 antiretroviral-naive individuals recently diagnosed with HIV, and the overall transmitted drug resistance (TDR) prevalence was 9.5% (150 sequences). The regional prevalence of resistance according to Brazilian geographical regions was 9.4% in the northeast, 11.2% in the southeast, 6.8% in the central region, 10.2% in the north and 8.8% in the south. The inhibitor-specific TDR prevalence was 3.6% for nucleoside reverse transcriptase inhibitors (NRTIs), 5.8% for non-nucleoside reverse transcriptase inhibitors (NNRTIs) and 1.6% for protease inhibitors (PIs); 1.0% of individuals presented resistance to more than one class of inhibitors. Overall, subtype B was more prevalent in every region except for the southern, where subtype C prevails. CONCLUSIONS: To the best of our knowledge, this is the first TDR study conducted in Brazil with nationwide representative sampling. The TDR prevalence revealed a moderate rate in the five Brazilian geographical regions, although some cities presented higher TDR prevalence rates, reaching 14% in São Paulo, for example. These results further illustrate the importance of surveillance studies for designing future strategies in primary antiretroviral therapy, aiming to mitigate TDR, as well as for predicting future trends in other regions of the globe where mass antiretroviral (ARV) treatment was implemented.

The spectrum of neuropathological changes associated with congenital Zika virus infection.

A major concern associated with ZIKV infection is the increased incidence of microcephaly with frequent calcifications in infants born from infected mothers. To date, postmortem analysis of the central nervous system (CNS) in congenital infection is limited to individual reports or small series. We report a comprehensive neuropathological study in ten newborn babies infected with ZIKV during pregnancy, including the spinal cords and dorsal root ganglia (DRG), and also muscle, pituitaries, eye, systemic organs, and placentas. Using in situ hybridization (ISH) and electron microscopy, we investigated the role of direct viral infection in the pathogenesis of the lesions. Nine women had Zika symptoms between the 4th and 18th and one in the 28th gestational week. Two babies were born at 32, one at 34 and 36 weeks each and six at term. The cephalic perimeter was reduced in four, and normal or enlarged in six patients, although the brain weights were lower than expected. All had arthrogryposis, except the patient infected at 28 weeks gestation. We defined three patterns of CNS lesions, with different patterns of destructive, calcification, hypoplasia, and migration disturbances. Ventriculomegaly was severe in the first pattern due to midbrain damage with aqueduct stenosis/distortion. The second pattern had small brains and mild/moderate (ex-vacuo) ventriculomegaly. The third pattern, a well-formed brain with mild calcification, coincided with late infection. The absence of descending fibres resulted in hypoplastic basis pontis, pyramids, and cortico-spinal tracts. Spinal motor cell loss explained the intrauterine akinesia, arthrogryposis, and neurogenic muscle atrophy. DRG, dorsal nerve roots, and columns were normal. Lympho-histiocytic inflammation was mild. ISH showed meningeal, germinal matrix, and neocortical infection, consistent with neural progenitors death leading to proliferation and migration disorders. A secondary ischemic process may explain the destructive lesions. In conclusion, we characterized the destructive and malformative consequences of ZIKV in the nervous system, as reflected in the topography and severity of lesions, anatomic localization of the virus, and timing of infection during gestation. Our findings indicate a developmental vulnerability of the immature CNS, and shed light on possible mechanisms of brain injury of this newly recognized public health threat.

Congenital Zika Virus Infection: Beyond Neonatal Microcephaly.

IMPORTANCE: Recent studies have reported an increase in the number of fetuses and neonates with microcephaly whose mothers were infected with the Zika virus (ZIKV) during pregnancy. To our knowledge, most reports to date have focused on select aspects of the maternal or fetal infection and fetal effects. OBJECTIVE: To describe the prenatal evolution and perinatal outcomes of 11 neonates who had developmental abnormalities and neurological damage associated with ZIKV infection in Brazil. DESIGN, SETTING, AND PARTICIPANTS: We observed 11 infants with congenital ZIKV infection from gestation to 6 months in the state of Paraíba, Brazil. Ten of 11 women included in this study presented with symptoms of ZIKV infection during the first half of pregnancy, and all 11 had laboratory evidence of the infection in several tissues by serology or polymerase chain reaction. Brain damage was confirmed through intrauterine ultrasonography and was complemented by magnetic resonance imaging. Histopathological analysis was performed on the placenta and brain tissue from infants who died. The ZIKV genome was investigated in several tissues and sequenced for further phylogenetic analysis. MAIN OUTCOMES AND MEASURES: Description of the major lesions caused by ZIKV congenital infection. RESULTS: Of the 11 infants, 7 (63.6%) were female, and the median (SD) maternal age at delivery was 25 (6) years. Three of 11 neonates died, giving a perinatal mortality rate of 27.3%. The median (SD) cephalic perimeter at birth was 31 (3) cm, a value lower than the limit to consider a microcephaly case. In all patients, neurological impairments were identified, including microcephaly, a reduction in cerebral volume, ventriculomegaly, cerebellar hypoplasia, lissencephaly with hydrocephalus, and fetal akinesia deformation sequence (ie, arthrogryposis). Results of limited testing for other causes of microcephaly, such as genetic disorders and viral and bacterial infections, were negative, and the ZIKV genome was found in both maternal and neonatal tissues (eg, amniotic fluid, cord blood, placenta, and brain). Phylogenetic analyses showed an intrahost virus variation with some polymorphisms in envelope genes associated with different tissues. CONCLUSIONS AND RELEVANCE: Combined findings from clinical, laboratory, imaging, and pathological examinations provided a more complete picture of the severe damage and developmental abnormalities caused by ZIKV infection than has been previously reported. The term congenital Zika syndrome is preferable to refer to these cases, as microcephaly is just one of the clinical signs of this congenital malformation disorder.

Lopinavir Resistance Classification with Imbalanced Data Using Probabilistic Neural Networks.

Resistance to antiretroviral drugs has been a major obstacle for long-lasting treatment of HIV-infected patients. The development of models to predict drug resistance is recognized as useful for helping the decision of the best therapy for each HIV+ individual. The aim of this study was to develop classifiers for predicting resistance to the HIV protease inhibitor lopinavir using a probabilistic neural network (PNN). The data were provided by the Molecular Virology Laboratory of the Health Sciences Center, Federal University of Rio de Janeiro (CCS-UFRJ/Brazil). Using bootstrap and stepwise techniques, ten features were selected by logistic regression (LR) to be used as inputs to the network. Bootstrap and cross-validation were used to define the smoothing parameter of the PNN networks. Four balanced models were designed and evaluated using a separate test set. The accuracies of the classifiers with the test set ranged from 0.89 to 0.94, and the area under the receiver operating characteristic (ROC) curve (AUC) ranged from 0.96 to 0.97. The sensitivity ranged from 0.94 to 1.00, and the specificity was between 0.88 and 0.92. Four classifiers showed performances very close to three existing expert-based interpretation systems, the HIVdb, the Rega and the ANRS algorithms, and to a k-Nearest Neighbor.

Prevalence of etravirine-associated mutations in clinical samples with genotypic resistance to nevirapine and efavirenz in Brazilian clinics.

Nonnucleoside reverse transcriptase inhibitors (NNRTIs) are potent and well tolerated. In Brazil, the first-generation NNRTI efavirenz is included in the majority of first-line antiretroviral treatment regimens. In this study, we evaluated the effectiveness of etravirine, a new second-generation NNRTI, among patients failing antiretroviral regimens containing first-generation NNRTIs. We assessed single resistance mutations to etravirine as well as complex resistance mutations profile and discuss the potential of introducing etravirine as salvage therapy.

Accumulation of P(T/S)AP late domain duplications in HIV type 1 subtypes B, C, and F derived from individuals failing ARV therapy and ARV drug-naive patients.

HIV-1 budding requires short peptide motifs in p6(Gag), known as late domains, that promote the release of infectious virions. The primary late domain of HIV-1 is a Pro-(Thr/Ser)-Ala-Pro (hereafter referred to as a PTAP) motif. This motif may be completely or partially duplicated. In this work we analyzed p6(Gag) sequences from 547 isolates from drug-naive patients and 213 isolates from patients failing HAART therapy. Complete duplications within PTAP were selected during HAART therapy in all HIV-1 subtypes analyzed: B (p = 0.0338), F1 (p = 0.0294), and C (p = 0.0001). Nevertheless, the patterns of these duplications were different; subtype C isolates accumulated longer duplications and displayed a higher frequency of duplications in both treated (54%) and drug-naive isolates (23%). Accumulation of PTAP duplications within subtypes B, F1, and C during therapy suggests a potential role of the duplications in antiretroviral drug resistance.

Brazilian Network for HIV Drug Resistance Surveillance: a survey of individuals recently diagnosed with HIV.

Use of antiretrovirals is widespread in Brazil, where more than 200,000 individuals are under treatment. Although general prevalence of primary antiretroviral resistance in Brazil is low, systematic sampling in large metropolitan areas has not being performed.The HIV Threshold Survey methodology (HIV-THS, WHO) was utilized, targeting Brazil's four major regions and selecting the six most populated state capitals: Sao Paulo, Rio de Janeiro, Salvador, Porto Alegre, Brasilia and Belem. We were able to sequence samples from 210 individuals with recent HIV diagnosis, 17 of them (8.1%) carrying HIV isolates with primary antiretroviral resistance mutations. Five, nine and four isolates showed mutations related to resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs) and protease inhibitors (PIs), respectively. Using HIV-THS, we could find an intermediate level of transmitted resistance (5% to 15%) in Belem/Brasilia, Sao Paulo and Rio de Janeiro. Lower level of transmitted resistance (<5%) were observed in the other areas. Despite the extensive antiretroviral exposure and high rates of virologic antiretroviral failure in Brazil, the general prevalence of primary resistance is still low. However, an intermediate level of primary resistance was found in the four major Brazilian cities, confirming the critical need to start larger sampling surveys to better define the risk factors associated with transmission of resistant HIV.

Genotypic and phenotypic characterization of human immunodeficiency virus type 1 isolates circulating in pregnant women from Mozambique.

This work evaluated HIV-1 subtypes from different geographic regions and phenotypic data from drug-naïve HIV-positive pregnant women from Mozambique. We analyzed 75 pol sequences from patients and the distribution of the subtypes in three regions of Mozambique and found that the majority of samples analyzed clustered with subtype C. In the northern region, multiple variants were found 5 (approximately 18%) subtype A, 3 (approximately 11%) subtype D and 2 (approximately 7.1%) mosaics (A/C/D and C/D), whereas 18 (64.3%) isolates were subtype C, from a total of 28 samples. Already in the southern region, only one (5%) isolate of 20 samples was subtype D, and the other 19 (95%) isolates were subtype C. All 27 (100%) isolates from the central region grouped within clade C. No primary resistance mutations to IP, NNRTI or NRTI were found. There was no evidence of phenotypic resistance in any of the isolates tested, suggesting that neither the polymorphism in the protease, nor the one found at codon 215 of the RT gene caused an increase in phenotypic resistance. This finding suggests that HAART regimens indicated by WHO will probably be successful in Mozambique.

Biological characterization of human immunodeficiency virus type 1 subtype C protease carrying indinavir drug-resistance mutations.

Human immunodeficiency virus type 1 subtype C isolates belong to one of the most prevalent strains circulating worldwide and are responsible for the majority of new infections in the sub-Saharan region and other highly populated areas of the globe. In this work, the impact of drug-resistance mutations in the protease gene of subtype C viruses was analysed and compared with that of subtype B counterparts. A series of recombinant subtype C and B viruses was constructed carrying indinavir (IDV)-resistance mutations (M46V, I54V, V82A and L90M) and their susceptibility to six FDA-approved protease inhibitor compounds (amprenavir, indinavir, lopinavir, ritonavir, saquinavir and nelfinavir) was determined. A different impact of these mutations was found when nelfinavir and lopinavir were tested. The IDV drug-resistance mutations in the subtype C protease backbone were retained for a long period in culture without selective pressure when compared with those in subtype B counterparts in washout experiments.

Low accumulation of L90M in protease from subtype F HIV-1 with resistance to protease inhibitors is caused by the L89M polymorphism.

BACKGROUND: This work evaluates the role of subtype F human immunodeficiency virus type 1 (HIV-1) protease (PR) substitutions L89M and L90M in viral replication and resistance to PR inhibitors (PIs). METHODS: Subtype B and F PR genes were subjected to site-directed mutagenesis, to create and reverse the methionine at positions 89 and 90. Viruses were re-created in cell culture, and their replicative capacity was assessed by fitness assay. Generated viruses were also phenotyped for PI resistance. RESULTS: The subtype F clone (89M90L) showed a replicative capacity comparable to that of the PI-susceptible subtype B clone (89L90L) and was more fit than the L89M mutated subtype B clone (89M90L). Both 89M90M subtype B and F clones presented the lowest fitness s values. The L89M mutation impacted phenotypic resistance to all PIs in half of the subtype F isolates but not in the subtype B isolates. Subtype F isolates presented a phenotypic profile similar to that of subtype B isolates when the M89L mutation was introduced. CONCLUSION: The L89M mutation in subtype F viruses is a high genetic barrier to the accumulation of the L90M resistance mutation and can function as a resistance mutation, depending on the presence of other polymorphisms in the subtype F PR backbone.