Use of Sanger protocols to identify variants of concern, key mutations and track evolution of SARS-CoV-2.

Vaccination and the emergence of SARS-CoV-2 variants mark the second year of the pandemic. Variants have amino acid mutations at the spike region, a viral protein central in the understanding of COVID-19 pathogenesis and vaccine response. Variants may dominate local epidemics, as Gamma (P.1) in Brazil, emerging in 2020 and prevailing until mid-2021. Different obstacles hinder a wider use of Next-Generation Sequencing for genomic surveillance. We describe Sanger based sequencing protocols: i) Semi-nested RT-PCR covering up to 3.684 kb (>96 %) spike gene; ii) One-Step RT-PCR for key Receptor Binding Domain (RBD) mutations (codons 417-501); iii) One-Step RT-PCR of partial N region to improve genomic capability. Protocols use leftovers of RNA extracted from nasopharyngeal swabs for quantitative RT-PCR diagnosis; with retro-transcribed DNA sequenced at ABI 3500 using dye termination chemistry. Analyses of sequences from 95 individuals (late 2020/early 2021) identified extensive amino acid variation, 57 % with at least one key mutation at the Receptor Binding Domain, with B.1.1.28 lineage most prevalent, followed by Gamma and Zeta variants, with no Delta variant observed. The relatively low cost and simplicity may provide an accessible tool to improve surveillance of SARS-CoV-2 evolution, monitor new variants and vaccinated breakthroughs.

Prevalence of HIV-1 transmitted drug resistance and viral suppression among recently diagnosed adults in São Paulo, Brazil.

HIV-1 transmitted drug resistance (TDR) mutations may reduce the efficacy of antiretroviral therapy (ART), but pre-treatment testing to determine the virus genotype can improve the efficacy of ART. Unfortunately, issues related to cost and logistics of pre-treatment testing limit its use in resource-limited settings. We studied 596 ART-naive individuals who were newly diagnosed from 2014 to 2016 in São Paulo, Brazil, to evaluate TDR and virological outcome after 48 weeks of genotype-guided therapy. One or more TDR (based on the WHO surveillance list) was observed in 10.9% (CI 95%, 8.6-13.6) of the sequences, the most common of which was the K103 N mutation, which confers resistance to first-generation drugs of the non-nucleoside reverse transcriptase inhibitor (NNRTI) antiretroviral drug class. Dual-class (1%, 6/596) and triple-class (0.34%, 2/596) resistance were uncommon. After 48 weeks of treatment with ART, infection was suppressed to below 200 copies/mL in most patients (95%), with full suppression (RNA target not detected) in 65%. The following characteristics at patient enrollment were independently associated with a lack of full suppression: CD4 T cell counts below 500 cells/µL, viremia above 100,000 copies/mL, older age, and TDR to NNRTI. The rates of resistance were intermediate, but genotype-guided therapy resulted in high rates of viral suppression. The observed resistance profile should not be an obstacle to the use of the dolutegravir-based regimen now recommended in Brazil, but genotype testing may be warranted before initiating first-generation NNRTI-based regimens.

HIV-1-infected patients with advanced disease failing a raltegravir-containing salvage regimen in São Paulo, Brazil.

Raltegravir (RAL) is the first licensed antiretroviral integrase inhibitor that may be used both for treatment-naïve human immunodeficiency virus type 1 (HIV-1) patients and for salvage therapy. The Brazilian public free access programme limits its use for salvage therapy, with scarce information regarding RAL resistance from patients failing a RAL-containing salvage regimen. This study evaluated RAL resistance mutations detected by population sequencing in 69 HIV-infected patients with advanced disease failing a RAL-containing regimen in a real-world setting. RAL resistance mutations were identified in 47/69 patients (68%). The most common salvage regimen, used by 56/69 patients (81%), included lamivudine, tenofovir, darunavir/ritonavir and RAL. At failure, major RAL resistance mutations included Q148H/R/K (21/47; 45%), N155H (14/47; 30%), Y143R/H/C (3/47; 6%) and E92Q (1/47; 2%). Most samples with Q148H/R/K also showed G140S/A/C (21/47; 45%). RAL resistance was significantly associated with less than two active drugs in the optimised background therapy regimen at failure [39/39 (100%) vs. 9/17 (53%); P<0.001] and with a longer cumulative duration with detectable viraemia (viral load >50 copies/mL) (86 weeks vs. 32 weeks; P=0.001). A high frequency of RAL mutations was observed in this study. In addition, these results reinforce the importance of close monitoring of RAL-containing regimens to reduce the time of failure and consequent resistance accumulation.

Discrepancies of HIV-1 reverse transcriptase resistance interpretation of insertions and deletions between two genotypic algorithms.

BACKGROUND: Bioinformatics algorithms have been developed for the interpretation of resistance from sequence submission, which supports clinical decision making. This study evaluated divergences of the interpretation of the genotyping in two commonly used algorithms, using sequences with indels of reverse transcriptase genes. METHODS: Sequences were obtained from virus RNA of patients failing highly active antiretroviral therapy from 2004 to 2011. Alignments were obtained using Clustal W including subtype B consensus and HXB2. Sequences with evidence of indels were submitted to the Stanford Resistance Database and to the Geno2Pheno to locate indel positioning and determine the resistance profile. RESULTS: A total of 1,959 partial reverse transcriptase sequences were assessed, mostly subtype B (74%). Insertions and deletions were observed in 0.9 and 0.6% of sequences, respectively. Discordant insert positioning was assigned for most (90%) insertion sequences, with 27% discordances for deletions. Susceptibility differed for some antiretroviral drugs, predominantly for TDF, d4T and ETV, when sequences with deletions were evaluated. CONCLUSION: Both indel positioning and its impact on drug susceptibility varies depending on the algorithm, a fact that might influence the clinical decision. Critical analysis of indel sequences with manual alignments is important, and its use alongside different algorithms may be important to better understand the outcomes of genotypic resistance prediction.

Prevalence of transmitted HIV-1 drug resistance mutations in children and adolescents in São Paulo, Brazil.

HIV drug resistance genotype testing, performed on 39 HIV-infected treatment-naive children from 2000 to 2011, identified 5 children (12.8%) with drug resistance mutations: 5.3% to nucleoside reverse transcriptase inhibitors, 5.3% to nonnucleoside reverse transcriptase inhibitors and 7.7% to protease inhibitors. There was a trend for increasing prevalence of drug resistance mutations during the 11-year study period.

In-vivo selection of the mutation F121Y in a patient failing raltegravir containing salvage regimen.

Raltegravir is an integrase inhibitor (INI) licensed for clinical use and other INI are in advanced stage of development. Different resistance mutations in HIV integrase from patients using these antiretroviral drugs have been described and G148H/R/K, N155H and less frequently Y143C/H/R are considered major resistant mutations to raltegravir. Both Stanford Database and Geno2Pheno list F121Y as conferring intermediate resistance "in vitro" both to raltegravir and elvitegravir. We report for the first time the "in vivo" selection F121Y and evolution to Y143R in a 31years old male clade B HIV-1 infected patient failing a raltegravir-containing salvage regimen. Plasma samples nine months prior to raltegravir (RAL-Naïve) and at weeks 32, 40 and 88 after RAL-containing regimen were analyzed. Antiretroviral susceptibility was evaluated at Stanford and Geno2Pheno from sequences obtained with RT-PCR. After a Viral load at week 12 below 50 copies/mL, viremia raised at week 20 to 4.5log10. The emergence of F121Y was observed at week 32 and 40, alongside with L74I, T97A, Q137H and V151I. At week 88 F121Y was no longer detected, L74I and T97A were maintained and Y143R emerged. F121Y might be an alternative pathway to Y143R. Changing of RAL-containing regimen upon the identification of F121Y might avoid the evolution of raltegravir resistance.

High-resolution phylogenetics and phylogeography of human immunodeficiency virus type 1 subtype C epidemic in South America.

Human immunodeficiency virus type 1 subtype C (HIV-1C) represents 30-65% of HIV infections in southern Brazil, and isolated cases of HIV-1C infection have also been reported in Argentina, Uruguay, Paraguay and Venezuela. Phylogenetic studies have suggested that the Brazilian subtype C epidemic was initiated by the introduction of closely related strains. Nevertheless, because of sampling limitations, the point of entry and the timing of subtype C introduction into Brazil, as well as the origin of the founder lineage, remain controversial. The present study investigated the origin, spread and phylogeography of HIV-1C in South America. Phylogenetic analysis showed a well-supported monophyletic clade including all available strains from Brazil, Uruguay and Argentina. Only one lineage from Venezuela was unrelated to the epidemic involving the other three countries. Molecular clock and likelihood mapping analysis showed that HIV-1C introduction in Brazil dated back to the period 1960-1970, much earlier than previously thought, and was followed by a nearly simultaneous star-like outburst of viral lineages, indicating a subsequent rapid spread. Phylogeographic patterns suggested Paraná or Rio Grande do Sul as the possible entrance points of subtype C and an asymmetrical gene flow from Paraná to Sao Paulo, Santa Catarina and Rio Grande do Sul, as well as from Rio Grande do Sul to Sao Paulo fostered by the strong inter-connectivity between population centres in southern Brazil. The study illustrates how coupling phylogeography inference with geographical information system data is critical to understand the origin and dissemination of viral pathogens and potentially predict their future spread.

Long terminal repeat sequence analysis of HTLV-2 molecular variants identified in Southern Brazil.

In Brazil, human T-lymphotropic virus type 2 (HTLV-2) is endemic in Amerindians and epidemic in intravenous drug users (IDUs). The long terminal repeat (LTR) is the most divergent genomic region of HTLV-2, therefore useful to characterize subtypes. Nucleotide sequence and restriction fragment length polymorphism (RFLP) analysis of LTR genomic segments of fourteen HTLV-2 strains isolated from HIV-infected patients of Londrina, Southern Brazil, were carried out. Molecular analysis disclosed that all HTLV-2 strains belonged to 2a subtype, and RFLP detected the presence of the a4, a5, and a6 subgroups according to Switzer's nomenclature. RFLP correlated with nucleotide sequence, and phylogenetic analysis clustered HTLV-2 sequences of IDUs into subgroups a5 and a6. HTLV-2 sequences from individuals of sexual risk factor clustered into the a4 subgroup. These results extend the knowledge of the genetic diversity of HTLV-2 circulating in Brazil and provide insights into HTLV-2 transmission and virus movement in this geographic area.

Young pregnant women living with HIV/AIDS in Criciuma, Southern Brazil, are infected almost exclusively with HIV type 1 clade C.

Southern Brazil has the highest prevalence rate of AIDS in the country and is the only region in the Americas where HIV-1 C prevails. Metropolitan areas and harbor cities have been evaluated, but limited information is available for small towns and specific populations. We studied women attending the obstetric outpatient clinic of Criciuma, State of Santa Catarina in 2007 to evaluate the molecular epidemiology of HIV-1 among pregnant women living with HIV/AIDS. Forty-two cases had partial pol gene sequenced and additional partial gag and/or env genes from nine women. HIV subtyping was evaluated by phylogenetic methods and antiretroviral (ARV) drug resistance mutations (DRMs) at the Stanford Database. DRMs to one or more ARV class was observed in 20/42, 48% of cases, with 15/41, 37% with viral load <500 copies/ml. Subtype C at pol was identified in 33/42, 78.6% (95% CI: 64-89%), C mosaics (CB, CF) in 2, 4.8% (95% CI: 0.8-19%), F in 4, 9.5% (95% CI: 3-21%), and B in 3, 7.1% (95% CI: 1.8-18%). Discordance in concatenated gag/pol/env or intraregion mosaic was observed in 1/9, 11% of HIV-1 C genomes. The proportion of HIV-1 C in this study is the highest rate described in the Americas. Molecular surveillance in specific populations is instrumental for a better understanding of the Brazilian HIV epidemic.

High frequency of BF mosaic genomes among HIV-1-infected children from Sao Paulo, Brazil.

HIV-1 genetic diversity information from a pediatric population is scarce. This study enrolled 128 children living with HIV/AIDS, 103 antiretroviral-treated and 25 naive, from the Sao Paulo metropolitan area. Gag, pol and env regions were amplified, and drug resistance mutations, V3 loop, tropism and viral clades were evaluated. Drug resistance mutations among naïve children infected by vertical transmission were uncommon (4.2%), whereas most ARV-experienced children showed extensive mutation patterns. Clade B predominated at the pol region, but the analysis of the three regions concatenated showed 28% with BF mosaic structures. The most common V3 motif was GPGR, followed by GWGR in clade B samples and GPGQ in clade F samples. A predicted X4 phenotype was observed in 27%, without correlation to HIV clade. These findings expand the limited information on molecular characteristics of HIV-1 among children living with HIV/AIDS in the area and may provide information useful for monitoring the epidemic.

Rate and incidence estimates of recent human immunodeficiency virus type 1 infections among pregnant women in Sao Paulo, Brazil, from 1991 to 2002.

The serological testing algorithm for recent human immunodeficiency virus (HIV) seroconversion (STARHS) was employed to estimate HIV incidence among pregnant women from Sao Paulo, Brazil. A cross-sectional study (1999 to 2002) showed an incidence of infection of 0.2 per 100 pregnant women per year (95% confidence interval, 0.041 to 0.608). Western blot profiles suggested an association between results of the STARHS analysis and gp41/gp31 bands.