The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Data Availability Nucleotide sequences reported in the paper have been deposited in the GenBank repository (https://www.ncbi.nlm.nih.gov/genbank/, Accession Figures: MH605452-MH605505).. found, followed by HIV-1 is the most prevalent variant in several West African countries and accounts for 39C83% of the infections in this area [11C13]. Three major HIV subtypes/CRFs have been explained in Guinea-Bissau: and referred to as [12, 13]. This may be important in the HIV epidemic since different viral variants have been linked to differences in viral weight [14C16], disease progression rate [12], vertical transmission rate [17] and propensity to develop resistance to ART [18]. Documenting the profile of DR in HIV-1-infected pregnant women is crucial for improving the efficacy of maternal ART and prophylaxis in infants, and is also a preferred approach to estimate pretreatment DR (19). This can also help policy makers in the process of designing future national HIV treatment guidelines. Thus, in the context of increasing prevalence of acquired DR, and to gain an understanding of the effectiveness of contemporary ART in Guinea-Bissau, the aim of the current study was to estimate the level of pretreatment DR among pregnant women in the country. Moreover, since resistance data linked to information regarding HIV-1 subtypes and recombinants circulating among pregnant women have not been reported in Guinea-Bissau previously, this was also studied. Methods Study design and participants Pregnant women who tested positive for HIV-1 in antenatal screening at four antenatal care clinics in the capital Bissau: Bairro Militar Health Centre, Antula Health Centre, Quelele Health Centre and Plack-II Health Centre, were asked for participation in the study. All participants finalized a questionnaire regarding previous HIV screening, antiretroviral treatment/mother-to-child prophylaxis as well as questions of a socio-economical nature. The survey aimed to follow the World Health Organization (WHO) recommended threshold survey methodology [19]. However, the WHO threshold survey methodology was under revision at the time of this study, and hence, we used the pre-revised guidelines. Original inclusion criteria were laboratory confirmation of HIV infection, age 25 years and no previous pregnancies. Due to frequent stock outs of HIV tests and a slower inclusion rate as a Rabbit Polyclonal to CDC2 result, and in order not to prolong the study period, we omitted the age limit of 25 years and also included women with previous pregnancies. A total of 52 reportedly antiretroviral-na? ve HIV-infected pregnant women were enrolled from October 2016 to November 2017. All participants that tested HIV positive were counselled and informed about antiretroviral treatment (ART), and were offered ART through the local health centre or at centralised services within Bissau City. Sample management Determine (Abbott Diagnostic Division, Hoofddorp, Holland) was used URAT1 inhibitor 1 for pretreatment HIV diagnosis at the antenatal care clinics. Samples were transported to the laboratory for national health (LNSP) and analyzed for CD4 absolute count, CD4% and haemoglobin count using FACSPrestoNear Patient CD4 counter (Becton Dickinson, NYSE:BDX, USA). A confirmatory HIV-1 discriminatory test was performed using Geenius HIV 1/2 confirmatory assay (Bio-RAD). Plasma was separated from whole blood by centrifugation and stored at -20 C until transported on dry ice for further storing in -80 C and genotyping at the Clinical Virology section at Lund University, Sweden. Drug resistance genotyping RNA was extracted from plasma using the QIAamp Viral RNA Mini Kit (Qiagen). Reverse transcription and PCR amplification of HIV-1 gene were done using One-Step SuperScript III RT/Platinum Taq High Fidelity Enzyme Mix (ThermoFisher Scientific), using JA269 and JA272 primers [20]. For nested PCR, High Fidelity Platinum Taq DNA Polymerase, (ThermoFisher URAT1 inhibitor 1 Scientific) was used, with primers JA270 and JA271 [20], resulting in a PCR fragment of 1086 bases. The PCR products were sequenced in both directions with six primers described by Zhou et al. [21] using the BigDye terminator kit v 1.1 (Applied Biosystem) followed by sequence analysis on an ABI PRISM 3130 l genetic analyzer (Applied Biosystem). Sequence assembly and editing were performed using RECall V 2.0 HIV sequencing analysis tool [22]. The final length of all the sequences following removal of regions corresponding to the primers, editing and alignment was 1035 bases, corresponding to nucleotide positions 2268C3302 of HXB2 (GenBank accession number “type”:”entrez-nucleotide”,”attrs”:”text”:”K03455″,”term_id”:”1906382″,”term_text”:”K03455″K03455). Sequence quality control was performed using the quality control program of the Los Alamos HIV sequence database [23]. The presence of drug resistance mutations (DRM) was assessed using the Stanford Genotypic Resistance Interpretation Algorithm [24]. DRM were examined according to the calibrated population resistance (CPR) tool v6.0 beta [25] (http://cpr.stanford.edu/cpr/servlet/CPR), based on the WHO surveillance transmitted drug resistance mutation list of 2009 [26]. Pretreatment DR was referred to as low ( 5%), moderate (5C15%), or high ( 15%) [27]. HIV subtyping All sequences were screened for recombination using RDP v.3.44 [28]. The sequences were subtyped through phylogenetic analysis with group M.Sequences were aligned using ClustalX2 and then edited to a final length of 1035 bases for each sequence using BioEdit [32, 33]. the HIV epidemic since different viral variants have been linked to differences in viral load [14C16], disease progression rate [12], vertical transmission rate [17] and propensity to develop resistance to ART [18]. Documenting the profile of DR URAT1 inhibitor 1 in HIV-1-infected pregnant women is crucial for improving the efficacy of maternal ART and prophylaxis in infants, and is also a preferred approach to estimate pretreatment DR (19). This can also help policy makers in the process of designing future national HIV treatment guidelines. Thus, in the context of increasing prevalence of acquired DR, and to gain an understanding of the effectiveness of contemporary ART in Guinea-Bissau, the aim of the current study was to estimate the level of pretreatment DR among pregnant women in the country. Moreover, since resistance data linked to information regarding HIV-1 subtypes and recombinants circulating among pregnant women have not been reported in Guinea-Bissau previously, this was also studied. Methods Study design and participants Pregnant women who tested positive for HIV-1 in antenatal screening at four URAT1 inhibitor 1 antenatal care clinics in the capital Bissau: Bairro Militar Health Centre, Antula Health Centre, Quelele Health Centre and Plack-II Health Centre, were asked for participation in the study. All participants finalized a questionnaire regarding previous HIV testing, antiretroviral treatment/mother-to-child prophylaxis as well as questions of a socio-economical nature. The survey aimed to follow the World Health Organization (WHO) recommended threshold survey methodology [19]. However, the WHO threshold survey methodology was under revision at the time of this study, and hence, we used the pre-revised guidelines. Original inclusion criteria were laboratory confirmation of HIV infection, age 25 years and no previous pregnancies. Due to frequent stock outs of HIV tests and a slower inclusion rate as a result, and in order not to prolong the study period, we omitted the age limit of 25 years and also included women with previous pregnancies. A total of 52 reportedly antiretroviral-na?ve HIV-infected pregnant women were enrolled from October 2016 to November 2017. All participants that tested HIV positive were counselled and informed about antiretroviral treatment (ART), and were offered ART through the local health centre or at centralised solutions within Bissau City. Sample management Determine (Abbott Diagnostic Division, Hoofddorp, Holland) was utilized for pretreatment HIV analysis in the antenatal care clinics. Samples were transported to the laboratory for national health (LNSP) and analyzed for CD4 absolute count, CD4% and haemoglobin count using FACSPrestoNear Patient CD4 counter (Becton Dickinson, NYSE:BDX, USA). A confirmatory HIV-1 discriminatory test was performed using Geenius HIV 1/2 confirmatory assay (Bio-RAD). Plasma was separated from whole blood by centrifugation and stored at -20 C until transferred on dry snow for further storing in -80 C and genotyping in the Clinical Virology section at Lund University or college, Sweden. Drug resistance genotyping RNA was extracted from plasma using the QIAamp Viral RNA Mini Kit (Qiagen). Reverse transcription and PCR amplification of HIV-1 gene were carried out using One-Step SuperScript III RT/Platinum Taq Large Fidelity Enzyme Blend (ThermoFisher Scientific), using JA269 and JA272 primers [20]. For nested PCR, Large Fidelity Platinum Taq DNA Polymerase, (ThermoFisher Scientific) was used, with primers JA270 and JA271 [20], resulting in a PCR fragment of 1086 bases. The PCR products were sequenced in both directions with six primers explained by Zhou et al. [21] using the BigDye terminator kit v.