HIV diagnosis in Equatorial Guinea. Keys to reduce the diagnostic and therapeutic delay.

BACKGROUND: In Equatorial Guinea, only 54 % of people living with HIV know their HIV status. There are no confirmatory or molecular diagnostic techniques for early diagnosis or monitoring of infection in the country. Rapid diagnostic tests can induce false-positive diagnoses if used as a confirmatory technique. Our study aimed to identify the challenges of early HIV diagnosis in Equatorial Guinea by analyzing the rate of false positive diagnoses, diagnostic and therapeutic delays, and treatment failures among those on antiretroviral therapy. METHODS: From 2019-2022, dried blood from 341 children, adolescents and adults diagnosed in Equatorial Guinea as HIV-positive by rapid diagnostic testing, and from 54 HIV-exposed infants were collected in Bata and sent to Madrid to confirm HIV-infection by molecular (Xpert HIV-1Qual, Cepheid) and/or serological confirmatory assays (Geenius-HIV-1/2, BioRad). HIV diagnostic delay (CD4 <350cells/mm3), advanced disease at diagnosis (CD4 <200cells/mm3) and antiretroviral treatment delay and failure (viraemia >1,000RNA-HIV-1-copies/ml) were also studied after viral quantification (XpertVL HIV-1, Cepheid). RESULTS: False-positive diagnoses were identified in 5 % of analysed samples. HIV infection was confirmed in 90.5 % of previously diagnosed patients in Equatorial Guinea and 3.7 % of HIV-exposed children undiagnosed in the field. Two-thirds of each new HIV patient had delayed diagnosis, and one-third had advanced disease. Treatment delay occurred in 28.3 % of patients, being around four times more likely in adolescents/adults than children. More than half (56 %) of 232 treated patients presented treatment failure, being significantly higher in children/adolescents than in adults (82.9 %/90 % vs. 45.6 %, p < 0.001). CONCLUSION: We identified some challenges of early HIV diagnosis in Equatorial Guinea, revealing a high rate of false positive diagnoses, diagnostic/treatment delays, and treatment failures that need to be addressed. The implementation of more accurate rapid diagnostic techniques and confirmatory tests, along with improving access to care, treatment, awareness, and screening, would contribute to controlling the spread of HIV in the country.

High Drug Resistance Levels Compromise the Control of HIV Infection in Pediatric and Adult Populations in Bata, Equatorial Guinea.

A lack of HIV viral load (VL) and HIV drug resistance (HIVDR) monitoring in sub-Saharan Africa has led to an uncontrolled circulation of HIV-strains with drug resistance mutations (DRM), compromising antiretroviral therapy (ART). This study updates HIVDR data and HIV-1 variants in Equatorial Guinea (EG), providing the first data on children/adolescents in the country. From 2019−2020, 269 dried blood samples (DBS) were collected in Bata Regional Hospital (EG) from 187 adults (73 ART-naïve/114 ART-treated) and 82 children/adolescents (25 HIV-exposed-ART-naïve/57 ART-treated). HIV-1 infection was confirmed in Madrid by molecular/serological confirmatory tests and ART-failure by VL quantification. HIV-1 pol region was identified as transmitted/acquired DRM, predicted antiretroviral susceptibility (Stanfordv9.0) and HIV-1 variants (phylogeny). HIV infection was confirmed in 88.1% of the individuals and virological failure (VL > 1000 HIV-1-RNA copies/mL) in 84.2/88.9/61.9% of 169 ART-treated children/adolescents/adults. Among the 167 subjects with available data, 24.6% suffered a diagnostic delay. All 125 treated had experienced nucleoside retrotranscriptase inhibitors (NRTI); 95.2% were non-NRTI (NNRTI); 22.4% had experienced integrase inhibitors (INSTI); and 16% had experienced protease inhibitors (PI). At sampling, they had received 1 (37.6%), 2 (32%), 3 (24.8%) or 4 (5.6%) different ART-regimens. Among the 43 treated children−adolescents/37 adults with sequence, 62.8/64.9% carried viruses with major-DRM. Most harbored DRM to NNRTI (68.4/66.7%), NRTI (55.3/43.3%) or NRTI+NNRTI (50/33.3%). One adult and one child carried major-DRM to PI and none carried major-DRM to INSTI. Most participants were susceptible to INI and PI. DRM was absent in 36.2% of treated patients with VL > 1000 cp/mL, suggesting adherence failure. TDR prevalence in 59 ART-naïve adults was high (20.3%). One-half (53.9%) of the 141 subjects with pol sequence carried CRF02_AG. The observed high rate of ART-failure and transmitted/acquired HIVDR could compromise the 95-95-95-UNAIDS targets in EG. Routine VL and resistance monitoring implementation are mandatory for early detection of ART-failure and optimal rescue therapy selection ART regimens based on PI, and INSTI can improve HIV control in EG.

Temporal evolution of the resistance genotypes of Plasmodium falciparum in isolates from Equatorial Guinea during 20 years (1999 to 2019).

BACKGROUND: Malaria is one of the deadliest diseases in the world, particularly in Africa. As such, resistance to anti-malarial drugs is one of the most important problems in terms of global malaria control. This study assesses the evolution of the different resistance markers over time and the possible influence of interventions and treatment changes that have been made in Equatorial Guinea. METHODS: A total of 1223 biological samples obtained in the period 1999 to 2019 were included in the study. Screening for mutations in the pfdhfr, pfdhps, pfmdr1, and pfcrt genes was carried out by nested PCR and restriction-fragment length polymorphisms (RFLPs), and the study of pfk13 genes was carried out by nested PCR, followed by sequencing to determine the presence of mutations. RESULTS: The partially and fully resistant haplotypes (pfdhfr + pfdhps) were found to increase over time. Moreover, in 2019, the fully resistant haplotype was found to be increasing, although its super-resistant counterpart remains much less prevalent. A continued decline in pfmdr1 and pfcrt gene mutations over time was also found. The number of mutations detected in pfk13 has increased since 2008, when artemisinin-based combination therapy (ACT) were first introduced, with more mutations being observed in 2019, with two synonymous and five non-synonymous mutations being detected, although these are not related to resistance to ACT. In addition, the non-synonymous A578S mutation, which is the most frequent on the African continent, was detected in 2013, although not in the following years. CONCLUSIONS: Withdrawal of the use of chloroquine (CQ) as a treatment in Equatorial Guinea has been shown to be effective over time, as wild-type parasite populations outnumber mutant populations. The upward trend observed in sulfadoxine-pyrimethamine (SP) resistance markers suggest its misuse, either alone or in combination with artesunate (AS) or amodiaquine (AQ), in some areas of the country, as was found in a previous study conducted by this group, which allows selective pressure from SP to continue. Single nucleotide polymorphisms (SNPs) 540E and 581G do not exceed the limit of 50 and 10%, respectively, thus meaning that SP is still effective as an intermittent preventive treatment (IPT) in this country. As for the pfk13 gene, no mutations have been detected in relation to resistance to ACT. However, in 2019 there is a greater accumulation of non-synonymous mutations compared to years prior to 2008.

High drug resistance levels could compromise the control of HIV infection in paediatric and adolescent population in Kinshasa, the Democratic Republic of Congo.

BACKGROUND: The inadequacy of HIV viraemia and resistance monitoring in Africa leads to uncontrolled circulation of HIV strains with drug resistance mutations (DRM), compromising antiretroviral therapy (ART) effectiveness. This study describes the DRM prevalence and its therapeutic impact in HIV-infected pediatric patients from Kinshasa (Democratic Republic of Congo, DRC). METHODS: From 2016-2018, dried blood were collected from 71 HIV-infected children and adolescents under ART in two hospitals in Kinshasa for HIV-1 DRM pol analysis, predicted ARV-susceptibility by Stanford and phylogenetic characterization. RESULTS: HIV-1 sequences were recovered from 55 children/adolescents with 14 years of median-age. All had received nucleoside and non-nucleoside reverse transcriptase inhibitors (NRTI, NNRTI), 9.1% protease inhibitors (PI) and only one integrase inhibitor (INI). Despite the use of ART, 89.1% showed virological failure and 67.3% carried viruses with major-DRM to one (12.7%), two (47.3%), or three (5.5%) ARV-families. Most children/adolescents harbored DRM to NNRTI (73.5%) or NRTI (61.2%). Major-DRM to PI was present in 8.3% and minor-DRM to INI in 15%. Dual-class-NRTI+NNRTI resistance appeared in 53.1% of patients. Viruses presented high/intermediate resistance to nevirapine (72.9% patients), efavirenz (70.9%), emtricitabine/lamivudine (47.9%), rilpivirine (41.7%), etravirine (39.6%), doravidine (33.3%), zidovudine (22.9%), among others. Most participants were susceptible to INI and PI. Great diversity of variants was found, with a high rate (40%) of unique recombinants. CONCLUSION: The high DRM prevalence observed among HIV-infected children and adolescents in Kinshasa could compromise the 95-95-95-UNAIDS targets in the DRC. It also reinforces the need for routine resistance monitoring for optimal rescue therapy election in this vulnerable population to control the spread of resistant HIV in the country.

Evaluation of bias induced by viral enrichment and random amplification protocols in metagenomic surveys of saliva DNA viruses.

BACKGROUND: Viruses are key players regulating microbial ecosystems. Exploration of viral assemblages is now possible thanks to the development of metagenomics, the most powerful tool available for studying viral ecology and discovering new viruses. Unfortunately, several sources of bias lead to the misrepresentation of certain viruses within metagenomics workflows, hindering the shift from merely descriptive studies towards quantitative comparisons of communities. Therefore, benchmark studies on virus enrichment and random amplification protocols are required to better understand the sources of bias. RESULTS: We assessed the bias introduced by viral enrichment on mock assemblages composed of seven DNA viruses, and the bias from random amplification methods on human saliva DNA viromes, using qPCR and deep sequencing, respectively. While iodixanol cushions and 0.45 µm filtration preserved the original composition of nuclease-protected viral genomes, low-force centrifugation and 0.22 µm filtration removed large viruses. Comparison of unamplified and randomly amplified saliva viromes revealed that multiple displacement amplification (MDA) induced stochastic bias from picograms of DNA template. However, the type of bias shifted to systematic using 1 ng, with only a marginal influence by amplification time. Systematic bias consisted of over-amplification of small circular genomes, and under-amplification of those with extreme GC content, a negative bias that was shared with the PCR-based sequence-independent, single-primer amplification (SISPA) method. MDA based on random priming provided by a DNA primase activity slightly outperformed those based on random hexamers and SISPA, which may reflect differences in ability to handle sequences with extreme GC content. SISPA viromes showed uneven coverage profiles, with high coverage peaks in regions with low linguistic sequence complexity. Despite misrepresentation of certain viruses after random amplification, ordination plots based on dissimilarities among contig profiles showed perfect overlapping of related amplified and unamplified saliva viromes and strong separation from unrelated saliva viromes. This result suggests that random amplification bias has a minor impact on beta diversity studies. CONCLUSIONS: Benchmark analyses of mock and natural communities of viruses improve understanding and mitigate bias in metagenomics surveys. Bias induced by random amplification methods has only a minor impact on beta diversity studies of human saliva viromes.