Human cytochrome P450 2B6 genetic variability in Botswana: a case of haplotype diversity and convergent phenotypes

Identification of inter-individual variability for drug metabolism through cytochrome P450 2B6 (CYP2B6) enzyme is important for understanding the di erences in clinical responses to malaria and HIV. This study evaluates the distribution of CYP2B6 alleles, haplotypes and inferred metabolic phenotypes among subjects with di erent ethnicity in Botswana. A total of 570 subjects were analyzed for CYP2B6 polymorphisms at position 516G>T (rs3745274), 785A>G (rs2279343) and 983T>C (rs28399499). Samples were collected in three districts of Botswana where the population belongs to Bantu (Serowe/Palapye and Chobe) and San-related (Ghanzi) ethnicity. The three districts showed di erent haplotype composition according to the ethnic background but similar metabolic inferred phenotypes, with 59.12%, 34.56%, 2.10% and 4.21% of the subjects having, respectively, an extensive, intermediate, slow and rapid metabolic pro le. The results hint at the possibility of a convergent adaptation of detoxifying metabolic phenotypes despite a di erent haplotype structure due to the di erent genetic background. The main implication is that, while there is substantial homogeneity of metabolic inferred phenotypes among the country, the response to drugs metabolized via CYP2B6 could be individually associated to an increased risk of treatment failure and toxicity. These are important facts since Botswana is facing malaria elimination and a very high HIV prevalence

Genetic diversity in L1 ORF of human papillomavirus in women with cervical cancer with and without human immunodeficiency virus in Botswana and Kenya

Background: The variation of human papillomavirus (HPV) genotypes shapes the risks of cervical cancer and these variations are not well defined in Africa. Nucleotide changes within the L1 gene, nucleotide variability, and phylogeny were explored in relation to HIV in samples from Botswana and Kenya. Methods: A total of 98 HPV-positive cervical samples were sequenced to identify diferent HPV variants. Phylogenetic inferences were used to determine HPV genotypes and investigate the clustering of sequences between women living with HIV (WLWHIV) and -women not living with HIV (WNLWHIV). Results: Out of 98 generated sequences, 83.7% (82/98) participants had high-risk (HR) HPV genotypes while 16.3% (16/98) had low-risk (LR) HPV genotypes. Among participants with HR-HPV genotypes, 47.6% (39/82) were coinfected with HIV. The prevalence of HR-HPV genotypes was statistically higher in the Botswana population compared to Kenya (p-valu

Preliminary survey on Anopheles species distribution in Botswana shows the presence of Anopheles gambiae and Anopheles funestus complexes

Background: Botswana is one of the four front line malaria elimination countries in Southern Africa, with malaria control activities that include routine vector control. Past and recent studies have shown that Anopheles arabiensis is the only known vector of Plasmodium parasites in the country. This report presents a preliminary evaluation on Anopheles species composition in seven districts of Botswana with some inferences on their vectorial role. Results: Overall, 404 Anopheles mosquito females were collected, of which 196 were larvae collected from several breeding sites, and 208 were adults obtained from indoor pyrethrum spray catches (PSC). Anopheles arabiensis (58.9%) accounted for the highest relative frequency in 5 out of 7 districts sampled. The other species collected, among those identified, were barely represented: Anopheles longipalpis type C (16.3%), Anopheles parensis (8.9%), Anopheles quadriannulatus (5.4%), and Anopheles leesoni (0.2%). PCR test for human β-globin on mosquitoes collected by PSC showed that An. arabiensis and An. parensis had bitten human hosts. Moreover, An. arabiensis showed a non-negligible Plasmodium falciparum infection rate in two sites (3.0% and 2.5% in Chobe and Kweneng West districts, respectively). Conclusions: This work provides first time evidence of Anopheles diversity in several areas of Botswana. Anopheles arabiensis is confirmed to be widespread in all the sampled districts and to be vector of P. falciparum. Moreover, the presence of Anopheles funestus group in Botswana has been assessed. Further research, entomological surveillance activities and possibly vector control programmes need to be better developed and implemented as well as targeting outdoors resting vectors

Association of CYP2B6 Genetic Variation with Efavirenz and Nevirapine Drug Resistance in HIV-1 Patients from Botswana

CYP2B6 liver enzyme metabolizes the two non-nucleoside reverse transcriptase inhibitors Efavirenz (EFV) and Nevirapine (NVP) used in the antiretroviral therapy (ART) regimens for HIV-infected individuals. Polymorphisms of the CYP2B6 gene influence drug levels in plasma and possibly virological outcomes. The aim of this study was to explore the potential impact of CYP2B6 genotype and haplotype variation on the risk of developing EFV/NVP drug resistance mutations (DRMs) in HIV-1 patients receiving EFV-/NVP-containing regimens in Botswana

Evaluation of a Novel Semiquantitative Cryptococcal Antigen Lateral Flow Assay in Patients with Advanced HIV Disease.

Higher cryptococcal antigen (CrAg) titers are strongly associated with mortality risk in individuals with HIV-associated cryptococcal disease. Rapid tests to quantify CrAg levels may provide important prognostic information and enable treatment stratification. We performed a laboratory-based validation of the IMMY semiquantitative cryptococcal antigen (CrAgSQ) lateral flow assay (LFA) against the current gold standard CrAg tests. We assessed the diagnostic accuracy of the CrAgSQ in HIV-positive individuals undergoing CrAg screening, determined the relationship between CrAgSQ scores and dilutional CrAg titers, assessed interrater reliability, and determined the clinical correlates of CrAgSQ scores. A total of 872 plasma samples were tested using both the CrAgSQ LFA and the conventional IMMY CrAg LFA, of which 692 were sequential samples from HIV-positive individuals undergoing CrAg screening and an additional 180 were known CrAg-positive plasma samples archived from prior studies. Interrater agreement in CrAgSQ reading was excellent (98.17% agreement, Cohen's kappa 0.962, P?<?0.001). Using the IMMY CrAg LFA as a reference standard, CrAgSQ was 93.0% sensitive (95% confidence interval [CI] 80.9% to 98.5%) and 93.8% specific (95% CI, 91.7% to 95.6%). After reclassification of discordant results using CrAg enzyme immunoassay testing, the sensitivity was 98.1% (95% CI, 90.1% to 100%) and specificity 95.8% (95% CI, 93.9% to 97.2%). The median CrAg titers for semiquantitative score categories (1+ to 4+) were 1:10 (interquartile range [IQR], 1:5 to 1:20) in the CrAgSQ 1+ category, 1:40 (IQR, 1:20 to 1:80) in the CrAgSQ 2+ category, 1:640 (IQR, 1:160 to 1:2,560) in the CrAgSQ 3+ category, and 1:5,120 (IQR, 1:2,560 to 1:30,720) in the CrAgSQ 4+ category. Increasing CrAgSQ scores were strongly associated with 10-week mortality. The IMMY CrAgSQ test had high sensitivity and specificity compared to the results for the IMMY CrAg LFA and provided CrAg scores that were associated with both conventional CrAg titers and clinical outcomes

Genetic diversity in L1 ORF of human papillomavirus in women with cervical cancer with and without human immunodeficiency virus in Botswana and Kenya

Background The variation of human papillomavirus (HPV) genotypes shapes the risks of cervical cancer and these variations are not well defined in Africa. Nucleotide changes within the L1 gene, nucleotide variability, and phylogeny were explored in relation to HIV in samples from Botswana and Kenya. Methods A total of 98 HPV-positive cervical samples were sequenced to identify different HPV variants. Phylogenetic inferences were used to determine HPV genotypes and investigate the clustering of sequences between women living with HIV (WLWHIV) and -women not living with HIV (WNLWHIV). Results Out of 98 generated sequences, 83.7% (82/98) participants had high-risk (HR) HPV genotypes while 16.3% (16/98) had low-risk (LR) HPV genotypes. Among participants with HR-HPV genotypes, 47.6% (39/82) were coinfected with HIV. The prevalence of HR-HPV genotypes was statistically higher in the Botswana population compared to Kenya (p-value < 0.001). Multiple amino acid mutations were identified in both countries. Genetic diversity differed considerably among WLWHIV and WNLWHIV. The mean pairwise distances between HPV-16 between HIV and HIV/HPV as well as for HPV-18 were statistically significant. Six (6) new deleterious mutations were identified in the HPV genotypes based on the sequencing of the L1 region, HPV-16 (L441P, S343P), HPV-18 (S424P), HPV-45 (Q366H, Y365F), and HPV-84 (F458L). The majority of the patients with these mutations were co-infected with HIV. Conclusions Genomic diversity and different genomic variants of HPV sequences were demonstrated. Candidate novel mutations within the L1 gene were identified in both countries which can be further investigated using functional assays

Cytochrome P450 2C8*2 allele in Botswana: human genetic diversity and public health implications

Human cytochrome P450 2C8 is a highly polymorphic gene and shows variation according to ethnicity. The CYP2C8*2 is a slow drug metabolism allele and shows 10–24% frequency in Black populations. The objective of this study was to assess the prevalence of CYP2C8*2 allele in Botswana among the San (or Bushmen) and the Bantu ethnic groups. For that purpose we recruited 544 children of the two ethnicities in three districts of Botswana from primary schools, collected blood samples, extracted DNA and genotyped them through PCR-based restriction fragment length polymorphism analysis. The results demonstrated that in the San the prevalence of the CYP2C8*2 allele is significantly higher than among the Bantu-related ethnic groups (17.5% and 8.5% for San and Bantu, respectively; P = 0.00002). These findings support the evidence of a different genetic background of the San with respect to Bantu-related populations, and highlight a possible higher risk of longer drug clearance or poor level of activation of pro-drugs among the San group

Molecular detection of human papillomavirus (HPV) in highly fragmented DNA from cervical cancer biopsies using double-nested PCR

Archived Formalin-Fixed Paraffin-Embedded (FFPE) tissue specimens can be a valuable source of human papillomavirus (HPV) nucleic acids for molecular biological analyses in retrospective studies. Although successful amplification with polymerase chain reaction (PCR) is essential for analysis of HPV DNA extracted from cervical FFPE specimens, extensive DNA damage due to cross-linking and fragmentation results in poor yield. Therefore, techniques to improve the diagnostic rate and sensitivity from FFPE tissues through PCR is highly desired and of wider interest. To overcome this, a highly sensitive double-nested PCR methodology was designed and optimized for limited-resource laboratories coupled with an organic extraction of DNA. This method allows the detection of a broad range of HPV genotypes and also allowing the sequencing of the final amplicon. Validation of the new approach developed was done with an automated DNA extraction coupled with Real Time PCR. Results showed that the proposed method achieves 96.3% of HPV detection as compared to 100% Abbott m2000rt used as ‘gold standard’. Moreover, the concordance rate between the two methods was equal for detecting HPV -16 or -18 genotypes. Nevertheless, the newly introduced assay has an advantage of: • Simultaneously identifying broad range of HPV genotypes besides HPV-16 and -18 from clinical samples. • It is an easy and cost-effective method that can be beneficial in resource-limited setting and can be employed for various molecular applications. • The method is indicated for highly degraded FFPE samples. Method name: Double-nested PCR for detecting and genotyping HPV, Keywords: HPV, FFPE, DNA, Double-nested PCR, RFLP, DNA sequencin

Cytochrome P450 2C8*2 allele in Botswana: human genetic diversity and public health implications

Human cytochrome P450 2C8 is a highly polymorphic gene and shows variation according to ethnicity. The CYP2C8*2 is a slow drug metabolism allele and shows 10–24% frequency in Black populations. The objective of this study was to assess the prevalence of CYP2C8*2 allele in Botswana among the San (or Bushmen) and the Bantu ethnic groups. For that purpose we recruited 544 children of the two ethnicities in three districts of Botswana from primary schools, collected blood samples, extracted DNA and genotyped them through PCR-based restriction fragment length polymorphism analysis. The results demonstrated that in the San the prevalence of the CYP2C8*2 allele is significantly higher than among the Bantu-related ethnic groups (17.5% and 8.5% for San and Bantu, respectively; P = 0.00002). These findings support the evidence of a different genetic background of the San with respect to Bantu-related populations, and highlight a possible higher risk of longer drug clearance or poor level of activation of pro-drugs among the San group