Introduction Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. Methods P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2and glurp data were obtained from the original clinical study. Results The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (<1h) and slow (>5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. Conclusions This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately.
Chronic hepatitis B infection (CHB) is a significant problem worldwide with around 300 million people infected. Ambitious goals have been set towards elimination as a public health threat by 2030. However, accurate seroprevalence estimates in many countries are lacking or fail to provide representative population estimates, particularly in the WHO African Region (AFRO). This means the full extent of HBV infection here is not well described, leading to a lack of investment in diagnostics, treatment and disease prevention. Clinical trials in the WHO AFRO region have been increasing over time and many test for infectious diseases including hepatitis B virus (HBV) to determine baseline eligibility for participants, however these screening data are not reported. Here we review data from six clinical trials completed at KEMRI-Wellcome Trust Research Programme between 2016 - 2023 that screened for HBV using hepatitis B surface antigen (HBsAg) as part of the trial exclusion criteria. 1727 people had HBsAg results available, of which 60 tested positive. We generated a crude period HBV prevalence estimate of 3.5% (95% CI 2.6-4.5%), and after standardisation for sex and age to account for the population structure of the Kilifi Health Demographics Surveillance System (KHDSS), the prevalence estimate increased to 5.0% (95% CI 3.4-6.6%). The underrepresentation of women in these trials was striking with 1263/1641 (77%) of participants being male. Alanine aminotransferase (ALT) was significantly higher in the HBsAg positive group but was not outside the normal range. We argue that routine collation and publishing of data from clinical trials could increase precision and geographical representation of global HBV prevalence estimates, enabling evidence-based provision of clinical care pathways and public health interventions to support progress towards global elimination targets. We do acknowledge when using clinical trials data for seroprevalence estimates, local population structure data is necessary to allow standardisation of results, and the point of care tests used here are limited in sensitivity and specificity.
Introduction Antimalarial therapeutic efficacy studies are routinely conducted in malaria-endemic countries to assess the effectiveness of antimalarial treatment strategies. Targeted amplicon sequencing (AmpSeq) uniquely identifies and quantifies genetically distinct parasites within an infection. In this study, AmpSeq of Plasmodium falciparum apical membrane antigen 1 (ama1), and multidrug resistance gene 1 (mdr1), were used to characterise the complexity of infection (COI) and drug-resistance genotypes, respectively. Methods P. falciparum-positive samples were obtained from a triple artemisinin combination therapy clinical trial conducted in 30 children under 13 years of age between 2018 and 2019 in Kilifi, Kenya. Nine of the 30 participants presented with recurrent parasitemia from day 26 (624h) onwards. The ama1 and mdr1 genes were amplified and sequenced, while msp1, msp2 and glurp data were obtained from the original clinical study. Results The COI was comparable between ama1 and msp1, msp2 and glurp; overall, ama1 detected more microhaplotypes. Based on ama1, a stable number of microhaplotypes were detected throughout treatment until day 3. Additionally, a recrudescent infection was identified with an ama1 microhaplotype initially observed at 30h and later in an unscheduled follow-up visit. Using the relative frequencies of ama1 microhaplotypes and parasitemia, we identified a fast (<1h) and slow (>5h) clearing microhaplotype. As expected, only two mdr1 microhaplotypes (NF and NY) were identified based on the combination of amino acid polymorphisms at codons 86 and 184. Conclusions This study highlights AmpSeq as a tool for highly-resolution tracking of parasite microhaplotypes throughout treatment and can detect variation in microhaplotype clearance estimates. AmpSeq can also identify slow-clearing microhaplotypes, a potential early sign of selection during treatment. Consequently, AmpSeq has the capability of improving the discriminatory power to distinguish recrudescences from reinfections accurately.
Background: Men who have sex with men (MSM) have a higher prevalence of Chlamydia trachomatis (CT) and Neisseria gonorrhoeae (NG) infections compared to the rest of the population, often remaining undiagnosed. In Kenya, prevalence of rectal CT and NG infection and NG antimicrobial sensitivity are poorly described.Methods: MSM who reported receptive anal intercourse (RAI) were recruited from an ongoing human immunodeficiency virus acquisition and treatment study in coastal Kenya in 2016-2017. Rectal swabs were collected at two time points 6 months apart to estimate prevalence and incidence of CT/NG infection using a molecular point-of-care assay. Participants positive for CT or NG were treated according to national guidelines. NG culture and antimicrobial susceptibility testing was performed. Participant and risk behaviour characteristics were collected and association with baseline CT/NG prevalence assessed by multivariable regression analysis.Results: Prevalence of CT/NG in 104 MSM was 21.2% (CT 13.5%, NG 9.6%, dual infection 1.9%) at baseline and 25.9% in 81 MSM at follow-up (CT 14.8%, NG 14.8%, dual infection 3.7%). CT/NG incidence was estimated at 53.0 (95% CI, 34.5-81.3) per 100 person-years. Most CT/NG positive participants were asymptomatic: 95.5% at baseline and 100% at follow-up. CT/NG infection was associated with being paid for sex [adjusted odds ratio (aOR)=6.2, 95% CI (1.7-22.9)] and being in formal employment [aOR=7.5, 95% CI (1.1-49.2)]. Six NG isolates were obtained at follow-up; all were susceptible to ceftriaxone and cefixime and all were resistant to penicillin, tetracycline and ciprofloxacin.Conclusions: There is a high prevalence and incidence of asymptomatic rectal CT and NG in MSM reporting RAI in coastal Kenya. MSM who were paid for sex or had formal employment were more likely to be infected with CT/NG suggesting increased risk behaviour during transactional sex. Antimicrobial susceptibility results suggest that current antibiotic choices in Kenya are appropriate for NG treatment.
These extended data accompany the manuscript: Targeted Amplicon deep sequencing of ama1 and mdr1 to track within-host P. falciparum diversity throughout treatment in a clinical drug trial Table S1. List of PCR and deep sequencing primers. This table shows the list of forward and reverse primers used for deep sequencing. In boldface are the MID tags while in the regular face are the forward primers. Table S2. The relative frequencies of each ama1 variant and the number of samples with each variant. The relative frequencies (%) of the 33 AMA1 variants in pre-and post-treatment samples (n = 330) are shown as a 33 amino acid sequence. The frequencies were calculated by dividing the number of reads of each haplotype by the total number of reads obtained (116,187,131). Figure S1. Performance of TADS on the sequencing controls made up of a mixture of laboratory isolates. Each control set was done in 6 replicates to ensure sufficient control data in the event of potential amplicon or sequencing failure. The median read depth in the lab controls was 5658 (range 4,310 – 12,603) and 658 (- 1,676). The x-axis represents the replicate identifier across the five mixtures, starting from 1 to 6 while the y-axis represents the proportions of each variant across all replicates. For ama1 (A), 2 variants (3D7 and Dd2) were detected whereas in MDR1 (B), 3 variants were detected YY, FY and NY following amplification of Dd2 Copy I, Dd2 Copy II and 3D7, respectively. For ama1, sequencing failed for replicate 6 of control set 1, while for mdr1, sequencing failed for replicate 2 and 6 of control set 3, replicates 1 and 6 of control set 4 and replicates 1 and 5 of control set 5. Under the MDR1 control set 4, the Dd2 copy II (86F, 184Y) was not identified possibly due to having very low concentrations that were not picked up in this replicate. Based on our control mixtures, the minimum variant frequency we were able to detect was 0.5%. Figure S2. Heatmaps of the successfully PCR amplified and sequenced samples for ama1 (A) and mdr1 (B). The rows represent the study participants while the columns represent time in hours. Successfully sequenced samples are shown in green, those that failed PCR in red and those that failed sequencing are in yellow. The timepoint Rec, represent unscheduled visits where a recurrent sample was collected. The unshaded areas with ‘-‘ are timepoints where samples were not collected. For each time-point, the number of samples successfully sequenced, n, is indicated in the last row of each panel. The table in panel C shows the groupings of samples based on parasitemia, high (> 5,000), moderate (100-5,000) and low (< 100 parasites per microlitre). Many samples collected between 0h-12h had high parasitemia, samples collected between 18h-30h had moderate parasitemia while samples collected after 30h were primarily of low parasitemia. Figure S3. The mean complexity of infection (COI) by AMA1 throughout treatment. The mean COI (red diamonds) appeared to be stable (between 1.5 - 2) from baseline (0h) up to 72h and thereafter fluctuates due to the small sample sizes (<5) in the post-treatment samples. The black dots represent the COI per sample.
Background: Men who have sex with men (MSM) experience a high burden of Chlamydia trachomatis (CT) and Neisseria gonorrhoea (NG) infections. These remain largely undiagnosed in the context of syndromic treatment. In Kenya, prevalence of rectal CT and NG infection and NG antimicrobial sensitivity are poorly described.Methods: MSM who reported receptive anal intercourse (RAI) were recruited from an ongoing human immunodeficiency virus acquisition and treatment study in coastal Kenya in 2016-2017. Rectal swabs were collected at two time points 6 months apart to estimate prevalence of CT/NG using a molecular point-of-care assay. Participants positive for CT or NG were treated according to national guidelines. NG culture and antimicrobial susceptibility testing was performed. Participant and risk behaviour characteristics were collected and association with baseline CT/NG prevalence was assessed by multivariable regression analysis.Results: Prevalence of CT/NG in 104 MSM was 21.2% (CT 13.5%, NG 9.6%, dual infection 1.9%) at baseline and 25% in 84 MSM at follow-up (CT 14.3%, NG 14.3%, dual infection 3.6%). Most CT/NG positive participants were asymptomatic: 95.5% at baseline and 100% at follow-up. CT/NG infection was associated with being paid for sex (aOR=6.2, 95%CI (1.7-22.9)) and being in formal employment (aOR=7.5, 95%CI (1.14-49.2)). Six NG isolates were obtained at follow-up; all isolates were susceptible to ceftriaxone and cefixime (1st line treatment for NG) and all were resistant to penicillin, tetracycline and ciprofloxacin.Conclusions: The high prevalence of asymptomatic rectal CT and NG in MSM reporting RAI demonstrates the need for frequent screening or presumptive treatment. MSM who were paid for sex or had formal employment were more likely to be infected with CT/NG, suggesting increased risk behaviour during transactional sex. Antimicrobial susceptibility results suggest that current antibiotic choices in Kenya are appropriate for NG treatment.
Point of-care (POC) HIV-1 RNA tests which are accurate and easy to use with limited infrastructure are needed in resource-limited settings (RLS). We systematically reviewed evidence of POC test performance compared to laboratory-based HIV-1 RNA assays and the potential utility of these tests for diagnosis and care in RLS.
Abstract Coronavirus Disease-2019 tests require a Nasopharyngeal (NP) and/or Oropharyngeal (OP) specimen from the upper airway, from which virus RNA is extracted and detected through quantitative reverse transcription-Polymerase Chain Reaction (qRT-PCR). The viability of the virus is maintained after collection by storing the NP/OP swabs in Viral Transport Media (VTM). We evaluated the performance of four transport media: locally manufactured (“REVITAL”) Viral Transport Media (RVTM), Standard Universal Transport Media (SUTM), PBS and 0.9% (w/v) NaCl (normal saline). We used laboratory cultured virus to evaluate: i) viral recovery and maintaining integrity at different time periods and temperatures; ii) stability in yielding detectable RNA consistently for all time points and conditions; and iii) their overall accuracy. Four vials of SARS-CoV-2 cultured virus (2 high and 2 low concentration samples) and 1 negative control sample were prepared for each media type (SUTM, RVTM, PBS and normal saline) and stored at the following temperatures, -80°C, 4°C, room temperature (25°C) and 37°C for 7 days. Viral Ribonucleic acid (RNA) extractions and qRT-PCR were done on the following days after inoculation with the cultured virus, days 1, 2, 3, 4 and 7 to assess virus stability and viral recovery. C T values fell over time at room temperature, but normal saline, PBS, RVTM and SUTM all showed comparable performance in maintaining virus integrity and stability allowing for the detection of SARS-CoV-2 viral RNA. Overall, this study demonstrated that normal saline, PBS and the locally manufactured VTM can be used for COVID-19 sample collection and testing, thus expanding the range of SARS-CoV-2 viral collection media.
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