Targeted amplicon deep sequencing for monitoring antimalarial resistance markers in western Kenya

Molecular surveillance of Plasmodium falciparum parasites is important to track emerging and new mutations and trends in established mutations and should serve as an early warning system for antimalarial resistance. Dried blood spots were obtained from a Plasmodium falciparum malaria survey in school children conducted across eight counties in western Kenya in 2019. Real-time PCR identified 500 P. falciparum-positive samples that were amplified at five drug resistance loci for targeted amplicon deep sequencing (TADS). The absence of important kelch 13 mutations was similar to previous findings in Kenya pre-2019, and low-frequency mutations were observed in codons 569 and 578. The chloroquine resistance transporter gene codons 76 and 145 were wild type, indicating that the parasites were chloroquine and piperaquine sensitive, respectively. The multidrug resistance gene 1 haplotypes based on codons 86, 184, and 199 were predominantly present in mixed infections with haplotypes NYT and NFT, driven by the absence of chloroquine pressure and the use of lumefantrine, respectively. The sulfadoxine-pyrimethamine resistance profile was a "superresistant" combination of triple mutations in both <i>Pfdhfr</i> (51I 59R 108N) and <i>Pfdhps</i> (436H 437G 540E), rendering sulfadoxine-pyrimethamine ineffective. TADS highlighted the low-frequency variants, allowing the early identification of new mutations, <i>Pfmdr1</i> codon 199S and <i>Pfdhfr</i> codon 85I and emerging 164L mutations. The added value of TADS is its accuracy in identifying mixed-genotype infections and for high-throughput monitoring of antimalarial resistance markers

Benefits of enhanced infection prophylaxis at antiretroviral therapy initiation by cryptococcal antigen status

Objectives: To assess baseline prevalence of cryptococcal antigen (CrAg) positivity; and its contribution to reductions in all-cause mortality, deaths from cryptococcus and unknown causes, and new cryptococcal disease in the REALITY trial. Design: Retrospective CrAg testing of baseline and week-4 plasma samples in all 1805 African adults/children with CD4+ cell count less than 100 cells/μl starting antiretroviral therapy who were randomized to receive 12-week enhanced-prophylaxis (fluconazole 100 mg/day, azithromycin, isoniazid, cotrimoxazole) vs. standard-prophylaxis (cotrimoxazole). Methods: Proportional hazards models were used to estimate the relative impact of enhanced-prophylaxis vs. standard-cotrimoxazole on all, cryptococcal and unknown deaths, and new cryptococcal disease, through 24 weeks, by baseline CrAg positivity. Results: Excluding 24 (1.4%) participants with active/prior cryptococcal disease at enrolment (all treated for cryptococcal disease), 133/1781 (7.5%) participants were CrAg-positive. By 24 weeks, 105 standard-cotrimoxazole vs. 78 enhanced-prophylaxis participants died. Of nine standard-cotrimoxazole and three enhanced-prophylaxis cryptococcal deaths, seven and two, respectively, were CrAg-positive at baseline. Among deaths of unknown cause, only 1/46 standard-cotrimoxazole and 1/28 enhanced-prophylaxis were CrAg-positive at baseline. There was no evidence that relative reductions in new cryptococcal disease associated with enhanced-prophylaxis varied between baseline CrAg-positives [hazard-ratio = 0.36 (95% confidence interval 0.13–0.98), incidence 19.5 vs. 56.5/100 person-years] and CrAg-negatives [hazard-ratio = 0.33 (0.03–3.14), incidence 0.3 vs. 0.9/100 person-years; Pheterogeneity = 0.95]; nor for all deaths, cryptococcal deaths or unknown deaths (Pheterogeneity > 0.3). Conclusions: Relative reductions in cryptococcal disease/death did not depend on CrAg status. Deaths of unknown cause were unlikely to be cryptococcus-related; plausibly azithromycin contributed to their reduction. Findings support including 100 mg fluconazole in an enhanced-prophylaxis package at antiretroviral therapy initiation where CrAg screening is unavailable/impractical.</p