Methods: A combination of active and passive detection of infection was carried out among communities in Batubara, Langkat, and South Nias regencies. Finger-prick blood samples from consenting individuals of all ages provided blood films for microscopic examination and blood spots on filter paper. Plasmodium species were identified using nested polymerase chain reaction (PCR) of ribosomal RNA genes and a novel assay that amplifies a conserved sequence specific for the sicavar gene family of Plasmodium knowlesi.
Results: Of 3731 participants, 614 (16.5%) were positive for malaria parasites by microscopy. PCR detected parasite DNA in samples from 1169 individuals (31.3%). In total, 377 participants (11.8%) harbored P. knowlesi. Also present were Plasmodium vivax (14.3%), Plasmodium falciparum (10.5%) and Plasmodium malariae (3.4%).
Conclusions: Amplification of sicavar is a specific and sensitive test for the presence of P. knowlesi DNA in humans. Subpatent and asymptomatic multispecies parasitemia is relatively common in North Sumatera, so PCR-based surveillance is required to support control and elimination activities.
METHODS: Three SNPs involved in most cases of resistance to the most widespread anti-malarial treatments have been analysed by PCR plus sequencing and by KASP (C580Y of the Kelch13 gene, Y86N of the Pfmdr1 gene and M133I of the Pfcytb gene). A total of 113 P. falciparum positive samples and 24 negative samples, previously analysed by PCR and sequencing, were selected for this assay. Likewise, the samples were genotyped for the MSP-1 and MSP-2 genes, and the Multiplicity of Infection (MOI) and parasitaemia were measured to observe their possible influence on the KASP method.
RESULTS: The KASP results showed the same expected mutations and wild type genotypes as the reference method, with few exceptions that correlated with very low parasitaemia samples. In addition, two cases of heterozygotes that had not been detected by sequencing were found. No correlation was found between the MOI or parasitaemia and the KASP values of the sample. The reproducibility of the technique shows no oscillations between repetitions in any of the three SNPs analysed.
CONCLUSIONS: The KASP assays developed in this study were efficient and versatile for the determination of the Plasmodium genotypes related to resistance. The method is simple, fast, reproducible with low cost in personnel, material and equipment and scalable, being able to core KASP arrays, including numerous SNPs, to complete the main pattern of mutations associated to P. falciparum resistance.
METHODS: We set out to assess the genetic variants of sulfadoxine-pyrimethamine resistance and the effectiveness of its treatment in eastern India prior to, during, and 6 to 8 years following the introduction of the new pharmacological regime. In 2008-2009, 318 P. falciparum-positive patients got the recommended doses of sulfadoxine-pyrimethamine. We used 379 additional isolates from 2015 to 2017 in addition to the 106 isolates from 2010. All 803 isolates from two study sites underwent in vitro sulfadoxine-pyrimethamine sensitivity testing and genomic characterisation of sulfadoxine-pyrimethamine resistance (pfdhfr and pfdhps).
RESULTS: In Kolkata and Purulia, we observed early treatment failure in 30.7 and 14.4% of patients, respectively, whereas recrudescence was found in 8.1 and 13.4% of patients, respectively, in 2008-2009. In 2017, the proportion of in vitro pyrimethamine and sulfadoxine resistance steadily grew in Kolkata and Purulia despite a single use of sulfadoxine-pyrimethamine. Treatment failures with sulfadoxine-pyrimethamine were linked to quintuple or quadruple pfdhfr- pfdhps mutations (AICII-AGKAT, AICII-AGKAA, AICII-SGKGT, AICII-AGKAA, AICNI-AGKAA) in 2008-2009 (p < 0.001). The subsequent spread of mutant-haplotypes with higher in vitro sulfadoxine-pyrimethamine resistance (p < 0.001), such as the sextuple (dhfr-AIRNI+dhps-AGEAA, dhfr-ANRNL+dhps-AGEAA) and septuple (dhfr-AIRNI+dhps-AGEAT), mutations were observed in 2015-2017.
DISCUSSION: This successive spread of mutations with high in vitro sulfadoxine-pyrimethamine resistance confirmed the progressive increase in antifolate resistance even after an 8-year withdrawal of sulfadoxine-pyrimethamine.