Kulpa, M. R. | bioRxiv Science

bioRxiv2026

Detection of Plasmodium infections in macaques from areas endemic for brugian filariasis in Belitung District, Indonesia

Background Malaria caused by Plasmodium knowlesi and lymphatic filariasis caused by Brugia malayi are mosquito-borne infections with non-human primates as reservoirs. P. knowlesi has emerged as a significant cause of human malaria in Southeast Asia over the past two decades. Belitung district, Indonesia, was until recently assumed to have eliminated B. malayi until infections were detected in humans and long-tailed macaques. To investigate whether the local reservoir of B. malayi is also a reservoir for malaria we screened macaques from 4 areas in Belitung for malaria parasites. Methods and findings Blood samples from 163 long-tailed macaques (Macaca fascicularis) that had been tested for B. malayi were examined by quantitative PCR assays specific for Plasmodium spp., P. knowlesi, P. inui, P. coatneyi and P. cynomolgi. A total of 130 macaques (79.8%) tested positive in the pan-Plasmodium qPCR assay. Plasmodium inui was most prevalent (41.7%), followed by P. knowlesi (38.7%), P. coatneyi (24.5%) and P. cynomolgi (13.5%). Multiple species infections, with 2-3 Plasmodium species were detected in 37% of macaques. Notably, 20 (91%) of 22 B. malayi-positive macaques were co-infected with at least one Plasmodium species. We sequenced the complete mitochondrion from 9 samples diagnosed by qPCR as mono-infections. Phylogenetic analysis confirmed 7 as P. knowlesi, and the other two as P. inui and P. coatneyi. Phylogenetic and pairwise analysis revealed that P. knowlesi isolates from Belitung were closely related to each other and to P. knowlesi from humans and monkeys from Thailand, Malaysia and Indonesia. Conclusions Molecular evidence shows high prevalence of zoonotic malaria parasites in macaques from Belitung, emphasizing the risk of human transmission. Increased surveillance, improved diagnostics, and targeted interventions are needed to prevent zoonotic spillover of P. knowlesi as it has been observed for B. malayi in Belitung.

microbiology↗

bioRxiv2025

Using deep amplicon sequencing as a molecular xenomonitoring approach for detecting filarial nematodes in biting arthropod vectors

Filarial nematodes are an important group of parasites that impact public, veterinary, and wildlife health globally. In order to understand these impacts and minimize their effects, scientists use molecular xenomonitoring techniques to understand their distribution and track elimination efforts. However, these molecular techniques can have narrow diagnostic capacity due to their species-specific approach which limits our understanding of important co-endemic filarial nematodes. Next-generation sequencing offers the ability to detect multiple species of coinfecting filarial nematodes and thus improve are ability to monitor, treat, and eliminate these pathogens. In this paper, we have developed a deep amplicon sequencing approach using filarial nematode primers targeting the cytochrome oxidase c subunit 1 (coxI) gene. To replicate molecular xenomonitoring conditions, third stage larvae (L3) of three species of filarioid nematodes (Brugia malayi, Brugia pahangi, Dirofilaria immitis) were spiked in different proportions to pools comprising various amounts of female Aedes aegypti mosquitoes (0, 10, 50, 100). Each pool was subjected to DNA extraction and Oxford Nanopore Technologies (ONT) deep amplicon sequencing protocols. Two sets of demultiplexing pipelines were utilized to optimize this novel approach, each reaching, 92.71% and 97.92% accuracy in identification of species composition across mock pools. However, in heterogenous pools, filarial species D. immitis exhibited an overrepresentation of reads and B. pahangi an underrepresentation of reads. We discuss reasons for recount biases and how this new molecular xenomonitoring tool could be implemented to serve public health, veterinary medicine, and scientific advancement. Note: Supplementary data associated with this article Author summaryHuman and animal diseases caused by filarial nematodes affect millions of people worldwide, particularly in low-income countries. These parasites are transmitted by blood-feeding arthropod vectors, such as mosquitoes and black flies. Thus, a major sector of public health research focuses on how to monitor, treat, and eliminate these harmful pathogens. An effective way is to capture these arthropod vectors and molecularly test these for filarial DNA in large sample pools. However, these pools can comprise multiple filarial species which targeted genetic analysis can miss. This proof-of-concept study seeks to circumvent these issues by using new next-generation sequencing approaches to capture the wider filarial diversity that may be contained in a single vector pool. We believe this tool could largely be beneficial to governments and organizations seeking to eliminate these filarial nematodes and become certified as a region free of certain devastating filarial species. Furthermore, we know very little about filarial diversity and this could be an integral tool to define their geographic distribution and future emerging threats to both human and animal health.

microbiology↗