Hypnozoite depletion in successive Plasmodium vivax relapses.

Genotyping Plasmodium vivax relapses can provide insights into hypnozoite biology. We performed targeted amplicon sequencing of 127 relapses occurring in Indonesian soldiers returning to malaria-free Java after yearlong deployment in malarious Eastern Indonesia. Hepatic carriage of multiple hypnozoite clones was evident in three-quarters of soldiers with two successive relapses, yet the majority of relapse episodes only displayed one clonal population. The number of clones detected in relapse episodes decreased over time and through successive relapses, especially in individuals who received hypnozoiticidal therapy. Interrogating the multiplicity of infection in this P. vivax relapse cohort reveals evidence of independent activation and slow depletion of hypnozoites over many months by multiple possible mechanisms, including parasite senescence and host immunity.

Molecular surveillance over 14 years confirms reduction of Plasmodium vivax and falciparum transmission after implementation of Artemisinin-based combination therapy in Papua, Indonesia.

Genetic epidemiology can provide important insights into parasite transmission that can inform public health interventions. The current study compared long-term changes in the genetic diversity and structure of co-endemic Plasmodium falciparum and P. vivax populations. The study was conducted in Papua Indonesia, where high-grade chloroquine resistance in P. falciparum and P. vivax led to a universal policy of Artemisinin-based Combination Therapy (ACT) in 2006. Microsatellite typing and population genetic analyses were undertaken on available isolates collected between 2004 and 2017 from patients with uncomplicated malaria (n = 666 P. falciparum and n = 615 P. vivax). The proportion of polyclonal P. falciparum infections fell from 28% (38/135) before policy change (2004-2006) to 18% (22/125) at the end of the study (2015-2017); p<0.001. Over the same period, polyclonal P. vivax infections fell from 67% (80/119) to 35% (33/93); p<0.001. P. falciparum strains persisted for up to 9 years compared to 3 months for P. vivax, reflecting higher rates of outbreeding in the latter. Sub-structure was observed in the P. falciparum population, but not in P. vivax, confirming different patterns of outbreeding. The P. falciparum population exhibited 4 subpopulations that changed in frequency over time. Notably, a sharp rise was observed in the frequency of a minor subpopulation (K2) in the late post-ACT period, accounting for 100% of infections in late 2016-2017. The results confirm epidemiological evidence of reduced P. falciparum and P. vivax transmission over time. The smaller change in P. vivax population structure is consistent with greater outbreeding associated with relapsing infections and highlights the need for radical cure to reduce recurrent infections. The study emphasizes the challenge in disrupting P. vivax transmission and demonstrates the potential of molecular data to inform on the impact of public health interventions.

Laboratory challenges of Plasmodium species identification in Aceh Province, Indonesia, a malaria elimination setting with newly discovered P. knowlesi.

The discovery of the life-threatening zoonotic infection Plasmodium knowlesi has added to the challenges of prompt and accurate malaria diagnosis and surveillance. In this study from Aceh Province, Indonesia, a malaria elimination setting where P. knowlesi endemicity was not previously known, we report the laboratory investigation and difficulties encountered when using molecular detection methods for quality assurance of microscopically identified clinical cases. From 2014 to 2015, 20 (49%) P. falciparum, 16 (39%) P. vivax, 3 (7%) P. malariae, and 2 (5%) indeterminate species were identified by microscopy from four sentinel health facilities. At a provincial-level reference laboratory, loop-mediated isothermal amplification (LAMP), a field-friendly molecular method, was performed and confirmed Plasmodium in all samples though further species-identification was limited by the unavailability of non-falciparum species-specific testing with the platform used. At a national reference laboratory, several molecular methods including nested PCR (nPCR) targeting the 18 small sub-unit (18S) ribosomal RNA, nPCR targeting the cytochrome-b (cytb) gene, a P. knowlesi-specific nPCR, and finally sequencing, were necessary to ultimately classify the samples as: 19 (46%) P. knowlesi, 8 (20%) P. falciparum, 14 (34%) P. vivax. Microscopy was unable to identify or mis-classified up to 56% of confirmed cases, including all cases of P. knowlesi. With the nPCR methods targeting the four human-only species, P. knowlesi was missed (18S rRNA method) or showed cross-reactivity for P. vivax (cytb method). To facilitate diagnosis and management of potentially fatal P. knowlesi infection and surveillance for elimination of human-only malaria in Indonesia and other affected settings, new detection methods are needed for testing at the point-of-care and in local reference laboratories.

Passively versus Actively Detected Malaria: Similar Genetic Diversity but Different Complexity of Infection.

The surveillance of malaria is generally undertaken on the assumption that samples passively collected at health facilities are comparable to or representative of the broader Plasmodium reservoir circulating in the community. Further characterization and comparability of the hidden asymptomatic parasite reservoir are needed to inform on the potential impact of sampling bias. This study explores the impact of sampling strategy on molecular surveillance by comparing the genetic make-up of Plasmodium falciparum and Plasmodium vivax isolates collected by passive versus active case detection. Sympatric isolates of P. falciparum and P. vivax were collected from a large community survey and ongoing clinical surveillance studies undertaken in the hypomesoendemic setting of Mimika District (Papua, Indonesia). Plasmodium falciparum isolates were genotyped at nine microsatellite loci and P. vivax at eight loci. Measures of diversity and differentiation were used to compare different patient and parasitological sample groups. The results demonstrated that passively detected cases (symptomatic) had comparable population diversity to those circulating in the community (asymptomatic) in both species. In addition, asymptomatic patent infections were as diverse as subpatent infections. However, a significant difference in multiplicity of infection (MOI) and percentage of polyclonal infections was observed between actively and passively detected P. vivax cases (mean MOI: 1.7 ± 0.7 versus 1.4 ± 1.4, respectively; P = 0.001). The study findings infer that, in hypomesoendemic settings, passive sampling is appropriate for molecular parasite surveillance strategies using the predominant clone in any given infection; however, the findings suggest caution when analyzing complexity of infection. Further evaluation is required in other endemic settings.

Genetic micro-epidemiology of malaria in Papua Indonesia: Extensive P. vivax diversity and a distinct subpopulation of asymptomatic P. falciparum infections.

BACKGROUND: Genetic analyses of Plasmodium have potential to inform on transmission dynamics, but few studies have evaluated this on a local spatial scale. We used microsatellite genotyping to characterise the micro-epidemiology of P. vivax and P. falciparum diversity to inform malaria control strategies in Timika, Papua Indonesia. METHODS: Genotyping was undertaken on 713 sympatric P. falciparum and P. vivax isolates from a cross-sectional household survey and clinical studies conducted in Timika. Standard population genetic measures were applied, and the data was compared to published data from Kalimantan, Bangka, Sumba and West Timor. RESULTS: Higher diversity (HE = 0.847 vs 0.625; p = 0.017) and polyclonality (46.2% vs 16.5%, p<0.001) were observed in P. vivax versus P. falciparum. Distinct P. falciparum substructure was observed, with two subpopulations, K1 and K2. K1 was comprised solely of asymptomatic infections and displayed greater relatedness to isolates from Sumba than to K2, possibly reflecting imported infections. CONCLUSIONS: The results demonstrate the greater refractoriness of P. vivax versus P. falciparum to control measures, and risk of distinct parasite subpopulations persisting in the community undetected by passive surveillance. These findings highlight the need for complimentary new surveillance strategies to identify transmission patterns that cannot be detected with traditional malariometric methods.

Contrasting Transmission Dynamics of Co-endemic Plasmodium vivax and P. falciparum: Implications for Malaria Control and Elimination.

BACKGROUND: Outside of Africa, P. falciparum and P. vivax usually coexist. In such co-endemic regions, successful malaria control programs have a greater impact on reducing falciparum malaria, resulting in P. vivax becoming the predominant species of infection. Adding to the challenges of elimination, the dormant liver stage complicates efforts to monitor the impact of ongoing interventions against P. vivax. We investigated molecular approaches to inform the respective transmission dynamics of P. falciparum and P. vivax and how these could help to prioritize public health interventions. METHODOLOGY/PRINCIPAL FINDINGS: Genotype data generated at 8 and 9 microsatellite loci were analysed in 168 P. falciparum and 166 P. vivax isolates, respectively, from four co-endemic sites in Indonesia (Bangka, Kalimantan, Sumba and West Timor). Measures of diversity, linkage disequilibrium (LD) and population structure were used to gauge the transmission dynamics of each species in each setting. Marked differences were observed in the diversity and population structure of P. vivax versus P. falciparum. In Bangka, Kalimantan and Timor, P. falciparum diversity was low, and LD patterns were consistent with unstable, epidemic transmission, amenable to targeted intervention. In contrast, P. vivax diversity was higher and transmission appeared more stable. Population differentiation was lower in P. vivax versus P. falciparum, suggesting that the hypnozoite reservoir might play an important role in sustaining local transmission and facilitating the spread of P. vivax infections in different endemic settings. P. vivax polyclonality varied with local endemicity, demonstrating potential utility in informing on transmission intensity in this species. CONCLUSIONS/SIGNIFICANCE: Molecular approaches can provide important information on malaria transmission that is not readily available from traditional epidemiological measures. Elucidation of the transmission dynamics circulating in a given setting will have a major role in prioritising malaria control strategies, particularly against the relatively neglected non-falciparum species.