Burden of Submicroscopic Plasmodium Infections and Detection of kelch13 Mutant Parasites in Military and Civilian Populations in Papua New Guinea.

Malaria remains a major public health problem in Papua New Guinea (PNG) and an important force health protection issue for both PNG and Australian Defence Forces. To investigate the malaria burden in the military and civilians residing on military bases, a cross-sectional survey was conducted in April 2019 at three military bases in Wewak, Manus Island, and Vanimo, PNG. A total of 1,041 participants were enrolled; 235 military personnel from three bases and 806 civilians from Wewak and Vanimo. Polymerase chain reaction (PCR) revealed an overall high prevalence of Plasmodium infection in both the military and civilians. Among the military, the infection prevalence was significantly higher in Wewak (35.5%) and Vanimo (33.3%) bases than on Manus Island (11.8%). Among civilians, children (<16 years old) had significantly higher odds of being PCR positive than adults (≥16 years old). At Wewak and Vanimo, Plasmodium vivax accounted for 85.4%, 78.2%, and 66.2% of infections in military, children, and adult populations. Overall, 87.3%, 41.3%, and 61.3% of Plasmodium infections in the military, children, and adults, respectively, were detected only by PCR, not by microscopy (submicroscopic [SM] infections). Children had a significantly lower proportion of SM infections than adults and Papua New Guinea Defence Force personnel. Infection status was not associated with hemoglobin levels in these populations at the time of the survey. Mutant kelch13 (C580Y) parasites were identified in 5/68 Plasmodium falciparum-infected individuals. The survey results indicate extensive malaria transmission on these bases, especially in Wewak and Vanimo. More intensified interventions are required to reduce malaria transmission on PNG military bases.

A Health Survey Revealing Prevalence of Vector-Borne Diseases and Tuberculosis in Papua New Guinea Defence Force Personnel and Families.

The Papua New Guinean Defence Force (PNGDF) and the Australian Defence Force (ADF) work and train closely. Infectious diseases pose a health threat to both forces, but recent knowledge about the risk at military bases in Papua New Guinea is limited. To improve understanding, a collaborative cross-sectional survey was conducted (March-April 2019) at Lombrum Naval base (Manus province) and Moem Army barracks (Wewak, East Sepik province) plus its Vanimo outpost (Sandaun province). Clinical data, venous blood, and sputum were collected from PNGDF personnel (DF) from the three sites, with point-of-care testing conducted for malaria (microscopy and rapid diagnostic test [RDT]), lymphatic filariasis (RDT), glucose-6-phosphate-dehydrogenase (G6PD) deficiency (RDT), tuberculosis (GeneXpert), and hemoglobin level. Finger prick blood collected from family members residing at the Wewak base was tested for malaria and hemoglobin level. Overall, 235 DF and 793 family members completed the survey. Microscopy revealed malaria prevalence as 0.4% Plasmodium falciparum and 3.1% Plasmodium vivax among DF and 3.5% P. falciparum, 14.3% P. vivax, and 0.3% mixed P. falciparum/P. vivax among family members. Among DF, 3.9% were G6PD deficient and none tested positive for tuberculosis or for lymphatic filariasis antigen. Anemia was present in 6.5% of DF and 47.3% of family members, predominantly females. Results suggest ongoing exposure to malaria, particularly P. vivax, at study sites, whereas infections of lymphatic filariasis and tuberculosis were not detected. Survey results will inform the PNGDF and the ADF regarding vector-borne disease risk for future sustainable health and disease control interventions.

Prevalence of Barmah Forest Virus, Chikungunya Virus and Ross River Virus Antibodies among Papua New Guinea Military Personnel before 2019.

Barmah Forest virus (BFV), Chikungunya virus (CHIKV) and Ross River virus (RRV) belong to the Alphavirus genus of the family Togaviridae. All three virus infections have been reported in Papua New Guinea (PNG) previously, but the exact prevalence and distribution of these three alphaviruses in PNG has not been established. Sera collected from 204 PNG Military Personnel (PNGMP) study participants in April 2019 was tested for the presence of anti-BFV, anti-CHIKV and anti-RRV immunoglobulin G (IgG) antibodies using commercially available enzyme-linked immunosorbent assay (ELISA) IgG detection kits, as well as for specific neutralizing antibodies (NAb) against individual viruses. Overall, sero-positivity of the sera was anti-BFV IgG 12.3% (25/204), anti-BFV NAb 8.3% (17/204); anti-CHIKV IgG 47.1% (96/204), anti-CHIKV NAb 34.8% (71/204); and anti-RRV IgG 93.1% (190/204), anti-RRV NAb 56.4% (115/204), respectively. Of the 137/204 participants that were Nab-positive for at least one virus, we identified 4 BFV, 40 CHIKV and 73 RRV single infections, and 9 RRV+CHIKV and 11 BFV+RRV double infections. The lower proportion of NAb sero-positive compared to the ELISA IgG sero-positive assay samples suggests that the currently available commercial ELISA detection kits for these three alphaviruses may not be suitable for diagnostic/surveillance purposes in endemic areas such as PNG, due to serological cross-reactivity among these three alphaviruses. Laboratory testing using known positive control sera indicated no cross-neutralization between BFV and RRV; however, some RRV or BFV single infection human sera demonstrated low-level cross-neutralization against CHIKV (the ratio of RRV/CHIKV NAb titers or BFV/CHIKV ≥ 4). Our preliminary results indicate that the majority of PNGMP have previously been exposed to RRV, with mild exposure to CHIKV and low-level exposure to BFV, suggesting that multiple alphaviruses have been circulating among PNGMP. The transmission landscapes of these three alphaviruses across PNG should be prioritized for further investigation, including identification of specific vectors and hosts that mediate human spillover in order to mitigate future outbreaks. Ongoing education regarding precautionary and protective measures are needed to better protect individuals who travel to PNG.

Serological evidence of possible high levels of undetected transmission of Zika virus among Papua New Guinea military personnel, 2019.

Objectives: The Papua New Guinea (PNG) Health Department retrospectively reported six cases of Zika virus (ZIKV) from a cohort of febrile patients during outbreaks of dengue and malaria in 2016. However, the transmission of ZIKV remains unclear due to lack of testing capability. This study aimed to determine the level of immunity to ZIKV among PNG military personnel (PNGMP) in 2019. Methods: Sera of 208 PNGMP recruited in April 2019 was tested for the presence of anti-ZIKV immunoglobulin G (IgG) and M (IgM) antibodies using Euroimmun IgG/IgM detection kits, and anti-ZIKV neutralizing antibody (Nab) against a ZIKV African strain on all anti-ZIKV-IgG/IgM+ samples. Results: Anti-ZIKV seropositivity of these sera was as follows: IgG, 67%; IgM, 9%; and Nab, 65%. Five of 19 anti-ZIKV-IgM+ samples had anti-ZIKV-Nab titres ≥20, as well as an anti-ZIKV-Nab titre ratio ≥4 compared with the Nab titres of four anti-dengue serotypes, so met the criteria of the World Health Organization (WHO) for confirmed ZIKV infection. Conclusions: The prevalence of anti-ZIKV-Nab of 65% suggests that there are high levels of ZIKV exposure among PNGMP. Five of the 19 anti-ZIKV-IgM+ samples met the WHO criteria for confirmed ZIKV infection, suggesting a recent undetected outbreak in PNGMP. These results provide better understanding of the current ZIKV epidemic status in PNGMP.

Seroprevalence of chikungunya virus among military personnel in Papua New Guinea, 2019.

Objectives: The first outbreak of chikungunya virus (CHIKV) was reported in West Sepik, Papua New Guinea (PNG) in June 2012, and spread rapidly throughout PNG. CHIKV imported from PNG to Queensland has been reported occasionally, but transmission of CHIKV in PNG remains unclear due to the lack of testing capability. This study investigated the degree of CHIKV exposure among PNG military personnel (PNGMP) in 2019, 7 years after its first emergence. Methods: Sera of 204 PNGMP recruited in April 2019 was tested for the presence of anti-CHIKV immunoglobulin G (IgG) antibodies using a commercially available IgG detection kit, and anti-CHIKV neutralizing antibodies against a CHIKV Reunion strain using a neutralizing assay. Results: Anti-CHIKV seropositivity of the sera was 47% and 35%, respectively, using the enzyme-linked immunosorbent assay (ELISA) and neutralizing assay. Five percent (n=11) of samples were found to be IgG negative or borderline, but neutralizing antibody positive. Conclusions: The prevalence of anti-CHIKV neutralizing antibody of 35% suggests that CHIKV infection has become endemic among PNGMP. Current commercially available CHIKV ELISA detection kits may not be suitable for diagnostic purposes in multiple alphavirus endemic areas such as PNG, due to serological cross-reactivity among alphaviruses. Re-emergence of CHIKV in PNGMP is possible.

Cytochrome P450 2D6 profiles and their relationship with outcomes of primaquine anti-relapse therapy in Australian Defence Force personnel deployed to Papua New Guinea and East Timor.

BACKGROUND: Primaquine, an 8-aminoquinoline with anti-hypnozoite activity against Plasmodium vivax, is metabolized by human cytochrome P450 2D6 (CYP2D6) to its active metabolite. Human CYP2D6 activities may influence the metabolism of primaquine and the risk of experiencing Plasmodium relapses following primaquine anti-relapse therapies (PART). In this study, the CYP2D6 profile and its relationship with outcomes of PART in Australian Defence Force (ADF) personnel is retrospectively investigated. METHODS: Genomic DNA was isolated from stored and de-identified serum or blood samples from ADF personnel deployed on peacekeeping duties to Papua New Guinea (PNG) (1999) and East Timor (1999-2000) who received PART before returning to Australia and after experiencing relapses. CYP2D6 allelic type was determined by PCR and Sanger sequencing. CYP2D6 allele frequency, predicted phenotypes and activity scores were compared among personnel who did not experience P. vivax (ADF-NR, n = 48) and those who experience at least one (ADF-R, n = 109) relapse, as well as between those who experienced 1 (n = 79), 2 (n = 21) and 3-5 (n = 9) relapses within the ADF-R group. RESULTS: 16 CYP2D6 alleles were observed in 157 ADF personnel. Alleles *1, *4, *2 and *41 were major alleles (> 5%). The CYP2D6 allele frequency profile in the ADF-NR group matched that of a European population. There was an increased proportion of non-functional CYP2D6 alleles in the ADF-R group compared to the European population and ADF-NR group. However, frequencies of predicted CYP2D6 phenotype and activity score were not different between the ADF-R and ADF-NR groups, nor among sub-groups experiencing multiple relapses within the ADF-R group. CONCLUSIONS: CYP2D6 phenotype or activity score based on the allele classification was not a major contributor to P. vivax relapse in this ADF cohort. Other factors such as adherence and/or parasite tolerance to primaquine are likely contributing factors to P. vivax relapses in this cohort.

Functional analysis of Plasmodium vivax dihydrofolate reductase-thymidylate synthase genes through stable transformation of Plasmodium falciparum.

Mechanisms of drug resistance in Plasmodium vivax have been difficult to study partially because of the difficulties in culturing the parasite in vitro. This hampers monitoring drug resistance and research to develop or evaluate new drugs. There is an urgent need for a novel method to study mechanisms of P. vivax drug resistance. In this paper we report the development and application of the first Plasmodium falciparum expression system to stably express P. vivax dhfr-ts alleles. We used the piggyBac transposition system for the rapid integration of wild-type, single mutant (117N) and quadruple mutant (57L/58R/61M/117T) pvdhfr-ts alleles into the P. falciparum genome. The majority (81%) of the integrations occurred in non-coding regions of the genome; however, the levels of pvdhfr transcription driven by the P. falciparum dhfr promoter were not different between integrants of non-coding and coding regions. The integrated quadruple pvdhfr mutant allele was much less susceptible to antifolates than the wild-type and single mutant pvdhfr alleles. The resistance phenotype was stable without drug pressure. All the integrated clones were susceptible to the novel antifolate JPC-2067. Therefore, the piggyBac expression system provides a novel and important tool to investigate drug resistance mechanisms and gene functions in P. vivax.

Defining the role of mutations in Plasmodium vivax dihydrofolate reductase-thymidylate synthase gene using an episomal Plasmodium falciparum transfection system.

Plasmodium vivax resistance to antifolates is prevalent throughout Australasia and is caused by point mutations within the parasite dihydrofolate reductase (DHFR)-thymidylate synthase. Several unique mutations have been reported in P. vivax DHFR, and their roles in resistance to classic and novel antifolates are not entirely clear due, in part, to the inability to culture P. vivax in vitro. In this study, we use a homologous system to episomally express both wild-type and various mutant P. vivax dhfr (pvdhfr) alleles in an antifolate-sensitive line of P. falciparum and to assess their influences on the susceptibility of the recipient P. falciparum line to commonly used and new antifolate drugs. Although the wild-type pvdhfr-transfected P. falciparum line was as susceptible to antifolate drugs as the P. falciparum parent line, the single (117N), double (57L/117T and 58R/117T), and quadruple (57L/58R/61M/117T) mutant pvdhfr alleles conferred a marked reduction in their susceptibilities to antifolates. The resistance index increased with the number of mutations in these alleles, indicating that these mutations contribute to antifolate resistance directly. In contrast, the triple mutant allele (58R/61M/117T) significantly reversed the resistance to all antifolates, indicating that 61M may be a compensatory mutation. These findings help elucidate the mechanism of antifolate resistance and the effect of existing mutations in the parasite population on the current and new generation of antifolate drugs. It also demonstrates that the episomal transfection system has the potential to provide a rapid screening system for drug development and for studying drug resistance mechanisms in P. vivax.

Multiple origins of resistance-conferring mutations in Plasmodium vivax dihydrofolate reductase.

BACKGROUND: In order to maximize the useful therapeutic life of antimalarial drugs, it is crucial to understand the mechanisms by which parasites resistant to antimalarial drugs are selected and spread in natural populations. Recent work has demonstrated that pyrimethamine-resistance conferring mutations in Plasmodium falciparum dihydrofolate reductase (dhfr) have arisen rarely de novo, but spread widely in Asia and Africa. The origin and spread of mutations in Plasmodium vivax dhfr were assessed by constructing haplotypes based on sequencing dhfr and its flanking regions. METHODS: The P. vivax dhfr coding region, 792 bp upstream and 683 bp downstream were amplified and sequenced from 137 contemporary patient isolates from Colombia, India, Indonesia, Papua New Guinea, Sri Lanka, Thailand, and Vanuatu. A repeat motif located 2.6 kb upstream of dhfr was also sequenced from 75 of 137 patient isolates, and mutational relationships among the haplotypes were visualized using the programme Network. RESULTS: Synonymous and non-synonymous single nucleotide polymorphisms (SNPs) within the dhfr coding region were identified, as was the well-documented in-frame insertion/deletion (indel). SNPs were also identified upstream and downstream of dhfr, with an indel and a highly polymorphic repeat region identified upstream of dhfr. The regions flanking dhfr were highly variable. The double mutant (58R/117N) dhfr allele has evolved from several origins, because the 58R is encoded by at least 3 different codons. The triple (58R/61M/117T) and quadruple (57L/61M/117T/173F, 57I/58R/61M/117T and 57L/58R/61M/117T) mutant alleles had at least three independent origins in Thailand, Indonesia, and Papua New Guinea/Vanuatu. CONCLUSION: It was found that the P. vivax dhfr coding region and its flanking intergenic regions are highly polymorphic and that mutations in P. vivax dhfr that confer antifolate resistance have arisen several times in the Asian region. This contrasts sharply with the selective sweep of rare antifolate resistant alleles observed in the P. falciparum populations in Asia and Africa. The finding of multiple origins of resistance-conferring mutations has important implications for drug policy.

A novel Plasmodium falciparum expression system for assessing antifolate resistance caused by mutant P. vivax dihydrofolate reductase-thymidylate synthase.

With the emergence of drug-resistant vivax malaria, in vitro studies are urgently needed to examine resistance mechanisms and for drug development. Currently, Plasmodium vivax culturing is inadequate for addressing these needs; therefore, surrogate biological systems have been developed. Although these systems are informative, they do not address Plasmodium species-specific mechanisms, such as drug delivery through erythrocytes and parasite membranes. Here, we demonstrate that P. falciparum is an excellent biological system for expression of P. vivax dhfr-ts alleles to assess dihydrofolate reductase (DHFR)-thymidylate synthase interactions with antifolates. Our results show that the P. vivax dhfr-ts quadruple-mutant allele AMRU1, expressed in P. falciparum, provides significant protection against pyrimethamine, cycloguanil, and clocicguanil. Moreover, the PvDHFR quadruple mutant confers greater resistance to cycloguanil, clociguanil, and WR99210 than the PfDHFR quadruple mutant. Modeling of both P. vivax and P. falciparum DHFR quadruple mutants suggests that mutations unique to P. vivax DHFR are responsible for differences seen in parasite susceptibility to antifolates.

Relapses of Plasmodium vivax infection result from clonal hypnozoites activated at predetermined intervals.

Plasmodium vivax infections are characterized by varying numbers of relapses occurring at different intervals as a result of activation of liver-stage hypnozoites. Parasite or host factors that determine the number and timing of relapses are unclear. In the present article, we report the analysis of relapse patterns and molecular characterization of parasites collected from Australian soldiers experiencing relapses of vivax malaria after exposure in East Timor. Although high molecular diversity was observed, a single allelic type was identified in association with 99% of relapses. Importantly, in 71% of patients experiencing >1 relapse, the allelic types were clonal and different in the 2 different relapses. These results, combined with those from a computer simulation model, suggest that a single hypnozoite clone was activated, causing a relapse, and that multiple relapses most likely arose from coordinated activation of hypnozoites originating from different parasite strains. These findings suggest remarkable regulation of relapse intervals in vivax malaria.

Amino acid mutations in Plasmodium vivax DHFR and DHPS from several geographical regions and susceptibility to antifolate drugs.

The increasing use of sulfadoxine-pyrimethamine (SP) for the treatment of chloroquine-resistant Plasmodium falciparum has resulted in increased reports of SP resistance of P. falciparum worldwide. Selection of SP-resistant Plasmodium vivax in areas where P. falciparum and P. vivax co-exist is not entirely clear. We examined the prevalence and extent of point mutations in pvdhfr and pvdhps in 70 P. vivax isolates from China, East Timor, Papua New Guinea (PNG), Philippines, Vanuatu, and Vietnam. Mutations in seven codon positions were found in pvdhfr, with the majority of isolates having double mutations (S58R/S117N). The greatest range of mutations was observed in the PNG and Vanuatu isolates, ranging from single to quadruple mutations (F57L/S58R/T61M/S117T). Single mutations in pvdhps were observed only in parasites with mutations in corresponding pvdhfr. Parasites with the S58R/S117N dhfr allelic type showed an MIC level for pyrimethamine and cycloguanil comparable to that previously reported, but were susceptible to WR99210.

Efficacy of sulfadoxine-pyrimethamine in the treatment of uncomplicated Plasmodium falciparum malaria in East Timor.

The efficacy of sulfadoxine-pyrimethamine (SP) in East Timor is unknown. We treated 38 individuals with uncomplicated Plasmodium falciparum malaria with SP and monitored the outcome for 28 days. Recrudescent parasitemia, confirmed by genotyping, were detected in three individuals resulting in a late treatment failure rate of 7.9% (95% confidence interval = 1.7-21.4%). The results suggest that SP is still efficacious in treating uncomplicated P. falciparum malaria in East Timor. However, the useful life of SP in East Timor may be limited because 80% of the parasites in our samples were found to already carry double mutations in P. falciparum dihydrofolate reductase (S108N/C59R). The data from this study also highlights that the presence of gametocytes may significantly influence the estimate of SP efficacy determined by genotyping.

Survey of personal protection measures against mosquitoes among Australian defense force personnel deployed to East Timor.

A questionnaire was completed by 955 Australian Defense Force soldiers from two battalion groups to determine their usage of mosquito repellents and bed nets during peacekeeping duties in East Timor. The survey showed that most soldiers (84%) used repellents, but only 19% used them daily. The soldiers used a number of repellent formulations; however, few soldiers used the Australian Defense Force deet (diethyl methylbenzamide) formulation containing 35% deet in a gel. Most soldiers preferred several commercial formulations, which contained 7 to 80% deet. The occurrence of mosquito-borne disease in soldiers was not affected by repellent usage, as the use of repellents was comparable between infected and noninfected individuals. The overall frequency of bed net usage differed in the two battalion groups. The occurrence of malaria in soldiers from one battalion group who did not sleep under a bed net every night of their deployment was significantly (p = 0.007) higher than those who did.