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Background Newly emerged mutations within the Plasmodium falciparum chloroquine resistance transporter (PfCRT) can confer piperaquine resistance in the absence of amplified plasmepsin II (pfpm2). In this study, we estimated the prevalence of co-circulating piperaquine resistance mutations in P. falciparum isolates collected in northern Cambodia from 2009 to 2017. Methods The sequence of pfcrt was determined for 410 P. falciparum isolates using PacBio amplicon sequencing or whole genome sequencing. Quantitative polymerase chain reaction was used to estimate pfpm2 and pfmdr1 copy number. Results Newly emerged PfCRT mutations increased in prevalence after the change to dihydroartemisinin-piperaquine in 2010, with >98% of parasites harboring these mutations by 2017. After 2014, the prevalence of PfCRT F145I declined, being outcompeted by parasites with less resistant, but more fit PfCRT alleles. After the change to artesunate-mefloquine, the prevalence of parasites with amplified pfpm2 decreased, with nearly half of piperaquine-resistant PfCRT mutants having single-copy pfpm2. Conclusions The large proportion of PfCRT mutants that lack pfpm2 amplification emphasizes the importance of including PfCRT mutations as part of molecular surveillance for piperaquine resistance in this region. Likewise, it is critical to monitor for amplified pfmdr1 in these PfCRT mutants, as increased mefloquine pressure could lead to mutants resistant to both drugs.

Background While human cases of Plasmodium knowlesi are now regularly recognized in Southeast Asia, infections with other simian malaria species, such as Plasmodium cynomolgi, are still rare. There has been a handful of clinical cases described, all from Malaysia, and retrospective studies of archived blood samples in Thailand and Cambodia have discovered the presence P. cynomolgi in isolates using polymerase chain reaction (PCR) assays. Case presentation In Thailand, an ongoing malaria surveillance study enrolled two patients from Yala Province diagnosed with Plasmodium vivax by blood smear, but who were subsequently found to be negative by PCR. Expanded PCR testing of these isolates detected mono-infection with P. cynomolgi, the first time this has been reported in Thailand. Upon re-testing of 60 isolates collected from Yala, one other case was identified, a co-infection of P. cynomolgi and P. vivax. The clinical course for all three was relatively mild, with symptoms commonly seen in malaria: fever, chills and headaches. All infections were cured with a course of chloroquine and primaquine. Conclusion In malaria-endemic areas with macaque populations, cases of simian malaria in humans are being reported at an increasing rate, although still comprise a very small percentage of total cases. Plasmodium cynomolgi and P. vivax are challenging to distinguish by blood smear; therefore, PCR can be employed when infections are suspected or as part of systematic malaria surveillance. As Thai MoPH policy schedules regular follow-up visits after each malaria infection, identifying those with P. cynomolgi will allow for monitoring of treatment efficacy, although at this time P. cynomolgi appears to have an uncomplicated clinical course and good response to commonly used anti-malarials.

Background In April 2017, the Thai Ministry of Public Health (MoPH) was alerted to a potential malaria outbreak among civilians and military personnel in Sisaket Province, a highly forested area bordering Cambodia. The objective of this study was to present findings from the joint civilian-military outbreak response. Methods A mixed-methods approach was used to assess risk factors among cases reported during the 2017 Sisaket malaria outbreak. Routine malaria surveillance data from January 2013 to March 2018 obtained from public and military medical reporting systems and key informant interviews (KIIs) (n = 72) were used to develop hypotheses about potential factors contributing to the outbreak. Joint civilian-military response activities included entomological surveys, mass screen and treat (MSAT) and vector control campaigns, and scale-up of the “1–3–7” reactive case detection approach among civilians alongside a pilot “1–3–7” study conducted by the Royal Thai Army (RTA). Results Between May–July 2017, the monthly number of MoPH-reported cases surpassed the epidemic threshold. Outbreak cases detected through the MoPH mainly consisted of Thai males (87%), working as rubber tappers (62%) or military/border police (15%), and Plasmodium vivax infections (73%). Compared to cases from the previous year (May–July 2016), outbreak cases were more likely to be rubber tappers (OR = 14.89 [95% CI: 5.79–38.29]; p P. vivax (OR=2.32 [1.27–4.22]; p = 0.006). Themes from KIIs were congruent with findings from routine surveillance data. Though limited risk factor information was available from military cases, findings from RTA’s “1–3–7” study indicated transmission was likely occurring outside military bases. Data from entomological surveys and MSAT campaigns support this hypothesis, as vectors were mostly exophagic and parasite prevalence from MSAT campaigns was very low (range: 0-0.7% by PCR/microscopy). Conclusions In 2017, an outbreak of mainly P. vivax occurred in Sisaket Province, affecting mainly military and rubber tappers. Vector control use was limited to the home/military barracks, indicating that additional interventions were needed during high-risk forest travel periods. Importantly, this outbreak catalyzed joint civilian-military collaborations and integration of the RTA into the national malaria elimination strategy (NMES). The Sisaket outbreak response serves as an example of how civilian and military public health systems can collaborate to advance national malaria elimination goals in Southeast Asia and beyond.

We determined the prevalence of Kelch 13 mutations and pfmdr1 copy number in samples collected from the Thailand–Myanmar border, the Thailand–Cambodia border, and southern Thailand from 2002 to 2007. C580Y was the most prevalent in Trat (Thailand–Cambodia border) and Ranong (Thailand–Myanmar border) at 42% (24/57) and 13% (6/48), respectively. Less predominant mutations were also identified including R539T (7%, 4/57) and Y493H (2%, 1/57) in Trat, P574L (6%, 3/48) and P553L (2%, 1/48) in Ranong, and N537I and D452E (7%, 1/15) in Sangkhlaburi (Thailand–Myanmar border). Samples from Mae sot (33%, 11/33) harbored the highest percentage of multiple pfmdr1 copies, followed by Trat (18%, 10/57), Chiang Dao in 2003 (13%, 4/30), Phang Nga (5%, 2/44), and Chiang Dao in 2002 (4%, 1/26). This retrospective study provides geographic diversity of K13 and pfmdr1 copies and the emergence of these molecular markers in Thailand, an important background information for future surveillance in the region.

Background High rates of dihydroartemisinin–piperaquine (DHA–PPQ) treatment failures have been documented for uncomplicated Plasmodium falciparum in Cambodia. The genetic markers plasmepsin 2 (pfpm2), exonuclease (pfexo) and chloroquine resistance transporter (pfcrt) genes are associated with PPQ resistance and are used for monitoring the prevalence of drug resistance and guiding malaria drug treatment policy. Methods To examine the relative contribution of each marker to PPQ resistance, in vitro culture and the PPQ survival assay were performed on seventeen P. falciparum isolates from northern Cambodia, and the presence of E415G-Exo and pfcrt mutations (T93S, H97Y, F145I, I218F, M343L, C350R, and G353V) as well as pfpm2 copy number polymorphisms were determined. Parasites were then cloned by limiting dilution and the cloned parasites were tested for drug susceptibility. Isobolographic analysis of several drug combinations for standard clones and newly cloned P. falciparum Cambodian isolates was also determined. Results The characterization of culture-adapted isolates revealed that the presence of novel pfcrt mutations (T93S, H97Y, F145I, and I218F) with E415G-Exo mutation can confer PPQ-resistance, in the absence of pfpm2 amplification. In vitro testing of PPQ resistant parasites demonstrated a bimodal dose–response, the existence of a swollen digestive vacuole phenotype, and an increased susceptibility to quinine, chloroquine, mefloquine and lumefantrine. To further characterize drug sensitivity, parental parasites were cloned in which a clonal line, 14-B5, was identified as sensitive to artemisinin and piperaquine, but resistant to chloroquine. Assessment of the clone against a panel of drug combinations revealed antagonistic activity for six different drug combinations. However, mefloquine-proguanil and atovaquone–proguanil combinations revealed synergistic antimalarial activity. Conclusions Surveillance for PPQ resistance in regions relying on DHA–PPQ as the first-line treatment is dependent on the monitoring of molecular markers of drug resistance. P. falciparum harbouring novel pfcrt mutations with E415G-exo mutations displayed PPQ resistant phenotype. The presence of pfpm2 amplification was not required to render parasites PPQ resistant suggesting that the increase in pfpm2 copy number alone is not the sole modulator of PPQ resistance. Genetic background of circulating field isolates appear to play a role in drug susceptibility and biological responses induced by drug combinations. The use of latest field isolates may be necessary for assessment of relevant drug combinations against P. falciparum strains and when down-selecting novel drug candidates.

Previously, ivermectin (1 to 10 mg/kg of body weight) was shown to inhibit the liver-stage development of Plasmodium berghei in orally dosed mice. Here, ivermectin showed inhibition of the in vitro development of Plasmodium cynomolgi schizonts (50% inhibitory concentration [IC50], 10.42 μM) and hypnozoites (IC50, 29.24 μM) in primary macaque hepatocytes when administered as a high dose prophylactically but not when administered in radical cure mode., Previously, ivermectin (1 to 10 mg/kg of body weight) was shown to inhibit the liver-stage development of Plasmodium berghei in orally dosed mice. Here, ivermectin showed inhibition of the in vitro development of Plasmodium cynomolgi schizonts (50% inhibitory concentration [IC50], 10.42 μM) and hypnozoites (IC50, 29.24 μM) in primary macaque hepatocytes when administered as a high dose prophylactically but not when administered in radical cure mode. The safety, pharmacokinetics, and efficacy of oral ivermectin (0.3, 0.6, and 1.2 mg/kg) with and without chloroquine (10 mg/kg) administered for 7 consecutive days were evaluated for prophylaxis or radical cure of P. cynomolgi liver stages in rhesus macaques. No inhibition or delay to blood-stage P. cynomolgi parasitemia was observed at any ivermectin dose (0.3, 0.6, and 1.2 mg/kg). Ivermectin (0.6 and 1.2 mg/kg) and chloroquine (10 mg/kg) in combination were well-tolerated with no adverse events and no significant pharmacokinetic drug-drug interactions observed. Repeated daily ivermectin administration for 7 days did not inhibit ivermectin bioavailability. It was recently demonstrated that both ivermectin and chloroquine inhibit replication of the novel severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in vitro. Further ivermectin and chloroquine trials in humans are warranted to evaluate their role in Plasmodium vivax control and as adjunctive therapies against COVID-19 infections.

In addition to evidence for a protective role of antibodies to the malaria blood stage antigen merozoite surface protein 1 (MSP1), MSP1 antibodies are also considered as a marker of past malaria exposure in sero-epidemiological studies. In order to better assess the potential use of MSP1 serology in malaria chemoprophylaxis trials in endemic areas, an analysis for the prevalence of antibodies to both Plasmodium falciparum and Plasmodium vivax MSP142 in healthy Cambodian adults was conducted at two sites as part of an active, observational cohort evaluating the efficacy of dihydroartemisinin-piperaquine (DP) for uncomplicated malaria ( ClinicalTrials.gov identifier NCT01280162). Rates of baseline sero-positivity were high (59 and 73 % for PfMSP142 and PvMSP142, respectively), and titers higher in those who lived in a higher transmission area, although there was little correlation in titers between the two species. Those volunteers who subsequently went on to develop malaria had higher baseline MSP142 titers than those who did not for both species. Titers to both antigens remained largely stable over the course of the 4–6 month study, except in those infected with P. falciparum who had multiple recurrences. These findings illuminate the difficulties in using MSP142 serology as either a screening criterion and/or biomarker of exposure in chemoprophylaxis studies. Further work remains to identify useful markers of malarial infection and/or immunity.

We assessed contamination by antibiotic-resistant bacteria in chicken meat obtained from supermarkets in Bangkok, Thailand. The prevalence of Salmonella enterica and Escherichia coli was 18.7% (14/75) and 53% (106/200), respectively. Most probable number (MPN) analysis showed that 56.7% of the samples (34/60) were in violation of the limit of allowable coliform bacteria in chicken meat, for which the maximum is 46,000 MPN/g. Multidrug-resistant phenotypes of both S. enterica and E. coli were found. The presence of class 1 integrons was demonstrated by polymerase chain reaction (PCR) and dot-blot hybridization. PCR showed that class 1 integrons were present in 42.9% (6/14) and 37.7% (40/106) of S. enterica and E. coli isolates, respectively. Resistance genes identified in this study were aadA2, aadA4, aadA22, and aadA23 (for aminoglycoside resistance); dfrA5 (for trimethoprim resistance), and lnuF (for lincosamide resistance). Four S. enterica isolates underwent multilocus sequence typing and the results were sequence type (ST) 50, ST 96, ST 1543, and ST 1549, which matched well with strains from many countries and reflected an international spread. Our study revealed that class 1 integrons have spread into community sources and might play an important role in horizontal antibiotic resistance gene transfer.