Safety, pharmacokinetics, and potential neurological interactions of ivermectin, tafenoquine, and chloroquine in Rhesus macaques.

Ivermectin (IVM) could be used for malaria control as treated individuals are lethal to blood-feeding Anopheles, resulting in reduced transmission. Tafenoquine (TQ) is used to clear the liver reservoir of Plasmodium vivax and as a prophylactic treatment in high-risk populations. It has been suggested to use ivermectin and tafenoquine in combination, but the safety of these drugs in combination has not been evaluated. Early derivatives of 8-aminoquinolones (8-AQ) were neurotoxic, and ivermectin is an inhibitor of the P-glycoprotein (P-gp) blood brain barrier (BBB) transporter. Thus, there is concern that co-administration of these drugs could be neurotoxic. This study aimed to evaluate the safety and pharmacokinetic interaction of tafenoquine, ivermectin, and chloroquine (CQ) in Rhesus macaques. No clinical, biochemistry, or hematological outcomes of concern were observed. The Cambridge Neuropsychological Test Automated Battery (CANTAB) was employed to assess potential neurological deficits following drug administration. Some impairment was observed with tafenoquine alone and in the same monkeys with subsequent co-administrations. Co-administration of chloroquine and tafenoquine resulted in increased plasma exposure to tafenoquine. Urine concentrations of the 5,6 orthoquinone TQ metabolite were increased with co-administration of tafenoquine and ivermectin. There was an increase in ivermectin plasma exposure when co-administered with chloroquine. No interaction of tafenoquine on ivermectin was observed in vitro. Chloroquine and trace levels of ivermectin, but not tafenoquine, were observed in the cerebrospinal fluid. The 3''-O-demethyl ivermectin metabolite was observed in macaque plasma but not in urine or cerebrospinal fluid. Overall, the combination of ivermectin, tafenoquine, and chloroquine did not have clinical, neurological, or pharmacological interactions of concern in macaques; therefore, this combination could be considered for evaluation in human trials.

Superior protection in a relapsing Plasmodium cynomolgi rhesus macaque model by a chemoprophylaxis with sporozoite immunization regimen with atovaquone-proguanil followed by primaquine.

BACKGROUND: To gain a deeper understanding of protective immunity against relapsing malaria, this study examined sporozoite-specific T cell responses induced by a chemoprophylaxis with sporozoite (CPS) immunization in a relapsing Plasmodium cynomolgi rhesus macaque model. METHODS: The animals received three CPS immunizations with P. cynomolgi sporozoites, administered by mosquito bite, while under two anti-malarial drug regimens. Group 1 (n = 6) received artesunate/chloroquine (AS/CQ) followed by a radical cure with CQ plus primaquine (PQ). Group 2 (n = 6) received atovaquone-proguanil (AP) followed by PQ. After the final immunization, the animals were challenged with intravenous injection of 104 P. cynomolgi sporozoites, the dose that induced reliable infection and relapse rate. These animals, along with control animals (n = 6), were monitored for primary infection and subsequent relapses. Immunogenicity blood draws were done after each of the three CPS session, before and after the challenge, with liver, spleen and bone marrow sampling and analysis done after the challenge. RESULTS: Group 2 animals demonstrated superior protection, with two achieving protection and two experiencing partial protection, while only one animal in group 1 had partial protection. These animals displayed high sporozoite-specific IFN-γ T cell responses in the liver, spleen, and bone marrow after the challenge with one protected animal having the highest frequency of IFN-γ+ CD8+, IFN-γ+ CD4+, and IFN-γ+ γδ T cells in the liver. Partially protected animals also demonstrated a relatively high frequency of IFN-γ+ CD8+, IFN-γ+ CD4+, and IFN-γ+ γδ T cells in the liver. It is important to highlight that the second animal in group 2, which experienced protection, exhibited deficient sporozoite-specific T cell responses in the liver while displaying average to high T cell responses in the spleen and bone marrow. CONCLUSIONS: This research supports the notion that local liver T cell immunity plays a crucial role in defending against liver-stage infection. Nevertheless, there is an instance where protection occurs independently of T cell responses in the liver, suggesting the involvement of the liver's innate immunity. The relapsing P. cynomolgi rhesus macaque model holds promise for informing the development of vaccines against relapsing P. vivax.

Strong positive selection biases identity-by-descent-based inferences of recent demography and population structure in Plasmodium falciparum.

Malaria genomic surveillance often estimates parasite genetic relatedness using metrics such as Identity-By-Decent (IBD), yet strong positive selection stemming from antimalarial drug resistance or other interventions may bias IBD-based estimates. In this study, we use simulations, a true IBD inference algorithm, and empirical data sets from different malaria transmission settings to investigate the extent of this bias and explore potential correction strategies. We analyze whole genome sequence data generated from 640 new and 3089 publicly available Plasmodium falciparum clinical isolates. We demonstrate that positive selection distorts IBD distributions, leading to underestimated effective population size and blurred population structure. Additionally, we discover that the removal of IBD peak regions partially restores the accuracy of IBD-based inferences, with this effect contingent on the population's background genetic relatedness and extent of inbreeding. Consequently, we advocate for selection correction for parasite populations undergoing strong, recent positive selection, particularly in high malaria transmission settings.

Force Protection Risks in AFRICOM, INDOPACOM and SOUTHCOM Due to Rapid Diagnostic Test Failures for Falciparum Malaria, 2016-2022.

Malaria, caused by various species of the Plasmodium parasite, remains a significant health threat in most U.S. military regions-AFRICOM, CENT-COM, INDOPACOM, and SOUTHCOM-and although less prevalent, also poses periodic risks to military personnel in NORTHCOM through imported cases. Early diagnosis is crucial for effective malaria chemotherapy, and rapid diagnostic tests (RDTs) have proven valuable in resource-poor settings and operational environments. The BinaxNow Malaria RDT is currently the sole U.S. Food and Drug Administration (FDA)-approved test for use on U.S. military personnel. This simple RDT targets Plasmodium falciparum, the deadliest malaria species, by detecting the histidine-rich protein 2 (HRP2), as well as pan-Plasmodium species by detecting aldolase. The emergence of mutant P. falciparum parasites lacking pfhrp2/pfhrp3 genes and thus not expressing HRP2/HRP3 proteins poses a significant challenge in many malaria-endemic areas. This genetic variation has led to false-negative results in all HRP2-detecting RDTs including BinaxNow, undermining its utility. Current U.S. military force health protection (FHP) measures for preventing malaria, including chemoprophylaxis, permethrin-treated uniforms, and DEET application to exposed skin, are effective, but breakthrough infections still occur. The use of portable and user-friendly malaria diagnostics is necessary in remote locations that lack microscopy or nucleic acid-based diagnostic capabilities. The alarmingly high prevalence of mutant pfhrp2/3-deleted parasites poses a threat to malaria diagnosis in all Combatant Commands where point-of-care testing is vital. This review emphasizes the importance of ongoing monitoring to determine the frequency and distribution of mutant parasites. Urgent attention is needed to develop alternative RDTs that can effectively detect malaria infections caused by these mutant strains. These findings confirm that mutant pfhrp2/3-deleted parasites are highly prevalent in SOUTHCOM and parts of AFRICOM, rendering HRP2-based RDTs such as BinaxNow an unsuitable diagnostic tool for malaria in many of the SOUTHCOM and AFRICOM countries surveyed: Peru (14.3-62% between 2011-2018), Eritrea (62% in 2016 and 9.4% in 2020), Nigeria (13.3%), Sudan (11.2%), South Sudan (17.7%), and Uganda (3.3%). In INDOPACOM countries surveyed, no prevalence greater than 5% pfhrp2 deletions were observed. It is critical to continue surveillance on the frequency and distribution of these mutant parasites and develop alternative RDTs. WHO recommends that countries switch to non-HRP2-based RDTs when prevalence of pfhrp2/3 deletions that cause false-negative RDT results exceed 5%. Current prevalence of mutant pfhrp2/3-deleted parasites causing false-negative RDT results has exceeded this threshold in most parts of SOUTHCOM and several areas of AFRICOM. If alternative diagnostic tests are not utilized in areas affected, life-saving malaria treatment for U.S. military personnel could be delayed. Continuous mapping of the frequency and distribution of mutant parasites directly informs FHP protection policy decisions for alternative diagnostic tool utilization.

Strong Positive Selection Biases Identity-By-Descent-Based Inferences of Recent Demography and Population Structure in Plasmodium falciparum.

Malaria genomic surveillance often estimates parasite genetic relatedness using metrics such as Identity-By-Decent (IBD). Yet, strong positive selection stemming from antimalarial drug resistance or other interventions may bias IBD-based estimates. In this study, we utilized simulations, a true IBD inference algorithm, and empirical datasets from different malaria transmission settings to investigate the extent of such bias and explore potential correction strategies. We analyzed whole genome sequence data generated from 640 new and 4,026 publicly available Plasmodium falciparum clinical isolates. Our findings demonstrated that positive selection distorts IBD distributions, leading to underestimated effective population size and blurred population structure. Additionally, we discovered that the removal of IBD peak regions partially restored the accuracy of IBD-based inferences, with this effect contingent on the population's background genetic relatedness. Consequently, we advocate for selection correction for parasite populations undergoing strong, recent positive selection, particularly in high malaria transmission settings.

Co-Infection with Plasmodium vivax and COVID-19 in Thailand.

With the emergence of SARS-CoV-2, healthcare systems not only had to address the pressing clinical needs of the COVID-19 pandemic but anticipate the effect on and of other conditions and diseases. This was of particular concern in areas of the world endemic with malaria, a disease which takes hundreds of thousands of lives each year. This case report from Thailand describes a 25-year-old man diagnosed with Plasmodium vivax, who was then found to be co-infected with COVID-19. Both conditions can have overlapping acute febrile illness symptoms which may delay or complicate diagnoses. He had no prior history of malaria and had received two vaccinations against COVID-19. His clinical course was mild with no pulmonary complications or oxygen requirement, and he responded well to treatments for both conditions. Three months after cure, he again contracted COVID-19 but did not experience any P. vivax relapse. Review of the available literature produced less than 10 publications describing co-infections with P. vivax and COVID-19; nonetheless, in endemic areas, vigilance for both diseases should continue, as co-infections could significantly alter the course of clinical management and prognosis as well as affect the healthcare staff caring for these patients.

Case series of three malaria patients from Thailand infected with the simian parasite, Plasmodium cynomolgi.

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.

A single point mutation in the Plasmodium falciparum 3'-5' exonuclease does not alter piperaquine susceptibility.

BACKGROUND: The rise in Plasmodium falciparum resistance to dihydroartemisinin-piperaquine (DHA-PPQ) treatment has been documented in the Greater Mekong Subregion with associations with mutations in the P. falciparum chloroquine resistance transporter (pfcrt) and plasmepsin 2 (pfpm2) genes. However, it is unclear whether other genes also play a role with PPQ resistance, such as the E415G mutation in the exonuclease (pfexo) gene. The aim of this study was to investigate the role of this mutation in PPQ resistance by generating transgenic parasites expressing the pfexo-E415G mutant allele. METHODS: Transgenic parasite clones carrying the E415G mutation in PfEXO of the B5 isolate were derived by CRISPR-Cas9 gene editing and verified using PCR and gene sequencing. Polymorphisms of pfkelch-13, pfcrt, and pfexo were examined by PCR while the copy number variations of pfpm2 were examined by both relative quantitative real-time PCR and the duplication breakpoint assay. Drug sensitivity against a panel of antimalarials, the ring-stage survival assay (RSA), the PPQ survival assay (PSA), and bimodal dose-response curves were used to evaluate antimalarial susceptibility. RESULTS: The transgenic line, B5-rexo-E415G-B8, was successfully generated. The PPQ-IC90, %PPQ survival, and the bimodal dose-response clearly showed that E415G mutation in PfEXO of B5 isolate remained fully susceptible to PPQ. Furthermore, growth assays demonstrated that the engineered parasites grew slightly faster than the unmodified parental isolates whereas P. falciparum isolates harbouring pfkelch-13, pfcrt, and pfexo mutations with multiple copies of pfpm2 grew much more slowly. CONCLUSIONS: Insertion of the E415G mutation in PfEXO did not lead to increased PPQ-IC90 and %PPQ survival, suggesting that this mutation alone may not be associated with PPQ resistance, but could still be an important marker if used in conjunction with other markers for monitoring PPQ-resistant parasites. The results also highlight the importance of monitoring and evaluating suspected genetic mutations with regard to parasite fitness and resistance.

Nationwide Seroprevalence of Scrub Typhus, Typhus, and Spotted Fever in Young Thai Men.

Scrub typhus group (STG), typhus group (TG), and spotted fever group (SFG) rickettsiae are pathogens distributed worldwide and are important causes of febrile illnesses in southeast Asia. The levels of rickettsioses burden and distribution in Thai communities are still unclear. Nonspecific symptoms, limit diagnostic capacity and underdiagnoses contribute to the absence of clarity. The objective of this study was to determine the nationwide IgG seroprevalence of STG, TG, and SFG by ELISA in repository sera from the Royal Thai Army recruits collected during 2007-2008 and 2012 to estimate rickettsiae exposure in young Thai men to better understand rickettsiae exposure distribution in the Thai population. IgG seroprevalence of STG, Orientia tsutsugamushi; TG, Rickettsia typhi; and SFG, R. rickettsii was 12.4%, 6.8%, and 3.3% in 2007-2008 and 31.8%, 4.2%, and 4.5% in 2012, respectively. The STG had the highest seroprevalence of Rickettsia assessed, with the highest regional seroprevalence found in southern Thailand. The STG seroprevalence changed significantly from 2007 to 2008 (P value < 0.05), which corresponds with morbidity rate of scrub typhus from the last decade in Thailand. We were unable to determine the causality for seroprevalence changes between the two periods due to the limitation in sample numbers for intervening years and limited information available for archived specimens. Additional research would be required to determine agency. However, study results do confirm Rickettsia endemicity in Thailand lends weight to reports of increasing STG seroprevalence. It also corroborates the need to raise rickettsial disease awareness and educate the general public in prevention measures.

Chronic Wound Infection Model of Acinetobacter baumannii in Outbred Mice.

INTRODUCTION: We established a murine wound infection model with doxycycline treatment against multidrug-resistant Acinetobacter baumannii (AB5075) in Institute of Cancer Research (ICR) outbred mice. METHODS: Using three groups of neutropenic ICR mice, two full-thickness dorsal dermal wounds (6 mm diameter) were made on each mouse. In two groups, wounds were inoculated with 7.0 × 104 colony-forming units of AB5075. Of these two groups, one received a 6-day regimen of doxycycline while the other was sham treated with phosphate-buffered saline as placebo control. Another uninfected/untreated group served as a control. Wound closure, clinical symptoms, bacterial burden in wound beds and organs, and wound histology were investigated. RESULTS: Doxycycline-treated wounds completely healed by day 21, but untreated, infected wounds failed to heal. Compared to controls, wound infections without treatment resulted in significant reductions in body weight and higher bacterial loads in wound beds, lung, liver, and spleen by day 7. Histological evaluation of wounds on day 21 revealed ulcerated epidermis, muscle necrosis, and bacterial presence in untreated wounds, while wounds treated with doxycycline presented intact epidermis. CONCLUSIONS: Compared to the previously developed BALB/c dermal wound model, this study demonstrates that the mouse strain selected impacts wound severity and resolution. Furthermore, this mouse model accommodates two dorsal wounds rather than only one. These variations offer investigators increased versatility when designing future studies of wound infection. In conclusion, ICR mice are a viable option as a model of dermal wound infection. They accommodate two simultaneous dorsal wounds, and upon infection, these wounds follow a different pattern of resolution compared to BALB/c mice.

Measurements of 5,6-Orthoquinone, a Surrogate for the Presumed Active Primaquine Metabolite 5-Hydroxyprimaquine, in the Urine of Cambodian Adults.

The active metabolites of primaquine, in particular 5-hydroxyprimaquine, likely responsible for the clearance of dormant hypnozoites, are produced through the hepatic CYP450 2D6 (CYP2D6) enzymatic pathway. With the inherent instability of 5-hydroxyprimaquine, a stable surrogate, 5,6-orthoquinone, can now be detected and measured in the urine as part of primaquine pharmacokinetic studies. This study performed CYP450 2D6 genotyping and primaquine pharmacokinetic testing, to include urine 5,6-orthoquinone, in 27 healthy adult Cambodians, as a preliminary step to prepare for future clinical studies assessing primaquine efficacy for Plasmodium vivax infections. The CYP2D6 *10 reduced activity allele was found in 57% of volunteers, and the CYP2D6 genotypes were dominated by *1/*10 (33%) and *10/*10 (30%). Predicted phenotypes were evenly split between Normal Metabolizer (NM) and Intermediate Metabolizer (IM) except for one volunteer with a gene duplication and unclear phenotype, classifying as either IM or NM. Median plasma primaquine (PQ) area under the curve (AUC) was lower in the NM group (460 h*ng/mL) compared to the IM group (561 h*ng/mL), although not statistically significant. Similar to what has been found in the US study, no 5,6-orthoquinone was detected in the plasma. The urine creatinine-corrected 5,6-orthoquinone AUC in the NM group was almost three times higher than in the IM group, with peak measurements (Tmax) at 4 h. Although there is variation among individuals, future studies examining the relationship between the levels of urine 5,6-orthoquinone and primaquine radical cure efficacy could result in a metabolism biomarker predictive of radical cure.

Antimicrobial Susceptibility of Neisseria gonorrhoeae in Adult Patients Seeking Care at Military Hospitals in Thailand From 2014 to 2020.

INTRODUCTION: The effective dual antibiotics ceftriaxone (CRO) and azithromycin (AZM) have successfully treated Neisseria gonorrhoeae (GC) infection, however, the CRO- and AZM-resistant strains have been sporadically detected globally and in Thailand. Furthermore, there are no currently antimicrobial susceptibility profiles of the GC isolates obtained from soldiers reported in Thailand. Hence, this is the first study to describe the antimicrobial susceptibility profiles of GC isolates obtained from predominately soldiers who seeking care at Military Camp Hospitals, in Thailand from 2014 to 2020. MATERIALS AND METHODS: A total of 624 symptomatic gonococcal samples were received from 10 military hospitals during 2014-2020. They were collected from urethral swabs and inoculated into selective media. The suspected GC isolates were subcultured and presumptively identified using conventional microbiology techniques. Antimicrobial susceptibility test was performed by Etest to determine minimal inhibitory concentration (µg/mL) against AZM, benzylpenicillin, cefepime, cefixime, ceftriaxone (CRO), ciprofloxacin, spectinomycin, and tetracycline using the criteria outlined in the Clinical and Laboratory Standards Institute guidelines. This study was approved by Institutional Review Board, Royal Thai Army Medical Department under protocol number S036b/56 and Walter Reed Army Institute of Research, and Silver Spring, MD under protocol number WR #2039. RESULTS: A total of 624 samples were collected from symptomatic gonococcal infectious patients with 91.5% (571/624) of samples obtained from soldiers. Of those, 78% (488/624) were identified as GC and 92% (449/488) of them were isolated from soldiers. All GC samples collected were susceptible to CRO (first-line treatment) with only one GC isolate identified as non-susceptible to cefepime and three isolates identified as non-susceptible to AZM. CONCLUSION: The recommended dual treatment of GC infections with CRO and AZM is currently an effective empirical treatment for patients who are seeking care at military hospitals in Thailand. Nevertheless, cefepime is a fourth-generation cephalosporin with documented high activity against GC strains equal to other "third-generation" cephalosporins such as CRO. Due to the active duty of military personnel, they concerned about the confidentiality and frequently seek treatment at civilian clinics. Additionally, due to the availability of antibiotics over the counter in Thailand, many choose the option to self-medicate without a physician's prescription. These could be subsequently driven the gradual increase of multidrug-resistant gonococcal strains throughout the country. Thus, the GC surveillance would be needed for further Force Health Protection and public health authorities in response to the drug-resistant GC threats.

First Report: Colistin Resistance Gene mcr-3.1 in Salmonella enterica Serotype Choleraesuis Isolated from Human Blood Sample from Thailand.

This study describes the first finding of Salmonella enterica serotype Choleraesuis (Salmonella Choleraesuis) isolate harboring mobile colistin resistance (mcr)-3.1 obtained from human blood sample. The clinical relevant blood sample was collected during October 2018. The phenotypic identification and antimicrobial susceptibility testing (AST) were studied by using automate microbiology platform (Phoenix M50, BD), and in-depth characterization by whole genome sequencing. The phenotypic identification was reported Salmonella Choleraesuis. AST result demonstrated that this isolate had high minimum inhibitory concentrations (MICs) against colistin, fluoroquinolone, and cephalosporin III and IV, which are first-line antibiotic treatment choices for Gram-negative bacterial pathogen infections. This Salmonella Choleraesuis is harboring mcr-3.1 and presented a diversity carbapenemase including blaTEM and blactx-m-55. Regarding the multilocus sequence typing result, this Salmonella presented ST139 that related to the Choleraesuis variant sensu stricto. Swine is not the host specific for the Salmonella Choleraesuis since it also causes enteric and other diseases in human. Hence, the presence of the mobile plasmid colistin mcr-3.1 resistant gene in human sample is resulting to the public health concerns due to the fact that it is enable to transmit to other hosts and distribute into an environment.

Update on Shigella and Nontyphoidal Salmonella Antimicrobial Drug Resistance: Implications on Empirical Treatment of Acute Infectious Diarrhea in Cambodia.

Information on causative diarrheal pathogens and their associated antimicrobial susceptibility remains limited for Cambodia. This study describes antimicrobial resistance patterns for Shigella and nontyphoidal Salmonella isolates collected in Cambodia over a 5-year period. Multidrug resistance was shown in 98% of Shigella isolates, with 70%, 11%, and 29% of isolates being resistant to fluoroquinolones, azithromycin, and cephalosporin, respectively. As many as 11% of Shigella isolates were resistant to nearly all oral and parenteral drugs typically used for shigellosis, demonstrating extreme drug resistance phenotypes. Although a vast majority of nontyphoidal Salmonella isolates remained susceptible to cephalosporins (99%) and macrolides (98%), decreased susceptibility to ciprofloxacin was found in 67% of isolates, which is notably higher than previous reports. In conclusion, increasing antimicrobial resistance of Shigella and nontyphoidal Salmonella is a major concern for selecting empirical treatment of acute infectious diarrhea in Cambodia. Treatment practices should be updated and follow local antimicrobial resistance data for the identified pathogens.

Molecular Detection of Mutations in the Propeller Domain of Kelch 13 and pfmdr1 Copy Number Variation in Plasmodium falciparum Isolates from Thailand Collected from 2002 to 2007.

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.

Plasmodium falciparum phenotypic and genotypic resistance profile during the emergence of Piperaquine resistance in Northeastern Thailand.

Malaria remains a public health problem in Thailand, especially along its borders where highly mobile populations can contribute to persistent transmission. This study aimed to determine resistant genotypes and phenotypes of 112 Plasmodium falciparum isolates from patients along the Thai-Cambodia border during 2013-2015. The majority of parasites harbored a pfmdr1-Y184F mutation. A single pfmdr1 copy number had CVIET haplotype of amino acids 72-76 of pfcrt and no pfcytb mutations. All isolates had a single pfk13 point mutation (R539T, R539I, or C580Y), and increased % survival in the ring-stage survival assay (except for R539I). Multiple copies of pfpm2 and pfcrt-F145I were detected in 2014 (12.8%) and increased to 30.4% in 2015. Parasites containing either multiple pfpm2 copies with and without pfcrt-F145I or a single pfpm2 copy with pfcrt-F145I exhibited elevated IC90 values of piperaquine. Collectively, the emergence of these resistance patterns in Thailand near Cambodia border mirrored the reports of dihydroartemisinin-piperaquine treatment failures in the adjacent province of Cambodia, Oddar Meanchey, suggesting a migration of parasites across the border. As malaria elimination efforts ramp up in Southeast Asia, host nations militaries and other groups in border regions need to coordinate the proposed interventions.

Combination of Subtherapeutic Doses of Tretazicar and Liposomal Amphotericin B Suppresses and Cures Leishmania major-Induced Cutaneous Lesions in Murine Models.

Cutaneous leishmaniasis (CL) is the most common form of leishmaniasis affecting human populations, yet CL remains largely ignored in drug discovery programs. CL causes disfiguring skin lesions and often relapses after "clinical cure" using existing therapeutics. To expand the pool of anti-CL lead candidates, we implemented an integrated screening platform comprising three progressive Leishmania parasite life cycle forms. We identified tretazicar (CB1954, 5-(aziridin-1-yl)-2,4-dinitrobenzamide) as a potent inhibitor of Leishmania parasite viability across multiple Leishmania species, which translated into complete and prolonged in vivo suppression of CL lesion formation in BALB/c mice when used as a monotherapy and which was superior to liposomal amphotericin B. In addition, oral twice a day administration of tretazicar healed the majority of existing Leishmania major (L. major) cutaneous lesions. In drug combination studies, there was a strong potentiation when subtherapeutic doses of liposomal amphotericin B and tretazicar were simultaneously administered. This drug combination decreased L. major lesion size in mice earlier than individual monotherapy drug treatments and maintained all animals lesion free for up to 64 days after treatment cessation. In contrast, administration of subtherapeutic doses of tretazicar or amphotericin B as monotherapies resulted in no or partial lesion cures, respectively. We propose that tretazicar should be explored as a component of a systemic CL combination therapy and potentially for other diseases where amphotericin B is a first line therapy.

Characterization of Arcobacter spp. Isolated from human diarrheal, non-diarrheal and food samples in Thailand.

Arcobacter butzleri is an emerging zoonotic food-borne and water-borne pathogen that can cause diarrhea in humans. The global prevalence of A. butzleri infection is underestimated, and little is known about their phenotypic and genotypic characterization. The aim of this study was to determine antimicrobial susceptibility (AST) profiles, detect related virulence genes, and classify sequence type (ST) of A. butzleri isolates obtained from human stool and food samples. A total of 84 A. butzleri isolates were obtained from human diarrheal (n = 25), non-diarrheal (n = 24) stool, and food (n = 35) samples in Thailand. They were evaluated for phenotypic identification by conventional microbiological procedures and AST by Kirby-Bauer disc diffusion method as well as virulence genes detection. Representative isolates from each origin were selected based on the presence of virulence genes and AST profiles to analyze genetic diversity by multilocus sequence typing (MLST). All isolates showed resistance to nalidixic acid 40.5% (34/84), ciprofloxacin 11.9% (10/84), azithromycin 8.3% (7/84), and erythromycin 3.6% (3/84). Regarding the ten virulence genes detected, cj1349, mviN and pldA had the highest prevalence 100% (84/84), followed by tlyA 98.8% (83/84), cadF 97.6% (82/84), ciaB 71.4% (60/84), hecA and hecB 22.6% (19/84), iroE 15.5% (13/84) and irgA 10.7% (9/84), respectively. Three virulence genes were present among A. butzleri isolates of human diarrheal stool and food samples, with a significant difference observed among isolates; hecB [36% (9/25) and 8.6% (3/35)], hecA [36% (9/25) and 5.7% (2/35)], and irgA [24% (6/25) and 2.9% (1/35)] (p < 0.05), respectively. The hecA and hecB virulence genes functions are related to the mechanism of hemolysis, while irgA supports a bacterial nutritional requirement. MLST analysis of 26 A. butzleri isolates revealed that 16 novel STs exhibited high genetic diversity. The results of this study is useful for understanding potentially pathogenic and antimicrobial-resistant A. butzleri in Thailand. The pathogenic virulence markers hecB, hecA, and irgA have the potential to be developed for rapid diagnostic detection in human diarrheal stool. No significant relationships among STs and sources of origin were observed. Little is known about A. butzleri, the mechanism of action of these virulence genes, is a topic that needs further investigation.

Distribution and Temporal Dynamics of Plasmodium falciparum Chloroquine Resistance Transporter Mutations Associated With Piperaquine Resistance in Northern Cambodia.

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.

Implementation of external quality assessment of microscopy for improved parasite detection and confirmatory diagnosis of malaria in Tanzanian Military health facilities.

OBJECTIVE: Good quality microscopy is critical for accurate detection and confirmation of malaria parasite infections. Microscopy relies on the skills of technicians to prepare and read slides, high quality reagents, and a good program of internal and external quality control (EQA), which are lacking in most malaria endemic settings. This study was undertaken between January 2016 and December 2018 to pilot an EQA of microscopy for improved diagnosis of malaria and patient care in Tanzanian Military health facilities. RESULTS: Of all blood smears crosschecked (n = 4000) at baseline, only 38.5% were incorrectly diagnosed by laboratory staff with false positive and negative rates of 46.7% and 16.4%, respectively. During the implementation of EQA, false positive and negative results decreased due to increased quality index of slide preparation and reading through supportive supervision, and retraining of laboratory personnel. There was a gradual increase of quarterly and annual total quality index for all laboratories, from 60% in 2016 to 78% in 2017 and 90% in 2018. The mean proficiency testing performance scores also increased from 75% in 2016 to 82% in 2017 and to 90% in 2018. Poor blood smear preparation and staining contributed to high false positive and negative rates while EQA helped in improvement of diagnostics.