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.

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.

Understanding work-related travel and its relation to malaria occurrence in Thailand using geospatial maximum entropy modelling.

BACKGROUND: Estimating malaria risk associated with work locations and travel across a region provides local health officials with information useful to mitigate possible transmission paths of malaria as well as understand the risk of exposure for local populations. This study investigates malaria exposure risk by analysing the spatial pattern of malaria cases (primarily Plasmodium vivax) in Ubon Ratchathani and Sisaket provinces of Thailand, using an ecological niche model and machine learning to estimate the species distribution of P. vivax malaria and compare the resulting niche areas with occupation type, work locations, and work-related travel routes. METHODS: A maximum entropy model was trained to estimate the distribution of P. vivax malaria for a period between January 2019 and April 2020, capturing estimated malaria occurrence for these provinces. A random simulation workflow was developed to make region-based case data usable for the machine learning approach. This workflow was used to generate a probability surface for the ecological niche regions. The resulting niche regions were analysed by occupation type, home and work locations, and work-related travel routes to determine the relationship between these variables and malaria occurrence. A one-way analysis of variance (ANOVA) test was used to understand the relationship between predicted malaria occurrence and occupation type. RESULTS: The MaxEnt (full name) model indicated a higher occurrence of P. vivax malaria in forested areas especially along the Thailand-Cambodia border. The ANOVA results showed a statistically significant difference between average malaria risk values predicted from the ecological niche model for rubber plantation workers and farmers, the two main occupation groups in the study. The rubber plantation workers were found to be at higher risk of exposure to malaria than farmers in Ubon Ratchathani and Sisaket provinces of Thailand. CONCLUSION: The results from this study point to occupation-related factors such as work location and the routes travelled to work, being risk factors in malaria occurrence and possible contributors to transmission among local populations.

First-in-human assessment of safety and immunogenicity of low and high doses of Plasmodium falciparum malaria protein 013 (FMP013) administered intramuscularly with ALFQ adjuvant in healthy malaria-naïve adults.

The global burden of malaria remains substantial. Circumsporozoite protein (CSP) has been demonstrated to be an effective target antigen, however, improvements that offer more efficacious and more durable protection are still needed. In support of research and development of next-generation malaria vaccines, Walter Reed Army Institute of Research (WRAIR) has developed a CSP-based antigen (FMP013) and a novel adjuvant ALFQ (Army Liposome Formulation containing QS-21). We present a single center, open-label, dose-escalation Phase 1 clinical trial to evaluate the safety and immunogenicity of the FMP013/ALFQ malaria vaccine candidate. In this first-in-human evaluation of both the antigen and adjuvant, we enrolled ten subjects; five received 20 µg FMP013 / 0.5 mL ALFQ (Low dose group), and five received 40 µg FMP013 / 1.0 mL ALFQ (High dose group) on study days 1, 29, and 57. Adverse events and immune responses were assessed during the study period. The clinical safety profile was acceptable and there were no serious adverse events. Both groups exhibited robust humoral and cellular immunological responses, and compared favorably with historical responses reported for RTS,S/AS01. Based on a lower reactogenicity profile, the 20 µg FMP013 / 0.5 mL ALFQ (Low dose) was selected for follow-on efficacy testing by controlled human malaria infection (CHMI) with a separate cohort. Trial Registration:Clinicaltrials.gov Identifier NCT04268420 (Registered February 13, 2020).

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.

Human papillomavirus seroprevalence in young Thai men.

Human papillomavirus (HPV) is one of the most common sexually transmitted infections in men and women. Most HPV studies have focused on vaccination toward women to prevent consequences of developing cervical cancer. However, persistent infections can cause penile, anal, and oropharyngeal cancers in men. Therefore, recent public health recommendations toward vaccination in men have been raised. There is limited HPV prevalence data among men in many countries, including Thailand. We conducted HPV sera IgG ELISA testing on a repository sera of Thai men (average age 21 years old) entering the Royal Thai Army as recruits in 2013 (n = 1000). HPV IgG antibodies against virus-like particles of HPV- type 6, 11, 16e, and 18 were evaluated using a commercial ELISA kit. Overall, the anti-HPV IgG was 47% (95% CI: 44%-50%). HPV seroprevalence was significantly associated with residence regions with the highest prevalence in South (64%), but not associated with educational level, marital status, or type of residence. This data suggested that almost half of the Thai men in this cohort were exposed to HPV by the age of 21. Thus, HPV vaccination provided to male adolescents should be considered for disease prevention and minimizing transmission to sexual partners.

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.

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.

Chemoprotective antimalarials identified through quantitative high-throughput screening of Plasmodium blood and liver stage parasites.

The spread of Plasmodium falciparum parasites resistant to most first-line antimalarials creates an imperative to enrich the drug discovery pipeline, preferably with curative compounds that can also act prophylactically. We report a phenotypic quantitative high-throughput screen (qHTS), based on concentration-response curves, which was designed to identify compounds active against Plasmodium liver and asexual blood stage parasites. Our qHTS screened over 450,000 compounds, tested across a range of 5 to 11 concentrations, for activity against Plasmodium falciparum asexual blood stages. Active compounds were then filtered for unique structures and drug-like properties and subsequently screened in a P. berghei liver stage assay to identify novel dual-active antiplasmodial chemotypes. Hits from thiadiazine and pyrimidine azepine chemotypes were subsequently prioritized for resistance selection studies, yielding distinct mutations in P. falciparum cytochrome b, a validated antimalarial drug target. The thiadiazine chemotype was subjected to an initial medicinal chemistry campaign, yielding a metabolically stable analog with sub-micromolar potency. Our qHTS methodology and resulting dataset provides a large-scale resource to investigate Plasmodium liver and asexual blood stage parasite biology and inform further research to develop novel chemotypes as causal prophylactic antimalarials.

A Phase IIa Controlled Human Malaria Infection and Immunogenicity Study of RTS,S/AS01E and RTS,S/AS01B Delayed Fractional Dose Regimens in Malaria-Naive Adults.

BACKGROUND: A previous RTS,S/AS01B vaccine challenge trial demonstrated that a 3-dose (0-1-7-month) regimen with a fractional third dose can produce high vaccine efficacy (VE) in adults challenged 3 weeks after vaccination. This study explored the VE of different delayed fractional dose regimens of adult and pediatric RTS,S/AS01 formulations. METHODS: A total of 130 participants were randomized into 5 groups. Four groups received 3 doses of RTS,S/AS01B or RTS,S/AS01E on a 0-1-7-month schedule, with the final 1 or 2 doses being fractional (one-fifth dose volume). One group received 1 full (month 0) and 1 fractional (month 7) dose of RTS,S/AS01E. Immunized and unvaccinated control participants underwent Plasmodium falciparum-infected mosquito challenge (controlled human malaria infection) 3 months after immunization, a timing chosen to potentially discriminate VEs between groups. RESULTS: The VE of 3-dose formulations ranged from 55% (95% confidence interval, 27%-72%) to 76% (48%-89%). Groups administered equivalent formulations of RTS,S/AS01E and RTS,S/AS01B demonstrated comparable VE. The 2-dose group demonstrated lower VE (29% [95% confidence interval, 6%-46%]). All regimens were well tolerated and immunogenic, with trends toward higher anti-circumsporozoite antibody titers in participants protected against infection. CONCLUSIONS: RTS,S/AS01E can provide VE comparable to an equivalent RTS,S/AS01B regimen in adults, suggesting a universal formulation may be considered. Results also suggest that the 2-dose regimen is inferior to the 3-dose regimens evaluated. CLINICAL TRIAL REGISTRATION: NCT03162614.