Etiology and risk factors for diarrheal disease amongst rural and peri-urban populations in Cambodia, 2012-2018.

Diarrheal diseases are a leading cause of mortality and morbidity, disproportionally affecting persons residing in low and middle-income countries. Accessing high-resolution surveillance data to understand community-level etiology and risk remains challenging, particularly in remote and resource limited populations. A multi-year prospective cohort study was conducted in two rural and two peri-urban villages in Cambodia from 2012 to 2018 to describe the epidemiology and etiology of acute diarrheal diseases within the population. Suspected diarrheal episodes among participants were self-reported or detected via routine weekly household visits. Fresh stool and fecal swabs were tested, and acute-illness and follow-up participant questionnaires collected. Of 5027 enrolled participants, 1450 (28.8%) reported at least one diarrheal incident. A total of 4266 individual diarrhea case events were recorded. Diarrhea incidence rate was calculated to be 281.5 persons per 1000 population per year, with an event rate of 664.3 individual diarrhea events occurring per 1000 population per year. Pathogenic Escherichia coli, Aeromonas spp., and Plesiomonas shigelloides were the most prevalent bacterial infections identified. Hookworm and Strongyloides stercoralis were the predominant helminth species, while Blastocystis hominis and Giardia lamblia were the predominant protozoan species found. Norovirus genotype 2 was the predominant virus identified. Mixed infections of two or more pathogens were detected in 36.2% of positive cases. Risk analyses identified unemployed status increased diarrhea risk by 63% (HR = 1.63 [95% CI 1.46, 1.83]). Individuals without access to protected water sources or sanitation facilities were 59% (HR = 1.59 [95% CI 1.49, 1.69]) and 19% (HR = 1.19 [95% CI 1.12, 1.28]) greater risk of contracting diarrhea, respectively. Patient-level surveillance data captured in this long-term study has generated a unique spatiotemporal profile of diarrheal disease in Cambodia. Understanding etiologies, together with associated epidemiological and community-level risk, provides valuable public health insight to support effective planning and delivery of appropriate local population-targeted interventions.

Leptospirosis infections among hospital patients, Sarawak, Malaysia.

BACKGROUND: Leptospirosis diagnoses have increased in Sarawak, Malaysia in recent years. METHODS: To better understand the burden of disease and associated risk factors, we evaluated 147 patients presenting with clinical leptospirosis to local hospitals in Sarawak, Malaysia for the presence of Leptospira and associated antibodies. Sera and urine specimens collected during the acute illness phase were assessed via a commercially available rapid diagnostic test (Leptorapide, Linnodee Ltd., Antrim, Northern Ireland), an ELISA IgM assay (Leptospira IgM ELISA, PanBio, Queensland, Australia) and a pan-Leptospira real-time PCR (qPCR) assay to estimate disease prevalence and diagnostic accuracy of each method. Microagglutination testing was performed on a subset of samples. RESULTS: Overall, 45 out of 147 patients (30.6%) showed evidence of leptospires through qPCR in either one or both sera (20 patients) or urine (33 patients), and an additional ten (6.8%) were considered positive through serological testing, for an overall prevalence of 37.4% within the study population. However, each diagnostic method individually yielded disparate prevalence estimates: rapid test 42.2% for sera and 30.5% for urine, ELISA 15.0% for sera, qPCR 13.8% for sera and 23.4% for urine. Molecular characterization of a subset of positive samples by conventional PCR identified the bacterial species as Leptospira interrogans in 4 specimens. A multivariate risk factor analysis for the outcome of leptospirosis identified having completed primary school (OR = 2.5; 95 CI% 1.0-6.4) and weekly clothes-washing in local rivers (OR = 10.6; 95 CI% 1.4-214.8) with increased likelihood of leptospirosis when compared with those who had not. CONCLUSION: Overall, the data suggest a relatively high prevalence of leptospirosis in the study population. The low sensitivities of the rapid diagnostic test and ELISA assay against qPCR highlight a need for better screening tools.

Genetic mechanisms and correlated risk factors of antimicrobial-resistant ESKAPEE pathogens isolated in a tertiary hospital in Malaysia.

BACKGROUND: Knowledge on the epidemiology, genotypic and phenotypic features of antimicrobial-resistant (AMR) ESKAPEE pathogens (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, Enterobacter spp., and Escherichia coli) and their association with hospital-acquired infections (HAIs) are limited in Malaysia. Therefore, we evaluated the AMR features and resistance mechanisms of the ESKAPEE pathogens collected in a tertiary hospital located in the capital of Malaysia. METHODS: A total of 378 AMR-ESKAPEE strains were obtained based on convenience sampling over a nine-month study period (2019-2020). All strains were subjected to disk diffusion and broth microdilution assays to determine the antimicrobial susceptibility profiles. Polymerase chain reaction (PCR) and DNA sequence analyses were performed to determine the AMR genes profiles of the non-susceptible strains. Chi-square test and logistic regression analyses were used to correlate the AMR profiles and clinical data to determine the risk factors associated with HAIs. RESULTS: High rates of multidrug resistance (MDR) were observed in A. baumannii, K. pneumoniae, E. coli, and S. aureus (69-89%). All organisms except E. coli were frequently associated with HAIs (61-94%). Non-susceptibility to the last-resort drugs vancomycin (in Enterococcus spp. and S. aureus), carbapenems (in A. baumannii, P. aeruginosa, and Enterobacteriaceae), and colistin (in Enterobacteriaceae) were observed. Both A. baumannii and K. pneumoniae harbored a wide array of extended-spectrum ß-lactamase genes (blaTEM, blaSHV, blaCTX-M, blaOXA). Metallo-ß-lactamase genes (blaVEB, blaVIM, blaNDM) were detected in carbapenem-resistant strains, at a higher frequency compared to other local reports. We detected two novel mutations in the quinolone-resistant determining region of the gyrA in fluoroquinolone-resistant E. coli (Leu-102-Ala; Gly-105-Val). Microbial resistance to ampicillin, methicillin, and cephalosporins was identified as important risk factors associated with HAIs in the hospital. CONCLUSION: Overall, our findings may provide valuable insight into the microbial resistance pattern and the risk factors of ESKAPEE-associated HAIs in a tertiary hospital located in central Peninsular Malaysia. The data obtained in this study may contribute to informing better hospital infection control in this region.

An Observational Study of Sepsis in Takeo Province Cambodia: An in-depth examination of pathogens causing severe infections.

The world's most consequential pathogens occur in regions with the fewest diagnostic resources, leaving the true burden of these diseases largely under-represented. During a prospective observational study of sepsis in Takeo Province Cambodia, we enrolled 200 patients over an 18-month period. By coupling traditional diagnostic methods such as culture, serology, and PCR to Next Generation Sequencing (NGS) and advanced statistical analyses, we successfully identified a pathogenic cause in 46.5% of our cohort. In all, we detected 25 infectious agents in 93 patients, including severe threat pathogens such as Burkholderia pseudomallei and viral pathogens such as Dengue virus. Approximately half of our cohort remained undiagnosed; however, an independent panel of clinical adjudicators determined that 81% of those patients had infectious causes of their hospitalization, further underscoring the difficulty of diagnosing severe infections in resource-limited settings. We garnered greater insight as to the clinical features of severe infection in Cambodia through analysis of a robust set of clinical data.

Evaluation of the Immunogenicity and Protective Efficacy of an Enterotoxigenic Escherichia coli CFA/I Adhesin-Heat-Labile Toxin Chimera.

Recent efforts to develop an enterotoxigenic Escherichia coli (ETEC) vaccine have focused on the antigenically conserved tip adhesins of colonization factors. We showed previously that intranasal immunization with dsc19CfaE, a soluble variant of the in cis donor strand-complemented tip adhesin of a colonization factor of the class 5 family (CFA/I) fimbria, is highly immunogenic and protects against oral challenge with CFA/I-positive (CFA/I+) ETEC strain H10407 in the Aotus nancymaae nonhuman primate. We also reported a cholera toxin (CT)-like chimera (called dsc19CfaE-CTA2/CTB) in which the CTA1 domain of CT was replaced by dsc19CfaE that was strongly immunogenic when administered intranasally or orogastrically in mice. Here, we evaluate the immunogenicity and protective efficacy (PE) of a refined and more stable chimera comprised of a pentameric B subunit of ETEC heat-labile toxin (LTB) in lieu of the CTB pentamer and a donor strand truncation (dsc14) of CfaE. The refined chimera, dsc14CfaE-sCTA2/LTB, was highly immunogenic in mice when administered intranasally or intradermally, eliciting serum and fecal antibody responses against CfaE and LTB, as well as strong hemagglutination inhibition titers, a surrogate for neutralization of intestinal adhesion mediated by CfaE. Moreover, the chimera was safe and highly immunogenic when administered intradermally to guinea pigs. In A. nancymaae, intradermal (i.d.) immunization with chimera plus single-mutant heat-labile toxin [LT(R192G)] elicited strong serum anti-CfaE and anti-LTB antibody responses and conferred significant reduction of diarrhea compared to phosphate-buffered saline (PBS) controls (PE = 84.1%; P < 0.02). These data support the further evaluation of dsc14CfaE-sCTA2/LTB as an ETEC vaccine in humans.

Tracking Antimicrobial Resistance Determinants in Diarrheal Pathogens: A Cross-Institutional Pilot Study.

Infectious diarrhea affects over four billion individuals annually and causes over a million deaths each year. Though not typically prescribed for treatment of uncomplicated diarrheal disease, antimicrobials serve as a critical part of the armamentarium used to treat severe or persistent cases. Due to widespread over- and misuse of antimicrobials, there has been an alarming increase in global resistance, for which a standardized methodology for geographic surveillance would be highly beneficial. To demonstrate that a standardized methodology could be used to provide molecular surveillance of antimicrobial resistance (AMR) genes, we initiated a pilot study to test 130 diarrheal pathogens (Campylobacter spp., Escherichia coli, Salmonella, and Shigella spp.) from the USA, Peru, Egypt, Cambodia, and Kenya for the presence/absence of over 200 AMR determinants. We detected a total of 55 different determinants conferring resistance to ten different categories of antimicrobials: genes detected in ≥ 25 samples included blaTEM, tet(A), tet(B), mac(A), mac(B), aadA1/A2, strA, strB, sul1, sul2, qacEΔ1, cmr, and dfrA1. The number of determinants per strain ranged from none (several Campylobacter spp. strains) to sixteen, with isolates from Egypt harboring a wider variety and greater number of genes per isolate than other sites. Two samples harbored carbapenemase genes, blaOXA-48 or blaNDM. Genes conferring resistance to azithromycin (ere(A), mph(A)/mph(K), erm(B)), a first-line therapeutic for severe diarrhea, were detected in over 10% of all Enterobacteriaceae tested: these included >25% of the Enterobacteriaceae from Egypt and Kenya. Forty-six percent of the Egyptian Enterobacteriaceae harbored genes encoding CTX-M-1 or CTX-M-9 families of extended-spectrum ß-lactamases. Overall, the data provide cross-comparable resistome information to establish regional trends in support of international surveillance activities and potentially guide geospatially informed medical care.

A Survey of Antimicrobial Resistance Determinants in Category A Select Agents, Exempt Strains, and Near-Neighbor Species.

A dramatic increase in global antimicrobial resistance (AMR) has been well documented. Of particular concern is the dearth of information regarding the spectrum and prevalence of AMR within Category A Select Agents. Here, we performed a survey of horizontally and vertically transferred AMR determinants among Category A agents and their near neighbors. Microarrays provided broad spectrum screening of 127 Francisella spp., Yersinia spp., and Bacillus spp. strains for the presence/absence of 500+ AMR genes (or families of genes). Detecting a broad variety of AMR genes in each genus, microarray analysis also picked up the presence of an engineered plasmid in a Y. pestis strain. High resolution melt analysis (HRMA) was also used to assess the presence of quinolone resistance-associated mutations in 100 of these strains. Though HRMA was able to detect resistance-causing point mutations in B. anthracis strains, it was not capable of discriminating these point mutations from other nucleotide substitutions (e.g., arising from sequence differences in near neighbors). Though these technologies are well-established, to our knowledge, this is the largest survey of Category A agents and their near-neighbor species for genes covering multiple mechanisms of AMR.

Distribution of Capsular Types of Campylobacter jejuni Isolates from Symptomatic and Asymptomatic Children in Peru.

Campylobacter jejuni is the leading bacterial cause of diarrhea worldwide. A capsular polysaccharide (CPS) conjugate vaccine is under development and requires determination of the valency. However, distribution of CPS types circulating globally is presently poorly described. We aimed to determine whether CPS type distribution in Peru differs from that in other endemic regions. We used a multiplex polymerase chain reaction (PCR) assay for the detection of CPS encoding genes capable of distinguishing all 35 CPS types on Campylobacter isolates in two prospective communities based studies conducted in cohorts of children less than 59 months of age in Peru. Results showed that CPS type HS4 complex was the most prevalent, followed by HS3 complex and HS15. Differences in CPS type for symptomatology were not statistically significant. Most subjects demonstrated repeated infections over time with different CPS types, suggesting that CPS types may confer of a level of homologous protective immunity. In this dataset, some differences in CPS type distribution were observed in comparison to other low-middle income countries. Further studies need to be conducted in endemic areas to increase our knowledge of CPS type distribution and guide vaccine development.

The other Campylobacters: Not innocent bystanders in endemic diarrhea and dysentery in children in low-income settings.

BACKGROUND: Campylobacter is one of the main causes of gastroenteritis worldwide. Most of the current knowledge about the epidemiology of this food-borne infection concerns two species, C. coli and C. jejuni. Recent studies conducted in developing countries and using novel diagnostic techniques have generated evidence of the increasing burden and importance of other Campylobacter species, i.e. non-C. coli/jejuni. We performed a nested case-control study to compare the prevalence of C. coli/jejuni and other Campylobacter in children with clinical dysentery and severe diarrhea as well as without diarrhea to better understand the clinical importance of infections with Campylobacter species other than C. coli/jejuni. METHODOLOGY/PRINCIPAL FINDINGS: Our nested case-control study of 439 stool samples included dysenteric stools, stools collected during severe diarrhea episodes, and asymptomatic stools which were systematically selected to be representative of clinical phenotypes from 9,160 stools collected during a birth cohort study of 201 children followed until two years of age. Other Campylobacter accounted for 76.4% of the 216 Campylobacter detections by qPCR and were more prevalent than C. coli/jejuni across all clinical groups. Other Campylobacter were also more prevalent than C. coli/jejuni across all age groups, with older children bearing a higher burden of other Campylobacter. Biomarkers of intestinal inflammation and injury (methylene blue, fecal occult test, myeloperoxidase or MPO) showed a strong association with dysentery, but mixed results with infection. MPO levels were generally higher among children infected with C. coli/jejuni, but Shigella-infected children suffering from dysentery recorded the highest levels (26,224 ng/mL); the lowest levels (10,625 ng/mL) were among asymptomatic children infected with other Campylobacter. Adjusting for age, sex, and Shigella infection, dysentery was significantly associated with C. coli/jejuni but not with other Campylobacter, whereas severe diarrhea was significantly associated with both C. coli/jejuni and other Campylobacter. Compared to asymptomatic children, children suffering from dysentery had a 14.6 odds of C. coli/jejuni infection (p-value < 0.001, 95% CI 5.5-38.7) but were equally likely to have other Campylobacter infections-odds ratio of 1.3 (0.434, 0.7-2.4). Children suffering from severe diarrhea were more likely than asymptomatic children to test positive for both C. coli/jejuni and other Campylobacter-OR of 2.8 (0.034, 1.1-7.1) and 1.9 (0.018, 1.1-3.1), respectively. Compared to the Campylobacter-free group, the odds of all diarrhea given C. coli/jejuni infection and other Campylobacter infection were 8.8 (<0.001, 3.0-25.7) and 2.4 (0.002, 1.4-4.2), respectively. Eliminating other Campylobacter in this population would eliminate 24.9% of the diarrhea cases, which is almost twice the population attributable fraction of 15.1% due to C. coli/jejuni. CONCLUSIONS/SIGNIFICANCE: Eighty-seven percent of the dysentery and 59.5% of the severe diarrhea samples were positive for Campylobacter, Shigella, or both, emphasizing the importance of targeting these pathogens to limit the impact of dysentery and severe diarrhea in children. Notably, the higher prevalence of other Campylobacter compared to C. coli/jejuni, their increasing burden during early childhood, and their association with severe diarrhea highlight the importance of these non-C. coli/jejuni Campylobacter species and suggest a need to clarify their importance in the etiology of clinical disease across different epidemiological contexts.

Melioidosis in lower provincial Cambodia: A case series from a prospective study of sepsis in Takeo Province.

Melioidosis is a severe infectious disease caused by the gram-negative soil bacterium Burkholderia pseudomallei. Melioidosis is well known to be a major cause of morbidity and mortality in Southeast Asia, particularly in Thailand. However, melioidosis remains underreported in surrounding areas such as Cambodia. We report a case series of melioidosis in seven patients from Takeo Province, Cambodia. The patients, aged 24-65 years, were enrolled from May 2014 to May 2015 during a one year prospective study of sepsis at Takeo Provincial Hospital. They presented with fever, rigors, dyspnea, fatigue, diaphoresis, productive cough, and skin abscesses. Six of the seven patients were also hyponatremic. B. pseudomallei was cultured from the blood of six patients and the sputum of one patient. In this manuscript, we provide a detailed description of the clinical presentation, case management and laboratory confirmation of B. pseudomallei, as well as discuss the difficulties of identifying and treating melioidosis in low resource settings.

Application of a broad-range resequencing array for detection of pathogens in desert dust samples from Kuwait and Iraq.

A significant percentage of the human population is exposed to high levels of naturally occurring airborne dusts. Although the link between airborne particulate inhalation and a variety of respiratory diseases has long been established, little is known about the pathogenic role of the microbial component of the dust. In this study, we applied highly multiplexed PCR and a high-density resequencing microarray (RPM-TEI version 1.0) to screen samples of fine topsoil particles and airborne dust collected in 19 locations in Iraq and Kuwait for the presence of a broad range of human pathogens. The results indicated the presence of potential human pathogens, including Mycobacterium, Brucella, Coxiella burnetii, Clostridium perfringens, and Bacillus. The presence of Coxiella burnetii, a highly infectious potential biowarfare agent, was confirmed and detected in additional samples by use of a more sensitive technique (real-time PCR), indicating a high prevalence of this organism in the analyzed samples. The detection of potentially viable pathogens in breathable dusts from arid regions of Iraq and Kuwait underscores the importance of further study of these environments.