Clinical Presentation, Risk Factors, and Comparison of Laboratory Diagnostics for Seasonal Influenza Virus Among Cambodians From 2007 to 2020.

Background: Despite its global significance, challenges associated with understanding the epidemiology and accurately detecting, measuring, and characterizing the true burden of seasonal influenza remain in many resource-poor settings. Methods: A prospective observational study was conducted in Cambodia at 28 health facilities between 2007 and 2020 utilizing passive surveillance data of patients presenting with acute undifferentiated febrile illness (AUFI) to describe the prevalence of influenza A and B and characterize associated risk factors and symptoms using a questionnaire. A comparison of rapid influenza diagnostic tests (RIDTs) and real-time reverse transcription polymerase chain reaction (rRT-PCR) results was also conducted. Results: Of 30 586 total participants, 5634 (18.4%) tested positive for either influenza A or B, with 3557 (11.6%) positive for influenza A and 2288 (7.5%) positive for influenza B during the study. Influenza A and B were strongly associated with the rainy season (odds ratio [OR], 2.30; P < .001) and being from an urban area (OR, 1.45; P < .001). Analysis of individual symptoms identified cough (OR, 2.8; P < .001), chills (OR, 1.4; P < .001), and sore throat (OR, 1.4; P < .001) as having the strongest positive associations with influenza among patients with AUFI. Analysis comparing RIDTs and rRT-PCR calculated the overall sensitivity of rapid tests to be 0.492 (95% CI, 0.479-0.505) and specificity to be 0.993 (95% CI, 0.992-0.994) for both influenza type A and B. Conclusions: Findings from this 14-year study include describing the epidemiology of seasonal influenza over a prolonged time period and identifying key risk factors and clinical symptoms associated with infection; we also demonstrate the poor sensitivity of RIDTs in Cambodia.

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.

Clinical and epidemiologic evaluation of a 2020 chikungunya outbreak in Cambodia.

BACKGROUND: In 2020, the Kingdom of Cambodia experienced a nationwide outbreak of chikungunya virus (CHIKV). Despite an increase in the frequency of outbreaks and expanding geographic range of CHIKV, diagnostic challenges remain, and limited surveillance data of sufficient granularity are available to characterize epidemiological profiles and disease dynamics of the virus. METHODS: An ongoing and long-standing cross-sectional study of acute undifferentiated febrile illness (AUFI) in Cambodia was leveraged to describe the disease epidemiology and characterize the clinical presentation of patients diagnosed with CHIKV during the 2020 outbreak. Participants presenting with AUFI symptoms at ten study locations provided acute and convalescent blood samples and were tested for CHIKV using a reverse transcription-polymerase chain reaction (RT-PCR) and serological diagnostic methods including IgM and IgG. Acute and follow-up clinical data were also collected. RESULTS: From 1194 participant blood samples tested, 331 (27.7%) positive CHIKV cases were detected. Most CHIKV positive individuals (280, 84.6%) reported having a fever 3 to 4 days prior to visiting a health facility. Symptoms including chills, joint pain, nausea, vomiting, and lesions were all statistically significant among CHIKV positive participants compared to CHIKV negative AUFI participants. Cough was negatively associated with CHIKV positive participants. Positivity proportions were significantly higher among adults compared to children. No significant difference was found in positivity proportion between rainy and dry seasons during the outbreak. Positive CHIKV cases were detected in all study site provinces, with the highest test positivity proportion recorded in the rural northeast province of Kratie. CONCLUSIONS: Surveillance data captured in this study provided a clinical and epidemiological characterization of positive CHIKV patients presenting at selected health facilities in Cambodia in 2020, and highlighted the widespread distribution of the outbreak, impacting both urban and rural locations. Findings also illustrated the importance of utilizing both RT-PCR and serological testing for effective CHIKV surveillance.

Retrospective Analysis of Fever and Sepsis Patients from Cambodia Reveals Serological Evidence of Melioidosis.

Burkholderia pseudomallei, the etiologic agent of melioidosis, is predicted to be ubiquitous in tropical regions of the world with areas of highest endemicity throughout Southeast Asia (SEA). Nevertheless, the distribution of B. pseudomallei and the burden of melioidosis in many SEA countries remain unclear. In Cambodia, only two human endemic cases of melioidosis were reported through 2008 and since then only a few hundred cases have been described in the literature. This is in sharp contrast to the annual burden of thousands of cases in surrounding areas. To further investigate the prevalence of melioidosis in Cambodia, we used a recently developed O-polysaccharide-based rapid enzyme-linked immunosorbent assay to detect B. pseudomallei-specific antibodies in serum samples obtained from 1,316 febrile illness or sepsis patients from 10 different provinces. Based on a cutoff value derived through culture-confirmed melioidosis cases, the proportion of positive samples in our cohort was approximately 12%. Regression analysis indicated that the odds of obtaining a positive result were 2.2 times higher for males than females controlling for age and province (95% confidence interval: 1.6-3.2, P < 0.001). Consistent with this, 9.2% of females were positive versus 18.2% of males (P < 0.001). Notably, 22.5% of grain or rice farmers were positive versus 10.1% of subjects with occupations not involving regular contact with soil. Positive results varied significantly by province. Collectively, the results of this study suggest that the true burden of melioidosis in Cambodia is greater than has previously been reported.

Drug resistance in bacteria isolated from patients presenting with wounds at a non-profit Surgical Center in Phnom Penh, Cambodia from 2011-2013.

BACKGROUND: Emerging antibiotic resistance amongst clinically significant bacteria is a public health issue of increasing significance worldwide, but it is relatively uncharacterized in Cambodia. In this study we performed standard bacterial cultures on samples from wounds at a Non-Governmental-Organization (NGO) Hospital in Phnom Penh, Cambodia. Testing was performed to elucidate pathogenic bacteria causing wound infections and the antibiotic resistance profiles of bacterial isolates. All testing was performed at the Naval Medical Research Unit, No.2 (NAMRU-2) main laboratory in Phnom Penh, Cambodia. METHODS: Between 2011-2013, a total of 251 specimens were collected from patients at the NGO hospital and analyzed for bacterial infection by standard bacterial cultures techniques. Specimens were all from wounds and anonymous. No specific clinical information accompanied the submitted specimens. Antibiotic susceptibility testing, and phenotypic testing for extended-spectrum beta-lactamase (ESBL) were performed and reported based on CLSI guidelines. Further genetic testing for CTX-M, TEM and SHV ESBLs was accomplished using PCR. RESULTS: One-hundred and seventy-six specimens were positive following bacterial culture (70 %). Staphlycoccus aureus was the most frequently isolated bacteria. Antibiotic drug resistance testing revealed that 52.5 % of Staphlycoccus aureus isolates were oxacillin resistant. For Escherichia coli isolates, 63.9 % were ciprofloxacin and levofloxacin resistant and 96 % were ESBL producers. Resistance to meropenem and imipenem was observed in one of three Acinetobacter spp isolates. CONCLUSIONS: This study is the first of its kind detailing the antibiotic resistance profiles of pathogenic bacteria causing wound infections at a single surgical hospital in Cambodia. The reported findings of this study demonstrate significant antibiotic resistance in bacteria from injured patients and should serve to guide treatment modalities in Cambodia.

Development of real-time PCR assays and evaluation of their potential use for rapid detection of Burkholderia pseudomallei in clinical blood specimens.

The early initiation of appropriate antimicrobial therapy is critical for improving the prognosis of patients with septicemic melioidosis. Thus, the use of a rapid molecular diagnosis may affect the outcome of this disease, which has a high mortality rate. We report the development of two TaqMan real-time PCR assays (designated 8653 and 9438) that detect the presence of two novel genes unique to Burkolderia pseudomallei. The analytical sensitivity and specificity of the assays were assessed with 91 different B. pseudomallei isolates, along with 96 isolates and strains representing 28 other bacterial species, including the closely related Burkholderia/Ralstonia. The two assays performed equally well with both purified DNA and crude cell lysates, with 100% analytical specificity for the detection of B. pseudomallei. The limit of detection was 50 fg of DNA (equivalent to six bacterial genomes) per PCR for both assay 8563 and 9438. We also evaluated these assays with DNA extracted from blood specimens taken from 45 patients with culture-confirmed septicemic melioidosis or other septicemias. Of the 28 melioidosis blood specimens, assays 8653 and 9438 gave sensitivities of 71% (20/28) and 54% (15/28), respectively. Effectively, all fatal cases of septicemic melioidosis were detected by 8653. For the 17 non-melioidosis blood specimens, specificities of 82% (14/17) and 88% (15/17) were obtained for assays 8653 and 9438, respectively. The real-time PCR assays developed in this study provide alternative, rapid molecular tools for the specific detection of B. pseudomallei, and this may be of particular use in the early diagnosis and treatment of septicemic melioidosis.