Antibiotic resistance profiles of sentinel bacteria isolated from aquaculture in Cambodia.

The misuse of antibiotics and the emergence of antimicrobial resistance (AMR) is a concern in the aquaculture industry because it contributes to global health risks and impacts the environment. This study analyzed the AMR of sentinel bacteria associated with striped catfish (Pangasisanodon hypophthalmus) and giant snakehead (Channa micropeltes), the two main fish species reared in the pond culture in Cambodia. Phenotypic and genotypic characterization of the recovered isolates from fish, water, and sediment samples revealed the presence of bacteria, such as 22 species belonging to families Aeromonadaceae, Enterobacteriaceae, and Pseudomonadaceae. Among 48 isolates, Aeromonas caviae (n = 2), Aeromonas hydrophila (n = 2), Aeromonas ichthiosmia (n = 1), Aeromonas salmonicida (n = 4) were detected. A. salmonicida and A. hydrophilla are known as fish pathogens that occur worldwide in both fresh and marine water aquaculture. Antibiotic susceptibility testing revealed antibiotic resistance patterns of 24 (50 %) isolates among 48 isolates with higher multiple antibiotic resistance index (> 0.2). All the isolates of Enterobacteriaceae were susceptible to ciprofloxacin. Ciprofloxacin is a frontline antibiotic that is not recommended to use in aquaculture. Therefore, its use has to be strictly controlled. This study expands our knowledge of the AMR status in aquaculture farms which is very limited in Cambodia.

Genomic insights into anthropozoonotic tuberculosis in captive sun bears (Helarctos malayanus) and an Asiatic black bear (Ursus thibetanus) in Cambodia.

Contact between humans and wildlife presents a risk for both zoonotic and anthropozoonotic disease transmission. In this study we report the detection of human strains of Mycobacterium tuberculosis in sun bears and an Asiatic black bear in a wildlife rescue centre in Cambodia, confirming for the first time the susceptibility of these bear species to tuberculosis when in close contact with humans. After genotyping revealed two different strains of M. tuberculosis from cases occurring between 2009 and 2019, 100 isolates from 30 sun bear cases, a single Asiatic black bear case, and a human case were subjected to whole genome sequencing. We combined single nucleotide polymorphism analysis and exploration of mixed base calls with epidemiological data to indicate the evolution of each outbreak. Our results confirmed two concurrent yet separate tuberculosis outbreaks and established a likely transmission route in one outbreak where the human case acted as an intermediatory between bear cases. In both outbreaks, we observed high rates of transmission and progression to active disease, suggesting that sun bears are highly susceptible to tuberculosis if exposed under these conditions. Overall, our findings highlight the risk of bi-directional transmission of tuberculosis between humans and captive bears in high human tuberculosis burden regions, with implied considerations for veterinary and public health. We also demonstrate the use of standard genomic approaches to better understand disease outbreaks in captive wildlife settings and to inform control and prevention measures.

Comparative Performance of Anyplex II HPV28 and Cobas 4800 Human Papillomavirus (HPV) Assays for High-Risk HPV Detection in Self-collected Anal Samples.

We compared 2 human papillomavirus (HPV) assays to detect the 14 high-risk HPV (hrHPV) genotypes in self-collected anal samples. We found a good agreement and similar performance to detect HPV-16, HPV-18, and the 12 other hrHPV genotypes. The global performance to detect the 14 hrHPV genotypes was not significantly different between the 2 assays.

Phylogenomic investigation of an outbreak of fluoroquinolone-resistant Salmonella enterica subsp. enterica serovar Paratyphi A in Phnom Penh, Cambodia.

In early 2020, the Medical Biology Laboratory of the Pasteur Institute of Cambodia isolated an unusually high number of fluoroquinolone-resistant Salmonella enterica subspecies enterica serovar Paratyphi A strains during its routine bacteriological surveillance activities in Phnom Penh, Cambodia. A public-health investigation was supported by genome sequencing of these Paratyphi A strains to gain insights into the genetic diversity and population structure of a potential outbreak of fluoroquinolone-resistant paratyphoid fever. Comparative genomic and phylodynamic analyses revealed the 2020 strains were descended from a previously described 2013-2015 outbreak of Paratyphi A infections. Our analysis showed sub-lineage 2.3.1 had remained largely susceptible to fluoroquinolone drugs until 2015, but acquired chromosomal resistance to these drugs during six separate events between late 2012 and 2015. The emergence of fluoroquinolone resistance was rapidly followed by the replacement of the original susceptible Paratyphi A population, which led to a dramatic increase of fluoroquinolone-resistant blood-culture-confirmed cases in subsequent years (2016-2020). The rapid acquisition of resistance-conferring mutations in the Paratyphi A population over a 3 year period is suggestive of a strong selective pressure on that population, likely linked with fluoroquinolone use. In turn, emergence of fluoroquinolone resistance has led to increased use of extended-spectrum cephalosporins like ceftriaxone that are becoming the drug of choice for empirical treatment of paratyphoid fever in Cambodia.

How a PCR Sequencing Strategy Can Bring New Data to Improve the Diagnosis of Ethionamide Resistance.

Ethionamide (ETH) is a second-line antituberculosis drug. ETH resistance (ETH-R) is mainly related to the mutations of the monooxygenase-activating ETH (EthA), the ETH target (InhA), and the inhA promoter. Nonetheless, diagnosing ETH-R is still challenging. We assessed the strategy used for detecting ETH-R at the French National Reference Center for Mycobacteria in 497 MDR-TB isolates received from 2008 to 2016. The genotypic ETH's resistance detection was performed by sequencing ethA, ethR, the ethA-ethR intergenic region, and the inhA promoter in the 497 multidrug-resistant isolates, whereas the phenotypic ETH susceptibility testing (PST) was performed using the reference proportion method. Mutations were found in up to 76% of the 387 resistant isolates and in up to 28% of the 110 susceptible isolates. Our results do not support the role of ethR mutations in ETH resistance. Altogether, the positive predictive value of our genotypic strategy to diagnose ETH-R was improved when only considering the variants included in the WHO catalogue and in other databases, such as TB-Profiler. Therefore, our work will help to update the list of mutations that could be graded as being associated with resistance to improve ETH-R diagnosis.

Non-toxigenic Corynebacterium diphtheriae in hallux ulceration.

INTRODUCTION: Toxigenic Corynebacterium diphtheriae causes classical diphtheria. Skin infections by toxigenic or non-toxigenic Corynebacterium diphtheriae are prevalent in the tropics but are rarely reported. CASE PRESENTATION: We report the identification of a non-toxigenic Corynebacterium diphtheriae (biovar Gravis) isolate in a 52-year-old Cambodian male. The patient presented purulent and non-healing ulcerations on the right hallux. The wound has healed after 7 days of antibiotic therapy with a favourable outcome. CONCLUSIONS: This case represents, to our knowledge, the first report of Corynebacterium diphtheriae in Cambodia in the last 10 years, and highlights the lack of diagnosis and notifications of diphtheria. It is important to raise awareness among clinicians and to set up diphtheria surveillance in Cambodia.

Rapid Generation of In-House Serological Assays Is Comparable to Commercial Kits Critical for Early Response to Pandemics: A Case With SARS-CoV-2.

Introduction: Accurate and sensitive measurement of antibodies is critical to assess the prevalence of infection, especially asymptomatic infection, and to analyze the immune response to vaccination during outbreaks and pandemics. A broad variety of commercial and in-house serological assays are available to cater to different laboratory requirements; however direct comparison is necessary to understand utility. Materials and Methods: We investigate the performance of six serological methods against SARS-CoV-2 to determine the antibody profile of 250 serum samples, including 234 RT-PCR-confirmed SARS-CoV-2 cases, the majority with asymptomatic presentation (87.2%) at 1-51 days post laboratory diagnosis. First, we compare to the performance of two in-house antibody assays: (i) an in-house IgG ELISA, utilizing UV-inactivated virus, and (ii) a live-virus neutralization assay (PRNT) using the same Cambodian isolate as the ELISA. In-house assays are then compared to standardized commercial anti-SARS-CoV-2 electrochemiluminescence immunoassays (Elecsys ECLIAs, Roche Diagnostics; targeting anti-N and anti-S antibodies) along with a flow cytometry based assay (FACS) that measures IgM and IgG against spike (S) protein and a multiplex microsphere-based immunoassay (MIA) determining the antibodies against various spike and nucleoprotein (N) antigens of SARS-CoV-2 and other coronaviruses (SARS-CoV-1, MERS-CoV, hCoVs 229E, NL63, HKU1). Results: Overall, specificity of assays was 100%, except for the anti-S IgM flow cytometry based assay (96.2%), and the in-house IgG ELISA (94.2%). Sensitivity ranged from 97.3% for the anti-S ECLIA down to 76.3% for the anti-S IgG flow cytometry based assay. PRNT and in-house IgG ELISA performed similarly well when compared to the commercial ECLIA: sensitivity of ELISA and PRNT was 94.7 and 91.1%, respectively, compared to S- and N-targeting ECLIA with 97.3 and 96.8%, respectively. The MIA revealed cross-reactivity of antibodies from SARS-CoV-2-infected patients to the nucleocapsid of SARS-CoV-1, and the spike S1 domain of HKU1. Conclusion: In-house serological assays, especially ELISA and PRNT, perform similarly to commercial assays, a critical factor in pandemic response. Selection of suitable immunoassays should be made based on available resources and diagnostic needs.

Resistance to Second-Line Anti-TB Drugs in Cambodia: A Phenotypic and Genetic Study.

BACKGROUND: Due to the emergence of Mycobacterium tuberculosis (M.tb) clinical isolates resistant to most potent first-line drugs (FLD), second-line drugs (SLD) are being prescribed more frequently. We explore the genetic characteristics and molecular mechanisms of M.tb isolates phenotypically resistant to SLD, including pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) isolates. METHODS: Drug-resistant (DR) M.tb isolates collected from 2012 to 2017 were tested using sequencing and phenotypic drug susceptibility testing. Genotypes were determined to explore their links with SLD resistance patterns. RESULTS: Of the 272 DR M.tb isolates, 6 non-multidrug resistant (non-MDR) isolates were fluoroquinolones (FQ)-resistant, 3 were XDR and 16 were pre-XDR (14 resistant to FQ and 2 to second-line injectable drugs). The most frequent mutations in FQ-resistant and second-line injectable drugs resistant isolates were gyrA D94G (15/23) and rrs a1401g (3/5), respectively. Seventy-five percent of pre-XDR isolates and 100% of XDR isolates harbored mutations conferring resistance to pyrazinamide. All XDR isolates belonged to the Beijing genotype, of which one, named XDR+, was resistant to all drugs tested. One cluster including pre-XDR and XDR isolates was observed. CONCLUSION: This is the first description of SLD resistance in Cambodia. The data suggest that the proportion of XDR and pre-XDR isolates remains low but is on the rise compared to previous reports. The characterization of the XDR+ isolate in a patient who refused treatment underlines the risk of transmission in the population. In addition, genotypic results show, as expected, that the Beijing family is the main involved in pre-XDR and XDR isolates and that the spread of the Beijing pre-XDR strain is capable of evolving into XDR strain. This study strongly indicates the need for rapid interventions in terms of diagnostic and treatment to prevent the spread of the pre-XDR and XDR strains and the emergence of more resistant ones.

Evaluation of Loopamp Assay for the Diagnosis of Pulmonary Tuberculosis in Cambodia.

The Loopamp™ MTBC kit (TB-LAMP) is recommended by WHO for Mycobacterium tuberculosis complex detection in low-income countries with a still low drug-resistant tuberculosis (TB) rate. This study is aimed at testing its feasibility in Cambodia on sputa collected from presumptive tuberculosis patients. 499 samples were tested at a smear microscopy center and 200 at a central-level mycobacteriology laboratory. Using mycobacterial cultures as reference, TB-LAMP results were compared with those of LED fluorescent microscopy (LED-FM) and Xpert® MTB/RIF. At the microscopy center, TB-LAMP sensitivity was higher than that of LED-FM (81.5% [95% CI, 74.5-87.6] versus 69.4% [95% CI, 62.2-76.6]), but lower than that of the Xpert assay (95.5% [95% CI 92.3-98.8]). At the central-level laboratory, TB-LAMP sensitivity (92.8% [95% CI, 87.6-97.9]) was comparable to that of Xpert (90.7% [95% CI, 85.0-96.5]) using stored sample. No significant difference in terms of specificity between TB-LAMP and Xpert assays was observed in both study sites. In conclusion, our data demonstrate that TB-LAMP could be implemented at microscopy centers in Cambodia to detect TB patients. In addition, TB-LAMP can be a better choice to replace smear microscopy for rapid TB diagnosis of new presumptive TB patients, in settings with relative low prevalence of drug-resistant TB and difficulties to implement Xpert assay.

Isolation of Nontuberculous Mycobacteria in Southeast Asian and African Human Immunodeficiency Virus-infected Children With Suspected Tuberculosis.

We enrolled 427 human immunodeficiency virus-infected children (median age, 7.3 years), 59.2% severely immunodeficient, with suspected tuberculosis in Southeast Asian and African settings. Nontuberculous mycobacteria were isolated in 46 children (10.8%); 45.7% of isolates were Mycobacterium avium complex. Southeast Asian origin, age 5-9 years, and severe immunodeficiency were independently associated with nontuberculous mycobacteria isolation. CLINICAL TRIALS REGISTRATION: NCT01331811.

Performance of Xpert MTB/RIF and Alternative Specimen Collection Methods for the Diagnosis of Tuberculosis in HIV-Infected Children.

BACKGROUND: The diagnosis of tuberculosis in human immunodeficiency virus (HIV)-infected children is challenging. We assessed the performance of alternative specimen collection methods for tuberculosis diagnosis in HIV-infected children using Xpert MTB/RIF (Xpert). METHODS: HIV-infected children aged ≤13 years with suspected intrathoracic tuberculosis were enrolled in 8 hospitals in Burkina Faso, Cambodia, Cameroon, and Vietnam. Gastric aspirates were taken for children aged <10 years and expectorated sputum samples were taken for children aged ≥10 years (standard samples); nasopharyngeal aspirate and stool were taken for all children, and a string test was performed if the child was aged ≥4 years (alternative samples). All samples were tested with Xpert. The diagnostic accuracy of Xpert for culture-confirmed tuberculosis was analyzed in intention-to-diagnose and per-protocol approaches. RESULTS: Of 281 children enrolled, 272 (96.8%) had ≥1 specimen tested with Xpert (intention-to-diagnose population), and 179 (63.5%) had all samples tested with Xpert (per-protocol population). Tuberculosis was culture-confirmed in 29/272 (10.7%) children. Intention-to-diagnose sensitivities of Xpert performed on all, standard, and alternative samples were 79.3% (95% confidence interval [CI], 60.3-92.0), 72.4% (95% CI, 52.8-87.3), and 75.9% (95% CI, 56.5-89.7), respectively. Specificities were ≥97.5%. Xpert combined on nasopharyngeal aspirate and stool had intention-to-diagnose and per-protocol sensitivities of 75.9% (95% CI, 56.5-89.7) and 75.0% (95% CI, 47.6-92.7), respectively. CONCLUSIONS: The combination of nasopharyngeal aspirate and stool sample is a promising alternative to methods usually recommended by national programs. Xpert performed on respiratory and stools samples enables rapid confirmation of tuberculosis diagnosis in HIV-infected children. CLINICAL TRIALS REGISTRATION: The ANRS (Agence Nationale de Recherche sur le Sida) 12229 PAANTHER (Pediatric Asian African Network for Tuberculosis and HIV Research) 01 study is registered at ClinicalTrials.gov (NCT01331811).

Acute lower respiratory infections on lung sequelae in Cambodia, a neglected disease in highly tuberculosis-endemic country.

BACKGROUND: Little is known about post-infectious pulmonary sequelae in countries like Cambodia where tuberculosis is hyper-endemic and childhood pulmonary infections are highly frequent. We describe the characteristics of hospitalized Cambodian patients presenting with community-acquired acute lower respiratory infections (ALRI) on post-infectious pulmonary sequelae (ALRIPS). METHODS: Between 2007 and 2010, inpatients ≥15 years with ALRI were prospectively recruited. Clinical, biological, radiological and microbiological data were collected. Chest radiographs were re-interpreted by experts to compare patients with ALRIPS, on previously healthy lungs (ALRIHL) and active pulmonary tuberculosis (TB). Patients without chest radiograph abnormality or with abnormality suggestive as other chronic respiratory diseases were excluded from this analysis. RESULTS: Among the 2351 inpatients with community-acquired ALRI, 1800 were eligible: 426 (18%) ALRIPS, 878 (37%) ALRIHL and 496 (21%) TB. ALRIPS patients had less frequent fever than other ALRI (p < 0.001) and more productive cough than ALRIHL (p < 0.001). Streptococcus pneumoniae, Haemophilus influenzae, and Pseudomonas aeruginosa accounted for 83% of ALRIPS group positive cultures. H. influenzae and P. aeruginosa were significantly associated with ALRIPS compared with ALRIHL. Treatment was appropriate in 58% of ALRIPS patients. Finally, 79% of ALRIPS were not recognized by local clinicians. In-hospital mortality was low (1%) but probably underestimated in the ALRIPS group. CONCLUSION: ALRIPS remains often misdiagnosed as TB with inappropriate treatment in low-income countries. Better-targeted training programs would help reduce the morbidity burden and financial costs.