Burkholderia pseudomallei Bacteria in Ornamental Fish Tanks, Vientiane, Laos, 2023.

In 2019, a melioidosis case in Maryland, USA, was shown to have been acquired from an ornamental fish tank contaminated with Burkholderia pseudomallei bacteria, likely derived from Southeast Asia. We investigated the presence of B. pseudomallei in ornamental fish tanks in the endemic area of Vientiane, Laos.

A multi-country study using MALDI-TOF mass spectrometry for rapid identification of Burkholderia pseudomallei.

BACKGROUND: Burkholderia pseudomallei is the bacterial causative agent of melioidosis, a difficult disease to diagnose clinically with high mortality if not appropriately treated. Definitive diagnosis requires isolation and identification of the organism. With the increased adoption of MALDI-TOF MS for the identification of bacteria, we established a method for rapid identification of B. pseudomallei using the Vitek MS, a system that does not currently have B. pseudomallei in its in-vitro diagnostic database. RESULTS: A routine direct spotting method was employed to create spectra and SuperSpectra. An initial B. pseudomallei SuperSpectrum was created at Shoklo Malaria Research Unit (SMRU) from 17 reference isolates (46 spectra). When tested, this initial SMRU SuperSpectrum was able to identify 98.2 % (54/55) of Asian isolates, but just 46.7 % (35/75) of Australian isolates. Using spectra (430) from different reference and clinical isolates, two additional SMRU SuperSpectra were created. Using the combination of all SMRU SuperSpectra with seven existing SuperSpectra from Townsville, Australia 119 (100 %) Asian isolates and 31 (100 %) Australian isolates were correctly identified. In addition, no misidentifications were obtained when using these 11 SuperSpectra when tested with 34 isolates of other bacteria including the closely related species Burkholderia thailandensis and Burkholderia cepacia. CONCLUSIONS: This study has established a method for identification of B. pseudomallei using Vitek MS, and highlights the impact of geographical differences between strains for identification using this technique.

Evaluation of Molecular Methods To Improve the Detection of Burkholderia pseudomallei in Soil and Water Samples from Laos.

Burkholderia pseudomallei is the cause of melioidosis, a severe and potentially fatal disease of humans and animals. It is endemic in northern Australia and Southeast Asia and is found in soil and surface water. The environmental distribution of B. pseudomallei worldwide and within countries where it is endemic, such as the Lao People's Democratic Republic (Laos), remains unclear. However, this knowledge is important to our understanding of the ecology and epidemiology of B. pseudomallei and to facilitate public health interventions. Sensitive and specific methods to detect B. pseudomallei in environmental samples are therefore needed. The aim of this study was to compare molecular and culture-based methods for the detection of B. pseudomallei in soil and surface water in order to identify the optimal approach for future environmental studies in Laos. Molecular detection by quantitative real-time PCR (qPCR) was attempted after DNA extraction directly from soil or water samples or after an overnight enrichment step. The positivity rates obtained by qPCR were compared to those obtained by different culture techniques. The rate of detection from soil samples by qPCR following culture enrichment was significantly higher (84/100) than that by individual culture methods and all culture methods combined (44/100; P < 0.001). Similarly, qPCR following enrichment was the most sensitive method for filtered river water compared with the sensitivity of the individual methods and all individual methods combined. In conclusion, molecular detection following an enrichment step has proven to be a sensitive and reliable approach for B. pseudomallei detection in Lao environmental samples and is recommended as the preferred method for future surveys.

Utility of InTray COLOREX Screen agar and InTray COLOREX ESBL agar for urine culture in the Lao PDR.

BACKGROUND: There is a need for simple microbiology diagnostics to enable antimicrobial resistance surveillance in low- and middle-income countries. OBJECTIVES: To investigate the field utility of InTray COLOREX plates for urine culture and ESBL detection. METHODS: Clinical urine samples from Mahosot Hospital, Vientiane, Lao PDR were inoculated onto chromogenic media and InTray COLOREX Screen plates between June and August 2020. Urine and isolates from other clinical specimens were inoculated onto COLOREX ESBL plates. A simulated field study investigating the field utility of the InTray COLOREX plates was also completed. RESULTS: In total, 355 urine samples were inoculated onto standard chromogenic agar and InTray COLOREX Screen plates, and 154 urine samples and 54 isolates from other clinical specimens on the COLOREX ESBL plates. Growth was similar for the two methods (COLOREX Screen 41%, standard method 38%) with 20% discordant results, mainly due to differences in colony counts or colonial appearance. Contamination occurred in 13% of samples, with the COLOREX Screen plates showing increased contamination rates, potentially due to condensation. ESBL producers were confirmed from 80% of isolates from the COLOREX ESBL plates, and direct plating provided rapid detection of presumptive ESBL producers. Burkholderia pseudomallei also grew well on the ESBL plates, a relevant finding in this melioidosis-endemic area. CONCLUSIONS: The InTray COLOREX Screen and ESBL plates were simple to use and interpret, permitting rapid detection of uropathogens and ESBLs, and have the potential for easy transport and storage from field sites and use in laboratories with low capacity.

Fatal Chromobacterium violaceum septicaemia in northern Laos, a modified oxidase test and post-mortem forensic family G6PD analysis.

BACKGROUND: Chromobacterium violaceum is a Gram negative facultative anaerobic bacillus, found in soil and stagnant water, that usually has a violet pigmented appearance on agar culture. It is rarely described as a human pathogen, mostly from tropical and subtropical areas. CASE PRESENTATION: A 53 year-old farmer died with Chromobacterium violaceum septicemia in Laos. A modified oxidase method was used to demonstrate that this violacious organism was oxidase positive. Forensic analysis of the glucose-6-phosphate dehydrogenase genotypes of his family suggest that the deceased patient did not have this possible predisposing condition. CONCLUSION: C. violaceum infection should be included in the differential diagnosis in patients presenting with community-acquired septicaemia in tropical and subtropical areas. The apparently neglected but simple modified oxidase test may be useful in the oxidase assessment of other violet-pigmented organisms or of those growing on violet coloured agar.

Evaluation of consensus method for the culture of Burkholderia pseudomallei in soil samples from Laos.

Background: We have previously shown that PCR following enrichment culture is the most sensitive method to detect Burkholderia pseudomallei in environmental samples. Here we report an evaluation of the published consensus method for the culture of B. pseudomallei from Lao soil in comparison with our conventional culture method and with PCR with or without prior broth enrichment. Methods: One hundred soil samples were collected from a field known to contain B. pseudomallei and processed by: (i) the conventional method, (ii-iii) the consensus method using media prepared in either Laos or Thailand, and (iv) the consensus method performed in Thailand, as well as by (v) PCR following direct extraction of DNA from soil and (vi) PCR following broth pre-enrichment. Results: The numbers of samples in which B. pseudomallei was detected were 42, 10, 7, 6, 6 and 84, respectively. However, two samples were positive by the consensus method but negative by conventional culture, and one sample was negative by PCR following enrichment although B. pseudomallei was isolated by the conventional culture method. Conclusions/Discussion: The results show that no single method will detect all environmental samples that contain B. pseudomallei. People conducting environmental surveys for this organism should be aware of the possibility of false-negative results using the consensus culture method. An approach that entails screening using PCR after enrichment, followed by the evaluation of a range of different culture methods on PCR-positive samples to determine which works best in each setting, is recommended.

The Utility of Blood Culture Fluid for the Molecular Diagnosis of Leptospira: A Prospective Evaluation.

Leptospirosis is an important zoonosis worldwide, with infections occurring after exposure to contaminated water. Despite being a global problem, laboratory diagnosis remains difficult with culture results taking up to 3 months, serology being retrospective by nature, and polymerase chain reaction showing limited sensitivity. Leptospira have been shown to survive and multiply in blood culture media, and we hypothesized that extracting DNA from incubated blood culture fluid (BCF), followed by quantitative real-time polymerase chain reaction (qPCR) could improve the accuracy and speed of leptospira diagnosis. We assessed this retrospectively, using preincubated BCF of Leptospira spp. positive (N= 109) and negative (N= 63) febrile patients in Vientiane, Lao PDR. The final method showed promising sensitivities of 66% (95% confidence interval [CI]: 55-76) and 59% (95% CI: 49-68) compared with direct or direct and indirect testing combined, as the respective reference standards (specificities > 95%). Despite these promising diagnostic parameters, a subsequent prospective evaluation in a Lao hospital population (N= 352) showed that the sensitivity was very low (∼30%) compared with qPCR on venous blood samples. The disappointingly low sensitivity does suggest that venous blood samples are preferable for the clinical microbiology laboratory, although BCF might be an alternative if leptospirosis is only suspected postadmission after antibiotics have been used.

An improved selective culture medium enhances the isolation of Burkholderia pseudomallei from contaminated specimens.

Burkholderia pseudomallei is a Gram-negative environmental bacterium found in tropical climates that causes melioidosis. Culture remains the diagnostic gold standard, but isolation of B. pseudomallei from heavily contaminated sites, such as fecal specimens, can be difficult. We recently reported that B. pseudomallei is capable of infecting the gastrointestinal tract of mice and suggested that the same may be true in humans. Thus, there is a strong need for new culture techniques to allow for efficient detection of B. pseudomallei in fecal and other specimens. We found that the addition of norfloxacin, ampicillin, and polymyxin B to Ashdown's medium (NAP-A) resulted in increased specificity without affecting the growth of 25 B. pseudomallei strains. Furthermore, recovery of B. pseudomallei from human clinical specimens was not affected by the three additional antibiotics. Therefore, we conclude that NAP-A medium provides a new tool for more sensitive isolation of B. pseudomallei from heavily contaminated sites.

Urine antibiotic activity in patients presenting to hospitals in Laos: implications for worsening antibiotic resistance.

Widespread use of antibiotics may be important in the spread of antimicrobial resistance. We estimated the proportion of Lao in- and outpatients who had taken antibiotics before medical consultation by detecting antibiotic activity in their urine added to lawns of Bacillus stearothermophilus, Escherichia coli, and Streptococcus pyogenes. In the retrospective (N = 2,058) and prospective studies (N = 1,153), 49.7% (95% confidence interval [CI] = 47.4-52.0) and 36.2% (95% CI = 33.4-38.9), respectively, of Vientiane patients had urinary antibiotic activity detected. The highest frequency of estimated antibiotic pre-treatment was found in patients recruited with suspected central nervous system infections and community-acquired septicemia (both 56.8%). In Vientiane, children had a higher frequency of estimated antibiotic pre-treatment than adults (60.0% versus 46.5%; P < 0.001). Antibiotic use based on patients histories was significantly less frequent than when estimated from urinary antibiotic activity (P < 0.0001).