Using Land Runoff to Survey the Distribution and Genetic Diversity of Burkholderia pseudomallei in Vientiane, Laos.

Melioidosis is a disease of significant public health importance that is being increasingly recognized globally. The majority of cases arise through direct percutaneous exposure to its etiological agent, Burkholderia pseudomallei In the Lao People's Democratic Republic (Laos), the presence and environmental distribution of B. pseudomallei are not well characterized, though recent epidemiological surveys of the bacterium have indicated that B. pseudomallei is widespread throughout the environment in the center and south of the country and that rivers can act as carriers and potential sentinels for the bacterium. The spatial and genetic distribution of B. pseudomallei within Vientiane Capital, from where the majority of cases diagnosed to date have originated, remains an important knowledge gap. We sampled surface runoff from drain catchment areas throughout urban Vientiane to determine the presence and local population structure of the bacterium. B. pseudomallei was detected in drainage areas throughout the capital, indicating it is widespread in the environment and that exposure rates in urban Vientiane are likely more frequent than previously thought. Whole-genome comparative analysis demonstrated that Lao B. pseudomallei isolates are highly genetically diverse, suggesting the bacterium is well-established and not a recent introduction. Despite the wide genome diversity, one environmental survey isolate was highly genetically related to a Lao melioidosis patient isolate collected 13?years prior to the study. Knowledge gained from this study will augment understanding of B. pseudomallei phylogeography in Asia and enhance public health awareness and future implementation of infection control measures within Laos.IMPORTANCE The environmental bacterium B. pseudomallei is the etiological agent of melioidosis, a tropical disease with one model estimating a global annual incidence of 165,000 cases and 89,000 deaths. In the Lao People's Democratic Republic (Laos), the environmental distribution and population structure of B. pseudomallei remain relatively undefined, particularly in Vientiane Capital from where most diagnosed cases have originated. We used surface runoff as a proxy for B. pseudomallei dispersal in the environment and performed whole-genome sequencing (WGS) to examine the local population structure. Our data confirmed that B. pseudomallei is widespread throughout Vientiane and that surface runoff might be useful for future environmental monitoring of the bacterium. B. pseudomallei isolates were also highly genetically diverse, suggesting the bacterium is well-established and endemic in Laos. These findings can be used to improve awareness of B. pseudomallei in the Lao environment and demonstrates the epidemiological and phylogeographical insights that can be gained from WGS

Orientia, rickettsia, and leptospira pathogens as causes of CNS infections in Laos: a prospective study

Background Scrub typhus (caused by Orientia tsutsugamushi), murine typhus (caused by Rickettsia typhi), and leptospirosis are common causes of febrile illness in Asia; meningitis and meningoencephalitis are severe complications. However, scarce data exist for the burden of these pathogens in patients with CNS disease in endemic countries. Laos is representative of vast economically poor rural areas in Asia with little medical information to guide public health policy. We assessed whether these pathogens are important causes of CNS infections in Laos. Methods Between Jan 10, 2003, and Nov 25, 2011, we enrolled 1112 consecutive patients of all ages admitted with CNS symptoms or signs requiring a lumbar puncture at Mahosot Hospital, Vientiane, Laos. Microbiological examinations (culture, PCR, and serology) targeted so-called conventional bacterial infections (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus infl uenzae, S suis) and O tsutsugamushi, Rickettsia typhi/Rickettsia spp, and Leptospira spp infections in blood or cerebrospinal fl uid (CSF). We analysed and compared causes and clinical and CSF characteristics between patient groups. Findings 1051 (95%) of 1112 patients who presented had CSF available for analysis, of whom 254 (24%) had a CNS infection attributable to a bacterial or fungal pathogen. 90 (35%) of these 254 infections were caused by O tsutsugamushi, R typhi/Rickettsia spp, or Leptospira spp. These pathogens were signifi cantly more frequent than conventional bacterial infections (90/1051 [9%] vs 42/1051 [4%]; p<0·0001) by use of conservative diagnostic defi nitions. CNS infections had a high mortality (236/876 [27%]), with 18% (13/71) for R typhi/Rickettsia spp, O tsutsugamushi, and Leptospira spp combined, and 33% (13/39) for conventional bacterial infections (p=0·076). Interpretation Our data suggest that R typhi/Rickettsia spp, O tsutsugamushi, and Leptospira spp infections are important causes of CNS infections in Laos. Antibiotics, such as tetracyclines, needed for the treatment of murine typhus and scrub typhus, are not routinely advised for empirical treatment of CNS infections. These severely neglected infections represent a potentially large proportion of treatable CNS disease burden across vast endemic areas and need more attention

Survival and Growth of Orientia tsutsugamushi in Conventional Hemocultures.

Orientia tsutsugamushi, which requires specialized facilities for culture, is a substantial cause of disease in Asia. We demonstrate that O. tsutsugamushi numbers increased for up to 5 days in conventional hemocultures. Performing such a culture step before molecular testing could increase the sensitivity of O. tsutsugamushi molecular diagnosis

Impact of delayed processing of positive blood cultures on organism detection: a prospective multi-centre study

Background Blood cultures remain the gold standard investigation for the diagnosis of bloodstream infections. In many locations, quality-assured processing of positive blood cultures is not possible. One solution is to incubate blood cultures locally, and then transport bottles that flag positive to a central reference laboratory for organism identification and antimicrobial susceptibility testing. However, the impact of delay between the bottle flagging positive and subsequent sub-culture on the viability of the isolate has received little attention. Methods This study evaluated the impact of delays to sub-culture (22 h to seven days) in three different temperature conditions (2–8 °C, 22–27 °C and 35 ± 2 °C) for bottles that had flagged positive in automated detection systems using a mixture of spiked and routine clinical specimens. Ninety spiked samples for five common bacterial causes of sepsis (Escherichia coli, Haemophilus influenzae, Staphylococcus aureus, Streptococcus agalactiae and Streptococcus pneumoniae) and 125 consecutive positive clinical blood cultures were evaluated at four laboratories located in Cambodia, Lao PDR and Thailand. In addition, the utility of transport swabs for preserving organism viability was investigated. Results All organisms were recoverable from all sub-cultures in all temperature conditions with the exception of S. pneumoniae, which was less likely to be recoverable after longer delays (> 46–50 h), when stored in hotter temperatures (35 °C), and from BacT/ALERT when compared with BACTEC blood culture bottles. Storage of positive blood culture bottles in cooler temperatures (22–27 °C or below) and the use of Amies bacterial transport swabs helped preserve viability of S. pneumoniae. Conclusions These results have practical implications for the optimal workflow for blood culture bottles that have flagged positive in automated detection systems located remotely from a central processing laboratory, particularly in tropical resource-constrained contexts

Orientia, rickettsia, and leptospira pathogens as causes of CNS infections in Laos: a prospective study

Background: Scrub typhus (caused by Orientia tsutsugamushi), murine typhus (caused by Rickettsia typhi), and leptospirosis are common causes of febrile illness in Asia; meningitis and meningoencephalitis are severe complications. However, scarce data exist for the burden of these pathogens in patients with CNS disease in endemic countries. Laos is representative of vast economically poor rural areas in Asia with little medical information to guide public health policy. We assessed whether these pathogens are important causes of CNS infections in Laos. Methods: Between Jan 10, 2003, and Nov 25, 2011, we enrolled 1112 consecutive patients of all ages admitted with CNS symptoms or signs requiring a lumbar puncture at Mahosot Hospital, Vientiane, Laos. Microbiological examinations (culture, PCR, and serology) targeted so-called conventional bacterial infections (Streptococcus pneumoniae, Neisseria meningitidis, Haemophilus influenzae, S suis) and O tsutsugamushi, Rickettsia typhi/Rickettsia spp, and Leptospira spp infections in blood or cerebrospinal fluid (CSF). We analysed and compared causes and clinical and CSF characteristics between patient groups. Findings: 1051 (95%) of 1112 patients who presented had CSF available for analysis, of whom 254 (24%) had a CNS infection attributable to a bacterial or fungal pathogen. 90 (35%) of these 254 infections were caused by O tsutsugamushi, R typhi/Rickettsia spp, or Leptospira spp. These pathogens were significantly more frequent than conventional bacterial infections (90/1051 [9%] vs 42/1051 [4%]; p<0·0001) by use of conservative diagnostic definitions. CNS infections had a high mortality (236/876 [27%]), with 18% (13/71) for R typhi/Rickettsia spp, O tsutsugamushi, and Leptospira spp combined, and 33% (13/39) for conventional bacterial infections (p=0·076). Interpretation: Our data suggest that R typhi/Rickettsia spp, O tsutsugamushi, and Leptospira spp infections are important causes of CNS infections in Laos. Antibiotics, such as tetracyclines, needed for the treatment of murine typhus and scrub typhus, are not routinely advised for empirical treatment of CNS infections. These severely neglected infections represent a potentially large proportion of treatable CNS disease burden across vast endemic areas and need more attention. Funding: Wellcome Trust UK

Management of Central Nervous System Infections, Vientiane, Laos, 2003–2011

During 2003–2011, we recruited 1,065 patients of all ages admitted to Mahosot Hospital (Vientiane, Laos) with suspected central nervous system (CNS) infection. Etiologies were laboratory confirmed for 42.3% of patients, who mostly had infections with emerging pathogens: viruses in 16.2% (mainly Japanese encephalitis virus [8.8%]); bacteria in 16.4% (including Orientia tsutsugamushi [2.9%], Leptospira spp. [2.3%], and Rickettsia spp. [2.3%]); and Cryptococcus spp. fungi in 6.6%. We observed no significant differences in distribution of clinical encephalitis and meningitis by bacterial or viral etiology. However, patients with bacterial CNS infection were more likely to have a history of diabetes than others. Death (26.3%) was associated with low Glasgow Coma Scale score, and the mortality rate was higher for patients with bacterial than viral infections. No clinical or laboratory variables could guide antibiotic selection. We conclude that high-dependency units and first-line treatment with ceftriaxone and doxycycline for suspected CNS infections could improve patient survival in Laos