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.

The Cholera Phone: Diarrheal Disease Surveillance by Mobile Phone in Bangladesh.

Existing methodologies to record diarrheal disease incidence in households have limitations due to a high-episode recall error outside a 48-hour window. Our objective was to use mobile phones for reporting diarrheal episodes in households to provide real-time incidence data with minimum resource consumption and low recall error. From June 2014 to June 2015, we enrolled 417 low-income households in Dhaka, Bangladesh, and asked them to report diarrheal episodes to a call center. A team of data collectors then visited persons reporting the episode to collect data. In addition, each month, the team conducted in-home surveys on diarrhea incidence for a preceding 48-hour period. The mobile phone surveillance reported an incidence of 0.16 cases per person-year (95% CI: 0.13-0.19), with 117 reported diarrhea cases, and the routine in-home survey detected an incidence of 0.33 cases per person-year (95% CI: 0.18-0.60), the incidence rate ratio was 2.11 (95% CI: 1.08-3.78). During focus group discussions, participants reported a lack in motivation to report diarrhea by phone because of the absence of provision of intervening treatment following reporting. Mobile phone technology can provide a unique tool for real-time disease reporting. The phone surveillance in this study reported a lower incidence of diarrhea than an in-home survey, possibly because of the absence of intervention and, therefore, a perceived lack of incentive to report. However, this study reports the untapped potential of mobile phones in monitoring infectious disease incidence in a low-income setting.

Transmission and Toxigenic Potential of Vibrio cholerae in Hilsha Fish (Tenualosa ilisha) for Human Consumption in Bangladesh.

Fish have been considered natural reservoirs of Vibrio cholerae, the deadly diarrheal pathogen. However, little is known about the role of fish in the transmission of V. cholerae from the Bay of Bengal to the households of rural and urban Bangladesh. This study analyzes the incidence and pathogenic potential of V. cholerae in Hilsha (Tenualosa ilisha), a commonly caught and consumed fish that exhibits a life cycle in both freshwater and marine environments in Bangladesh. During the period from October 2014 to October 2015, samples from the gills, recta, intestines, and scale swabs of a total of 48 fish were analyzed. The fish were collected both at local markets in the capital city Dhaka and directly from fishermen at the river. PCR analysis by targeting V. cholerae species-specific ompW gene revealed that 39 of 48 (81%) fish were positive in at least one of the sample types. Real-time PCR analysis demonstrated that the cholera-causing ctxA gene was detected in 20% (8 of 39) of V. cholerae-positive fish. A total of 158 V. cholerae isolates were obtained which were categorized into 35 genotypic groups. Altogether, 25 O1 and 133 non-O1/O139 strains were isolated, which were negative for the cholera toxin gene. Other pathogenic genes such as stn/sto, hlyA, chxA, SXT, rtxC, and HA-P were detected. The type three secretion system gene cluster (TTSS) was present in 18% (24 of 133) of non-O1/O139 isolates. The antibiotic susceptibility test revealed that the isolates conferred high resistance to sulfamethoxazole-trimethoprim and kanamycin. Both O1 and non-O1/O139 strains were able to accumulate fluid in rabbit ileal loops and caused distinctive cell death in HeLa cell. Multilocus sequence typing (MLST) showed clonal diversity among fish isolates with pandemic clones. Our data suggest a high prevalence of V. cholerae in Hilsha fish, which indicates that this fish could serve as a potential vehicle for V. cholerae transmission. Moreover, the indigenous V. cholerae strains isolated from Hilsha fish possess considerable virulence potential despite being quite diverse from current epidemic strains. This represents the first study of the population structure of V. cholerae associated with fish in Bangladesh.

A Prospective Hospital Study to Evaluate the Diagnostic Accuracy of Rapid Diagnostic Tests for the Early Detection of Leptospirosis in Laos.

Leptospirosis is a globally important cause of acute febrile illness, and a common cause of non-malarial fever in Asia, Africa, and Latin America. Simple rapid diagnostic tests (RDTs) are needed to enable health-care workers, particularly in low resource settings, to diagnose leptospirosis early and give timely targeted treatment. This study compared four commercially available RDTs to detect human IgM against Leptospira spp. in a head-to-head prospective evaluation in Mahosot Hospital, Lao PDR. Patients with an acute febrile illness consistent with leptospirosis (N = 695) were included in the study during the 2014 rainy season. Samples were tested with four RDTs: ("Test-it" [Life Assay, Cape Town, South Africa; N = 418]; "Leptorapide" [Linnodee, Ballyclare, Northern Ireland; N = 492]; "Dual Path Platform" [DPP] [Chembio, Medford, NY; N = 530]; and "SD-IgM" [Standard Diagnostics, Yongin, South Korea; N = 481]). Diagnostic performance characteristics were calculated and compared with a composite reference standard combining polymerase chain reaction (PCR) (rrs), microscopic agglutination tests (MATs), and culture. Of all patients investigated, 39/695 (5.6%) were positive by culture, PCR, or MAT. The sensitivity and specificity of the RDTs ranged greatly from 17.9% to 63.6% and 62.1% to 96.8%, respectively. None of the investigated RDTs reached a sensitivity or specificity of > 90% for detecting Leptospira infections on admission. In conclusion, our investigation highlights the challenges associated with Leptospira diagnostics, particularly in populations with multiple exposures. These findings emphasize the need for extensive prospective evaluations in multiple endemic settings to establish the value of rapid tools for diagnosing fevers to allow targeting of antibiotics.

Can Escherichia coli fly? The role of flies as transmitters of E. coli to food in an urban slum in Bangladesh.

OBJECTIVE: To investigate the transmission of faecal bacteria by flies to food under natural settings. METHODS: Over a period of 2 months, paired (exposed and non-exposed) containers with cooked rice were placed on the ground in kitchen areas in an urban slum area in Dhaka, Bangladesh, and the numbers of flies landing on the exposed rice were counted. Following exposure, the surface of the rice was microbiologically and molecularly analysed for the presence of Escherichia coli and genes of diarrhoeagenic E. coli and Shigella strains. RESULTS: Rice was at greater risk (P < 0·001) of being contaminated with E. coli if flies landed on the rice than if no flies landed on the rice (odds ratio 5·4 (P < 0·001, 95% CI: 2·5-11·7). Mean contamination in exposed rice samples (n = 60) was 3·1 × 103 CFU/g (95% CI: 2·2 × 103-4·0 × 103). Furthermore, for approximately half of the observed fly landings, the average CFU per fly landing was >0·6 × 103 CFU. Genes of diarrhoeagenic E. coli and Shigella species were detected in 39 of 60 (65%) of exposed rice samples. Two fly species were identified: the common housefly (Musca domestica) and the oriental latrine fly (Chrysomya megacephala). CONCLUSION: Flies may transmit large quantities of E. coli to food under field settings. The findings highlight the importance of implementing control measures to minimise exposure of food to flies to ensure food safety. Fly control measures should be considered for the prevention of diarrhoeal diseases caused by E. coli.

Development and Validation of a Novel Real-time Assay for the Detection and Quantification of Vibrio cholerae.

Vibrio cholerae O1 and O139 has been known for its ability to cause epidemics. These strains produce cholera toxin which is the main cause of secretory diarrhea. V. cholerae non-O1 and non-O139 strains are also capable of causing gastroenteritis as well as septicemia and peritonitis. It has been proven that virulence factors such as T6SS, hapA, rtxA, and hlyA are present in almost all V. cholerae strains. It is imperative that viable but non-culturable cells of V. cholerae are also detected since they are also known to cause diarrhea. Thus, the aim of this study was to develop an assay that detects all V. cholerae regardless of their serotype, culturable state, and virulence genes present, by targeting the species specific conserved ompW sequence. The developed assay meets these goals with 100% specificity and is capable of detecting as low as 5.46 copy number of V. cholerae. Detection is rapid since neither lengthy incubation period nor electrophoresis is required. The assay had excellent repeatability (CV%: 0.24-1.32) and remarkable reproducibility (CV%: 1.08-3.7). Amplification efficiencies in the 89-100% range were observed. The assay is more economical than Taqman-based multiplex real-time PCR assays. Compared to other real-time assays, the ompW assay is specific and sensitive, has better repeatability and reproducibility, and is more economical.

Measuring domestic water use: a systematic review of methodologies that measure unmetered water use in low-income settings.

OBJECTIVE: To present a systematic review of methods for measuring domestic water use in settings where water meters cannot be used. METHODS: We systematically searched EMBASE, PubMed, Water Intelligence Online, Water Engineering and Development Center, IEEExplore, Scielo, and Science Direct databases for articles that reported methodologies for measuring water use at the household level where water metering infrastructure was absent or incomplete. A narrative review explored similarities and differences between the included studies and provide recommendations for future research in water use. RESULTS: A total of 21 studies were included in the review. Methods ranged from single-day to 14-consecutive-day visits, and water use recall ranged from 12 h to 7 days. Data were collected using questionnaires, observations or both. Many studies only collected information on water that was carried into the household, and some failed to mention whether water was used outside the home. Water use in the selected studies was found to range from two to 113 l per capita per day. CONCLUSION: No standardised methods for measuring unmetered water use were found, which brings into question the validity and comparability of studies that have measured unmetered water use. In future studies, it will be essential to define all components that make up water use and determine how they will be measured. A pre-study that involves observations and direct measurements during water collection periods (these will have to be determined through questioning) should be used to determine optimal methods for obtaining water use information in a survey. Day-to-day and seasonal variation should be included. A study that investigates water use recall is warranted to further develop standardised methods to measure water use; in the meantime, water use recall should be limited to 24 h or fewer.

Survival of Vibrio cholerae O1 on fomites.

It is well established that the contamination sources of cholera causing bacteria, Vibrio cholerae, are water and food, but little is known about the transmission role of the fomites (surfaces that can carry pathogens) commonly used in households. In the absence of appropriate nutrients or growth conditions on fomites, bacteria have been known to assume a viable but non-culturable (VBNC) state after a given period of time. To investigate whether and when V. cholerae O1 assumes such a state, this study investigated the survival and viable quantification on a range of fomites such as paper, wood, glass, plastic, cloth and several types of metals under laboratory conditions. The fomites were inoculated with an outbreak strain of V. cholerae and its culturability was examined by drop plate count method at 30 min intervals for up to 6 h. For molecular detection, the viable/dead stain ethidium monoazide (EMA) which inhibits amplification of DNA from dead cells was used in combination with real-time polymerase chain reaction (EMA-qPCR) for direct quantitative analyses of viable V. cholerae at 2, 4, 6, 24 h and 7 day time intervals. Results showed that V. cholerae on glass and aluminum surfaces lost culturability within one hour after inoculation but remained culturable on cloth and wood for up to four hours. VBNC V. cholerae on dry fomite surfaces was detected and quantified by EMA-qPCR even 7 days after inoculation. In conclusion, the prolonged survival of V. cholerae on various household fomites may play vital role in cholera transmission and needs to be further investigated.

What is cholera? A preliminary study on caretakers' knowledge in Bangladesh.

BACKGROUND: Cholera has afflicted the Indian sub-continent for centuries, predominantly in West Bengal and modern-day Bangladesh. This preliminary study aims to understand the current level of knowledge of cholera in female Bangladeshi caretakers, which is important in the outcome of the disease and its spread. A pilot study was conducted among 85 women in Bangladesh using qualitative questionnaires to explore the ability of female caretakers in identifying cholera and its transmission. FINDINGS: The survey revealed that though all the female caretakers were aware of the term "cholera," nearly a third of the respondents did not associate diarrhea with cholera or mentioned symptoms that could not be caused by cholera (29 %). Approximately half of the respondents associated water with the cause of cholera (56 %) and only 8 % associated cholera with sanitation or hygiene. Shame and stigma (54 %) were more commonly described than death (47 %) as negative effects of cholera. CONCLUSIONS: The results from this study are suggestive of a need for reformulation of cholera and diarrhea communication. Messaging should be based on signs of dehydration, foregoing the use of medical terminology.

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.

Causes of Fever in Rural Southern Laos.

The etiology of fever in rural Lao People's Democratic Republic (Laos) has remained obscure until recently owing to the lack of laboratory facilities. We conducted a study to determine the causes of fever among 229 patients without malaria in Savannakhet Province, southern Laos; 52% had evidence of at least one diagnosis (45% with single and 7% with apparent multiple infections). Among patients with only one diagnosis, dengue (30.1%) was the most common, followed by leptospirosis (7.0%), Japanese encephalitis virus infection (3.5%), scrub typhus (2.6%), spotted fever group infection (0.9%), unspecified flavivirus infection (0.9%), and murine typhus (0.4%). We discuss the empirical treatment of fever in relation to these findings.

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.

Haemoglobin and red cell counts in leptospirosis patients infected with different serovars.

INTRODUCTION: The aim of the study was to compare haemoglobin and red cell counts between patients known to be infected with a range of leptospiral serovars. METHODS: The study retrospectively compared the haemoglobin and red cell count results from the first blood samples taken from 207 patients at presentation to a Queensland Health hospital. RESULTS: Significant differences were observed in haemoglobin and red cell counts in those infected with Leptospira interrogans serovars Szwajizak and Canicola when compared with most of the other serovars. CONCLUSIONS: These findings suggest that haemoglobin and red cell counts may be useful in differentiating leptospiral serovars in leptospirosis patients.

Haemoglobin and red cell counts in leptospirosis patients infected with different serovars

Introduction The aim of the study was to compare haemoglobin and red cell counts between patients known to be infected with a range of leptospiral serovars. Methods The study retrospectively compared the haemoglobin and red cell count results from the first blood samples taken from 207 patients at presentation to a Queensland Health hospital. Results Significant differences were observed in haemoglobin and red cell counts in those infected with Leptospira interrogans serovars Szwajizak and Canicola when compared with most of the other serovars. Conclusions These findings suggest that haemoglobin and red cell counts may be useful in differentiating leptospiral serovars in leptospirosis patients. .