Ladaporn Bodhidatta

Human Bocaviruses Are Highly Diverse, Dispersed, Recombination Prone, and Prevalent in Enteric Infections

Abstract A new species of parvovirus, tentatively named human bocavirus 4 (HBoV4), was genetically characterized. Among 641 feces samples obtained from children and adults, the most commonly detected bocavirus species were, in descending order, HBoV2, HBoV3, HBoV4, and HBoV1, with an HBoV2 prevalence of 21% and 26% in Nigerian and Tunisian children, respectively. HBoV3 or HBoV4 species were found in 12 of 192 patients with non-polio acute flaccid paralysis in Tunisia and Nigeria and 0 of 96 healthy Tunisian contacts (P= .01). Evidence of extensive recombination at the NP1 and VP1 gene boundary between and within bocavirus species was found. The high degree of genetic diversity seen among the human bocaviruses found in feces specimens, relative to the highly homogeneous HBoV1, suggest that this worldwide-distributed respiratory pathogen may have recently evolved from an enteric bocavirus after acquiring an expanded tropism favoring the respiratory tract. Elucidating the possible role of the newly identified enteric bocaviruses in human diseases, including acute flaccid paralysis and diarrhea, will require further epidemiological studies.

Pathogen-specific burdens of community diarrhoea in developing countries: a multisite birth cohort study (MAL-ED)

BACKGROUND Most studies of the causes of diarrhoea in low-income and middle-income countries have looked at severe disease in people presenting for care, and there are few estimates of pathogen-specific diarrhoea burdens in the community. METHODS We undertook a birth cohort study with not only intensive community surveillance for diarrhoea but also routine collection of non-diarrhoeal stools from eight sites in South America, Africa, and Asia. We enrolled children within 17 days of birth, and diarrhoeal episodes (defined as maternal report of three or more loose stools in 24 h, or one loose stool with visible blood) were identified through twice-weekly home visits by fieldworkers over a follow-up period of 24 months. Non-diarrhoeal stool specimens were also collected for surveillance for months 1-12, 15, 18, 21, and 24. Stools were analysed for a broad range of enteropathogens using culture, enzyme immunoassay, and PCR. We used the adjusted attributable fraction (AF) to estimate pathogen-specific burdens of diarrhoea. FINDINGS Between November 26, 2009, and February 25, 2014, we tested 7318 diarrhoeal and 24 310 non-diarrhoeal stools collected from 2145 children aged 0-24 months. Pathogen detection was common in non-diarrhoeal stools but was higher with diarrhoea. Norovirus GII (AF 5·2%, 95% CI 3·0-7·1), rotavirus (4·8%, 4·5-5·0), Campylobacter spp (3·5%, 0·4-6·3), astrovirus (2·7%, 2·2-3·1), and Cryptosporidium spp (2·0%, 1·3-2·6) exhibited the highest attributable burdens of diarrhoea in the first year of life. The major pathogens associated with diarrhoea in the second year of life were Campylobacter spp (7·9%, 3·1-12·1), norovirus GII (5·4%, 2·1-7·8), rotavirus (4·9%, 4·4-5·2), astrovirus (4·2%, 3·5-4·7), and Shigella spp (4·0%, 3·6-4·3). Rotavirus had the highest AF for sites without rotavirus vaccination and the fifth highest AF for sites with the vaccination. There was substantial variation in pathogens according to geography, diarrhoea severity, and season. Bloody diarrhoea was primarily associated with Campylobacter spp and Shigella spp, fever and vomiting with rotavirus, and vomiting with norovirus GII. INTERPRETATION There was substantial heterogeneity in pathogen-specific burdens of diarrhoea, with important determinants including age, geography, season, rotavirus vaccine usage, and symptoms. These findings suggest that although single-pathogen strategies have an important role in the reduction of the burden of severe diarrhoeal disease, the effect of such interventions on total diarrhoeal incidence at the community level might be limited.

Comparative antibiotic resistance of diarrheal pathogens from Vietnam and Thailand, 1996-1999.

Antimicrobial resistance rates for shigella, campylobacter, nontyphoidal salmonella, and enterotoxigenic Escherichia coli were compared for Vietnam and Thailand from 1996 to 1999. Resistance to trimethoprim-sulfamethoxazole, ampicillin, chloramphenicol, and tetracycline was common. Quinolone resistance remains low in both countries, except among campylobacter and salmonella organisms in Thailand. Nalidixic acid resistance among salmonellae has more than doubled since 1995 (to 21%) in Thailand but is not yet documented in Vietnam. Resistance to quinolones correlated with resistance to azithromycin in both campylobacter and salmonella in Thailand. This report describes the first identification of this correlation and its epidemiologic importance among clinical isolates. These data illustrate the growing magnitude of antibiotic resistance and important differences between countries in Southeast Asia.

High Variety of Known and New RNA and DNA Viruses of Diverse Origins in Untreated Sewage

ABSTRACT Deep sequencing of untreated sewage provides an opportunity to monitor enteric infections in large populations and for high-throughput viral discovery. A metagenomics analysis of purified viral particles in untreated sewage from the United States (San Francisco, CA), Nigeria (Maiduguri), Thailand (Bangkok), and Nepal (Kathmandu) revealed sequences related to 29 eukaryotic viral families infecting vertebrates, invertebrates, and plants (BLASTx E score, <10−4), including known pathogens (>90% protein identities) in numerous viral families infecting humans (Adenoviridae, Astroviridae, Caliciviridae, Hepeviridae, Parvoviridae, Picornaviridae, Picobirnaviridae, and Reoviridae), plants (Alphaflexiviridae, Betaflexiviridae, Partitiviridae, Sobemovirus, Secoviridae, Tombusviridae, Tymoviridae, Virgaviridae), and insects (Dicistroviridae, Nodaviridae, and Parvoviridae). The full and partial genomes of a novel kobuvirus, salivirus, and sapovirus are described. A novel astrovirus (casa astrovirus) basal to those infecting mammals and birds, potentially representing a third astrovirus genus, was partially characterized. Potential new genera and families of viruses distantly related to members of the single-stranded RNA picorna-like virus superfamily were genetically characterized and named Picalivirus, Secalivirus, Hepelivirus, Nedicistrovirus, Cadicistrovirus, and Niflavirus. Phylogenetic analysis placed these highly divergent genomes near the root of the picorna-like virus superfamily, with possible vertebrate, plant, or arthropod hosts inferred from nucleotide composition analysis. Circular DNA genomes distantly related to the plant-infecting Geminiviridae family were named Baminivirus, Nimivirus, and Niminivirus. These results highlight the utility of analyzing sewage to monitor shedding of viral pathogens and the high viral diversity found in this common pollutant and provide genetic information to facilitate future studies of these newly characterized viruses.

Development and assessment of molecular diagnostic tests for 15 enteropathogens causing childhood diarrhoea: a multicentre study

BACKGROUND Childhood diarrhoea can be caused by many pathogens that are difficult to assay in the laboratory. Molecular diagnostic techniques provide a uniform method to detect and quantify candidate enteropathogens. We aimed to develop and assess molecular tests for identification of enteropathogens and their association with disease. METHODS We developed and assessed molecular diagnostic tests for 15 enteropathogens across three platforms-PCR-Luminex, multiplex real-time PCR, and TaqMan array card-at five laboratories worldwide. We judged the analytical and clinical performance of these molecular techniques against comparator methods (bacterial culture, ELISA, and PCR) using 867 diarrhoeal and 619 non-diarrhoeal stool specimens. We also measured molecular quantities of pathogens to predict the association with diarrhoea, by univariate logistic regression analysis. FINDINGS The molecular tests showed very good analytical and clinical performance at all five laboratories. Comparator methods had limited sensitivity compared with the molecular techniques (20-85% depending on the target) but good specificity (median 97·3%, IQR 96·5-98·9; mean 95·2%, SD 9·1). Positive samples by comparator methods usually had higher molecular quantities of pathogens than did negative samples, across almost all platforms and for most pathogens (p<0·05). The odds ratio for diarrhoea at a given quantity (measured by quantification cycle, Cq) showed that for most pathogens associated with diarrhoea-including Campylobacter jejuni and Campylobacter coli, Cryptosporidium spp, enteropathogenic Escherichia coli, heat-stable enterotoxigenic E coli, rotavirus, Shigella spp and enteroinvasive E coli, and Vibrio cholerae-the strength of association with diarrhoea increased at higher pathogen loads. For example, Shigella spp at a Cq range of 15-20 had an odds ratio of 8·0 (p<0·0001), but at a Cq range of 25-30 the odds ratio fell to 1·7 (p=0·043). INTERPRETATION Molecular diagnostic tests can be implemented successfully and with fidelity across laboratories around the world. In the case of diarrhoea, these techniques can detect pathogens with high sensitivity and ascribe diarrhoeal associations based on quantification, including in mixed infections, providing rich and unprecedented measurements of infectious causes. FUNDING Bill & Melinda Gates Foundation Next Generation Molecular Diagnostics Project.

Traveler's Diarrhea in Thailand: Randomized, Double-Blind Trial Comparing Single-Dose and 3-Day Azithromycin-Based Regimens with a 3-Day Levofloxacin Regimen

BACKGROUND Travelers diarrhea in Thailand is frequently caused by Campylobacter jejuni. Rates of fluoroquinolone (FQ) resistance in Campylobacter organisms have exceeded 85% in recent years, and reduced fluoroquinolone efficacy has been observed. METHODS Azithromycin regimens were evaluated in a randomized, double-blind trial of azithromycin, given as a single 1-g dose or a 3-day regimen (500 mg daily), versus a 3-day regimen of levofloxacin (500 mg daily) in military field clinics in Thailand. Outcomes included clinical end points (time to the last unformed stool [TLUS] and cure rates) and microbiological end points (pathogen eradication). RESULTS A total of 156 patients with acute diarrhea were enrolled in the trial. Campylobacter organisms predominated (in 64% of patients), with levofloxacin resistance noted in 50% of Campylobacter organisms and with no azithromycin resistance noted. The cure rate at 72 h after treatment initiation was highest (96%) with single-dose azithromycin, compared with the cure rates of 85% noted with 3-day azithromycin and 71% noted with levofloxacin (P=.002). Single-dose azithromycin was also associated with the shortest median TLUS (35 h; P=.03, by log-rank test). Levofloxacins efficacy was inferior to azithromycins efficacy, except in patients with no pathogen identified during the first 24 h of treatment or in patients with levofloxacin-susceptible Campylobacter isolates, in whom it appeared to be equal to azithromycin. The rate of microbiological eradication was significantly better with azithromycin-based regimens (96%-100%), compared with levofloxacin (38%) (P=.001); however, this finding was poorly correlated with clinical outcome. A higher rate of posttreatment nausea in the 30 min after receipt of the first dose (14% vs. <6%; P=.06) was observed as a mild, self-limited complaint associated with single-dose azithromycin. CONCLUSIONS Single-dose azithromycin is recommended for empirical therapy of travelers diarrhea acquired in Thailand and is a reasonable first-line option for empirical management in general.

Ciprofloxacin and loperamide in the treatment of bacillary dysentery.

Antimotility drugs are not recommended in the treatment of dysentery. In guinea pigs, the motility of the small intestine is an important defense mechanism. Eight of 10 starved guinea pigs that were experimentally infected with Shigella and given opium died, whereas 10 similarly infected guinea pigs not given opium survived [1]. In a study of diphenoxylate-atropine (Lomotil, Searle and Company, Chicago, Illinois) used alone or in combination with oxolinic acid to treat experimental shigellosis in 25 volunteers [2], the authors concluded that drugs that reduced intestinal motility should not be used to treat invasive diarrhea. The report acknowledged that the results were inconclusive and suggested that larger trials should be done in naturally occurring illness to document the frequency and severity of prolongation of fever and pathogen carriage when patients are treated with Lomotil or paregoric [2]. Loperamide, a synthetic antidiarrheal agent, has not been studied in large numbers of patients with dysentery. Although it does not contain atropine (as does Lomotil), its primary action is to slow intestinal motility. In studies designed to look at nondysenteric diarrhea, loperamide has recently been used, either alone or in combination with antibiotics (cotrimazole or ciprofloxacin), to treat more than 25 adults with Shigella infections [3-7]. Although loperamide did not deliver a definable therapeutic advantage, the drug appeared to be safe in treating patients infected with Shigella. Because loperamide is frequently used in the treatment of diarrhea, which is often due to Shigella, a double-blind, placebo-controlled, randomized clinical trial of loperamide in the treatment of bacillary dysentery (primarily due to Shigella) was done. Methods Study Design Between 28 November 1990 and 29 February 1992, we studied patients with dysentery at Bamrasnaradura Hospital, Nonthaburi, Thailand. Dysentery was defined as three or more loose stools containing blood or mucus associated with at least one of the following symptoms: abdominal cramps, nausea, vomiting, or temperature greater than 38 C. Patients entered in the study had symptoms for fewer than 60 hours. Patients were excluded if they had received antimotility drugs or antibiotics in the previous 7 days, were pregnant, were immunosuppressed, or could not be followed for the next 10 days. Adults admitted to the Bamrasnaradura Hospital who met the study criteria and gave consent were treated with 500 mg of ciprofloxacin (Cipro, Miles Pharmaceuticals, West Haven, Connecticut) twice daily for 3 days. Patients were sequentially assigned a study number and an associated vial containing 16 caplets of loperamide (Imodium, McNeil Consumer Products Company, Fort Washington, Pennsylvania) or identical placebo. Numbered vials were randomized before the beginning of the study using a computer-generated random number table. Nurses, physicians, investigators, and patients were blinded to treatment regimen throughout the study. Patients received 4 mg (two caplets) of loperamide as an initial dose under the observation of a nurse. Patients were instructed to take one caplet after every loose stool (as many as eight caplets per day). Age, duration of diarrhea in hours, number of previous unformed stools, previous medication, and history of symptoms were recorded. Patients were examined by a physician, and stool specimens were collected at study entry. Patients were interviewed daily for 3 days (or until diarrheal symptoms had resolved) and again 10 days later. Symptoms, the time of the last unformed stool, and the number of unformed stools occurring during the previous 24 hours were recorded. Stools (or rectal swabs) were collected at the time of each interview. Laboratory Studies Stools were examined microscopically for fecal leukocytes and erythrocytes after staining with methylene blue [8] and were cultured promptly on MacConkey and Hektoen agar before and after inoculation in Selenite F broth (Difco, Detroit, Michigan). Bacterial, viral, and intestinal protozoa were identified as previously described [9, 10]. Up to 10 lactose-fermenting and up to 10 nonlactose-fermenting Escherichia coli, as identified on a MacConkey plate, were saved on Dorset egg yolk media slants. Escherichia coli isolates were tested within 1 month of isolation for heat-labile and heat-stable toxin production in the Y-1 adrenal cell [11] and suckling mouse [12] assays. Escherichia coli isolates were also fixed on Whatman 541 filters (Millipore Corporation, Bedford, Massachusetts) as described by Maas [13] and were tested for genes coding for Shiga-like toxin I and II as well as those with the 17-kilobase EcoRI fragment of pWR100 [14]. Isolates that hybridized with the 17-kilobase EcoRI probe were speciated and tested using the Sereny test [15]. Shigella, enteroinvasive E. coli, and enterotoxigenic E. coli as well as Aeromonas, Plesiomonas, and Vibrio species were tested for susceptibility to ampicillin, chloramphenicol, ciprofloxacin, nalidixic acid, trimethoprim-sulfamethoxazole, and tetracycline by disc diffusion [16] using CM471 agar (Oxoid Ltd., Basingstoke, United Kingdom). Enteroinvasive and enterotoxigenic E. coli were serotyped as described by Orskov and Orskov [17]. Intestinal parasites were identified by direct microscopy of saline suspension of stools after formalin-ether concentration, polyvinyl alcohol fixation of stools, and trichrome staining [18]. Cryptosporidium was identified microscopically with a modified dimethyl-sulfoxide method [19]. Rotavirus was identified using a monoclonal enzyme-linked immunosorbent assay (Pathfinder, Kallsted Laboratories, Austin, Texas) [20]. Data Analysis All investigators, nurses, and laboratory personnel were blinded to treatment group until all end points were determined at the end of the study. Data were entered from data collection forms and laboratory records into a computer database. Patients not followed until the resolution of diarrhea and those found not to have met the enrollment criteria were excluded from the analysis before unblinding. Infections with enteroinvasive E. coli or Shigella were evaluated together because of the similar pathogenicity of these organisms [21]. Statistical comparisons were made using Epi Info software [22]. A P value of less than 0.05 was considered statistically significant. The sample size necessary to show a difference of 24 hours in duration of diarrhea (80% power, P < 0.05) was 66 patients with Shigella or enteroinvasive E. coli. We planned to conduct this study over a 1-year period or until 66 patients with Shigella or enteroinvasive E. coli infections were entered. Data analysis included a chi-square with Yates correction (or two-tailed Fisher exact test if an expected cell was less than five) for the detection of proportional differences between the two treatment groups; the Student t-test for the comparison of mean ages and weights; and the Mann-Whitney-Wilcoxon test for the evaluation of non-normally distributed outcome variables (duration of diarrhea and number of diarrheal stools). Confidence intervals (CIs) of 95% are given where appropriate. Results From November 1990 to February 1992, patients with dysentery were screened by a nurse each morning, Monday through Friday. Ninety-two adults met the enrollment criteria. Two patients refused to submit additional specimens, one vomited his initial medication, and one could not be located after discharge from the hospital. Our final sample included 88 analyzable patients with dysentery. Forty-two patients received loperamide and 46 received placebo. Characteristics of the evaluable patients are shown in Table 1. Patients in the loperamide and placebo groups did not differ significantly in age, weight, sex, fecal leukocytes or erythrocytes, proportion who had fever at enrollment, duration of diarrhea before enrollment, or number of diarrheal stools during the 24 hours immediately preceding enrollment. Shigella species were isolated from 44 (50%) and enteroinvasive E. coli from 3 (3%) study participants (Table 2). The patients in whom Shigella or enteroinvasive E. coli were isolated had a lower mean weight (53 compared with 58 kg) and had a longer duration of diarrhea before enrollment (26 compared with 8 hours) than did the patients in whom these organisms were not cultured. Severity of illness was greater in the Shigella-enteroinvasive E. coli group, which had a higher proportion of volunteers with fecal leukocytes (87% compared with 46%), fecal erythrocytes (94% compared with 34%), and fever at enrollment (47% compared with 22%) than did other patients. The median number of stools passed in the 24 hours before enrollment was similar in the two groups (10 and 8 stools, respectively). Table 1. Patient Characteristics at Study Entry* Table 2. Enteric Pathogens Identified in 88 Patients with Dysentery Receiving Ciprofloxacin and Loperamide or Placebo* After treatment, the number of diarrheal stools was fewer for patients with dysentery who received loperamide (median, 4.5 and 7.0 stools, respectively; P = 0.030). This difference was caused by patients infected with Shigella or enteroinvasive E. coli (P = 0.016) (Table 3). In addition, the duration of diarrhea was less in patients infected with Shigella or enteroinvasive E. coli who received loperamide (P = 0.028) (Table 3). During the observation period after the start of treatment, the percentage of patients infected with Shigella or enteroinvasive E. coli receiving loperamide compared with placebo did not differ statistically in the following parameters: abdominal cramps (21% compared with 27%, P = 0.282), nausea (16% compared with 18%, P = 0.903), vomiting (16% compared with 19%, P = 0.770), or constipation (5% compared with 0%, P = 0.447). An oral temperature greater than 38 C was not found in any participant for more than 1 day after enrollment. Shigella or enteroinvasive E. coli were isolated 1 day after begin

Etiology of diarrhea in young children and patterns of antibiotic resistance in Cambodia.

Background: Little is known about diarrhea etiology and antibiotic resistance in developing countries where diarrhea is a major public health problem. Methods: To describe diarrhea etiology and antibiotic resistance patterns in Cambodia, 600 children aged 3 months to 5 years with acute diarrhea (cases) and 578 children without diarrhea (controls) were enrolled from a hospital in Phnom Penh. Stool samples were collected, and pathogens and antibiotic resistance patterns were described. Results: The most frequently isolated pathogens in these cases were enteroaggregative Escherichia coli (20%) and rotavirus (26%). Enterotoxigenic E. coli, enteroaggregative E. coli, Shigella, Aeromonas, rotavirus, and adenovirus were statistically significantly associated with diarrhea. Among cases, vomiting was associated with viral infections, whereas bloody stool was associated with Shigella. Enterotoxigenic E. coli isolates were highly resistant to ampicillin, sulfonamides, and tetracycline. Approximately 50% of Campylobacter coli and 30% of Campylobacter jejuni isolates were resistant to nalidixic acid and ciprofloxacin. Over 33% of Salmonella isolates were resistant to ampicillin and tetracycline, and almost 100% of Shigella isolates were resistant to trimethoprim/sulfamethoxazole. Conclusions: These data on the etiology of diarrhea and antibiotic resistance patterns in Cambodia will have significant effect on local public health policies and on local resource prioritization practices.

Comparison of novel MLB-clade, VA-clade and classic human astroviruses highlights constrained evolution of the classic human astrovirus nonstructural genes

Eight serotypes of human astroviruses (the classic human astroviruses) are causative agents of diarrhea. Recently, five additional astroviruses belonging to two distinct clades have been described in human stool, including astroviruses MLB1, MLB2, VA1, VA2 and VA3. We report the discovery in human stool of two novel astroviruses, astroviruses MLB3 and VA4. The complete genomes of these two viruses and the previously described astroviruses VA2 and VA3 were sequenced, affording seven complete genomes from the MLB and VA clades for comparative analysis to the classic human astroviruses. Comparison of the genetic distance, number of synonymous mutations per synonymous site (dS), number of non-synonymous mutations per non-synonymous site (dN) and the dN/dS ratio in the protease, polymerase and capsid of the classic human, MLB and VA clades suggests that the protease and polymerase of the classic human astroviruses are under distinct selective pressure.

Microbiologic Methods Utilized in the MAL-ED Cohort Study

A central hypothesis of The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) study is that enteropathogens contribute to growth faltering. To examine this question, the MAL-ED network of investigators set out to achieve 3 goals: (1) develop harmonized protocols to test for a diverse range of enteropathogens, (2) provide quality-assured and comparable results from 8 global sites, and (3) achieve maximum laboratory throughput and minimum cost. This paper describes the rationale for the microbiologic assays chosen and methodologies used to accomplish the 3 goals.