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Antimicrobial resistance is a major issue in the Shigellae, particularly as a specific multidrug-resistant (MDR) lineage of Shigella sonnei (lineage III) is becoming globally dominant. Ciprofloxacin is a recommended treatment for Shigella infections. However, ciprofloxacin-resistant S. sonnei are being increasingly isolated in Asia and sporadically reported on other continents. We hypothesized that Asia is a primary hub for the recent international spread of ciprofloxacin-resistant S. sonnei. We performed whole-genome sequencing on a collection of 60 contemporaneous ciprofloxacin-resistant S. sonnei isolated in four countries within Asia (Vietnam, n = 11; Bhutan, n = 12; Thailand, n = 1; Cambodia, n = 1) and two outside of Asia (Australia, n = 19; Ireland, n = 16). We reconstructed the recent evolutionary history of these organisms and combined these data with their geographical location of isolation. Placing these sequences into a global phylogeny, we found that all ciprofloxacin-resistant S. sonnei formed a single clade within a Central Asian expansion of lineage III. Furthermore, our data show that resistance to ciprofloxacin within S. sonnei may be globally attributed to a single clonal emergence event, encompassing sequential gyrA-S83L, parC-S80I, and gyrA-D87G mutations. Geographical data predict that South Asia is the likely primary source of these organisms, which are being regularly exported across Asia and intercontinentally into Australia, the United States and Europe. Our analysis was limited by the number of S. sonnei sequences available from diverse geographical areas and time periods, and we cannot discount the potential existence of other unsampled reservoir populations of antimicrobial-resistant S. sonnei. This study suggests that a single clone, which is widespread in South Asia, is likely driving the current intercontinental surge of ciprofloxacin-resistant S. sonnei and is capable of establishing endemic transmission in new locations. Despite being limited in geographical scope, our work has major implications for understanding the international transfer of antimicrobial-resistant pathogens, with S. sonnei acting as a tractable model for studying how antimicrobial-resistant Gram-negative bacteria spread globally.

Shigella sonnei increasingly dominates the international epidemiological landscape of shigellosis. Treatment options for S. sonnei are dwindling due to resistance to several key antimicrobials, including the fluoroquinolones. Here we analyse nearly 400  S. sonnei whole genome sequences from both endemic and non-endemic regions to delineate the evolutionary history of the recently emergent fluoroquinolone-resistant S. sonnei . We reaffirm that extant resistant organisms belong to a single clonal expansion event. Our results indicate that sequential accumulation of defining mutations ( gyrA -S83L, parC -S80I, and gyrA -D87G) led to the emergence of the fluoroquinolone-resistant S. sonnei population around 2007 in South Asia. This clone was then transmitted globally, resulting in establishments in Southeast Asia and Europe. Mutation analysis suggests that the clone became dominant through enhanced adaptation to oxidative stress. Experimental evolution reveals that under fluoroquinolone exposure in vitro, resistant S. sonnei develops further intolerance to the antimicrobial while the susceptible counterpart fails to attain complete resistance. Shigella sonnei is one of the main species causing shigellosis worldwide. Here the authors analyse nearly 400  S. sonnei genome sequences and carry out experimental evolution experiments to shed light into the evolutionary processes underlying the recent emergence of fluoroquinolone resistance in this pathogen.

Detection and quantification of enteropathogens in stool specimens is useful for diagnosing the cause of diarrhea but is technically challenging. Here we evaluate several important determinants of quantification: specimen collection, nucleic acid extraction, and extraction and amplification efficiency. First, we evaluate the molecular detection and quantification of pathogens in rectal swabs versus stool, using paired flocked rectal swabs and whole stool collected from 129 children hospitalized with diarrhea in Tanzania. Swabs generally yielded a higher quantification cycle (Cq) (average 29.7, standard deviation 3.5 vs. 25.3 ± 2.9 from stool, P<0.001) but were still able to detect 80% of pathogens with a Cq < 30 in stool. Second, a simplified total nucleic acid (TNA) extraction procedure was compared to separate DNA and RNA extractions and showed 92% (318/344) sensitivity and 98% (951/968) specificity, with no difference in Cq value for the positive results (ΔCq(DNA+RNA-TNA) = -0.01 ± 1.17, P = 0.972, N = 318). Third, we devised a quantification scheme that adjusts pathogen quantity to the specimen's extraction and amplification efficiency, and show that this better estimates the quantity of spiked specimens than the raw target Cq. In sum, these methods for enteropathogen quantification, stool sample collection, and nucleic acid extraction will be useful for laboratories studying enteric disease.

Individuals in the developing world live in conditions of intense exposure to enteric pathogens due to suboptimal water and sanitation. These environmental conditions lead to alterations in intestinal structure, function, and local and systemic immune activation that are collectively referred to as environmental enteropathy (EE). This condition, although poorly defined, is likely to be exacerbated by undernutrition as well as being responsible for permanent growth deficits acquired in early childhood, vaccine failure, and loss of human potential. This article addresses the underlying theoretical and analytical frameworks informing the methodology proposed by the Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study to define and quantify the burden of disease caused by EE within a multisite cohort. Additionally, we will discuss efforts to improve, standardize, and harmonize laboratory practices within the MAL-ED Network. These efforts will address current limitations in the understanding of EE and its burden on children in the developing world.

Scrub typhus is a vector-borne febrile illness caused by Orientia tsutsugamushi transmitted by the bite of Trombiculid mites. O. tsutsugamushi has a high genetic diversity and is increasingly recognized to have a wider global distribution than previously assumed. We evaluated the clinical outcomes and host immune responses of the two most relevant human pathogenic strains of O. tsutsugamushi; Karp (n = 4) and Gilliam (n = 4) in a time-course study over 80 days post infection (dpi) in a standardized scrub typhus non-human primate rhesus macaque model. We observed distinct features in clinical progression and immune response between the two strains; Gilliam-infected macaques developed more pronounced systemic infection characterized by an earlier onset of bacteremia, lymph node enlargement, eschar lesions and higher inflammatory markers during the acute phase of infection, when compared to the Karp strain. C-reactive protein (CRP) plasma levels, interferon gamma (IFN-γ, interleukin-1 receptor antagonist (IL-1ra), IL-15 serum concentrations, CRP/IL10- and IFN-γ/IL-10 ratios correlated positively with bacterial load in blood, implying activation of the innate immune response and preferential development of a T helper-type 1 immune response. The O. tsutsugamushi-specific immune memory responses in cells isolated from skin and lymph nodes at 80 dpi were more markedly elevated in the Gilliam-infected macaques than in the Karp-infected group. The comparative cytokine response dynamics of both strains revealed significant up-regulation of IFN-γ, tumor necrosis factor (TNF), IL-15, IL-6, IL-18, regulatory IL-1ra, IL-10, IL-8 and granulocyte-colony-stimulating factor (G-CSF). These data suggest that the clinical outcomes and host immune responses to scrub typhus could be associated with counter balancing effects of pro- and anti-inflammatory cytokine-mediated responses. Currently, no data on characterized time-course comparisons of O. tsutsugamushi strains regarding measures of disease severity and immune response is available. Our study provides evidence for the strain-specificity of host responses in scrub typhus, which supports our understanding of processes at the initial inoculation site (eschar), systemic disease progression, protective and/or pathogenic host immune mechanisms and cellular immune memory function. This study characterised an improved intradermal rhesus macaque challenge model for scrub typhus, whereby the Gilliam strain infection associated with higher disease severity in the rhesus macaque model than the previous Karp strain infection. Difficulties associated with inoculum quantitation for obligate-intracellular bacteria were overcome by using functional inoculum titrations in outbred mice. The Gilliam-based rhesus macaque model provides improved endpoint measurements and contributes towards the identification of correlates of protection for future vaccine development.

Campylobacter jejuni produces a polysaccharide capsule that is the major determinant of the Penner serotyping scheme. This passive slide agglutination typing system was developed in the early 1980's and was recognized for over two decades as the gold standard for C. jejuni typing. A preliminary multiplex PCR technique covering 17 serotypes was previously developed in order to replace this classic serotyping scheme. Here we report the completion of the multiplex PCR technology that is able to identify all the 47 Penner serotypes types known for C. jejuni. The number of capsule types represented within the 47 serotypes is 35. We have applied this method to a collection of 996 clinical isolates from Thailand, Cambodia and Nepal and were able to successfully determine capsule types of 98% of these.

The TaqMan® array card (TAC) was used to detect enteropathogens in stool samples from travelers' diarrhea cases and asymptomatic controls in a hospital-based surveillance in Thailand. Analyses were performed to determine pathogen-disease association to compare TAC and conventional results. Abstract Background Travelers' diarrhea (TD) is a common illness experienced by travelers from developed countries who visit developing countries. Recent questionnaire-based surveillance studies showed that approximately 6%-16% of travelers experienced TD while visiting Thailand; however, a majority of TD information was limited mainly to US military populations. Methods A TD surveillance study was conducted at Bumrungrad International Hospital in 2012-2014 in Bangkok, Thailand. Enteropathogens were identified using conventional methods and the TaqMan® array card (TAC), which uses real-time polymerase chain reaction for the simultaneous detection of multiple pathogens. Analyses to determine pathogen-disease and symptoms association were performed to elucidate the clinical relevance of each enteropathogen. Results TAC identified more pathogens per sample than conventional methods. Campylobacter spp. were the most prevalent, followed by the diarrheagenic Escherichia coli and norovirus GII. These agents had significant pathogen-disease associations as well as high attributable fractions among diarrheal cases. A wide range of pathogen loads for Campylobacter spp. was associated with TD, while heat-labile toxin enterotoxigenic Escherichia coli was associated with an increased pathogen load. Most cases were associated with inflammatory diarrhea, while Campylobacter spp. and Shigella spp. were associated with dysentery. Conclusions A pan-molecular diagnostic method such as TAC produces quantifiable and comparable results of all tested pathogens, thereby reducing the variability associated with multiple conventional methods. This allows better determination of the clinical relevance of each diarrhea etiologic agent, as well as their geographical relevance in Thailand.

The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) cohort study site in Nepal is located in the Bhaktapur municipality, 15 km east of Kathmandu, the capital city of Nepal. Bhaktapur, an ancient city famous for its traditional temples and buildings, is included on UNESCO's World Heritage List and is a major tourist attraction in Nepal. Nepal is a land-locked country located in South Asia between China and India with an area of 147 181 km 2 , ranging from sea-level plains to Mount Everest, the world's highest peak. The total population as of the 2011 census was 26.6 million, with an average annual population growth rate of 1.4. Nepal is one of the world's least developed countries and is ranked 157 of 186 in the 2013 Human Development Report; one-third of the Nepali population lives below the poverty line. The current under-5 mortality rate is 54 per 1000 live births, the infant mortality rate is 46 per 1000 live births, and the neonatal mortality rate is 33 per 1000 live births. Vaccine coverage for all Expanded Program on Immunization vaccines is >80%. Among children, the most common diseases contributing to significant morbidity and mortality are acute respiratory infection and dehydration from severe diarrhea. In this article, we report on the geographic, demographic, and socioeconomic features of the Bhaktapur MAL-ED site and describe the data that informed our cohort recruitment strategy.

Launched in 1974, the Expanded Program on Immunization (EPI) is estimated to prevent two-three million deaths annually from polio, diphtheria, tuberculosis, pertussis, measles, and tetanus. Additional lives could be saved through better understanding what influences adherence to the EPI schedule in specific settings. The Etiology, Risk Factors and Interactions of Enteric Infections and Malnutrition and the Consequences for Child Health and Development (MAL-ED) study followed cohorts in eight sites in South Asia, Africa, and South America and monitored vaccine receipt over the first two years of life for the children enrolled in the study. Vaccination histories were obtained monthly from vaccination cards, local clinic records and/or caregiver reports. Vaccination histories were compared against the prescribed EPI schedules for each country, and coverage rates were examined in relation to the timing of vaccination. The influence of socioeconomic factors on vaccine timing and coverage was also considered. Coverage rates for EPI vaccines varied between sites and by type of vaccine; overall, coverage was highest in the Nepal and Bangladesh sites and lowest in the Tanzania and Brazil sites. Bacillus Calmette-Guérin coverage was high across all sites, 87–100%, whereas measles vaccination rates ranged widely, 73–100%. Significant delays between the scheduled administration age and actual vaccination date were present in all sites, especially for measles vaccine where less than 40% were administered on schedule. A range of socioeconomic factors were significantly associated with vaccination status in study children but these results were largely site-specific. Our findings highlight the need to improve measles vaccination rates and reduce delayed vaccination to achieve EPI targets related to the establishment of herd immunity and reduction in disease transmission.