The Aetiologies and Impact of Fever in Pregnant Inpatients in Vientiane,Laos

Introduction

Laos has the highest maternal mortality ratio in mainland Southeast Asia and a high incidence of infectious diseases. Globally, malaria has been the pathogen most intensively investigated in relation to impact on pregnancy, but there has been relatively little research on the aetiology and impact of other diseases. We therefore aimed to determine the causes and impact of fever in pregnant women admitted to two central hospitals in Vientiane City, Lao PDR (Laos).

Materials and Methods

This hospital-based prospective study was conducted in Mahosot Hospital and the Mother and Child Hospital, Vientiane, between 2006 and 2010, with the aim to recruit 250 consenting pregnant women admitted with tympanic temperature ≥37.5°C. Primary outcome was the cause of fever and secondary outcomes were pregnancy outcomes. Specific investigations (culture, antigen, molecular and serological tests) were performed to investigate causes of fever. After discharge, all pregnant women were asked to return for review and convalescence serum on day 10–14 and were monitored until delivery.

Principle findings

250 pregnant women were recruited to this study between February 2006 and November 2010. Fifty percent were pregnant for the first time. Their median (range) gestational age on admission was 24 (4–43) weeks. The median (range) tympanic admission temperature was 38.5°C (37.5–40.5°C). Fifteen percent of patients stated that they had taken antibiotics before admission. Headache, myalgia, back pain and arthralgia were described by >60% of patients and 149 (60%) were given a laboratory diagnosis. Of those with confirmed diagnoses, 132 (53%) had a single disease and 17 (7%) had apparent mixed diseases. Among those who had a single disease, dengue fever was the most common diagnosis, followed by pyelonephritis, scrub typhus, murine typhus and typhoid. Patients were also diagnosed with tuberculosis, appendicitis, Staphylococcus aureus septicemia, leptospirosis, Japanese encephalitis virus infection and Plasmodium falciparum malaria. Severe consequences, including maternal death, miscarriage, stillbirth, low birth weight and preterm birth, were found among 28 (78%) mothers with dengue fever, rickettsioses and typhoid.

Conclusion

Fevers other than malaria, such as dengue, pyelonephritis, rickettsioses and typhoid are common causes of fever during pregnancy in the Asian tropics. Further investigations of their impact in the community on maternal death, fetal loss, vertical transmission, low birth weight and preterm birth are needed.     

Randomized soil survey of the distribution of Burkholderia pseudomallei in rice fields in Laos

Melioidosis is a major cause of morbidity and mortality in Southeast Asia, where the causative organism (Burkholderia pseudomallei) is present in the soil. In the Lao People's Democratic Republic (Laos), B. pseudomallei is a significant cause of sepsis around the capital, Vientiane, and has been isolated in soil near the city, adjacent to the Mekong River. We explored whether B. pseudomallei occurs in Lao soil distant from the Mekong River, drawing three axes across northwest, northeast, and southern Laos to create nine sampling areas in six provinces. Within each sampling area, a random rice field site containing a grid of 100 sampling points each 5 m apart was selected. Soil was obtained from a depth of 30 cm and cultured for B. pseudomallei. Four of nine sites (44%) were positive for B. pseudomallei, including all three sites in Saravane Province, southern Laos. The highest isolation frequency was in east Saravane, where 94% of soil samples were B. pseudomallei positive with a geometric mean concentration of 464 CFU/g soil (95% confidence interval, 372 to 579 CFU/g soil; range, 25 to 10,850 CFU/g soil). At one site in northwest Laos (Luangnamtha), only one sample (1%) was positive for B. pseudomallei, at a concentration of 80 CFU/g soil. Therefore, B. pseudomallei occurs in Lao soils beyond the immediate vicinity of the Mekong River, alerting physicians to the likelihood of melioidosis in these areas. Further studies are needed to investigate potential climatic, soil, and biological determinants of this heterogeneity.     

Orientia tsutsugamushi in human scrub typhus eschars shows tropism for dendritic cells and monocytes rather than endothelium

Scrub typhus is a common and underdiagnosed cause of febrile illness in Southeast Asia, caused by infection with Orientia tsutsugamushi. Inoculation of the organism at a cutaneous mite bite site commonly results in formation of a localized pathological skin reaction termed an eschar. The site of development of the obligate intracellular bacteria within the eschar and the mechanisms of dissemination to cause systemic infection are unclear. Previous postmortem and in vitro reports demonstrated infection of endothelial cells, but recent pathophysiological investigations of typhus patients using surrogate markers of endothelial cell and leucocyte activation indicated a more prevalent host leucocyte than endothelial cell response in vivo. We therefore examined eschar skin biopsies from patients with scrub typhus to determine and characterize the phenotypes of host cells in vivo with intracellular infection by O. tsutsugamushi, using histology, immunohistochemistry, double immunofluorescence confocal laser scanning microscopy and electron microscopy. Immunophenotyping of host leucocytes infected with O. tsutsugamushi showed a tropism for host monocytes and dendritic cells, which were spatially related to different histological zones of the eschar. Infected leucocyte subsets were characterized by expression of HLADR+, with an "inflammatory" monocyte phenotype of CD14/LSP-1/CD68 positive or dendritic cell phenotype of CD1a/DCSIGN/S100/FXIIIa and CD163 positive staining, or occasional CD3 positive T-cells. Endothelial cell infection was rare, and histology did not indicate a widespread inflammatory vasculitis as the cause of the eschar. Infection of dendritic cells and activated inflammatory monocytes offers a potential route for dissemination of O. tsutsugamushi from the initial eschar site. This newly described cellular tropism for O. tsutsugamushi may influence its interaction with local host immune responses.     

An Epidemic of Dengue-1 in a Remote Village in Rural Laos

In the Lao PDR (Laos), urban dengue is an increasingly recognised public health problem. We describe a dengue-1 virus outbreak in a rural northwestern Lao forest village during the cool season of 2008. The isolated strain was genotypically “endemic” and not “sylvatic,” belonging to the genotype 1, Asia 3 clade. Phylogenetic analyses of 37 other dengue-1 sequences from diverse areas of Laos between 2007 and 2010 showed that the geographic distribution of some strains remained focal overtime while others were dispersed throughout the country. Evidence that dengue viruses have broad circulation in the region, crossing country borders, was also obtained. Whether the outbreak arose from dengue importation from an urban centre into a dengue-naïve community or crossed into the village from a forest cycle is unknown. More epidemiological and entomological investigations are required to understand dengue epidemiology and the importance of rural and forest dengue dynamics in Laos.     

The Diversity and Geographical Structure of Orientia tsutsugamushi Strains from Scrub Typhus Patients in Laos

Orientia tsutsugamushi is the causative agent of scrub typhus, a disease transmitted by Leptotrombidium mites which is responsible for a severe and under-reported public health burden throughout Southeast Asia. Here we use multilocus sequence typing (MLST) to characterize 74 clinical isolates from three geographic locations in the Lao PDR (Laos), and compare them with isolates described from Udon Thani, northeast Thailand. The data confirm high levels of diversity and recombination within the natural O. tsutsugamushi population, and a rate of mixed infection of ~8%. We compared the relationships and geographical structuring of the strains and populations using allele based approaches (eBURST), phylogenetic approaches, and by calculating F-statistics (F_ST). These analyses all point towards low levels of population differentiation between isolates from Vientiane and Udon Thani, cities which straddle the Mekong River which defines the Lao/Thai border, but with a very distinct population in Salavan, southern Laos. These data highlight how land use, as well as the movement of hosts and vectors, may impact on the epidemiology of zoonotic infections.     

Genetic history of hepatitis C virus in East Asia

The hepatitis C virus (HCV), which currently infects an estimated 3% of people worldwide, has been present in some human populations for several centuries, notably HCV genotypes 1 and 2 in West Africa and genotype 6 in Southeast Asia. Here we use newly developed methods of sequence analysis to conduct the first comprehensive investigation of the epidemic and evolutionary history of HCV in Asia. Our analysis includes new HCV core (n = 16) and NS5B (n = 14) gene sequences, obtained from serum samples of jaundiced patients from Laos. These exceptionally diverse isolates were analyzed in conjunction with all available reference strains using phylogenetic and Bayesian coalescent methods. We performed statistical tests of phylogeographic structure and applied a recently developed “relaxed molecular clock” approach to HCV for the first time, which indicated an unexpectedly high degree of rate variation. Our results reveal a >1,000-year-long development of genotype 6 in Asia, characterized by substantial phylogeographic structure and two distinct phases of epidemic history, before and during the 20th century. We conclude that HCV lineages representing preexisting and spatially restricted strains were involved in multiple, independent local epidemics during the 20th century. Our analysis explains the generation and maintenance of HCV diversity in Asia and could provide a template for further investigations of HCV spread in other regions.     

Contrasting spatial distribution and risk factors for past infection with scrub typhus and murine typhus in Vientiane City, Lao PDR

Background

The aetiological diagnostic of fevers in Laos remains difficult due to limited laboratory diagnostic facilities. However, it has recently become apparent that both scrub and murine typhus are common causes of previous undiagnosed fever. Epidemiological data suggests that scrub typhus would be more common in rural areas and murine typhus in urban areas, but there is very little recent information on factors involved in scrub and murine typhus transmission, especially where they are sympatric - as is the case in Vientiane, the capital of the Lao PDR.

Methodology and Principal Findings

We therefore determined the frequency of IgG seropositivity against scrub typhus (Orientia tsutsugamushi) and murine typhus (Rickettsia typhi), as indices of prior exposure to these pathogens, in randomly selected adults in urban and peri-urban Vientiane City (n = 2,002, ≥35 years). Anti-scrub and murine typhus IgG were detected by ELISA assays using filter paper elutes. We validated the accuracy of ELISA of these elutes against ELISA using serum samples. The overall prevalence of scrub and murine typhus IgG antibodies was 20.3% and 20.6%, respectively. Scrub typhus seropositivity was significantly higher among adults living in the periphery (28.4%) than in the central zone (13.1%) of Vientiane. In contrast, seroprevalence of murine typhus IgG antibodies was significantly higher in the central zone (30.8%) as compared to the periphery (14.4%). In multivariate analysis, adults with a longer residence in Vientiane were at significant greater risk of past infection with murine typhus and at lower risk for scrub typhus. Those with no education, living on low incomes, living on plots of land with poor sanitary conditions, living in large households, and farmers were at higher risk of scrub typhus and those living in neighborhoods with high building density and close to markets were at greater risk for murine typhus and at lower risk of scrub typhus past infection.

Conclusions

This study underscores the intense circulation of both scrub and murine typhus in Vientiane city and underlines difference in spatial distribution and risk factors involved in the transmission of these diseases.