Antimicrobial Susceptibility Testing of Leptospira spp. in the Lao People's Democratic Republic Using Disk Diffusion.

Leptospirosis is a global zoonotic disease caused by pathogenic bacteria of the Leptospira genus, which are fastidious, slow-growing organisms. Antimicrobial susceptibility data are limited; traditionally, the organisms have not been culturable on solid media. The recent development of Leptospira Vanaporn Wuthiekanun (LVW) agar, which facilitates rapid growth of Leptospira spp., provides the opportunity for antimicrobial susceptibility testing. Eighty-three Leptospira spp. clinical isolates originating from patients in Laos between 2006 and 2016 were tested against six antimicrobials (azithromycin, ceftriaxone, ciprofloxacin, doxycycline, gentamicin, and penicillin G) using disk diffusion on LVW agar. Quality control was undertaken using American Type Culture Collection (ATCC) reference strains with known susceptibilities on both standard media and LVW agar. All Leptospira spp. isolates produced large zones of inhibition around each of the six antimicrobials. All zones were greater than 25 mm: gentamicin produced the smallest zones (median 35 mm; interquartile range 30 mm-37 mm) and azithromycin produced the largest zones (median 85 mm; interquartile range 85 mm-85 mm). Zones produced by non-leptospiral ATCC reference strains on LVW agar were within 2 mm of accepted strain-specific quality control range on standard media. Antimicrobial activity on LVW agar appears to be similar to that on standard media. As there are no published susceptibility guidelines for the Leptospira genus, zone interpretation was subjective. Leptospira Vanaporn Wuthiekanun agar enabled antimicrobial susceptibility testing of multiple Leptospira isolates on solid media; the large zone sizes observed suggest that resistance has not emerged to these six antimicrobials in Lao Leptospira spp

Causes of non-malarial fever in Laos: a prospective study

Background Because of reductions in the incidence of Plasmodium falciparum malaria in Laos, identifi cation of the causes of fever in people without malaria, and discussion of the best empirical treatment options, are urgently needed. We aimed to identify the causes of non-malarial acute fever in patients in rural Laos. Methods For this prospective study, we recruited 1938 febrile patients, between May, 2008, and December, 2010, at Luang Namtha provincial hospital in northwest Laos (n=1390), and between September, 2008, and December, 2010, at Salavan provincial hospital in southern Laos (n=548). Eligible participants were aged 5–49 years with fever (≥38°C) lasting 8 days or less and were eligible for malaria testing by national guidelines. Findings With conservative defi nitions of cause, we assigned 799 (41%) patients a diagnosis. With exclusion of infl uenza, the top fi ve diagnoses when only one aetiological agent per patient was identifi ed were dengue (156 [8%] of 1927 patients), scrub typhus (122 [7%] of 1871), Japanese encephalitis virus (112 [6%] of 1924), leptospirosis (109 [6%] of 1934), and bacteraemia (43 [2%] of 1938). 115 (32%) of 358 patients at Luang Namtha hospital tested infl uenza PCR-positive between June and December, 2010, of which infl uenza B was the most frequently detected strain (n=121 [87%]). Disease frequency diff ered signifi cantly between the two sites: Japanese encephalitis virus infection (p=0·04), typhoid (p=0·006), and leptospirosis (p=0·001) were more common at Luang Namtha, whereas dengue and malaria were more common at Salavan (all p<0·0001). With use of evidence from southeast Asia when possible, we estimated that azithromycin, doxycycline, ceftriaxone, and ofl oxacin would have had signifi cant effi cacy for 258 (13%), 240 (12%), 154 (8%), and 41 (2%) of patients, respectively. Interpretation Our fi ndings suggest that a wide range of treatable or preventable pathogens are implicated in nonmalarial febrile illness in Laos. Empirical treatment with doxycycline for patients with undiff erentiated fever and negative rapid diagnostic tests for malaria and dengue could be an appropriate strategy for rural health workers in Laos. Funding Wellcome Trust, WHO–Western Pacifi c Region, Foundation for Innovative New Diagnostics, US Centers for Disease Control and Prevention

An Extended Multilocus Sequence Typing (MLST) Scheme for Rapid Direct Typing of <i>Leptospira</i> from Clinical Samples

<div><p>Background</p><p>Rapid typing of <i>Leptospira</i> is currently impaired by requiring time consuming culture of leptospires. The objective of this study was to develop an assay that provides multilocus sequence typing (MLST) data direct from patient specimens while minimising costs for subsequent sequencing.</p><p>Methodology and Findings</p><p>An existing PCR based MLST scheme was modified by designing nested primers including anchors for facilitated subsequent sequencing. The assay was applied to various specimen types from patients diagnosed with leptospirosis between 2014 and 2015 in the United Kingdom (UK) and the Lao Peoples Democratic Republic (Lao PDR). Of 44 clinical samples (23 serum, 6 whole blood, 3 buffy coat, 12 urine) PCR positive for pathogenic <i>Leptospira</i> spp. at least one allele was amplified in 22 samples (50%) and used for phylogenetic inference. Full allelic profiles were obtained from ten specimens, representing all sample types (23%). No nonspecific amplicons were observed in any of the samples. Of twelve PCR positive urine specimens three gave full allelic profiles (25%) and two a partial profile. Phylogenetic analysis allowed for species assignment. The predominant species detected was <i>L</i>. <i>interrogans</i> (10/14 and 7/8 from UK and Lao PDR, respectively). All other species were detected in samples from only one country (Lao PDR: <i>L</i>. <i>borgpetersenii</i> [1/8]; UK: <i>L</i>. <i>kirschneri</i> [1/14], <i>L</i>. <i>santarosai</i> [1/14], <i>L</i>. <i>weilii</i> [2/14]).</p><p>Conclusion</p><p>Typing information of pathogenic <i>Leptospira</i> spp. was obtained directly from a variety of clinical samples using a modified MLST assay. This assay negates the need for time-consuming culture of <i>Leptospira</i> prior to typing and will be of use both in surveillance, as single alleles enable species determination, and outbreaks for the rapid identification of clusters.</p></div

Low Zika Virus Seroprevalence in Vientiane, Laos, 2003–2015

International audienceZika virus (ZIKV) has been presumed to be endemic in Southeast Asia (SEA), with a low rate of human infections. Although the first ZIKV evidence was obtained in the 1950s through serosurveys, the first laboratory-confirmed case was only detected in 2010 in Cambodia. The epidemiology of ZIKV in SEA remains uncertain because of the scarcity of available data. From 2016, subsequent to the large outbreaks in the Pacific and Latin America, several Asian countries started reporting increasing numbers of confirmed ZIKV patients, but no global epidemiological assessment is available to date. Here, with the aim of providing information on ZIKV circulation and population immunity, we conducted a seroprevalence study among blood donors in Vientiane, Laos. Sera from 359 asymptomatic consenting adult donors in 2003-2004 and 687 in 2015 were screened for anti-ZIKV IgG using NS1 ELISA assay (Euroimmun, Luebeck, Germany). Positive and equivocal samples were confirmed for anti-ZIKV-neutralizing antibodies by virus neutralization tests. Our findings suggest that ZIKV has been circulating in Vientiane over at least the last decade. Zika virus seroprevalence observed in the studied blood donors was low, 4.5% in 2003-2004 with an increase in 2015 to 9.9% (P = 0.002), possibly reflecting the increase of ZIKV incident cases reported over this period. We did not observe any significant difference in seroprevalence according to gender. With a low herd immunity in the Vientiane population, ZIKV represents a risk for future large-scale outbreaks. Implementation of a nationwide ZIKV surveillance network and epidemiological studies throughout the country is needed

Haemophilus influenzae serotype b seroprevalence in central Lao PDR before and after vaccine introduction

Introduction Vaccination has dramatically reduced invasive Haemophilus influenzae type b (Hib) disease worldwide. Hib vaccination was introduced in the Lao PDR in 2009, as part of the pentavalent vaccine. To contribute to the understanding of the epidemiology of Hib in Lao PDR and the protection levels before and after the introduction of the vaccination, we tested serum samples from existing cohorts of vaccine age-eligible children and unvaccinated adolescents for antibodies against Hib. Methods Serum samples from 296 adolescents born before vaccine introduction and from 1017 children under 5 years (vaccinated and unvaccinated) were tested for anti-Hib antibodies by ELISA. Bivariate analyses were performed to investigate factors associated with long-term protection. Results The vast majority of all participants showed evidence of short- (42.7%) or long-term (56.1%) protection against Hib. Almost all of the unvaccinated adolescents had antibody titers indicating short-term protection and almost half (45.6%) were long-term protected. Nearly all children (>99.0%) were at least short-term protected, even those that were unvaccinated or whose vaccination status was unknown. Among vaccinated children, participants vaccinated more than 1 or 2 years ago and with a mid-upper arm circumference z-score < -2 were less likely to be long-term protected. Discussion Nearly all adolescents born before the introduction of Hib vaccination in the Lao PDR had antibody titers corresponding to at least short-term protection, indicating a high burden of Hib disease at that time. After vaccine introduction, all but four children (>99%) showed at least short-term protection. Possible explanations for the proportion of protected, yet apparently unvaccinated children, may be past infections, cross-reacting antibodies or faulty vaccination documentation. Our results highlight the need for robust surveillance and reporting of invasive Hib disease to determine the burden of disease despite vaccination

Maximum likelihood phylogenetic tree based on concatenated MLST sequences.

<p>All species defining branches are fully supported by 500 bootstraps. Bar represents substitutions per site. Tips are labelled with <i>strain_ST</i> (WHO panel) or <i>sample ID_ST</i> (clinical samples) and coloured by species: marine = <i>L</i>. <i>interrogans</i>, red = <i>L</i>. <i>kirschneri</i>, green: <i>L</i>. <i>noguchii</i>, yellow: <i>L</i>. <i>santarosai</i>, pink: <i>L</i>. <i>weilii</i>, light blue: <i>L</i>. <i>borgpetersenii</i>. P = 2015, C = 2014, both UK; L = Lao PDR, 2014.</p

Results of nested MLST PCR for clinical specimens by sample type and full/ partial allelic profiles.

<p>Results of nested MLST PCR for clinical specimens by sample type and full/ partial allelic profiles.</p

MLST nested primers for all seven loci designed to accompany the MLST scheme by Boonsilp <i>et al</i>., 2013 [22].

<p>MLST nested primers for all seven loci designed to accompany the MLST scheme by Boonsilp <i>et al</i>., 2013 [<a href="http://www.plosntds.org/article/info:doi/10.1371/journal.pntd.0004996#pntd.0004996.ref022" target="_blank">22</a>].</p

Species assigned to clinical specimens based on phylogenetic analysis.

<p>Species assigned to clinical specimens based on phylogenetic analysis.</p

JEV MAC-ELISA results from DCS using CSF-substitutes, 903 filter paper and various protocols for sample spotting.

<p>JEV MAC-ELISA results from DCS using CSF-substitutes, 903 filter paper and various protocols for sample spotting.</p