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1.  Characterisation of Invasive Streptococcus pneumoniae Isolated from Cambodian Children between 2007 – 2012 
PLoS ONE  2016;11(7):e0159358.
Background
The 13-valent pneumococcal vaccine (PCV13) was introduced in Cambodia in January 2015. There are limited data concerning the common serotypes causing invasive pneumococcal disease (IPD). Knowledge of the circulating pneumococcal serotypes is important to monitor epidemiological changes before and after vaccine implementation.
Methods
All episodes of IPD defined by the isolation of Streptococcus pneumoniae from blood, cerebrospinal fluid or other sterile site in Cambodian children admitted to the Angkor Hospital for Children in Siem Reap, Northwestern Cambodia, between 1st January 2007 and 1st July 2012 were retrospectively studied. Streptococcus pneumoniae isolates that could be retrieved underwent phenotypic typing and whole genome sequencing.
Results
There were 90 Cambodian children hospitalized with IPD with a median (IQR) age of 2.3 years (0.9–6.2). The case fatality was 15.6% (95% CI 8–23). Of 50 Streptococcus pneumoniae isolates available for further testing, 46% were penicillin non-susceptible and 8% were ceftriaxone non-susceptible, 78% were cotrimoxazole resistant, 30% were erythromycin resistant and 30% chloramphenicol resistant. There were no significant changes in resistance levels over the five-year period. The most common serotypes were 1 (11/50; 22%), 23F (8/50; 16%), 14 (6/50; 12%), 5 (5/50; 10%) and 19A (3/50; 6%). Coverage by PCV7, PCV10 and PCV13 was 44%, 76% and 92% respectively. We identified novel multilocus sequence types and resistotypes using whole genome sequencing.
Conclusions
This study suggests IPD is an important disease in Cambodian children and can have a significant mortality. PCV13 coverage of the serotypes determined in studied strains was high and consistent with another recent study. The phenotypic resistance patterns observed were similar to other regional studies. The use of whole genome sequencing in the present study provides additional typing and resistance information together with the description of novel sequence types and resistotypes.
doi:10.1371/journal.pone.0159358
PMCID: PMC4957771  PMID: 27448096
2.  Molecular Characterization of Cryptosporidium Species and Giardia duodenalis from Symptomatic Cambodian Children 
PLoS Neglected Tropical Diseases  2016;10(7):e0004822.
Background
In a prospective study, 498 single faecal samples from children aged under 16 years attending an outpatient clinic in the Angkor Hospital for Children, northwest Cambodia, were examined for Cryptosporidium oocysts and Giardia cysts using microscopy and molecular assays.
Methodology/Principal Findings
Cryptosporidium oocysts were detected in 2.2% (11/498) of samples using microscopy and in 7.7% (38/498) with molecular tests. Giardia duodenalis cysts were detected in 18.9% (94/498) by microscopy and 27.7% (138/498) by molecular tests; 82% of the positive samples (by either method) were from children aged 1–10 years. Cryptosporidium hominis was the most common species of Cryptosporidium, detected in 13 (34.2%) samples, followed by Cryptosporidium meleagridis in 9 (23.7%), Cryptosporidium parvum in 8 (21.1%), Cryptosporidium canis in 5 (13.2%), and Cryptosporidium suis and Cryptosporidium ubiquitum in one sample each. Cryptosporidium hominis and C. parvum positive samples were subtyped by sequencing the GP60 gene: C. hominis IaA16R6 and C. parvum IIeA7G1 were the most abundant subtypes. Giardia duodenalis was typed using a multiplex real-time PCR targeting assemblages A and B. Assemblage B (106; 76.8% of all Giardia positive samples) was most common followed by A (12.3%) and mixed infections (5.1%). Risk factors associated with Cryptosporidium were malnutrition (AOR 9.63, 95% CI 1.67–55.46), chronic medical diagnoses (AOR 4.51, 95% CI 1.79–11.34) and the presence of birds in the household (AOR 2.99, 95% CI 1.16–7.73); specifically C. hominis (p = 0.03) and C. meleagridis (p<0.001) were associated with the presence of birds. The use of soap was protective against Giardia infection (OR 0.74, 95% CI 0.58–0.95).
Conclusions/Significance
This is the first report to describe the different Cryptosporidium species and subtypes and Giardia duodenalis assemblages in Cambodian children. The variety of Cryptosporidium species detected indicates both anthroponotic and zoonotic transmission in this population. Interventions to improve sanitation, increase hand washing after defecation and before preparing food and promote drinking boiled water may reduce the burden of these two parasites.
Author Summary
The parasites Cryptosporidium and Giardia frequently cause diarrhea in children worldwide. They can be transmitted from animals to humans and between humans, either through direct contact, from sewage, or by contamination of food and water. The importance of these infections in Cambodian children is unknown. Animals are an essential part of life in Southeast Asia with livestock as well as domestic animals living in close proximity to humans. We previously conducted a prospective study to determine the intestinal parasites causing disease in symptomatic children attending hospital in Siem Reap, Northwestern Cambodia. This study examines these two parasites in more detail using molecular methods which provide better detection and are more discriminating. We were able to determine the different”strains” of Cryptosporidium and Giardia and, along with risk factor data, concluded that most of the infections were spread between people or within households suggesting that public health programmes should concentrate on improved sanitation, community handwashing initiatives and better food and water hygiene practices.
doi:10.1371/journal.pntd.0004822
PMCID: PMC4936737  PMID: 27387755
3.  Clinical trials of artesunate plus sulfadoxine-pyrimethamine for Plasmodium falciparum malaria in Afghanistan: maintained efficacy a decade after introduction 
Malaria Journal  2016;15:121.
Background
Combination therapy with artesunate plus sulfadoxine-pyrimethamine (SP) was adopted as recommended treatment for Plasmodium falciparum infection in Afghanistan in 2003.
Methods
A series of prospective clinical studies examining the efficacy of artesunate plus sulfadoxine-pyrimethamine (AS + SP) against P. falciparum were undertaken in sentinel sites in Afghanistan from 2007 to 2014, accompanied by relevant molecular studies. The first study was a randomized trial of AS + SP versus dihydroartemisinin-piperaquine, while two subsequent studies were standard therapeutic efficacy studies of AS + SP.
Results
Three hundred and three patients were enrolled across four provinces in the north and east of the country. Curative efficacy was high in all the trials, with an adequate clinical and parasitological response (ACPR) of more than 95 % in all groups and trial stages. Genotyping for drug-resistance alleles at dhfr indicated fixation of the S108 N mutation and a prevalence of the C59R mutation of approximately 95 % across all sites. Other mutations in dhfr and dhps remained rare or absent entirely, although five isolates from the first trial carried the dhps triple mutant SGEGA haplotype. In the last study undertaken in 2012–2014 the K13 artemisinin resistance marker was examined; only two of 60 successfully sequenced samples carried a K13-propeller mutation.
Conclusions
These data confirm maintained efficacy 10 years after introduction of artesunate plus SP as combination treatment of P. falciparum in Afghanistan. The molecular data indicate that despite a substantial fall in incidence, resistance has not developed to artemisinins, or intensified to the ACT partner drug components.
Trial Registrationhttp://www.clinicaltrials.gov/ct NCT00682578, NCT01115439 and NCT01707199
doi:10.1186/s12936-016-1167-z
PMCID: PMC4766631  PMID: 26917051
Malaria; Falciparum; Afghanistan; Artesunate; Sulphadoxine; Pyrimethamine; Dihydrofolate reductase; Dihydropteroate synthase; Piperaquine; Kelch
4.  Utility of a Lateral Flow Immunoassay (LFI) to Detect Burkholderia pseudomallei in Soil Samples 
PLoS Neglected Tropical Diseases  2016;10(12):e0005204.
Background
Culture is the gold standard for the detection of environmental B. pseudomallei. In general, soil specimens are cultured in enrichment broth for 2 days, and then the culture broth is streaked on an agar plate and incubated further for 7 days. However, identifying B. pseudomallei on the agar plates among other soil microbes requires expertise and experience. Here, we evaluate a lateral flow immunoassay (LFI) developed to detect B. pseudomallei capsular polysaccharide (CPS) in clinical samples as a tool to detect B. pseudomallei in environmental samples.
Methodology/Principal Findings
First, we determined the limit of detection (LOD) of LFI for enrichment broth of the soil specimens. Soil specimens (10 grams/specimen) culture negative for B. pseudomallei were spiked with B. pseudomallei ranging from 10 to 105 CFU, and incubated in 10 ml of enrichment broth in air at 40°C. Then, on day 2, 4 and 7 of incubation, 50 μL of the upper layer of the broth were tested on the LFI, and colony counts to determine quantity of B. pseudomallei in the broth were performed. We found that all five soil specimens inoculated at 10 CFU were negative by LFI on day 2, but four of those five specimens were LFI positive on day 7. The LOD of the LFI was estimated to be roughly 3.8x106 CFU/ml, and culture broth on day 7 was selected as the optimal sample for LFI testing. Second, we evaluated the utility of the LFI by testing 105 soil samples from Northeast Thailand. All samples were also tested by standard culture and quantitative PCR (qPCR) targeting orf2. Of 105 soil samples, 35 (33%) were LFI positive, 25 (24%) were culture positive for B. pseudomallei, and 79 (75%) were qPCR positive. Of 11 LFI positive but standard culture negative specimens, six were confirmed by having the enrichment broth on day 7 culture positive for B. pseudomallei, and an additional three by qPCR. The LFI had 97% (30/31) sensitivity to detect soil specimens culture positive for B. pseudomallei.
Conclusions/Significance
The LFI can be used to detect B. pseudomallei in soil samples, and to select which samples should be sent to reference laboratories or proceed further for bacterial isolation and confirmation. This could considerably decrease laboratory workload and assist the development of a risk map for melioidosis in resource-limited settings.
Author Summary
Burkholderia pseudomallei is an environmental Gram-negative bacillus and the causative agent of melioidosis. Culture and PCR assays are standard diagnostic tools used to detect B. pseudomallei in the environment. However, those tests require experienced microbiologists and are regularly conducted only in a few research laboratories worldwide. In this study, we demonstrated that the prototype lateral flow immunoassay (LFI) developed to detect B. pseudomallei capsular polysaccharide (CPS) in clinical samples could be used to detect B. pseudomallei in environmental samples. We found that the LFI can be used to detect B. pseudomallei in experimentally spiked soil specimens. Next, we evaluated the sensitivity of LFI using 105 soil samples collected in Northeast Thailand. We found that the LFI had high sensitivity to detect B. pseudomallei in the soil. We propose that the LFI could be used to detect environmental B. pseudomallei in resource-limited settings. Soil samples positive for LFI could be sent to reference laboratories for confirmation with culture or molecular methods. The use of LFI could assist in the development of a global risk map for melioidosis.
doi:10.1371/journal.pntd.0005204
PMCID: PMC5156366  PMID: 27973567
6.  Optimal Cutoff and Accuracy of an IgM Enzyme-Linked Immunosorbent Assay for Diagnosis of Acute Scrub Typhus in Northern Thailand: an Alternative Reference Method to the IgM Immunofluorescence Assay 
Journal of Clinical Microbiology  2016;54(6):1472-1478.
The enzyme-linked immunosorbent assay (ELISA) has been proposed as an alternative serologic diagnostic test to the indirect immunofluorescence assay (IFA) for scrub typhus. Here, we systematically determine the optimal sample dilution and cutoff optical density (OD) and estimate the accuracy of IgM ELISA using Bayesian latent class models (LCMs). Data from 135 patients with undifferentiated fever were reevaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar and tested with a blood culture for Orientia tsutsugamushi, three different PCR assays, and an IgM IFA. The IgM ELISA was performed for every sample at sample dilutions from 1:100 to 1:102,400 using crude whole-cell antigens of the Karp, Kato, and Gilliam strains of O. tsutsugamushi developed by the Naval Medical Research Center. We used Bayesian LCMs to generate unbiased receiver operating characteristic curves and found that the sample dilution of 1:400 was optimal for the IgM ELISA. With the optimal cutoff OD of 1.474 at a sample dilution of 1:400, the IgM ELISA had a sensitivity of 85.7% (95% credible interval [CrI], 77.4% to 86.7%) and a specificity of 98.1% (95% CrI, 97.2% to 100%) using paired samples. For the ELISA, the OD could be determined objectively and quickly, in contrast to the reading of IFA slides, which was both subjective and labor-intensive. The IgM ELISA for scrub typhus has high diagnostic accuracy and is less subjective than the IgM IFA. We suggest that the IgM ELISA may be used as an alternative reference test to the IgM IFA for the serological diagnosis of scrub typhus.
doi:10.1128/JCM.02744-15
PMCID: PMC4879268  PMID: 27008880
7.  A retrospective analysis of melioidosis in Cambodian children, 2009–2013 
BMC Infectious Diseases  2016;16:688.
Background
Melioidiosis, infection by Burkholderia pseudomallei, is an important but frequently under-recognised cause of morbidity and mortality in Southeast Asia and elsewhere in the tropics. Data on the epidemiology of paediatric melioidosis in Cambodia are extremely limited.
Methods
Culture-positive melioidosis cases presenting to Angkor Hospital for Children, a non-governmental paediatric hospital located in Siem Reap, Northern Cambodia, between 1st January 2009 and 31st December 2013 were identified by searches of hospital and laboratory databases and logbooks.
Results
One hundred seventy-three evaluable cases were identified, presenting from eight provinces. For Siem Reap province, the median commune level incidence was estimated to be 28-35 cases per 100,000 children <15 years per year. Most cases presented during the wet season, May to October. The median age at presentation was 5.7 years (range 8 days–15.9 years). Apart from undernutrition, co-morbidities were rare. Three quarters (131/173) of the children had localised infection, most commonly skin/soft tissue infection (60 cases) or suppurative parotitis (51 cases). There were 39 children with B. pseudomallei bacteraemia: 29 (74.4%) of these had clinical and/or radiological evidence of pneumonia. Overall mortality was 16.8% (29/173) with mortality in bacteraemic cases of 71.8% (28/39). At least seven children did not receive an antimicrobial with activity against B. pseudomallei prior to death.
Conclusions
This retrospective study demonstrated a considerable burden of melioidosis in Cambodian children. Given the high mortality associated with bacteraemic infection, there is an urgent need for greater awareness amongst healthcare professionals in Cambodia and other countries where melioidosis is known or suspected to be endemic. Empiric treatment guidelines should ensure suspected cases are treated early with appropriate antimicrobials.
Electronic supplementary material
The online version of this article (doi:10.1186/s12879-016-2034-9) contains supplementary material, which is available to authorized users.
doi:10.1186/s12879-016-2034-9
PMCID: PMC5117556  PMID: 27871233
Cambodia; Melioidosis; Paediatric
8.  Soil Nutrient Depletion Is Associated with the Presence of Burkholderia pseudomallei 
Applied and Environmental Microbiology  2016;82(24):7086-7092.
ABSTRACT
Burkholderia pseudomallei is a soil-dwelling bacterium and the cause of melioidosis, which kills an estimated 89,000 people per year worldwide. Agricultural workers are at high risk of infection due to repeated exposure to the bacterium. Little is known about the soil physicochemical properties associated with the presence or absence of the organism. Here, we evaluated the soil physicochemical properties and presence of B. pseudomallei in 6,100 soil samples collected from 61 rice fields in Thailand. The presence of B. pseudomallei was negatively associated with the proportion of clay, proportion of moisture, level of salinity, percentage of organic matter, presence of cadmium, and nutrient levels (phosphorus, potassium, calcium, magnesium, and iron). The presence of B. pseudomallei was not associated with the level of soil acidity (P = 0.54). In a multivariable logistic regression model, the presence of B. pseudomallei was negatively associated with the percentage of organic matter (odds ratio [OR], 0.06; 95% confidence interval [CI], 0.01 to 0.47; P = 0.007), level of salinity (OR, 0.06; 95% CI, 0.01 to 0.74; P = 0.03), and percentage of soil moisture (OR, 0.81; 95% CI, 0.66 to 1.00; P = 0.05). Our study suggests that B. pseudomallei thrives in rice fields that are nutrient depleted. Some agricultural practices result in a decline in soil nutrients, which may impact the presence and amount of B. pseudomallei bacteria in affected areas.
IMPORTANCE Burkholderia pseudomallei is an environmental Gram-negative bacillus and the cause of melioidosis. Humans acquire the disease following skin inoculation, inhalation, or ingestion of the bacterium in the environment. The presence of B. pseudomallei in soil defines geographic regions where humans and livestock are at risk of melioidosis, yet little is known about the soil properties associated with the presence of the organism. We evaluated the soil properties and presence of B. pseudomallei in 61 rice fields in East, Central, and Northeast Thailand. We demonstrated that the organism was more commonly found in soils with lower levels of organic matter and nutrients, including phosphorus, potassium, calcium, magnesium, and iron. We also demonstrated that crop residue burning after harvest, which can reduce soil nutrients, was not uncommon. Some agricultural practices result in a decline in soil nutrients, which may impact the presence and amount of B. pseudomallei bacteria in affected areas.
doi:10.1128/AEM.02538-16
PMCID: PMC5118919  PMID: 27694236
9.  Community engagement and population coverage in mass anti-malarial administrations: a systematic literature review 
Malaria Journal  2016;15:523.
Background
Mass anti-malarial administration has been proposed as a key component of the malaria elimination strategy in South East Asia. The success of this approach depends on the local malaria epidemiology, nature of the anti-malarial regimen and population coverage. Community engagement is used to promote population coverage but little research has systematically analysed its impact. This systematic review examines population coverage and community engagement in programmes of mass anti-malarial drug administration.
Methods
This review builds on a previous review that identified 3049 articles describing mass anti-malarial administrations published between 1913 and 2011. Further search and application of a set of criteria conducted in the current review resulted in 51 articles that were retained for analysis. These 51 papers described the population coverage and/or community engagement in mass anti-malarial administrations. Population coverage was quantitatively assessed and a thematic analysis was conducted on the community engagement activities.
Results
The studies were conducted in 26 countries: in diverse healthcare and social contexts where various anti-malarial regimens under varied study designs were administered. Twenty-eight articles reported only population coverage; 12 described only community engagement activities; and 11 community engagement and population coverage. Average population coverage was 83% but methods of calculating coverage were frequently unclear or inconsistent. Community engagement activities included providing health education and incentives, using community structures (e.g. existing hierarchies or health infrastructure), mobilizing human resources, and collaborating with government at some level (e.g. ministries of health). Community engagement was often a process involving various activities throughout the duration of the intervention.
Conclusion
The mean population coverage was over 80% but incomplete reporting of calculation methods limits conclusions and comparisons between studies. Various community engagement activities and approaches were described, but many articles contained limited or no details. Other factors relevant to population coverage, such as the social, cultural and study context were scarcely reported. Further research is needed to understand the factors that influence population coverage and adherence in mass anti-malarial administrations and the role community engagement activities and approaches play in satisfactory participation.
Electronic supplementary material
The online version of this article (doi:10.1186/s12936-016-1593-y) contains supplementary material, which is available to authorized users.
doi:10.1186/s12936-016-1593-y
PMCID: PMC5093999  PMID: 27806717
Malaria; MDA; Community; Engagement; Population coverage
10.  Optimal health and disease management using spatial uncertainty: a geographic characterization of emergent artemisinin-resistant Plasmodium falciparum distributions in Southeast Asia 
Background
Artemisinin-resistant Plasmodium falciparum malaria parasites are now present across much of mainland Southeast Asia, where ongoing surveys are measuring and mapping their spatial distribution. These efforts require substantial resources. Here we propose a generic ‘smart surveillance’ methodology to identify optimal candidate sites for future sampling and thus map the distribution of artemisinin resistance most efficiently.
Methods
The approach uses the ‘uncertainty’ map generated iteratively by a geostatistical model to determine optimal locations for subsequent sampling.
Results
The methodology is illustrated using recent data on the prevalence of the K13-propeller polymorphism (a genetic marker of artemisinin resistance) in the Greater Mekong Subregion.
Conclusion
This methodology, which has broader application to geostatistical mapping in general, could improve the quality and efficiency of drug resistance mapping and thereby guide practical operations to eliminate malaria in affected areas.
Electronic supplementary material
The online version of this article (doi:10.1186/s12942-016-0064-6) contains supplementary material, which is available to authorized users.
doi:10.1186/s12942-016-0064-6
PMCID: PMC5078981  PMID: 27776514
Surveillance; Drug resistance; Malaria; Artemisinin; Greater Mekong Subregion
11.  Dynamic Transmission Economic Evaluation of Infectious Disease Interventions in Low‐ and Middle‐Income Countries: A Systematic Literature Review 
Health Economics  2016;25(Suppl Suppl 1):124-139.
Abstract
Economic evaluation using dynamic transmission models is important for capturing the indirect effects of infectious disease interventions. We examine the use of these methods in low‐ and middle‐income countries, where infectious diseases constitute a major burden. This review is comprised of two parts: (1) a summary of dynamic transmission economic evaluations across all disease areas published between 2011 and mid‐2014 and (2) an in‐depth review of mosquito‐borne disease studies focusing on health economic methods and reporting. Studies were identified through a systematic search of the MEDLINE database and supplemented by reference list screening. Fifty‐seven studies were eligible for inclusion in the all‐disease review. The most common subject disease was HIV/AIDS, followed by malaria. A diverse range of modelling methods, outcome metrics and sensitivity analyses were used, indicating little standardisation. Seventeen studies were included in the mosquito‐borne disease review. With notable exceptions, most studies did not employ economic evaluation methods beyond calculating a cost‐effectiveness ratio or net benefit. Many did not adhere to health care economic evaluations reporting guidelines, particularly with respect to full model reporting and uncertainty analysis. We present a summary of the state‐of‐the‐art and offer recommendations for improved implementation and reporting of health economic methods in this crossover discipline. © 2016 The Authors. Health Economics published by John Wiley & Sons Ltd.
doi:10.1002/hec.3303
PMCID: PMC5066646  PMID: 26778620
economic evaluation; dynamic transmission modelling; low income; infectious disease
12.  A prospective study of the importance of enteric fever as a cause of non-malarial febrile illness in patients admitted to Chittagong Medical College Hospital, Bangladesh 
BMC Infectious Diseases  2016;16:567.
Background
Fever is a common cause of hospital admission in Bangladesh but causative agents, other than malaria, are not routinely investigated. Enteric fever is thought to be common.
Methods
Adults and children admitted to Chittagong Medical College Hospital with a temperature of ≥38.0 °C were investigated using a blood smear for malaria, a blood culture, real-time PCR to detect Salmonella Typhi, S. Paratyphi A and other pathogens in blood and CSF and an NS1 antigen dengue ELISA.
Results
We enrolled 300 febrile patients with a negative malaria smear between January and June 2012: 156 children (aged ≤15 years) and 144 adults with a median (interquartile range) age of 13 (5–31) years and median (IQR) illness duration before admission of five (2–8) days. Clinical enteric fever was diagnosed in 52 patients (17.3 %), lower respiratory tract infection in 48 (16.0 %), non-specific febrile illness in 48 (16.0 %), a CNS infection in 37 patients (12.3 %), urinary sepsis in 23 patients (7.7 %), an upper respiratory tract infection in 21 patients (7.0 %), and diarrhea or dysentery in 21 patients (7.0 %). Malaria was still suspected in seven patients despite a negative microscopy test. S. Typhi was detected in blood by culture or PCR in 34 (11.3 %) of patients. Of note Rickettsia typhi and Orientia tsutsugamushi were detected by PCR in two and one patient respectively. Twenty-nine (9 %) patients died during their hospital admission (15/160 (9.4 %) of children and 14/144 (9.7 %) adults). Two of 52 (3.8 %) patients with enteric fever, 5/48 (10.4 %) patients with lower respiratory tract infections, and 12/37 (32.4 %) patients with CNS infection died.
Conclusion
Enteric fever was confirmed in 11.3 % of patients admitted to this hospital in Bangladesh with non-malaria fever. Lower respiratory tract and CNS infections were also common. CNS infections in this location merit more detailed study due to the high mortality.
doi:10.1186/s12879-016-1886-3
PMCID: PMC5064917  PMID: 27737634
Non-malaria febrile illness; Bangladesh; Enteric fever
13.  Opposite malaria and pregnancy effect on oral bioavailability of artesunate – a population pharmacokinetic evaluation 
Aim
The aim was to compare the pharmacokinetic properties of artesunate and dihydroartemisinin in the same women: i) pregnant with acute uncomplicated malaria on day 1 and 2, ii) pregnant with convalescent malaria on day 7 and iii) in a healthy state 3 months post-partum on day 1, 2 and 7.
Methods
Non-linear mixed-effects modelling was used to compare plasma concentration–time profiles of artesunate and dihydroartemisinin over 7 days of treatment following oral and intravenous artesunate administration to pregnant women with uncomplicated Plasmodium falciparum malaria during their second or third trimesters of pregnancy. The same women were restudied 3 months after delivery when fully recovered. Non-compartmental results of the same study have been published previously.
Results
Twenty pregnant patients on the Thailand-Myanmar border were studied and 15 volunteered to be restudied 3 months post-partum. Malaria and pregnancy had no effect on the pharmacokinetic properties of artesunate or dihydroartemisinin after intravenous artesunate administration. However, malaria and pregnancy had opposite effects on the absorption of orally administered artesunate. Malaria increased the absolute oral bioavailability of artesunate by 87%, presumably by inhibiting first pass effect, whereas pregnancy decreased oral bioavailability by 23%.
Conclusions
The population pharmacokinetic analysis demonstrated opposite effects of malaria and pregnancy on the bioavailability of orally administered artesunate. Lower drug exposures during the second and third trimesters of pregnancy may contribute to lower cure rates and thus the development of drug resistance. Dose optimization studies are required for artesunate containing artemisinin-based combination therapies (ACTs) in later pregnancy.
doi:10.1111/bcp.12660
PMCID: PMC4594700  PMID: 25877779
artesunate; dihydroartemisinin; nonmem; population pharmacokinetics; post-partum women; pregnant women
14.  Geographic distribution of amino acid mutations in DHFR and DHPS in Plasmodium vivax isolates from Lao PDR, India and Colombia 
Malaria Journal  2016;15:484.
Background
Non-synonymous mutations in dhfr and dhps genes in Plasmodium vivax are associated with sulfadoxine–pyrimethamine (SP) resistance. The present study aimed to assess the prevalence of point mutations in P. vivax dhfr (pvdhfr) and P. vivax dhps (pvdhps) genes in three countries: Lao PDR, India and Colombia.
Methods
Samples from 203 microscopically diagnosed vivax malaria were collected from the three countries. Five codons at positions 13, 57, 58, 61, and 117 of pvdhfr and two codons at positions 383 and 553 of pvdhps were examined by polymerase chain reaction-restriction fragment length polymorphism methodology.
Results
The largest number of 58R/117 N double mutations in pvdhfr was observed in Colombia (94.3 %), while the corresponding wild-type amino acids were found at high frequencies in Lao PDR during 2001–2004 (57.8 %). Size polymorphism analysis of the tandem repeats within pvdhfr revealed that 74.3 % of all the isolates carried the type B variant. Eighty-nine per cent of all the isolates examined carried wild-type pvdhps A383 and A553.
Conclusions
Although SP is not generally used to treat P. vivax infections, mutations in dhfr and dhps that confer antifolate resistance in P. vivax are common. The data strongly suggest that, when used primarily to treat falciparum malaria, SP can exert a substantial selective pressure on P. vivax populations, and this can lead to point mutations in dhfr and dhps. Accurate data on the global geographic distribution of dhfr and dhps genotypes should help to inform anti-malarial drug-use policies.
doi:10.1186/s12936-016-1543-8
PMCID: PMC5031260  PMID: 27654047
Plasmodium vivax; Sulfadoxine–pyrimethamine; dhfr; dhps
15.  Diagnostic Accuracy Assessment of Immunochromatographic Tests for the Rapid Detection of Antibodies against Orientia tsutsugamushi Using Paired Acute and Convalescent Specimens 
We assessed the diagnostic accuracy of two immunochromatographic tests (ICTs), the Access Bio CareStart Scrub Typhus test (Somerset, NJ) (IgM), and the SD BIOLINE Tsutsugamushi test (Kyonggi-do, Republic of Korea) (IgG, IgM, or IgA) compared with indirect immunofluorescence assay (IFA) and real-time PCR results as reference tests using 86 paired acute and convalescent specimens from febrile patients. The sensitivity and specificity of the CareStart test were 23.3% (95% confidence interval [CI]: 11.8–38.6) and 81.4% (95% CI: 66.6–91.6), respectively, for acute specimens and 32.6% (95% CI: 19.1–48.5) and 79.1% (95% CI: 64.0–90.0), respectively, for convalescent specimens. For the SD BIOLINE test, sensitivity and specificity were 20.9% (95% CI: 10.0–36.0) and 74.4% (95% CI: 58.8–86.5), respectively, for acute specimens and 76.7% (95% CI: 61.4–88.2) and 76.7% (95% CI: 61.4–88.2), respectively, for convalescent specimens. The poor sensitivity obtained for both ICTs during this study when performed on acute specimens highlights the difficulties in prompt diagnosis of scrub typhus.
doi:10.4269/ajtmh.15-0435
PMCID: PMC4674230  PMID: 26458778
16.  Molecular Epidemiology of Group A Streptococcus Infections in Cambodian Children, 2007–2012 
Supplemental Digital Content is available in the text.
doi:10.1097/INF.0000000000000878
PMCID: PMC4643749  PMID: 26780027
17.  Evaluation of Polysaccharide-Based Latex Agglutination Assays for the Rapid Detection of Antibodies to Burkholderia pseudomallei 
Melioidosis is a severe disease caused by the Gram-negative bacterium Burkholderia pseudomallei. Diagnosis of melioidosis currently relies on the isolation of B. pseudomallei from clinical samples, which can take several days. An indirect hemagglutination assay (IHA) is widely used for serodiagnosis, but it has a short shelf life, is poorly standardized, and requires a viable bacteria culture performed in a biosafety level 3 (BSL-3) laboratory. To improve the diagnostic methods, we have developed two rapid latex agglutination tests based on purified B. pseudomallei O-polysaccharide (OPS) and capsular polysaccharide (CPS) antigens. The immunodiagnostic potential of these tests was evaluated using serum from culture-confirmed melioidosis patients (N = 143) and healthy donors from either endemic (N = 199) or non-endemic areas (N = 90). The sensitivity of the OPS-based latex agglutination assay (OPS-latex; 84.4%) was significantly higher than both the CPS-latex (69.5%) (P < 0.001) and IHA (69.5%) (P = 0.001). When evaluated with Thai donor serum, the OPS-latex had comparable specificity (56.9%) to the CPS-latex (63.8%) (P = 0.053), but was significantly lower than the IHA (67.6%) (P = 0.002). In contrast, all tests with U.S. donor serum were highly specific (≥ 97.8%). These results suggest that polysaccharide-based latex agglutination assays may be useful for serodiagnosis of melioidosis in non-endemic areas.
doi:10.4269/ajtmh.15-0114
PMCID: PMC4559694  PMID: 26123956
18.  Blood–Brain Barrier Function and Biomarkers of Central Nervous System Injury in Rickettsial versus Other Neurological Infections in Laos 
Blood–brain barrier (BBB) function and cerebrospinal fluid (CSF) biomarkers were measured in patients admitted to hospital with severe neurological infections in the Lao People's Democratic Republic (N = 66), including bacterial meningitis (BM; N = 9) or tuberculosis meningitis (TBM; N = 11), Japanese encephalitis virus (JEV; N = 25), and rickettsial infections (N = 21) including murine and scrub typhus patients. The albumin index (AI) and glial fibrillary acidic protein (GFAP) levels were significantly higher in BM and TBM than other diseases but were also raised in individual rickettsial patients. Total tau protein was significantly raised in the CSF of JEV patients. No differences were found between clinical or neurological symptoms, AI, or biomarker levels that allowed distinction between severe neurological involvement by Orientia tsutsugamushi compared with Rickettsia species.
doi:10.4269/ajtmh.15-0119
PMCID: PMC4530739  PMID: 26055741
19.  Antimicrobial Disk Susceptibility Testing of Leptospira spp. Using Leptospira Vanaporn Wuthiekanun (LVW) Agar 
Leptospira Vanaporn Wuthiekanun (LVW) agar was used to develop a disk diffusion assay for Leptospira spp. Ten pathogenic Leptospira isolates were tested, all of which were susceptible to 17 antimicrobial agents (amoxicillin/clavulanic acid, amoxicillin, azithromycin, cefoxitin, ceftazidime, ceftriaxone, chloramphenicol, ciprofloxacin, clindamycin, doripenem, doxycycline, gentamicin, linezolid, nitrofurantoin, penicillin, piperacillin/tazobactam, and tetracycline). All 10 isolates had no zone of growth inhibition for four antimicrobials (fosfomycin, nalidixic acid, rifampicin, and trimethoprim/sulfamethoxazole). Of the ten Leptospira, seven had a growth inhibition zone of ≤ 21 mm for aztreonam, the zone diameter susceptibility break point for Enterobacteriaceae. This assay could find utility as a simple screening method during the epidemiological surveillance of antimicrobial resistance in Leptospira spp.
doi:10.4269/ajtmh.15-0180
PMCID: PMC4530741  PMID: 26055750
20.  Declining Efficacy of Artemisinin Combination Therapy Against P. Falciparum Malaria on the Thai–Myanmar Border (2003–2013): The Role of Parasite Genetic Factors 
The pivotal factor leading to the declining efficacy of the artemisinin-based combination on the Thailand–Myanmar border (mefloquine–artesunate) to a clinically unacceptable level is the increasing local prevalence of K13 mutations superimposed onto a long-standing background of Pfmdr1 amplification.
Background. Deployment of mefloquine–artesunate (MAS3) on the Thailand–Myanmar border has led to a sustained reduction in falciparum malaria, although antimalarial efficacy has declined substantially in recent years. The role of Plasmodium falciparum K13 mutations (a marker of artemisinin resistance) in reducing treatment efficacy remains controversial.
Methods. Between 2003 and 2013, we studied the efficacy of MAS3 in 1005 patients with uncomplicated P. falciparum malaria in relation to molecular markers of resistance.
Results. Polymerase chain reaction (PCR)–adjusted cure rates declined from 100% in 2003 to 81.1% in 2013 as the proportions of isolates with multiple Pfmdr1 copies doubled from 32.4% to 64.7% and those with K13 mutations increased from 6.7% to 83.4%. K13 mutations conferring moderate artemisinin resistance (notably E252Q) predominated initially but were later overtaken by propeller mutations associated with slower parasite clearance (notably C580Y). Those infected with both multiple Pfmdr1 copy number and a K13 propeller mutation were 14 times more likely to fail treatment. The PCR-adjusted cure rate was 57.8% (95% confidence interval [CI], 45.4, 68.3) compared with 97.8% (95% CI, 93.3, 99.3) in patients with K13 wild type and Pfmdr1 single copy. K13 propeller mutation alone was a strong risk factor for recrudescence (P = .009). The combined population attributable fraction of recrudescence associated with K13 mutation and Pfmdr1 amplification was 82%.
Conclusions. The increasing prevalence of K13 mutations was the decisive factor for the recent and rapid decline in efficacy of artemisinin-based combination (MAS3) on the Thailand–Myanmar border.
doi:10.1093/cid/ciw388
PMCID: PMC4996140  PMID: 27313266
Plasmodium falciparum malaria; mefloquine–artesunate; Pfmdr1; K13 mutation; artemisinin resistance
21.  Revisiting doxycycline in pregnancy and early childhood – time to rebuild its reputation? 
Expert Opinion on Drug Safety  2016;15(3):367-382.
ABSTRACT
Introduction: Doxycycline is highly effective, inexpensive with a broad therapeutic spectrum and exceptional bioavailability. However these benefits have been overshadowed by its classification alongside the tetracyclines – class D drugs, contraindicated in pregnancy and in children under 8 years of age. Doxycycline-treatable diseases are emerging as leading causes of undifferentiated febrile illness in Southeast Asia. For example scrub typhus and murine typhus have an unusually severe impact on pregnancy outcomes, and current mortality rates for scrub typhus reach 12-13% in India and Thailand. The emerging evidence for these important doxycycline-treatable diseases prompted us to revisit doxycycline usage in pregnancy and childhood.
Areas Covered: A systematic review of the available literature on doxycycline use in pregnant women and children revealed a safety profile of doxycycline that differed significantly from that of tetracycline; no correlation between the use of doxycycline and teratogenic effects during pregnancy or dental staining in children was found.
Expert Opinion: The change of the US FDA pregnancy classification scheme to an evidence-based approach will enable adequate evaluation of doxycycline in common tropical illnesses and in vulnerable populations in clinical treatment trials, dosage-optimization pharmacokinetic studies and for the empirical treatment of undifferentiated febrile illnesses, especially in pregnant women and children.
doi:10.1517/14740338.2016.1133584
PMCID: PMC4898140  PMID: 26680308
Doxycycline; tetracycline; pregnancy; rickettsiosis; prenatal exposure; side effects; teratogenicity; major congenital anomalies; tooth discolouration; Orientia tsutsugamushi; Rickettsia typhi; scrub typhus; murine typhus; undifferentiated fever
22.  Molecular Epidemiology of Group A Streptococcus Infections in Cambodian Children, 2007 - 2012 
doi:10.1097/INF.0000000000000878
PMCID: PMC4643749  PMID: 26780027
Group A Streptococcus; emm-type; vaccine; children; epidemiology
23.  Antigenic Relationships among Human Pathogenic Orientia tsutsugamushi Isolates from Thailand 
PLoS Neglected Tropical Diseases  2016;10(6):e0004723.
Background
Scrub typhus is a common cause of undiagnosed febrile illness in certain tropical regions, but can be easily treated with antibiotics. The causative agent, Orientia tsutsugamushi, is antigenically variable which complicates diagnosis and efforts towards vaccine development.
Methodology/Principal Findings
This study aimed to dissect the antigenic and genetic relatedness of O. tsutsugamushi strains and investigate sero-diagnostic reactivities by titrating individual patient sera against their O. tsutsugamushi isolates (whole-cell antigen preparation), in homologous and heterologous serum-isolate pairs from the same endemic region in NE Thailand. The indirect immunofluorescence assay was used to titrate Orientia tsutsugamushi isolates and human sera, and a mathematical technique, antigenic cartography, was applied to these data to visualise the antigenic differences and cross-reactivity between strains and sera. No functional or antigen-specific analyses were performed. The antigenic variation found in clinical isolates was much less pronounced than the genetic differences found in the 56kDa type-specific antigen genes. The Karp-like sera were more broadly reactive than the Gilliam-like sera.
Conclusions/Significance
Antigenic cartography worked well with scrub typhus indirect immunofluorescence titres. The data from humoral responses suggest that a Karp-like strain would provide broader antibody cross-reactivity than a Gilliam-like strain. Although previous exposure to O. tsutsugamushi could not be ruled out, scrub typhus patient serum antibody responses were characterised by strong homologous, but weak heterologous antibody titres, with little evidence for cross-reactivity by Gilliam-like sera, but a broader response from some Karp-like sera. This work highlights the importance of antigenic variation in O. tsutsugamushi diagnosis and determination of new serotypes.
Author Summary
Scrub Typhus is a common and potentially severe febrile illness in certain tropical regions in Asia. This infection is treatable with specific antibiotics if diagnosed correctly, but there is no effective vaccine available at present. The bacterium causing this disease is called Orientia tsutsugamushi; it is transmitted by small mites and it has variable surface proteins, which make diagnosis and vaccine development difficult. In this study, we tested how well the antibodies in the blood of patients with scrub typhus recognise different strains of bacteria, and used these results to create a map of the relationships between the bacteria and sera. From examining this map we can see that some sera have activity against a wider range of bacteria than others. These methods and findings will help with selecting bacteria strains and evaluating immune responses, which will potentially help us to improve diagnosis and vaccine development.
doi:10.1371/journal.pntd.0004723
PMCID: PMC4889052  PMID: 27248711
24.  Detailed functional analysis of two clinical glucose-6-phosphate dehydrogenase (G6PD) variants, G6PDViangchan and G6PDViangchan + Mahidol: Decreased stability and catalytic efficiency contribute to the clinical phenotype 
Deficiency of glucose-6-phosphate dehydrogenase (G6PD) is an X-linked hereditary genetic defect that is the most common polymorphism and enzymopathy in humans. To investigate functional properties of two clinical variants, G6PDViangchan and G6PDViangchan + Mahidol, these two mutants were created by overlap-extension PCR, expressed in Escherichia coli and purified to homogeneity. We describe an overexpression and purification method to obtain substantial amounts of functionally active protein. The KM for G6P of the two variants was comparable to the KM of the native enzyme, whereas the KM for NADP+ was increased 5-fold for G6PDViangchan and 8-fold for G6PDViangchan + Mahidol when compared with the native enzyme. Additionally, kcat of the mutant enzymes was markedly reduced, resulting in a 10- and 18-fold reduction in catalytic efficiency for NADP+ catalysis for G6PDViangchan and G6PDViangchan + Mahidol, respectively. Furthermore, the two variants demonstrated significant reduction in thermostability, but similar susceptibility to trypsin digestion, when compared with the wild-type enzyme. The presence of NADP+ is shown to improve the stability of G6PD enzymes. This is the first report indicating that protein instability and reduced catalytic efficiency are responsible for the reduced catalytic activity of G6PDViangchan and G6PDViangchan + Mahidol and, as a consequence, contribute to the clinical phenotypes of these two clinical variants.
Highlights
•Recombinant human G6PD enzymes were expressed in E. coli and substantial amount of enzymes were obtained.•Detailed biochemical characterization of G6PD enzymes was performed.•Purified G6PD variants showed a significant decrease in catalytic efficiency and protein stability.•Data obtained provide further knowledge in phenotype-genotype relationship of G6PD deficiency.
doi:10.1016/j.ymgme.2016.03.008
PMCID: PMC4894296  PMID: 27053284
Glucose-6-phosphate dehydrogenase; G6PD deficiency; Variants; Steady state kinetics; Thermostability
25.  Asymptomatic Plasmodium infections in 18 villages of southern Savannakhet Province, Lao PDR (Laos) 
Malaria Journal  2016;15:296.
Background
A large fraction of Plasmodium infections do not cause clinical signs and symptoms of disease and persist at densities in blood that are not detectable by microscopy or rapid diagnostic tests. These infections may be critical as a transmission reservoir in areas of low malaria endemicity. Understanding the epidemiology of these infections would be helpful for malaria elimination.
Methods
A cross-sectional survey was conducted in Thapangthong and Nong Districts of Savannakhet Province, Lao PDR, to determine the prevalence of parasitaemia. A total of 888 blood samples were collected from afebrile volunteers aged ≥15 years in 18 villages during March and July 2015. Plasmodium infections were diagnosed by rapid diagnostic tests (RDT) and high volume, ultra-sensitive quantitative polymerase chain reaction (uPCR).
Results
uPCR detected Plasmodium infections in 175 of 888 samples (20 %). The species distribution was Plasmodiumfalciparum 3.6 % (32/888), Plasmodium vivax 11.1 % (99/888), mixed infections with P. falciparum and P. vivax 1.6 % (14/888) and Plasmodium of undetermined species 3.4 % (30/888). RDT identified only 2 % (18/888) positive cases. Using uPCR as reference, the sensitivity and specificity of RDTs were 28 and 100 %, respectively, in detecting P. falciparum infections, and 3 and 99 % in detecting asymptomatic P. vivax infections. The K13 kelch propeller domain C580Y mutation, associated with reduced susceptibility to artemisinin derivatives, was found in 75 % (12/18) of P. falciparum isolates from Thapangthong and in 7 % (2/28) from Nong (p < 0.001). In a multivariate analysis, males were more likely to have P. vivax infections [adjusted odds ratio (aOR) 4.76 (95 % CI 2.84–8.00)] while older villagers were at lower risk for parasitaemia [aOR for increasing age 0.98 (95 % CI 0.96–0.99)].
Conclusion
There is a high prevalence of asymptomatic Plasmodium infections in southern Savannakhet. Artemisinin-resistant P. falciparum strains form an increasing proportion of the parasite population in Thapangthong District and are already present in the more remote Nong District. This worrying trend has wider implications for Laos and could reverse the gains achieved by the successful control of malaria in Laos and the Greater Mekong Sub-region (GMS). Rapid elimination of P. falciparum has to be a top priority in Laos as well as in the wider GMS.
doi:10.1186/s12936-016-1336-0
PMCID: PMC4882819  PMID: 27234446
Asymptomatic; RDT; uPCR; Prevalence; Lao PDR

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