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1.  Absence of association between Plasmodium falciparum small sub-unit ribosomal RNA gene mutations and in vitro decreased susceptibility to doxycycline 
Malaria Journal  2015;14:348.
Doxycycline is an antibiotic used in combination with quinine or artesunate for malaria treatment or alone for malaria chemoprophylaxis. Recently, one prophylactic failure has been reported, and several studies have highlighted in vitro doxycycline decreased susceptibility in Plasmodium falciparum isolates from different areas. The genetic markers that contribute to detecting and monitoring the susceptibility of P. falciparum to doxycycline, the pfmdt and pftetQ genes, have recently been identified. However, these markers are not sufficient to explain in vitro decreased susceptibility of P. falciparum to doxycycline. In this paper, the association between polymorphism of the small sub-unit ribosomal RNA apicoplastic gene pfssrRNA (PFC10_API0057) and in vitro susceptibilities of P. falciparum isolates to doxycycline were investigated.
Doxycycline IC50 determinations using the hypoxanthine uptake inhibition assay were performed on 178 African and Thai P. falciparum isolates. The polymorphism of pfssrRNA was investigated in these samples by standard PCR followed by sequencing.
No point mutations were found in pfssrRNA in the Thai or African isolates, regardless of the determined IC50 values.
The pfssrRNA gene is not associated with in vitro decreased susceptibility of P. falciparum to doxycycline. Identifying new in vitro molecular markers associated with reduced susceptibility is needed, to survey the emergence of doxycycline resistance.
PMCID: PMC4574345  PMID: 26377329
Malaria; Plasmodium falciparum; In vitro; Anti-malarial; Molecular marker; Doxycycline; Small ribosomal sub-unit RNA gene; pfssrRNA; 16S rRNA
2.  Perceived Benefits, Harms, and Views About How to Share Data Responsibly 
The Thailand Major Overseas Programme coordinates large multi-center studies in tropical medicine and generates vast amounts of data. As the data sharing movement gains momentum, we wanted to understand attitudes and experiences of relevant stakeholders about what constitutes good data sharing practice. We conducted 15 interviews and three focus groups discussions involving 25 participants and found that they generally saw data sharing as something positive. Data sharing was viewed as a means to contribute to scientific progress and lead to better quality analysis, better use of resources, greater accountability, and more outputs. However, there were also important reservations including potential harms to research participants, their communities, and the researchers themselves. Given these concerns, several areas for discussion were identified: data standardization, appropriate consent models, and governance.
PMCID: PMC4547202  PMID: 26297749
Thailand; data sharing; consent; collaboration; research ethics
3.  The suitability of laboratory-bred Anopheles cracens for the production of Plasmodium vivax sporozoites 
Malaria Journal  2015;14:312.
A stenogamous colony of Anopheles cracens (A. dirus B) established 20 years ago in a Thai insectary proved susceptible to Plasmodium vivax. However, routine sporozoite production by feeding on field-collected blood samples has not been described. The setting-up of an A. cracens colony in an insectary on the Thai-Myanmar border and the process of using P. vivax field samples for the production of infectious sporozoites are described.
The colony was started in 2012 from egg batches that were sent from the Department of Parasitology, Faculty of Medicine, University of Chiang Mai, to the Shoklo Malaria Research Unit (SMRU), on wet filter paper in sealed Petri dishes. From May 2013 to December 2014, P. vivax-infected blood samples collected from patients seeking care at SMRU clinics were used for membrane feeding assays and sporozoite production.
Mosquitoes were fed on blood samples from 55 patients, and for 38 (69 %) this led to the production sporozoites. The average number of sporozoites obtained per mosquito was 26,112 (range 328–79,310). Gametocytaemia was not correlated with mosquito infectiousness (p = 0.82), or with the number of the sporozoites produced (Spearman’s ρ = −0.016, p = 0.905). Infectiousness did not vary with the date of collection or the age of the patient. Mosquito survival was not correlated with sporozoite load (Spearman’s ρ = 0.179, p = 0.282).
Consistent and routine P. vivax sporozoites production confirms that A. cracens is highly susceptible to P. vivax infection. Laboratory-bred colonies of this vector are suitable for experimental transmission protocols and thus constitute a valuable resource.
Electronic supplementary material
The online version of this article (doi:10.1186/s12936-015-0830-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4531391  PMID: 26259952
Anopheles cracens; Sporozoites; Plasmodium vivax; Transmission; Insectary
4.  Past and new challenges for malaria control and elimination: the role of operational research for innovation in designing interventions 
Malaria Journal  2015;14:279.
This meeting report presents the outcomes of a workshop held in Bangkok on December 1st 2014, where the following challenges were discussed: the threat of resistance to artemisinin and artemisinin-based combination therapy in the Greater Mekong Sub-region (GMS) and in Africa; access to treatment for most at risk and hard to reach population; insecticide resistance, residual and outdoors transmission. The role of operational research and the interactions between research institutions, National Malaria Control Programmes, Civil Society Organizations, and of financial and technical partners to address those challenges and to accelerate translation of research into policies and programmes were debated. The threat and the emergency of the artemisinin resistance spread and independent emergence in the GMS was intensely debated as it is now close to the border of India. The need for key messages, based on scientific evidence and information available and disseminated without delay, was highlighted as crucial for an effective and urgent response.
Electronic supplementary material
The online version of this article (doi:10.1186/s12936-015-0802-4) contains supplementary material, which is available to authorized users.
PMCID: PMC4504133  PMID: 26185098
5.  Estimating Gestational Age in Late Presenters to Antenatal Care in a Resource-Limited Setting on the Thai-Myanmar Border 
PLoS ONE  2015;10(6):e0131025.
Estimating gestational age in resource-limited settings is prone to considerable inaccuracy because crown-rump length measured by ultrasound before 14 weeks gestation, the recommended method for estimating gestational age, is often unavailable. Judgements regarding provision of appropriate obstetric and neonatal care are dependent on accurate estimation of gestational age. We determined the accuracy of the Dubowitz Gestational Age Assessment, a population-specific symphysis-fundal height formula, and ultrasound biometry performed between 16 and 40 weeks gestation in estimating gestational age using pre-existing data from antenatal clinics of the Shoklo Malaria Research Unit on the Thai-Myanmar border, where malaria is endemic. Two cohorts of women who gave birth to live singletons were analysed: 1) 250 women who attended antenatal care between July 2001 and May 2006 and had both ultrasound crown-rump length (reference) and a Dubowitz Gestational Age Assessment; 2) 975 women attending antenatal care between April 2007 and October 2010 who had ultrasound crown-rump length, symphysis-fundal measurements, and an additional study ultrasound (biparietal diameter and head circumference) randomly scheduled between 16 and 40 weeks gestation. Mean difference in estimated newborn gestational age between methods and 95% limits of agreement (LOA) were determined from linear mixed-effects models. The Dubowitz method and the symphysis-fundal height formula performed well in term newborns, but overestimated gestational age of preterms by 2.57 weeks (95% LOA: 0.49, 4.65) and 3.94 weeks (95% LOA: 2.50, 5.38), respectively. Biparietal diameter overestimated gestational age by 0.83 weeks (95% LOA: -0.93, 2.58). Head circumference underestimated gestational age by 0.39 weeks (95% LOA: -2.60, 1.82), especially if measured after 24 weeks gestation. The results of this study can be used to quantify biases associated with alternative methods for estimating gestational age in the absence of ultrasound crown-rump length to inform critical clinical judgements in this population, and as a point of reference elsewhere.
PMCID: PMC4482646  PMID: 26114295
6.  Intervals to Plasmodium falciparum recurrence after anti-malarial treatment in pregnancy: a longitudinal prospective cohort 
Malaria Journal  2015;14:221.
Plasmodium falciparum infections adversely affect pregnancy. Anti-malarial treatment failure is common. The objective of this study was to examine the duration of persistent parasite carriage following anti-malarial treatment in pregnancy.
The data presented here are a collation from previous studies carried out since 1994 in the Shoklo Malaria Research Unit (SMRU) on the Thailand-Myanmar border and performed using the same unique methodology detailed in the Materials and Methods section. Screening for malaria by microscopy is a routine part of weekly antenatal care (ANC) visits and therapeutic responses to anti-malarials were assessed in P. falciparum malaria cases. Women with microscopy confirmed P. falciparum malaria had a PCR blood spot from a finger-prick sample collected. Parasite DNA was extracted from the blood-spot samples using saponin lysis/Chelex extraction method and genotyped using polymorphic segments of MSP1, MSP2 and GLURP. Recurrent infections were classified by genotyping as novel, recrudescent or indeterminate. Factors associated with time to microscopy-detected recrudescence were analysed using multivariable regression techniques.
From December 1994 to November 2009, 700 women were treated for P. falciparum and there were 909 recurrent episodes (481 novel and 428 recrudescent) confirmed by PCR genotyping. Most of the recurrences, 85 % (770/909), occurred after treatment with quinine monotherapy, artesunate monotherapy or artesunate-clindamycin. The geometric mean number of days to recurrence was significantly shorter in women with recrudescent infection, 24.5 (95 %: 23.4-25.8), compared to re-infection, 49.7 (95 %: 46.9-52.7), P <0.001. The proportion of recrudescent P. falciparum infections that occurred after days 28, 42 and 63 from the start of treatment was 29.1 % (124/428), 13.3 % (57/428) and 5.6 % (24/428). Recrudescent infections ≥100 days after treatment occurred with quinine and mefloquine monotherapy, and quinine + clindamycin and artesunate + atovaquone-proguanil combination therapy. Treatments containing an artemisinin derivative or an intercalated Plasmodium vivax infection increased the geometric mean interval to recrudescence by 1.28-fold (95 % CI: 1.09-1.51) and 2.19-fold (1.77-2.72), respectively. Intervals to recrudescence were decreased 0.83-fold (0.73-0.95) if treatment was not fully supervised (suggesting incomplete adherence) and 0.98-fold (0.96-0.99) for each doubling in baseline parasitaemia.
Prolonged time to recrudescence may occur in pregnancy, regardless of anti-malarial treatment. Long intervals to recrudescence are more likely with the use of artemisinin-containing treatments and also observed with intercalated P. vivax infections treated with chloroquine. Accurate determination of drug efficacy in pregnancy requires longer duration of follow-up, preferably until delivery or day 63, whichever occurs last.
PMCID: PMC4449611  PMID: 26017553
7.  Underrecognized Arthropod-Borne and Zoonotic Pathogens in Northern and Northwestern Thailand: Serological Evidence and Opportunities for Awareness 
Although scrub typhus and murine typhus are well-described tropical rickettsial illnesses, especially in Southeast Asia, only limited evidence is available for rickettsia-like pathogens contributing to the burden of undifferentiated febrile illness. Using commercially available kits, this study measured immunoglobulin G (IgG) antibody seroprevalence for Coxiella burnetii, Ehrlichia chaffeensis, Bartonella henselae, Anaplasma phagocytophilum, and spotted fever group rickettsiae (SFGR) in 375 patients enrolled in undifferentiated febrile illness studies at Chiangrai (northern Thailand) and Mae Sot (Thai–Myanmar border). Ehrlichia and SFGR were the most common causes of IgG seropositivity. A distinct relationship between age and seropositivity was found in Chiangrai with acquisition of IgG titers against Ehrlichia, Bartonella, Anaplasma, and SFGR in young adulthood, suggesting cumulative exposure to these pathogens. At Mae Sot, high early IgG titers against Ehrlichia and SFGR were common, whereas Anaplasma and Bartonella IgG titers increased at 50–60 years. Q fever associated with low IgG positivity at both study sites, with significantly higher prevalence at 30 years of age in Chiangrai. These data suggest that other rickettsial illnesses could contribute to the burden of febrile illness in Thailand and possibly adjacent regions. Improved diagnostics and better understanding of antibody longevity and cross-reactivity will improve identification and management of these easily treatable infectious diseases.
PMCID: PMC4449621  PMID: 25988437
Orientia; Rickettsia; Coxiella; Ehrlichia; Bartonella; Anaplasma; Spotted fever group rickettsiae; Seroprevalence; Arthropod-borne; Zoonotic pathogens
8.  Defining the In Vivo Phenotype of Artemisinin-Resistant Falciparum Malaria: A Modelling Approach 
PLoS Medicine  2015;12(4):e1001823.
Artemisinin-resistant falciparum malaria has emerged in Southeast Asia, posing a major threat to malaria control. It is characterised by delayed asexual-stage parasite clearance, which is the reference comparator for the molecular marker ‘Kelch 13’ and in vitro sensitivity tests. However, current cut-off values denoting slow clearance based on the proportion of individuals remaining parasitaemic on the third day of treatment ('day-3'), or on peripheral blood parasite half-life, are not well supported. We here explore the parasite clearance distributions in an area of artemisinin resistance with the aim refining the in vivo phenotypic definitions.
Methods and Findings
Data from 1,518 patients on the Thai-Myanmar and Thai-Cambodian borders with parasite half-life assessments after artesunate treatment were analysed. Half-lives followed a bimodal distribution. A statistical approach was developed to infer the characteristics of the component distributions and their relative contribution to the composite mixture.
A model representing two parasite subpopulations with geometric mean (IQR) parasite half-lives of 3.0 (2.4-3.9) hours and 6.50 (5.7-7.4) hours was consistent with the data. For individual patients, the parasite half-life provided a predicted likelihood of an artemisinin-resistant infection which depends on the population prevalence of resistance in that area. Consequently, a half-life where the probability is 0.5 varied between 3.5 and 5.5 hours. Using this model, the current 'day-3' cut-off value of 10% predicts the potential presence of artemisinin-resistant infections in most but not all scenarios. These findings are relevant to the low-transmission setting of Southeast Asia. Generalisation to a high transmission setting as in regions of Sub-Saharan Africa will need additional evaluation.
Characterisation of overlapping distributions of parasite half-lives provides quantitative insight into the relationship between parasite clearance and artemisinin resistance, as well as the predictive value of the 10% cut-off in 'day-3' parasitaemia. The findings are important for the interpretation of in vitro sensitivity tests and molecular markers for artemisinin resistance and for contextualising the ‘day 3’ threshold to account for initial parasitaemia and sample size.
Lisa White and colleagues explore parasite clearance distributions in an area of artemisinin resistance with the aim of refining the phenotypic definition of resistance.
Editors' Summary
Artemisinin and its derivatives are powerful medicines that can quickly reduce the number of Plasmodium parasites in the blood of patients with malaria. Artemisinin combination therapies (ACTs) are recommended by the WHO as the first-line treatment for uncomplicated Plasmodium falciparum malaria. Expanding access to ACTs has been a key contributor to the recent success in reducing the global malaria burden. Several hundred million ACT treatment courses are currently distributed in malaria-endemic countries every year. However, recent findings of artemisinin-resistant malaria parasites have alarmed the global health community. According to the WHO, as of February 2015, artemisinin resistance had been confirmed in five countries in Southeast Asia, and its spread is seen only as a question of time by many experts. Such spread—or independent emergence of artemisinin resistance—in other parts of the world would pose a major health crisis because there are currently no other antimalarial drugs that are as effective and well-tolerated as ACTs.
Why Was This Study Done?
Surveillance systems to detect the spread or emergence of artemisinin resistance in malaria-endemic regions are crucial so that control and elimination measures can be undertaken rapidly. Tools for surveillance include clinical studies on parasite clearance from the blood, in vitro parasite testing for artemisinin sensitivity in the laboratory, and assessment of molecular markers. (Tests based on molecular markers of resistance are being developed based on the 2013 discovery of artemisinin-resistance mutations in a parasite gene called Kelch 13 that are associated with delayed parasite clearance after ACT treatment.) The best description of parasite clearance in the individual patient is the parasite half-life, which requires frequent blood sampling. Since this is difficult to do in resource-limited settings, the WHO uses the following working definition: artemisinin resistance in a population is suspected if more than 10% of patients are still carrying parasites three days after the start of ACT treatment. The researchers conducted this study to see how well this working definition matches actual data from individual patients in areas with artemisinin-resistant P. falciparum parasites.
What Did the Researchers Do and Find?
The researchers used data on blood samples from 1,518 patients collected along the Thai-Myanmar and Thai-Cambodian borders (where resistance first developed) from 2001 to 2012. All patients had been treated with ACT, and the time it took for the drugs to kill half of the parasites in their blood (the parasite half-life) had been measured. When the researchers used mathematical models to analyse these parasite half-life assessments after ACT treatment, they found that a model that assumed two different parasite subpopulations best explained the actual data. One of these subpopulations—and the only one present in the early years before resistance developed—had a shorter parasite half-life of about 3 hours (equivalent to sensitive parasites that still are killed efficiently by the ACT drugs). The second population began to appear around 2008 and had a longer half-life of about 6.5 hours, equivalent to parasites that had acquired resistance to artemisinin, and consistent with measurements of Kelch 13–mutant parasites. The parasite half-life predicted the likelihood of an artemisinin-resistant infection for individual patients, but this was influenced by how common resistance was in that area: the critical half-life varied between 3.5 hours (in areas where resistance is rare) and 5.5 hours (in areas where resistance is common). This means that there is not a universal cut-off value in parasite half-life to denote an infection as ‘sensitive’ or ‘resistant’ because intermediate half-lives can be part of the lower tail end of the resistant parasite half-life distribution or the top-end of the sensitive one. The model the researchers developed can estimate the background proportion of resistant infections, and this can then be used to calculate the probability for an individual infection to belong to a resistant or sensitive parasite half-life distribution, thereby providing a more accurate assessment of resistance. Taking this into account, the researchers found that the current WHO 'day-3' cut-off value of 10% predicts the potential presence of artemisinin-resistant infections in most but not all scenarios.
What Do these Findings Mean?
The findings are relevant to the situation in Southeast Asia, where malaria is less common than in so-called high-transmission areas, including much of Sub-Saharan Africa. Whether the model is useful for resistance surveillance in high-transmission areas will need further study. Nonetheless, the results suggest that a model that analyses data on parasite half-life as distributions of artemisinin-sensitive and artemisinin-resistant populations is a useful tool for surveillance of artemisinin resistance in a geographical area. The model also measures the probability that a parasite strain with a given parasite half-life is resistant to artemisinin, which should help with the evaluation of other surveillance methods, including those based on Kelch 13 mutations and possibly other molecular markers. The WHO—recommended definition of suspected artemisinin resistance in cases where more than 10% of patients still carry parasites on the third day of treatment is useful, but, according to this study, including the initial parasite load (i.e., the number of parasites in a patient’s blood before the start of treatment) would make it more informative. In addition, as the ‘day-3’ cut-off method lacks accuracy in predicting the actual proportion of artemisinin-resistant parasites, a positive result should be followed by a more detailed assessment.
Additional Information
This list of resources contains links that can be accessed when viewing the PDF on a device or via the online version of the article at The WHO issues regular updates about the status of artemisinin-resistant malariaThe WHO provides a list of questions and answers about artemisinin-resistant malariaWikipedia provides information on artemisinin (note that Wikipedia is a free online encyclopedia that anyone can edit; available in several languages)The Bill & Melinda Gates Foundation describes its global strategy to fight and eradicate malaria in the face of artemisinin resistanceThe Mahidol Oxford Research Unit, where several of the study’s authors work, has pages about malaria, including information about treatments and drug-resistance
PMCID: PMC4412633  PMID: 25919029
9.  Pregnant migrant and refugee women’s perceptions of mental illness on the Thai-Myanmar border: a qualitative study 
Mental illness is a significant contributor to the global burden of disease, with prevalence highest in low- and middle-income countries. Rates are high in women of childbearing age, especially during pregnancy and the first year post-partum. Migrant and refugee populations are at risk of developing mental illness due to the multiple stressors associated with migration. The Thai-Myanmar border area is home to large populations of migrants and refugees as a result of long-standing conflict, poverty and unemployment in Myanmar. This study aims to explore perceptions of mental illness among pregnant migrants and refugees and antenatal clinic staff living and working along the Thai-Myanmar border.
Thirteen focus group discussions were conducted with pregnant migrants, pregnant refugees and antenatal clinic staff. Focus groups were held in one large refugee camp and two migrant health clinics along the Thai-Myanmar border. Thematic analysis was used to identify and code themes emerging from the data.
A total of 92 pregnant women and 24 antenatal clinic staff participated. Discussions centered around five main themes: symptoms of mental illness; causes of mental illness; suicide; mental illness during pregnancy and the post-partum period; and managing mental illness. Symptoms of mental illness included emotional disturbances, somatic symptoms and socially inappropriate behavior. The main causes were described as current economic and family-related difficulties. Suicide was frequently attributed to shame. Mental illness was thought to be more common during and following pregnancy due to a lack of family support and worries about the future. Talking to family and friends, medication and hospitalization were suggested as means of helping those suffering from mental illness.
Mental illness was recognized as a concept by the majority of participants and there was a general willingness to discuss various aspects of it. More formal and systematic training including the development of assessment tools in the local languages would enable better ascertainment and treatment of mental illness in this population.
PMCID: PMC4464696  PMID: 25884681
Migration; Migrant; Refugee; Pregnancy; Mental health; Qualitative; Myanmar
10.  A tool to improve competence in the management of emergency patients by rural clinic health workers: a pilot assessment on the Thai-Myanmar border 
Conflict and Health  2015;9:11.
Shoklo Malaria Research Unit has been providing health care in remote clinics on the Thai-Myanmar border to refugee and migrant populations since 1986 and 1995, respectively. Clinics are staffed by local health workers with a variety of training and experience. The need for a tool to improve the competence of local health workers in basic emergency assessment and management was recognised by medical faculty after observing the case mix seen at the clinic and reviewing the teaching programme that had been delivered in the past year (Jan-13 to March-14).
To pilot the development and evaluation of a simple teaching tool to improve competence in the assessment and management of acutely unwell patients by local health workers that can be delivered onsite with minimal resources.
A structured approach to common emergencies presenting to rural clinics and utilizing equipment available in the clinics was developed. A prospective repeated-measures observed structured clinical examination (OSCE) assessment design was used to score participants in their competence to assess and manage a scenario based ‘emergency patient’ at baseline, immediately post-course, and 8 weeks after the delivery of the teaching course. The assessment was conducted at 3 clinic sites and staff participation was voluntary. Participants filled out questionnaires on their confidence with different scenario based emergency patients.
All staff who underwent the baseline assessment failed to carry out the essential steps in initial emergency assessment and management of an unconscious patient scenario. Following delivery of the teaching session, all groups showed improved competence in both objective assessment and subjective confidence levels.
Structured and practical teaching and learning with minimal theory in this resource limited setting had a positive short-term effect on the competence of individual staff to carry out an initial assessment and manage an acutely unwell patient. Health-worker confidence likewise improved. Workplace assessments are needed to determine if this type of skills training impacts upon mortality or near miss mortality patients at the clinic.
Electronic supplementary material
The online version of this article (doi:10.1186/s13031-015-0041-x) contains supplementary material, which is available to authorized users.
PMCID: PMC4395965  PMID: 25873993
Medical emergencies; Training; Paramedical staff; Low resource setting; Knowledge; Confidence
11.  Spread of artemisinin-resistant Plasmodium falciparum in Myanmar: a cross-sectional survey of the K13 molecular marker 
The Lancet. Infectious Diseases  2015;15(4):415-421.
Emergence of artemisinin resistance in southeast Asia poses a serious threat to the global control of Plasmodium falciparum malaria. Discovery of the K13 marker has transformed approaches to the monitoring of artemisinin resistance, allowing introduction of molecular surveillance in remote areas through analysis of DNA. We aimed to assess the spread of artemisinin-resistant P falciparum in Myanmar by determining the relative prevalence of P falciparum parasites carrying K13-propeller mutations.
We did this cross-sectional survey at malaria treatment centres at 55 sites in ten administrative regions in Myanmar, and in relevant border regions in Thailand and Bangladesh, between January, 2013, and September, 2014. K13 sequences from P falciparum infections were obtained mainly by passive case detection. We entered data into two geostatistical models to produce predictive maps of the estimated prevalence of mutations of the K13 propeller region across Myanmar.
Overall, 371 (39%) of 940 samples carried a K13-propeller mutation. We recorded 26 different mutations, including nine mutations not described previously in southeast Asia. In seven (70%) of the ten administrative regions of Myanmar, the combined K13-mutation prevalence was more than 20%. Geospatial mapping showed that the overall prevalence of K13 mutations exceeded 10% in much of the east and north of the country. In Homalin, Sagaing Region, 25 km from the Indian border, 21 (47%) of 45 parasite samples carried K13-propeller mutations.
Artemisinin resistance extends across much of Myanmar. We recorded P falciparum parasites carrying K13-propeller mutations at high prevalence next to the northwestern border with India. Appropriate therapeutic regimens should be tested urgently and implemented comprehensively if spread of artemisinin resistance to other regions is to be avoided.
Wellcome Trust–Mahidol University–Oxford Tropical Medicine Research Programme and the Bill & Melinda Gates Foundation.
PMCID: PMC4374103  PMID: 25704894
12.  Suitability of Capillary Blood for Quantitative Assessment of G6PD Activity and Performances of G6PD Point-of-Care Tests 
The use of primaquine and other 8-aminoquinolines for malaria elimination is hampered by, among other factors, the limited availability of point-of-care tests for the diagnosis of glucose-6-phosphate dehydrogenase (G6PD) deficiency. Historically, the most used source of blood for G6PD analyses is venous blood, whereas diagnostic devices used in the field require the use of capillary blood; data have shown that the two sources of blood often differ with respect to hemoglobin concentration and number of red blood cells. Therefore, we have analyzed, in both capillary and venous blood drawn from the same healthy donors, the correlation of G6PD activity assessed by two qualitative tests (the Fluorescent Spot test and the CareStart test) with the gold standard quantitative spectrophotometric assay. Results obtained on 150 subjects with normal, intermediate, and deficient G6PD phenotypes show that, although differences exist between the aforementioned characteristics in capillary and venous blood, these do not impact on the quantitative assessment of G6PD activity after corrected for hemoglobin concentration or red blood cell count. Furthermore, we have assessed the sensitivity and specificity of the two qualitative tests against the gold standard spectrophotometric assay at different activity thresholds of residual enzymatic activity in both blood sources.
PMCID: PMC4385780  PMID: 25646252
13.  Modeling the Dynamics of Plasmodium vivax Infection and Hypnozoite Reactivation In Vivo 
PLoS Neglected Tropical Diseases  2015;9(3):e0003595.
The dynamics of Plasmodium vivax infection is characterized by reactivation of hypnozoites at varying time intervals. The relative contribution of new P. vivax infection and reactivation of dormant liver stage hypnozoites to initiation of blood stage infection is unclear. In this study, we investigate the contribution of new inoculations of P. vivax sporozoites to primary infection versus reactivation of hypnozoites by modeling the dynamics of P. vivax infection in Thailand in patients receiving treatment for either blood stage infection alone (chloroquine), or the blood and liver stages of infection (chloroquine + primaquine). In addition, we also analysed rates of infection in a study in Papua New Guinea (PNG) where patients were treated with either artesunate, or artesunate + primaquine. Our results show that up to 96% of the P. vivax infection is due to hypnozoite reactivation in individuals living in endemic areas in Thailand. Similar analysis revealed the around 70% of infections in the PNG cohort were due to hypnozoite reactivation. We show how the age of the cohort, primaquine drug failure, and seasonality may affect estimates of the ratio of primary P. vivax infection to hypnozoite reactivation. Modeling of P. vivax primary infection and hypnozoite reactivation provides important insights into infection dynamics, and suggests that 90–96% of blood stage infections arise from hypnozoite reactivation. Major differences in infection kinetics between Thailand and PNG suggest the likelihood of drug failure in PNG.
Author Summary
Plasmodium vivax is one of two major parasite species causing human disease. This parasite can lie dormant in the liver as a hypnozoite, before later reactivating to cause blood-stage infection. Treatment to eliminate the dormant hypnozoite stage relies mostly on a single drug—primaquine. Understanding the rate of primary infection versus hypnozoite reactivation is important to understanding primaquine efficacy and drug resistance, as well as the development of new drugs targeting hypnozoites. Here we use mathematical modeling to analyse data from two clinical cohorts and show that up to 96% of infections may be caused by hypnozoite reactivation. We also use modeling to understand the impact of drug resistance, seasonal infection and subject age.
PMCID: PMC4364305  PMID: 25780913
14.  Intrahost Selection of Plasmodium falciparum pfmdr1 Alleles after Antimalarial Treatment on the Northwestern Border of Thailand 
The Journal of infectious diseases  2006;195(1):134-141.
Increased pfmdr1 copy number is associated with reduced susceptibility to structurally unrelated antimalarial drugs. We assessed how administration of different antimalarial drugs altered pfmdr1 polymorphism in parasites from patients who experienced treatment failure.
In studies conducted on the northwestern border of Thailand, amplifications and single-nucleotide polymorphisms in pfmdr1 were compared before and after antimalarial drug treatment.
Intrahost changes in pfmdr1 copy number were observed in 20% (26/132) of patients with recurrent infections. Among infections that recrudesced after mefloquine-containing regimens, increases in pfmdr1 copy number occurred in 68% (95% confidence interval [CI], 46%–85%), and decreases occurred in 2% (95% CI, 0.4%–11%) of isolates; corresponding proportions after artemether-lumefantrine were 25% (2/8) and 11% (2/19); after quinine, 50% (1/2) and 40% (4/10); and after artemisinins alone, 0% (0/10) and 19% (3/16) of isolates (overall P < .001).
Intrahost selection based on pfmdr1 copy number occurs frequently in parasite populations within individual patients. Amplification confers multidrug resistance but probably imposes a significant fitness cost to the parasites.
PMCID: PMC4337981  PMID: 17152017
15.  Molecular and Pharmacological Determinants of the Therapeutic Response to Artemether-Lumefantrine in Multidrug-Resistant Plasmodium falciparum Malaria 
Our study examined the relative contributions of host, pharmacokinetic, and parasitological factors in determining the therapeutic response to artemether-lumefantrine (AL).
On the northwest border of Thailand, patients with uncomplicated Plasmodium falciparum malaria were enrolled in prospective studies of AL treatment (4- or 6-dose regimens) and followed up for 42 days. Plasma lumefantrine concentrations were measured by high performance liquid chromatography; malaria parasite pfmdr1 copy number was quantified using a real-time polymerase chain reaction assay (PCR), and in vitro drug susceptibility was tested.
All treatments resulted in a rapid clinical response and were well tolerated. PCR-corrected failure rates at day 42 were 13% (95% confidence interval [CI], 9.6%–17%) for the 4-dose regimen and 3.2% (95% CI, 1.8%–4.6%) for the 6-dose regimen. Increased pfmdr1 copy number was associated with a 2-fold (95% CI, 1.8–2.4-fold) increase in lumefantrine inhibitory concentration50 (P = .001) and an adjusted hazard ratio for risk of treatment failure following completion of a 4-dose regimen, but not a 6-dose regimen, of 4.0 (95% CI, 1.4–11; P = .008). Patients who had lumefantrine levels below 175 ng/mL on day 7 were more likely to experience recrudescence by day 42 (adjusted hazard ratio, 17; 95% CI, 5.5–53), allowing prediction of treatment failure with 75% sensitivity and 84% specificity. The 6-dose regimen ensured that therapeutic levels were achieved in 91% of treated patients.
The lumefantrine plasma concentration profile is the main determinant of efficacy of artemether-lumefantrine. Amplification in pfmdr1 determines lumefantrine susceptibility and, therefore, treatment responses when plasma lumefantrine levels are subtherapeutic.
PMCID: PMC4337983  PMID: 16652314
The factors contributing to anemia in falciparum malaria were characterized in 4,007 prospectively studied patients on the western border of Thailand. Of these, 727 patients (18%) presented with anemia (haematocrit < 30%), and 1% (55 of 5,253) required blood transfusion. The following were found to be independent risk factors for anemia at admission: age < 5 years, a palpable spleen, a palpable liver, recrudescent infections, being female, a prolonged history of illness (> 2 days) before admission, and pure Plasmodium falciparum infections rather than mixed P. falciparum and Plasmodium vivax infections. The mean maximum fractional fall in hematocrit after antimalarial treatment was 14.1% of the baseline value (95% confidence interval [CI], 13.6–14.6). This reduction was significantly greater in young children (aged < 5 years) and in patients with a prolonged illness, high parasitemia, or delayed parasite clearance. Loss of parasitized erythrocytes accounted for < 10% of overall red blood cell loss. Hematological recovery was usually complete within 6 weeks, but it was slower in patients who were anemic at admission (adjusted hazards ratio [AHR], 1.9, 95% CI, 1.5–2.3), and those whose infections recrudesced (AHR, 1.2, 95% CI, 1.01–1.5). Half the patients with treatment failure were anemic at 6 weeks compared with 19% of successfully treated patients (relative risk, 2.8, 95% CI, 2.0–3.8). Patients coinfected with P. vivax (16% of the total) were 1.8 (95% CI, 1.2–2.6) times less likely to become anemic and recovered 1.3 (95% CI, 1.0–1.5) times faster than those with P. falciparum only. Anemia is related to drug resistance and treatment failure in uncomplicated malaria. Children aged < 5 years of age were more likely than older children or adults to become anemic. Coinfection with P. vivax attenuates the anemia of falciparum malaria, presumably by modifying the severity of the infection.
PMCID: PMC4337986  PMID: 11716124
17.  Mefloquine resistance in Plasmodium falciparum and increased pfmdr1 gene copy number 
Lancet  2004;364(9432):438-447.
The borders of Thailand harbour the world’s most multidrug resistant Plasmodium falciparum parasites. In 1984 mefloquine was introduced as treatment for uncomplicated falciparum malaria, but substantial resistance developed within 6 years. A combination of artesunate with mefloquine now cures more than 95% of acute infections. For both treatment regimens, the underlying mechanisms of resistance are not known.
The relation between polymorphisms in the P falciparum multidrug resistant gene 1 (pfmdr1) and the in-vitro and in-vivo responses to mefloquine were assessed in 618 samples from patients with falciparum malaria studied prospectively over 12 years. pfmdr1 copy number was assessed by a robust real-time PCR assay. Single nucleotide polymorphisms of pfmdr1, P falciparum chloroquine resistance transporter gene (pfcrt) and P falciparum Ca2+ ATPase gene (pfATP6) were assessed by PCR-restriction fragment length polymorphism.
Increased copy number of pfmdr1 was the most important determinant of in-vitro and in-vivo resistance to mefloquine, and also to reduced artesunate sensitivity in vitro. In a Cox regression model with control for known confounders, increased pfmdr1 copy number was associated with an attributable hazard ratio (AHR) for treatment failure of 6·3 (95% CI 2·9–13·8, p<0·001) after mefloquine monotherapy and 5·4 (2·0-14·6, p=0·001) after artesunate-mefloquine therapy. Single nucleotide polymorphisms in pfmdr1 were associated with increased mefloquine susceptibility in vitro, but not in vivo.
Amplification in pfmdr1 is the main cause of resistance to mefloquine in falciparum malaria.
Relevance to practice
Multidrug resistant P falciparum malaria is common in southeast Asia, but difficult to identify and treat. Genes that encode parasite transport proteins maybe involved in export of drugs and so cause resistance. In this study we show that increase in copy number of pfmdr1, a gene encoding a parasite transport protein, is the best overall predictor of treatment failure with mefloquine. Increase in pfmdr1 copy number predicts failure even after chemotherapy with the highly effective combination of mefloquine and 3 days’ artesunate. Monitoring of pfmdr1 copy number will be useful in epidemiological surveys of drug resistance in P falciparum, and potentially for predicting treatment failure in individual patients.
PMCID: PMC4337987  PMID: 15288742
18.  Quality of intrapartum care by skilled birth attendants in a refugee clinic on the Thai-Myanmar border: a survey using WHO Safe Motherhood Needs Assessment 
Increasing the number of women birthing with skilled birth attendants (SBAs) as one of the strategies to reduce maternal mortality and morbidity must be partnered with a minimum standard of care. This manuscript describes the quality of intrapartum care provided by SBAs in Mae La camp, a low resource, protracted refugee context on the Thai-Myanmar border.
In the obstetric department of Shoklo Malaria Research Unit (SMRU) the standardized WHO Safe Motherhood Needs Assessment tool was adapted to the setting and used: to assess the facility; interview SBAs; collect data from maternal records during a one year period (August 2007 – 2008); and observe practice during labour and childbirth.
The facility assessment recorded no ‘out of stock’ or ‘out of date’ drugs and supplies, equipment was in operating order and necessary infrastructure e.g. a stand-by emergency car, was present. Syphilis testing was not available. SBA interviews established that danger signs and symptoms were recognized except for sepsis and endometritis. All SBAs acknowledged receiving theoretical and ‘hands-on’ training and regularly attended deliveries. Scores for the essential elements of antenatal care from maternal records were high (>90%) e.g. providing supplements, recording risk factors as well as regular and correct partogram use. Observed good clinical practice included: presence of a support person; active management of third stage; post-partum monitoring; and immediate and correct neonatal care. Observed incorrect practice included: improper controlled cord traction; inadequate hand washing; an episiotomy rate in nulliparous women 49% (34/70) and low rates 30% (6/20) of newborn monitoring in the first hours following birth. Overall observed complications during labour and birth were low with post-partum haemorrhage being the most common in which case the SBAs followed the protocol but were slow to recognize severity and take action.
In the clinic of SMRU in Mae La refugee camp, SBAs were able to comply with evidence-based guidelines but support to improve quality of care in specific areas is required. The structure of the WHO Safe Motherhood Needs Assessment allowed significant insights into the quality of intrapartum care particularly through direct observation, identifying a clear pathway for quality improvement.
Electronic supplementary material
The online version of this article (doi:10.1186/s12884-015-0444-0) contains supplementary material, which is available to authorized users.
PMCID: PMC4332741  PMID: 25652646
Skilled birth attendant; Health care quality assessment; Pregnancy and childbirth care; Safe motherhood; Refugee setting
19.  Spiroindolone KAE609 for Falciparum and Vivax Malaria 
The New England journal of medicine  2014;371(5):403-410.
KAE609 (cipargamin; formerly NITD609, Novartis Institute for Tropical Diseases) is a new synthetic antimalarial spiroindolone analogue with potent, dose-dependent antimalarial activity against asexual and sexual stages of Plasmodium falciparum.
We conducted a phase 2, open-label study at three centers in Thailand to assess the antimalarial efficacy, safety, and adverse-event profile of KAE609, at a dose of 30 mg per day for 3 days, in two sequential cohorts of adults with uncomplicated P. vivax malaria (10 patients) or P. falciparum malaria (11). The primary end point was the parasite clearance time.
The median parasite clearance time was 12 hours in each cohort (interquartile range, 8 to 16 hours in patients with P. vivax malaria and 10 to 16 hours in those with P. falciparum malaria). The median half-lives for parasite clearance were 0.95 hours (range, 0.68 to 2.01; interquartile range, 0.85 to 1.14) in the patients with P. vivax malaria and 0.90 hours (range, 0.68 to 1.64; interquartile range, 0.78 to 1.07) in those with P. falciparum malaria. By comparison, only 19 of 5076 patients with P. falciparum malaria (<1%) who were treated with oral artesunate in Southeast Asia had a parasite clearance half-life of less than 1 hour. Adverse events were reported in 14 patients (67%), with nausea being the most common. The adverse events were generally mild and did not lead to any discontinuations of the drug. The mean terminal half-life for the elimination of KAE609 was 20.8 hours (range, 11.3 to 37.6), supporting a once-daily oral dosing regimen.
KAE609, at dose of 30 mg daily for 3 days, cleared parasitemia rapidly in adults with uncomplicated P. vivax or P. falciparum malaria. (Funded by Novartis and others; number, NCT01524341.)
PMCID: PMC4143746  PMID: 25075833
20.  Selection of Drug Resistance-Mediating Plasmodium falciparum Genetic Polymorphisms by Seasonal Malaria Chemoprevention in Burkina Faso 
Seasonal malaria chemoprevention (SMC), with regular use of amodiaquine plus sulfadoxine-pyrimethamine (AQ/SP) during the transmission season, is now a standard malaria control measure in the Sahel subregion of Africa. Another strategy under study is SMC with dihydroartemisinin plus piperaquine (DP). Plasmodium falciparum single nucleotide polymorphisms (SNPs) in P. falciparum crt (pfcrt), pfmdr1, pfdhfr, and pfdhps are associated with decreased response to aminoquinoline and antifolate antimalarials and are selected by use of these drugs. To characterize selection by SMC of key polymorphisms, we assessed 13 SNPs in P. falciparum isolated from children aged 3 to 59 months living in southwestern Burkina Faso and randomized to receive monthly DP or AQ/SP for 3 months in 2009. We compared SNP prevalence before the onset of SMC and 1 month after the third treatment in P. falciparum PCR-positive samples from 120 randomly selected children from each treatment arm and an additional 120 randomly selected children from a control group that did not receive SMC. The prevalence of relevant mutations was increased after SMC with AQ/SP. Significant selection was seen for pfcrt 76T (68.5% to 83.0%, P = 0.04), pfdhfr 59R (54.8% to 83.3%, P = 0.0002), and pfdhfr 108N (55.0% to 87.2%, P = 0.0001), with trends toward selection of pfmdr1 86Y, pfdhfr 51I, and pfdhps 437G. After SMC with DP, only borderline selection of wild-type pfmdr1 D1246 (mutant; 7.7% to 0%, P = 0.05) was seen. In contrast to AQ/SP, SMC with DP did not clearly select for known resistance-mediating polymorphisms. SMC with AQ/SP, but not DP, may hasten the development of resistance to components of this regimen. (This study has been registered at under registration no. NCT00941785.)
PMCID: PMC4068591  PMID: 24733476
21.  Preclinical Assessment of Viral Vectored and Protein Vaccines Targeting the Duffy-Binding Protein Region II of Plasmodium Vivax 
Malaria vaccine development has largely focused on Plasmodium falciparum; however, a reawakening to the importance of Plasmodium vivax has spurred efforts to develop vaccines against this difficult to treat and at times severe form of relapsing malaria, which constitutes a significant proportion of human malaria cases worldwide. The almost complete dependence of P. vivax red blood cell invasion on the interaction of the P. vivax Duffy-binding protein region II (PvDBP_RII) with the human Duffy antigen receptor for chemokines (DARC) makes this antigen an attractive vaccine candidate against blood-stage P. vivax. Here, we generated both preclinical and clinically compatible adenoviral and poxviral vectored vaccine candidates expressing the Salvador I allele of PvDBP_RII – including human adenovirus serotype 5 (HAdV5), chimpanzee adenovirus serotype 63 (ChAd63), and modified vaccinia virus Ankara (MVA) vectors. We report on the antibody and T cell immunogenicity of these vaccines in mice or rabbits, either used alone in a viral vectored prime-boost regime or in “mixed-modality” adenovirus prime – protein-in-­adjuvant boost regimes (using a recombinant PvDBP_RII protein antigen formulated in Montanide®ISA720 or Abisco®100 adjuvants). Antibodies induced by these regimes were found to bind to native parasite antigen from P. vivax infected Thai patients and were capable of inhibiting the binding of PvDBP_RII to its receptor DARC using an in vitro binding inhibition assay. In recent years, recombinant ChAd63 and MVA vectors have been quickly translated into human clinical trials for numerous antigens from P. falciparum as well as a growing number of other pathogens. The vectors reported here are immunogenic in small animals, elicit antibodies against PvDBP_RII, and have recently entered clinical trials, which will provide the first assessment of the safety and immunogenicity of the PvDBP_RII antigen in humans.
PMCID: PMC4495344  PMID: 26217340
malaria; vaccine; Plasmodium vivax; blood-stage; adenovirus; poxvirus; MVA; Duffy-binding protein
22.  Characterization of G6PD Genotypes and Phenotypes on the Northwestern Thailand-Myanmar Border 
PLoS ONE  2014;9(12):e116063.
Mutations in the glucose-6-phosphate dehydrogenase (G6PD) gene result in red blood cells with increased susceptibility to oxidative damage. Significant haemolysis can be caused by primaquine and other 8-aminoquinoline antimalarials used for the radical treatment of Plasmodium vivax malaria. The distribution and phenotypes of mutations causing G6PD deficiency in the male population of migrants and refugees in a malaria endemic region on the Thailand-Myanmar border were characterized. Blood samples for G6PD fluorescent spot test (FST), G6PD genotyping, and malaria testing were taken from 504 unrelated males of Karen and Burman ethnicities presenting to the outpatient clinics. The overall frequency of G6PD deficiency by the FST was 13.7%. Among the deficient subjects, almost 90% had the Mahidol variant (487G>A) genotype. The remaining subjects had Chinese-4 (392G>T), Viangchan (871G>A), Açores (595A>G), Seattle (844G>C) and Mediterranean (563C>T) variants. Quantification of G6PD activity was performed using a modification of the standard spectrophotometric assay on a subset of 24 samples with Mahidol, Viangchan, Seattle and Chinese-4 mutations; all samples showed a residual enzymatic activity below 10% of normal and were diagnosed correctly by the FST. Further studies are needed to characterise the haemolytic risk of using 8-aminoquinolines in patients with these genotypes.
PMCID: PMC4275285  PMID: 25536053
23.  Pooled Sequencing and Rare Variant Association Tests for Identifying the Determinants of Emerging Drug Resistance in Malaria Parasites 
Molecular Biology and Evolution  2014;32(4):1080-1090.
We explored the potential of pooled sequencing to swiftly and economically identify selective sweeps due to emerging artemisinin (ART) resistance in a South-East Asian malaria parasite population. ART resistance is defined by slow parasite clearance from the blood of ART-treated patients and mutations in the kelch gene (chr. 13) have been strongly implicated to play a role. We constructed triplicate pools of 70 slow-clearing (resistant) and 70 fast-clearing (sensitive) infections collected from the Thai–Myanmar border and sequenced these to high (∼150-fold) read depth. Allele frequency estimates from pools showed almost perfect correlation (Lin’s concordance = 0.98) with allele frequencies at 93 single nucleotide polymorphisms measured directly from individual infections, giving us confidence in the accuracy of this approach. By mapping genome-wide divergence (FST) between pools of drug-resistant and drug-sensitive parasites, we identified two large (>150 kb) regions (on chrs. 13 and 14) and 17 smaller candidate genome regions. To identify individual genes within these genome regions, we resequenced an additional 38 parasite genomes (16 slow and 22 fast-clearing) and performed rare variant association tests. These confirmed kelch as a major molecular marker for ART resistance (P = 6.03 × 10−6). This two-tier approach is powerful because pooled sequencing rapidly narrows down genome regions of interest, while targeted rare variant association testing within these regions can pinpoint the genetic basis of resistance. We show that our approach is robust to recurrent mutation and the generation of soft selective sweeps, which are predicted to be common in pathogen populations with large effective population sizes, and may confound more traditional gene mapping approaches.
PMCID: PMC4379400  PMID: 25534029
drug resistance; malaria; pooled sequencing; rare variants
24.  Pregnancy Outcome in Relation to Treatment of Murine Typhus and Scrub Typhus Infection: A Fever Cohort and a Case Series Analysis 
There is a paucity of published reports on pregnancy outcome following scrub and murine typhus despite these infections being leading causes of undifferentiated fever in Asia. This study aimed to relate pregnancy outcome with treatment of typhus.
Methodology/Principal Findings
Data were analyzed from: i) pregnant women with a diagnosis of scrub and/or murine typhus from a fever cohort studies; ii) case series of published studies in PubMed using the search terms “scrub typhus” (ST), “murine typhus” (MT), “Orientia tsutsugamushi”, “Rickettsia tsutsugamushi”, “Rickettsia typhi”, “rickettsiae”, “typhus”, or “rickettsiosis”; and “pregnancy”, until February 2014 and iii) an unpublished case series. Fever clearance time (FCT) and pregnancy outcome (miscarriage and delivery) were compared to treatment. Poor neonatal outcome was a composite measure for pregnancies sustained to 28 weeks or more of gestation ending in stillbirth, preterm birth, or delivery of a growth restricted or low birth weight newborn.
There were 26 women in the fever cohort. MT and ST were clinically indistinguishable apart from two ST patients with eschars. FCTs (median [range] hours) were 25 [16–42] for azithromycin (n = 5), 34 [20–53] for antimalarials (n = 5) and 92 [6–260] for other antibiotics/supportive therapy (n = 16). There were 36.4% (8/22) with a poor neonatal outcome.
In 18 years, 97 pregnancies were collated, 82 with known outcomes, including two maternal deaths. Proportions of miscarriage 17.3% (14/81) and poor neonatal outcomes 41.8% (28/67) were high, increasing with longer FCTs (p = 0.050, linear trend). Use of azithromycin was not significantly associated with improved neonatal outcomes (p = 0.610)
The published ST and MT world literature amounts to less than 100 pregnancies due to under recognition and under diagnosis. Evidence supporting the most commonly used treatment, azithromycin, is weak. Collaborative, prospective clinical trials in pregnant women are urgently required to reduce the burden of adverse maternal and newborn outcomes and to determine the safety and efficacy of antimicrobial treatment.
Author Summary
Typhus is an under-recognised and under-studied public health problem in Asia. In rural areas of Southeast Asia murine and scrub typhus are probably the most common treatable cause of fever. The estimated number of scrub typhus cases in Southeast Asia, more than 1 million yearly, results in approximately 50–80,000 deaths per year. Treatment delays due to lack of appropriate diagnostics and lack of awareness lead to a substantial health and economic impact in the one of the world's most densely populated regions. Only 97 cases in pregnancy are available from the published world literature over the past 18 years. Only 82 of these had known outcomes, including two maternal deaths. The proportion of poor neonatal outcome including stillbirth, prematurity and low birth weight was high occurring in more than 40% of pregnancies, and higher when the fever clearance time was longer. While poor neonatal outcomes were observed with all antibiotics prescribed, azithromycin appeared to be associated with shorter fever clearance times but this was not statistically significant. Evidence to support the use of azithromycin is weak. The correct antimicrobial or combination for undifferentiated fever in pregnant women in Southeast Asia is unknown.
PMCID: PMC4238995  PMID: 25412503
25.  Characterization of the Commercially-Available Fluorescent Chloroquine-BODIPY Conjugate, LynxTag-CQGREEN, as a Marker for Chloroquine Resistance and Uptake in a 96-Well Plate Assay 
PLoS ONE  2014;9(10):e110800.
Chloroquine was a cheap, extremely effective drug against Plasmodium falciparum until resistance arose. One approach to reversing resistance is the inhibition of chloroquine efflux from its site of action, the parasite digestive vacuole. Chloroquine accumulation studies have traditionally relied on radiolabelled chloroquine, which poses several challenges. There is a need for development of a safe and biologically relevant substitute. We report here a commercially-available green fluorescent chloroquine-BODIPY conjugate, LynxTag-CQGREEN, as a proxy for chloroquine accumulation. This compound localized to the digestive vacuole of the parasite as observed under confocal microscopy, and inhibited growth of chloroquine-sensitive strain 3D7 more extensively than in the resistant strains 7G8 and K1. Microplate reader measurements indicated suppression of LynxTag-CQGREEN efflux after pretreatment of parasites with known reversal agents. Microsomes carrying either sensitive- or resistant-type PfCRT were assayed for uptake; resistant-type PfCRT exhibited increased accumulation of LynxTag-CQGREEN, which was suppressed by pretreatment with known chemosensitizers. Eight laboratory strains and twelve clinical isolates were sequenced for PfCRT and Pgh1 haplotypes previously reported to contribute to drug resistance, and pfmdr1 copy number and chloroquine IC50s were determined. These data were compared with LynxTag-CQGREEN uptake/fluorescence by multiple linear regression to identify genetic correlates of uptake. Uptake of the compound correlated with the logIC50 of chloroquine and, more weakly, a mutation in Pgh1, F1226Y.
PMCID: PMC4208776  PMID: 25343249

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