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1.  Artemether-lumefantrine co-administration with antiretrovirals: population pharmacokinetics and dosing implications 
AIM
Drug–drug interactions between antimalarial and antiretroviral drugs may influence antimalarial treatment outcomes. The aim of this study was to investigate the potential drug–drug interactions between the antimalarial drugs, lumefantrine, artemether and their respective metabolites desbutyl-lumefantrine and dihydroartemisinin, and the HIV drugs efavirenz, nevirapine and lopinavir/ritonavir.
METHOD
Data from two clinical studies, investigating the influence of the HIV drugs efavirenz, nevirapine and lopinavir/ritonavir on the pharmacokinetics of the antimalarial drugs lumefantrine, artemether and their respective metabolites, in HIV infected patients were pooled and analyzed using a non-linear mixed effects modelling approach.
RESULTS
Efavirenz and nevirapine significantly decreased the terminal exposure to lumefantrine (decrease of 69.9% and 25.2%, respectively) while lopinavir/ritonavir substantially increased the exposure (increase of 439%). All antiretroviral drugs decreased the total exposure to dihydroartemisinin (decrease of 71.7%, 41.3% and 59.7% for efavirenz, nevirapine and ritonavir/lopinavir, respectively). Simulations suggest that a substantially increased artemether-lumefantrine dose is required to achieve equivalent exposures when co-administered with efavirenz (250% increase) and nevirapine (75% increase). When co-administered with lopinavir/ritonavir it is unclear if the increased lumefantrine exposure compensates adequately for the reduced dihydroartemisinin exposure and thus whether dose adjustment is required.
CONCLUSION
There are substantial drug interactions between artemether-lumefantrine and efavirenz, nevirapine and ritonavir/lopinavir. Given the readily saturable absorption of lumefantrine, the dose adjustments predicted to be necessary will need to be evaluated prospectively in malaria-HIV co-infected patients.
doi:10.1111/bcp.12529
PMCID: PMC4386948  PMID: 25297720
artemether-lumefantrine; drug–drug interaction; efavirenz; lopinavir/ritonavir; nevirapine; population pharmacokinetics
4.  How to Determine the Accuracy of an Alternative Diagnostic Test when It Is Actually Better than the Reference Tests: A Re-Evaluation of Diagnostic Tests for Scrub Typhus Using Bayesian LCMs 
PLoS ONE  2015;10(5):e0114930.
Background
The indirect immunofluorescence assay (IFA) is considered a reference test for scrub typhus. Recently, the Scrub Typhus Infection Criteria (STIC; a combination of culture, PCR assays and IFA IgM) were proposed as a reference standard for evaluating alternative diagnostic tests. Here, we use Bayesian latent class models (LCMs) to estimate the true accuracy of each diagnostic test, and of STIC, for diagnosing scrub typhus.
Methods/Principal Findings
Data from 161 patients with undifferentiated fever were re-evaluated using Bayesian LCMs. Every patient was evaluated for the presence of an eschar, and tested with blood culture for Orientia tsutsugamushi, three different PCR assays, IFA IgM, and the Panbio IgM immunochromatographic test (ICT). True sensitivity and specificity of culture (24.4% and 100%), 56kDa PCR assay (56.8% and 98.4%), 47kDa PCR assay (63.2% and 96.1%), groEL PCR assay (71.4% and 93.0%), IFA IgM (70.0% and 83.8%), PanBio IgM ICT (72.8% and 96.8%), presence of eschar (42.7% and 98.9%) and STIC (90.5% and 82.5%) estimated by Bayesian LCM were considerably different from those obtained when using STIC as a reference standard. The IgM ICT had comparable sensitivity and significantly higher specificity compared to IFA (p=0.34 and p<0.001, respectively).
Conclusions
The low specificity of STIC was caused by the low specificity of IFA IgM. Neither STIC nor IFA IgM can be used as reference standards against which to evaluate alternative diagnostic tests. Further evaluation of new diagnostic tests should be done with a carefully selected set of diagnostic tests and appropriate statistical models.
doi:10.1371/journal.pone.0114930
PMCID: PMC4449177  PMID: 26024375
5.  Microvascular obstruction and endothelial activation are independently associated with the clinical manifestations of severe falciparum malaria in adults: an observational study 
BMC Medicine  2015;13:122.
Background
Microvascular obstruction and endothelial dysfunction have both been linked to tissue hypoperfusion in falciparum malaria, but their relative contributions to the disease’s pathogenesis and outcome are unknown.
Methods
Microvascular blood flow was quantified in adults with severe falciparum malaria on their admission to hospital; plasma biomarkers of endothelial function were measured simultaneously. The relationship between these indices and the patients’ clinical findings and in-hospital course was examined.
Results
Microvascular obstruction was observed in 119/142 (84 %) patients; a median (interquartile range (IQR)) of 14.9 % (6.6–34.9 %) of capillaries were obstructed in patients that died versus 8.3 % (1.7–26.6 %) in survivors (P = 0.039). The proportion of obstructed capillaries correlated with the estimated parasite biomass (rs = 0.25, P = 0.004) and with plasma lactate (rs = 0.38, P <0.0001), the strongest predictor of death in the series. Plasma angiopoietin-2 (Ang-2) concentrations were markedly elevated suggesting widespread endothelial activation; the median (IQR) Ang-2 concentration was 21.9 ng/mL (13.4–29.4 ng/mL) in patients that died versus 14.9 ng/mL (9.8–29.3 ng/mL) in survivors (P = 0.035). Ang-2 concentrations correlated with estimated parasite biomass (rs = 0.35, P <0.001) and plasma lactate (rs = 0.37, P <0.0001). Microvascular obstruction and Ang-2 concentrations were not significantly correlated with each other (rs = 0.17, P = 0.06), but were independently associated with plasma lactate (P <0.001 and P = 0.002, respectively).
Conclusions
Microvascular obstruction and systemic endothelial activation are independently associated with plasma lactate, the strongest predictor of death in adults with falciparum malaria. This supports the hypothesis that the two processes make an independent contribution to the pathogenesis and clinical manifestations of the disease.
doi:10.1186/s12916-015-0365-9
PMCID: PMC4453275  PMID: 26018532
Falciparum malaria; Microcirculation; Pathophysiology; Endothelial dysfunction
6.  Mortality Attributable to Seasonal Influenza A and B Infections in Thailand, 2005–2009: A Longitudinal Study 
American Journal of Epidemiology  2015;181(11):898-907.
Influenza epidemiology differs substantially in tropical and temperate zones, but estimates of seasonal influenza mortality in developing countries in the tropics are lacking. We aimed to quantify mortality due to seasonal influenza in Thailand, a tropical middle-income country. Time series of polymerase chain reaction–confirmed influenza infections between 2005 and 2009 were constructed from a sentinel surveillance network. These were combined with influenza-like illness data to derive measures of influenza activity and relationships to mortality by using a Bayesian regression framework. We estimated 6.1 (95% credible interval: 0.5, 12.4) annual deaths per 100,000 population attributable to influenza A and B, predominantly in those aged ≥60 years, with the largest contribution from influenza A(H1N1) in 3 out of 4 years. For A(H3N2), the relationship between influenza activity and mortality varied over time. Influenza was associated with increases in deaths classified as resulting from respiratory disease (posterior probability of positive association, 99.8%), cancer (98.6%), renal disease (98.0%), and liver disease (99.2%). No association with circulatory disease mortality was found. Seasonal influenza infections are associated with substantial mortality in Thailand, but evidence for the strong relationship between influenza activity and circulatory disease mortality reported in temperate countries is lacking.
doi:10.1093/aje/kwu360
PMCID: PMC4445392  PMID: 25899091
Bayesian regression; burden; developing country; influenza; middle-income country; mortality; seasonal variation; tropics
7.  Artemether-lumefantrine co-administration with antiretrovirals: population pharmacokinetics and dosing implications 
AIM
Drug–drug interactions between antimalarial and antiretroviral drugs may influence antimalarial treatment outcomes. The aim of this study was to investigate the potential drug–drug interactions between the antimalarial drugs, lumefantrine, artemether and their respective metabolites desbutyl-lumefantrine and dihydroartemisinin, and the HIV drugs efavirenz, nevirapine and lopinavir/ritonavir.
METHOD
Data from two clinical studies, investigating the influence of the HIV drugs efavirenz, nevirapine and lopinavir/ritonavir on the pharmacokinetics of the antimalarial drugs lumefantrine, artemether and their respective metabolites, in HIV infected patients were pooled and analyzed using a non-linear mixed effects modelling approach.
RESULTS
Efavirenz and nevirapine significantly decreased the terminal exposure to lumefantrine (decrease of 69.9% and 25.2%, respectively) while lopinavir/ritonavir substantially increased the exposure (increase of 439%). All antiretroviral drugs decreased the total exposure to dihydroartemisinin (decrease of 71.7%, 41.3% and 59.7% for efavirenz, nevirapine and ritonavir/lopinavir, respectively). Simulations suggest that a substantially increased artemether-lumefantrine dose is required to achieve equivalent exposures when co-administered with efavirenz (250% increase) and nevirapine (75% increase). When co-administered with lopinavir/ritonavir it is unclear if the increased lumefantrine exposure compensates adequately for the reduced dihydroartemisinin exposure and thus whether dose adjustment is required.
CONCLUSION
There are substantial drug interactions between artemether-lumefantrine and efavirenz, nevirapine and ritonavir/lopinavir. Given the readily saturable absorption of lumefantrine, the dose adjustments predicted to be necessary will need to be evaluated prospectively in malaria-HIV co-infected patients.
doi:10.1111/bcp.12529
PMCID: PMC4386948  PMID: 25297720
artemether-lumefantrine; drug–drug interaction; efavirenz; lopinavir/ritonavir; nevirapine; population pharmacokinetics
8.  Evaluating Clinical Trial Designs for Investigational Treatments of Ebola Virus Disease 
PLoS Medicine  2015;12(4):e1001815.
Background
Experimental treatments for Ebola virus disease (EVD) might reduce EVD mortality. There is uncertainty about the ability of different clinical trial designs to identify effective treatments, and about the feasibility of implementing individually randomised controlled trials during an Ebola epidemic.
Methods and Findings
A treatment evaluation programme for use in EVD was devised using a multi-stage approach (MSA) with two or three stages, including both non-randomised and randomised elements. The probabilities of rightly or wrongly recommending the experimental treatment, the required sample size, and the consequences for epidemic outcomes over 100 d under two epidemic scenarios were compared for the MSA, a sequential randomised controlled trial (SRCT) with up to 20 interim analyses, and, as a reference case, a conventional randomised controlled trial (RCT) without interim analyses.
Assuming 50% 14-d survival in the population treated with the current standard of supportive care, all designs had similar probabilities of identifying effective treatments correctly, while the MSA was less likely to recommend treatments that were ineffective. The MSA led to a smaller number of cases receiving ineffective treatments and faster roll-out of highly effective treatments. For less effective treatments, the MSA had a high probability of including an RCT component, leading to a somewhat longer time to roll-out or rejection. Assuming 100 new EVD cases per day, the MSA led to between 6% and 15% greater reductions in epidemic mortality over the first 100 d for highly effective treatments compared to the SRCT. Both the MSA and SRCT led to substantially fewer deaths than a conventional RCT if the tested interventions were either highly effective or harmful. In the proposed MSA, the major threat to the validity of the results of the non-randomised components is that referral patterns, standard of care, or the virus itself may change during the study period in ways that affect mortality. Adverse events are also harder to quantify without a concurrent control group.
Conclusions
The MSA discards ineffective treatments quickly, while reliably providing evidence concerning effective treatments. The MSA is appropriate for the clinical evaluation of EVD treatments.
Ben Cooper and colleagues model different trial designs, including a multi-stage approach that contains non-randomized and randomized elements.
Editors' Summary
Background
The current outbreak of Ebola virus disease (EVD)—a frequently fatal disease that first appeared in human populations in 1976 in remote villages in central Africa—has infected more than 24,000 people and killed more than 10,000 people in Guinea, Sierra Leone, and Liberia since early 2014. Ebola virus is transmitted to people from wild animals and spreads in human populations through direct contact with the bodily fluids (including blood, saliva, and urine) or with the organs of infected people or through contact with bedding and other materials contaminated with bodily fluids. The symptoms of EVD which start 2–21 days after infection, include fever, headache, vomiting, diarrhea, and internal and external bleeding. Infected individuals are not infectious until they develop symptoms but remain infectious as long as their bodily fluids contain virus. There is no proven treatment or vaccine for EVD, but supportive care—given under strict isolation conditions to prevent the spread of the disease to other patients or to healthcare workers—improves survival.
Why Was This Study Done?
Potential treatments for EVD include several antiviral drugs and injections of antibodies against Ebola virus from patients who have survived EVD. Before such therapies can be used clinically, their safety and effectiveness need to be evaluated, but experts disagree about how to undertake this evaluation. Drugs for clinical use are usually evaluated by undertaking a series of clinical trials. A phase I trial establishes the safety of the treatment and how the human body copes with it by giving several healthy volunteers the drug. Next, a phase II trial provides early indications of the drug’s efficacy by giving a few patients the drug. Finally, a large-scale multi-arm phase III randomized controlled trial (RCT) confirms the drug’s efficacy by comparing outcomes in patients randomly chosen to receive the investigational drug or standard care. This evaluation process is very lengthy. Moreover, it is hard to ethically justify undertaking an RCT in which only some patients receive a potentially life-saving drug during an Ebola epidemic. Here, the researchers evaluate a multi-stage approach (MSA) to EVD drug evaluation that comprises a single-arm phase II study followed by one or two phase III trials, one of which may be a sequential RCT (SRCT), a type of RCT that allows for multiple interim analyses, each of which may lead to study termination.
What Did the Researchers Do and Find?
The researchers used analytic methods and computer simulations to compare EVD drug evaluation using the MSA, an SRCT, and a conventional RCT without interim analyses. Specifically, they estimated the probabilities of rightly or wrongly recommending the experimental treatment and the consequences for epidemic outcomes over 100 days for the three approaches. Assuming 50% survival at 14 days after symptom development in patients treated with supportive care only, all three trial designs were equally likely to identify effective treatments, but the MSA was less likely than the other designs to incorrectly recommend an ineffective treatment. Notably, the MSA led to fewer patients receiving ineffective treatments and faster roll-out of highly effective treatments. In an epidemic where 100 new cases occurred per day, for highly effective treatments, the MSA led to between 6% and 15% larger reductions in epidemic mortality over the first 100 days of the epidemic than the SRCT did. Finally, both the MSA and the SRCT led to fewer deaths than the conventional RCT if the tested interventions were either highly effective or harmful.
What Do These Findings Mean?
These findings suggest that for experimental treatments that offer either no clinically significant benefit or large reductions in mortality the MSA can provide useful information about drug effectiveness faster than the other approaches tested. Thus, the MSA has the potential to reduce patient harm and the time to roll-out of an effective treatment for EVD. Although alternative evaluation designs are possible, the researchers suggest that including a non-randomized design in phase II is the quickest way to triage potential treatments and to decide how to test them further. For treatments that show strong evidence of benefit, it might even be possible to recommend the treatment without undertaking an RCT, they suggest. Moreover, for treatments that show only modest benefit in phase II, it should be easier (and more ethical) to set up RCTs to test the treatment further.
Additional Information
Please access these websites via the online version of this summary at http://dx.doi.org/10.1371/journal.pmed.1001815.
The World Health Organization (WHO) provides information about EVD, information about potential EVD therapies, and regular updates on the current EVD epidemic; a summary of the discussion of a WHO Ethics Working Group Meeting on the ethical issues related to study design for EVD drug trials is available; the WHO website also provides information about efforts to control Ebola in the field and personal stories from people who have survived EVD
The UK National Health Service Choices website provides detailed information on EVD
The US Centers for Disease Control and Prevention also provides information about EVD
Wikipedia provides information about adaptive clinical trial design; a Lancet Global Health blog argues why adaptive trial designs should be used to evaluate drugs for the treatment of EVD
doi:10.1371/journal.pmed.1001815
PMCID: PMC4397078  PMID: 25874579
9.  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.
Summary
Background
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.
Methods
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.
Findings
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.
Interpretation
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.
Funding
Wellcome Trust–Mahidol University–Oxford Tropical Medicine Research Programme and the Bill & Melinda Gates Foundation.
doi:10.1016/S1473-3099(15)70032-0
PMCID: PMC4374103  PMID: 25704894
10.  Prognosis of neonatal tetanus in the modern management era: an observational study in 107 Vietnamese infants 
Highlights
•Large contemporary case-series in a setting of improved critical care facilities.•Analysis of variables, some not analysed in previous studies/meta-analyses.•Age and weight were associated with a poor outcome.•Delay in admission to hospital and leukocytosis were also associated with a poor outcome.
Summary
Objectives
Most data regarding the prognosis in neonatal tetanus originate from regions where limited resources have historically impeded management. It is not known whether recent improvements in critical care facilities in many low- and middle-income countries have affected indicators of a poor prognosis in neonatal tetanus. We aimed to determine the factors associated with worse outcomes in a Vietnamese hospital with neonatal intensive care facilities.
Methods
Data were collected from 107 cases of neonatal tetanus. Clinical features on admission were analyzed against mortality and a combined endpoint of ‘death or prolonged hospital stay’.
Results
Multivariable analysis showed that only younger age (odds ratio (OR) for mortality 0.69, 95% confidence interval (CI) 0.48–0.98) and lower weight (OR for mortality 0.06, 95% CI 0.01–0.54) were significantly associated with both the combined endpoint and death. A shorter period of onset (OR 0.94, 95% CI 0.88–0.99), raised white cell count (OR 1.17, 95% CI 1.02–1.35), and time between first symptom and admission (OR 3.77, 95% CI 1.14–12.51) were also indicators of mortality.
Conclusions
Risk factors for a poor outcome in neonatal tetanus in a setting with critical care facilities include younger age, lower weight, delay in admission, and leukocytosis.
doi:10.1016/j.ijid.2014.12.011
PMCID: PMC4396701  PMID: 25499039
Neonatal tetanus; Management; Prognosis; Outcome
11.  Public Awareness of Melioidosis in Thailand and Potential Use of Video Clips as Educational Tools 
PLoS ONE  2015;10(3):e0121311.
Background
Melioidosis causes more than 1,000 deaths in Thailand each year. Infection occurs via inoculation, ingestion or inhalation of the causative organism (Burkholderia pseuodmallei) present in soil and water. Here, we evaluated public awareness of melioidosis using a combination of population-based questionnaire, a public engagement campaign to obtain video clips made by the public, and viewpoints on these video clips as potential educational tools about the disease and its prevention.
Methods
A questionnaire was developed to evaluate public awareness of melioidosis, and knowledge about its prevention. From 1 March to 31 April 2012, the questionnaire was delivered to five randomly selected adults in each of 928 districts in Thailand. A video clip contest entitled “Melioidosis, an infectious disease that Thais must know” was run between May and October 2012. The best 12 video clips judged by a contest committee were shown to 71 people at risk from melioidosis (diabetics). Focus group interviews were used to evaluate their perceptions of the video clips.
Results
Of 4,203 Thais who completed our study questionnaire, 74% had never heard of melioidosis, and 19% had heard of the disease but had no further knowledge. Most participants in all focus group sessions felt that video clips were beneficial and could positively influence them to increase adherence to recommended preventive behaviours, including drinking boiled water and wearing protective gear if in contact with soil or environmental water. Participants suggested that video clips should be presented in the local dialect with simple words rather than medical terms, in a serious manner, with a doctor as the one presenting the facts, and having detailed pictures of each recommended prevention method.
Conclusions
In summary, public awareness of melioidosis in Thailand is very low, and video clips could serve as a useful medium to educate people and promote disease prevention.
Presented in Part
World Melioidosis Congress 2013, Bangkok, Thailand, 18–20 September 2013 (abstract OS VII-04).
doi:10.1371/journal.pone.0121311
PMCID: PMC4372587  PMID: 25803048
12.  Clinically and Microbiologically Derived Azithromycin Susceptibility Breakpoints for Salmonella enterica Serovars Typhi and Paratyphi A 
Azithromycin is an effective treatment for uncomplicated infections with Salmonella enterica serovar Typhi and serovar Paratyphi A (enteric fever), but there are no clinically validated MIC and disk zone size interpretative guidelines. We studied individual patient data from three randomized controlled trials (RCTs) of antimicrobial treatment in enteric fever in Vietnam, with azithromycin used in one treatment arm, to determine the relationship between azithromycin treatment response and the azithromycin MIC of the infecting isolate. We additionally compared the azithromycin MIC and the disk susceptibility zone sizes of 1,640 S. Typhi and S. Paratyphi A clinical isolates collected from seven Asian countries. In the RCTs, 214 patients who were treated with azithromycin at a dose of 10 to 20 mg/ml for 5 to 7 days were analyzed. Treatment was successful in 195 of 214 (91%) patients, with no significant difference in response (cure rate, fever clearance time) with MICs ranging from 4 to 16 μg/ml. The proportion of Asian enteric fever isolates with an MIC of ≤16 μg/ml was 1,452/1,460 (99.5%; 95% confidence interval [CI], 98.9 to 99.7) for S. Typhi and 207/240 (86.3%; 95% CI, 81.2 to 90.3) (P < 0.001) for S. Paratyphi A. A zone size of ≥13 mm to a 5-μg azithromycin disk identified S. Typhi isolates with an MIC of ≤16 μg/ml with a sensitivity of 99.7%. An azithromycin MIC of ≤16 μg/ml or disk inhibition zone size of ≥13 mm enabled the detection of susceptible S. Typhi isolates that respond to azithromycin treatment. Further work is needed to define the response to treatment in S. Typhi isolates with an azithromycin MIC of >16 μg/ml and to determine MIC and disk breakpoints for S. Paratyphi A.
doi:10.1128/AAC.04729-14
PMCID: PMC4394775  PMID: 25733500
13.  Rapid Diagnostic Tests for Dengue Virus Infection in Febrile Cambodian Children: Diagnostic Accuracy and Incorporation into Diagnostic Algorithms 
PLoS Neglected Tropical Diseases  2015;9(2):e0003424.
Background
Dengue virus (DENV) infection is prevalent across tropical regions and may cause severe disease. Early diagnosis may improve supportive care. We prospectively assessed the Standard Diagnostics (Korea) BIOLINE Dengue Duo DENV rapid diagnostic test (RDT) to NS1 antigen and anti-DENV IgM (NS1 and IgM) in children in Cambodia, with the aim of improving the diagnosis of DENV infection.
Methodology and principal findings
We enrolled children admitted to hospital with non-localised febrile illnesses during the 5-month DENV transmission season. Clinical and laboratory variables, and DENV RDT results were recorded at admission. Children had blood culture and serological and molecular tests for common local pathogens, including reference laboratory DENV NS1 antigen and IgM assays. 337 children were admitted with non-localised febrile illness over 5 months. 71 (21%) had DENV infection (reference assay positive). Sensitivity was 58%, and specificity 85% for RDT NS1 and IgM combined. Conditional inference framework analysis showed the additional value of platelet and white cell counts for diagnosis of DENV infection. Variables associated with diagnosis of DENV infection were not associated with critical care admission (70 children, 21%) or mortality (19 children, 6%). Known causes of mortality were melioidosis (4), other sepsis (5), and malignancy (1). 22 (27%) children with a positive DENV RDT had a treatable other infection.
Conclusions
The DENV RDT had low sensitivity for the diagnosis of DENV infection. The high co-prevalence of infections in our cohort indicates the need for a broad microbiological assessment of non-localised febrile illness in these children.
Author Summary
DENV infection first manifests as an undifferentiated fever before either settling without complications, or progressing to severe disease requiring inpatient admission and careful supportive intravenous fluid management. The ability to differentiate DENV infection from other febrile illnesses, and to predict those at risk of severe disease is likely to be important. We assessed the diagnostic accuracy of a commercially available DENV rapid diagnostic test (RDT) for children admitted with febrile illness to a hospital in Cambodia during the DENV transmission season. We found sensitivity of the DENV RDT to be 58% and specificity to be 85% versus reference assay DENV serology. We then modelled the ability of clinical features, basic laboratory parameters, and DENV RDT result at presentation of the child to distinguish DENV infection from other febrile illness, and determine the need for critical care admission. We found that the DENV RDT did not increase the accuracy with which we diagnosed DENV infection, and was not helpful in deciding which children required critical care admission. Indeed, the relatively high prevalence of serious bacterial disease in the cohort of children indicated a broad microbiological differential diagnosis in all febrile children, regardless of their DENV infection status.
doi:10.1371/journal.pntd.0003424
PMCID: PMC4340051  PMID: 25710684
14.  A Simple Score to Predict the Outcome of Severe Malaria in Adults 
Background
World Health Organization treatment guidelines recommend that adults with severe malaria be admitted to an intensive care unit (ICU). However, ICU facilities are limited in the resource-poor settings where most malaria occurs. Identification of patients at greater risk of complications may facilitate their triage and resource allocation.
Methods
With use of data from a trial conducted in Southeast Asia (n = 868), a logistic regression model was built to identify independent predictors of mortality among adults with severe malaria. A scoring system based on this model was tested in the original dataset and then validated in 2 series from Bangladesh (n = 188) and Vietnam (n = 292).
Results
Acidosis (base deficit) and cerebral malaria (measured as Glasgow Coma Score) were the main independent predictors of outcome. The 5-point Coma Acidosis Malaria (CAM) score was simply derived from these 2 variables. Mortality increased steadily with increasing score. A CAM score <2 predicted survival with a positive predictive value (PPV) of 95.8% (95% confidence interval [CI], 93%–97.7%). Of the 14 of 331 patients who died with a CAM score <2, 11 (79%) had renal failure and death occurred late after hospital admission (median, 108 h; range, 40–360 h). Substitution of plasma bicarbonate as the measure of acidosis only slightly reduced the prognostic value of the model. Use of respiratory rate was inferior, but a score <2 still predicted survival with a PPV of 92.2% (95% CI, 89.1%–94.7%).
Conclusions
Patients with a CAM score <2 at hospital admission may be safely treated in a general ward, provided that renal function can be monitored.
doi:10.1086/649928
PMCID: PMC4313369  PMID: 20105074
15.  A Nonhuman Primate Scrub Typhus Model: Protective Immune Responses Induced by pKarp47 DNA Vaccination in Cynomolgus Macaques 
We developed an intradermal (ID) challenge cynomolgus macaque (Macaca fascicularis) model of scrub typhus, the leading cause of treatable undifferentiated febrile illness in tropical Asia, caused by the obligate intracellular bacterium, Orientia tsutsugamushi. A well-characterized animal model is required for the development of clinically relevant diagnostic assays and evaluation of therapeutic agents and candidate vaccines. We investigated scrub typhus disease pathophysiology and evaluated two O. tsutsugamushi 47-kDa, Ag-based candidate vaccines, a DNA plasmid vaccine (pKarp47), and a virus-vectored vaccine (Kp47/47-Venezuelan equine encephalitis virus replicon particle) for safety, immunogenicity, and efficacy against homologous ID challenge with O. tsutsugamushi Karp. Control cynomolgus macaques developed fever, classic eschars, lymphadenopathy, bacteremia, altered liver function, increased WBC counts, pathogen-specific Ab (IgM and IgG), and cell-mediated immune responses. Vaccinated macaques receiving the DNA plasmid pKarp47 vaccine had significantly increased O. tsutsugamushi–specific, IFN-γ–producing PBMCs (p = 0.04), reduced eschar frequency and bacteremia duration (p ≤ 0.01), delayed bacteremia onset (p < 0.05), reduced circulating bacterial biomass (p = 0.01), and greater reduction of liver transaminase levels (p < 0.03) than controls. This study demonstrates a vaccine-induced immune response capable of conferring sterile immunity against high-dose homologous ID challenge of O. tsutsugamushi in a nonhuman primate model, and it provides insight into cell-mediated immune control of O. tsutsugamushi and dissemination dynamics, highlights the importance of bacteremia indices for evaluation of both natural and vaccine-induced immune responses, and importantly, to our knowledge, has determined the first phenotypic correlates of immune protection in scrub typhus. We conclude that this model is suitable for detailed investigations into vaccine-induced immune responses and correlates of immunity for scrub typhus.
doi:10.4049/jimmunol.1402244
PMCID: PMC4319312  PMID: 25601925
16.  Failure of Burkholderia pseudomallei to Grow in an Automated Blood Culture System 
We compared the organisms isolated from 30,210 pairs of blood culture bottles by using BacT/Alert system and the conventional system. Overall, 2,575 (8.5%) specimens were culture positive for pathogenic organisms. The sensitivity for detection of pathogenic organisms with the BACT/Alert system (85.6%, 2,203 of 2,575) was significantly higher than that with the conventional method (74.1%, 1,908 of 2,575; P < 0.0001). However, Burkholderia pseudomallei was isolated less often with the BacT/ALERT system (73.5%, 328 of 446) than with the conventional system (90.3%, 403 of 446; P < 0.0001). This finding suggests that use of the conventional culture method in conjunction with the BacT/Alert system may improve the isolation rate for B. pseudomallei in melioidosis-endemic areas.
doi:10.4269/ajtmh.14-0018
PMCID: PMC4257642  PMID: 25311697
17.  Open-Label Crossover Study of Primaquine and Dihydroartemisinin-Piperaquine Pharmacokinetics in Healthy Adult Thai Subjects 
Antimicrobial Agents and Chemotherapy  2014;58(12):7340-7346.
Dihydroartemisinin-piperaquine is an artemisinin-based combination treatment (ACT) recommended by the WHO for uncomplicated Plasmodium falciparum malaria, and it is being used increasingly for resistant vivax malaria where combination with primaquine is required for radical cure. The WHO recently reinforced its recommendations to add a single dose of primaquine to ACTs to reduce P. falciparum transmission in low-transmission settings. The pharmacokinetics of primaquine and dihydroartemisinin-piperaquine were evaluated in 16 healthy Thai adult volunteers in a randomized crossover study. Volunteers were randomized to two groups of three sequential hospital admissions to receive 30 mg (base) primaquine, 3 tablets of dihydroartemisinin-piperaquine (120/960 mg), and the drugs together at the same doses. Blood sampling was performed over 3 days following primaquine and 36 days following dihydroartemisinin-piperaquine dosing. Pharmacokinetic assessment was done with a noncompartmental approach. The drugs were well tolerated. There were no statistically significant differences in dihydroartemisinin and piperaquine pharmacokinetics with or without primaquine. Dihydroartemisinin-piperaquine coadministration significantly increased plasma primaquine levels; geometric mean ratios (90% confidence interval [CI]) of primaquine combined versus primaquine alone for maximum concentration (Cmax), area under the concentration-time curve from 0 h to the end of the study (AUC0–last), and area under the concentration-time curve from 0 h to infinity (AUC0–∞) were 148% (117 to 187%), 129% (103 to 163%), and 128% (102 to 161%), respectively. This interaction is similar to that described recently with chloroquine and may result in an enhanced radical curative effect. (This study has been registered at ClinicalTrials.gov under registration no. NCT01525511.)
doi:10.1128/AAC.03704-14
PMCID: PMC4249579  PMID: 25267661
18.  Maintenance of Leptospira Species in Leptospira Vanaporn Wuthiekanun Agar 
Journal of Clinical Microbiology  2014;52(12):4350-4352.
The maintenance of Leptospira species in liquid or semisolid medium is time-consuming and at risk of contamination due to the needs of routine subculture and dark field microscopy. Using Leptospira Vanaporn Wuthiekanun (LVW) agar, we maintained 100 pathogenic Leptospira isolates for 12 months without the need for subculture and confirmed the viability of all isolates by the naked eye.
doi:10.1128/JCM.02273-14
PMCID: PMC4313312  PMID: 25253789
19.  Burkholderia pseudomallei in Water Supplies, Southern Thailand 
Emerging Infectious Diseases  2014;20(11):1947-1949.
doi:10.3201/eid2011.140832
PMCID: PMC4215545  PMID: 25340393
melioidosis; B. pseudomallei; water; Phangan; Thailand; bacteria; Koh Phangan
20.  Plasma Concentration of Parasite DNA as a Measure of Disease Severity in Falciparum Malaria 
The Journal of Infectious Diseases  2014;211(7):1128-1133.
In malaria-endemic areas, Plasmodium falciparum parasitemia is common in apparently healthy children and severe malaria is commonly misdiagnosed in patients with incidental parasitemia. We assessed whether the plasma Plasmodium falciparum DNA concentration is a useful datum for distinguishing uncomplicated from severe malaria in African children and Asian adults. P. falciparum DNA concentrations were measured by real-time polymerase chain reaction (PCR) in 224 African children (111 with uncomplicated malaria and 113 with severe malaria) and 211 Asian adults (100 with uncomplicated malaria and 111 with severe malaria) presenting with acute falciparum malaria. The diagnostic accuracy of plasma P. falciparum DNA concentrations in identifying severe malaria was 0.834 for children and 0.788 for adults, similar to that of plasma P. falciparum HRP2 levels and substantially superior to that of parasite densities (P < .0001). The diagnostic accuracy of plasma P. falciparum DNA concentrations plus plasma P. falciparum HRP2 concentrations was significantly greater than that of plasma P. falciparum HRP2 concentrations alone (0.904 for children [P = .004] and 0.847 for adults [P = .003]). Quantitative real-time PCR measurement of parasite DNA in plasma is a useful method for diagnosing severe falciparum malaria on fresh or archived plasma samples.
doi:10.1093/infdis/jiu590
PMCID: PMC4354984  PMID: 25344520
Plasmodium falciparum; malaria; severe disease; plasma DNA; diagnostic accuracy
21.  Serosurveillance of Orientia tsutsugamushi and Rickettsia typhi in Bangladesh 
Scrub and murine typhus infections are under-diagnosed causes of febrile illness across the tropics, and it is not known how common they are in Bangladesh. We conducted a prospective seroepidemiologic survey across six major teaching hospitals in Bangladesh by using an IgM enzyme-linked immunosorbent assay. Results indicated recent exposure (287 of 1,209, 23.7% seropositive for Orientia tsutsugamushi and 805 of 1,209, 66.6% seropositive for Rickettsia typhi). Seropositive rates were different in each region. However, there was no geographic clustering of seropositive results for both organisms. There was no difference between those from rural or urban areas. Rickettsia typhi seroreactivity was positively correlated with age. Scrub typhus and murine typhus should be considered as possible causes of infection in Bangladesh.
doi:10.4269/ajtmh.13-0570
PMCID: PMC4155564  PMID: 25092819
22.  High-Throughput Ultrasensitive Molecular Techniques for Quantifying Low-Density Malaria Parasitemias 
Journal of Clinical Microbiology  2014;52(9):3303-3309.
The epidemiology of malaria in “low-transmission” areas has been underestimated. Molecular detection methods have revealed higher prevalences of malaria than conventional microscopy or rapid diagnostic tests, but these typically evaluate finger-prick capillary blood samples (∼5 μl) and therefore cannot detect parasite densities of <200/ml. Their use underestimates true parasite carriage rates. To characterize the epidemiology of malaria in low-transmission settings and plan elimination strategies, more sensitive quantitative PCR (qPCR) is needed to identify and quantify low-density malaria parasitemias. A highly sensitive “high-volume” quantitative PCR (qPCR) method based on Plasmodium sp. 18S RNA was adapted for blood sample volumes of ≥250 μl and scaled for high throughput. The methods were validated by assessment of the analytical sensitivity and specificity, diagnostic sensitivity, and specificity, efficiency, precision, analytical and diagnostic accuracies, limit of detection, root cause analysis of false positives, and robustness. The high-volume qPCR method based on Plasmodium sp. 18S RNA gave high PCR efficiency of 90 to 105%. Concentrations of parasite DNA from large volumes of blood gave a consistent analytical detection limit (LOD) of 22 parasites/ml (95% CI, 21.79 to 74.9), which is some 2,500 times more sensitive than conventional microscopy and 50 times more sensitive than currently used PCR methods from filter paper blood spots. The diagnostic specificity was 99.75%. Using automated procedures it was possible to process 700 blood samples per week. A very sensitive and specific high-throughput high-volume qPCR method for the detection of low-density parasitemias (>20 parasites/ml) was developed and validated.
doi:10.1128/JCM.01057-14
PMCID: PMC4313154  PMID: 24989601
23.  Ethics, Economics, and the Use of Primaquine to Reduce Falciparum Malaria Transmission in Asymptomatic Populations 
PLoS Medicine  2014;11(8):e1001704.
Yoel Lubell and colleagues consider ethical and economic perspectives on mass drug administration of primaquine to limit transmission of P. falciparum malaria.
Please see later in the article for the Editors' Summary
doi:10.1371/journal.pmed.1001704
PMCID: PMC4137981  PMID: 25137246
24.  Fatal Melioidosis in Goats in Bangkok, Thailand 
Bangkok, Thailand, is a city considered to be at low risk for melioidosis. We describe 10 goats that died of melioidosis in Bangkok. Half of them were born and reared in the city. Multilocus sequence typing ruled out an outbreak. This finding challenges the assumption that melioidosis is rarely acquired in central Thailand.
doi:10.4269/ajtmh.14-0115
PMCID: PMC4125250  PMID: 24891468
25.  Statistical Power Calculations for Mixed Pharmacokinetic Study Designs Using a Population Approach 
The AAPS Journal  2014;16(5):1110-1118.
Simultaneous modelling of dense and sparse pharmacokinetic data is possible with a population approach. To determine the number of individuals required to detect the effect of a covariate, simulation-based power calculation methodologies can be employed. The Monte Carlo Mapped Power method (a simulation-based power calculation methodology using the likelihood ratio test) was extended in the current study to perform sample size calculations for mixed pharmacokinetic studies (i.e. both sparse and dense data collection). A workflow guiding an easy and straightforward pharmacokinetic study design, considering also the cost-effectiveness of alternative study designs, was used in this analysis. Initially, data were simulated for a hypothetical drug and then for the anti-malarial drug, dihydroartemisinin. Two datasets (sampling design A: dense; sampling design B: sparse) were simulated using a pharmacokinetic model that included a binary covariate effect and subsequently re-estimated using (1) the same model and (2) a model not including the covariate effect in NONMEM 7.2. Power calculations were performed for varying numbers of patients with sampling designs A and B. Study designs with statistical power >80% were selected and further evaluated for cost-effectiveness. The simulation studies of the hypothetical drug and the anti-malarial drug dihydroartemisinin demonstrated that the simulation-based power calculation methodology, based on the Monte Carlo Mapped Power method, can be utilised to evaluate and determine the sample size of mixed (part sparsely and part densely sampled) study designs. The developed method can contribute to the design of robust and efficient pharmacokinetic studies.
doi:10.1208/s12248-014-9641-4
PMCID: PMC4147042  PMID: 25011414
mixed pharmacokinetic study designs; Monte Carlo Mapped Power; optimal pharmacokinetic study design; statistical power calculations

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