No abstract available

Paul Newton and colleagues propose guidelines for conducting and reporting field surveys of the quality of medicines.

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

The availability of falsified antimalarial drugs can be reduced with effective drug regulatory agencies and proper enforcement. Fundamental to these agencies taking action, rapid identification must be made as soon as they appear in the market place. Since falsified antimalarials occur mostly in developing countries, performing drug analysis presents itself with unique challenges. A fundamental factor in choosing a useful technique is affordability and simplicity. Therefore, we suggest a three-tiered drug evaluation strategy for identifying a falsified drug in resource-poor areas. Tier I is a simple comparison of a tablet's weight and dimensions with official specifications. Tier II uses inexpensive photometric devices (laser and fluorescence) to evaluate a tablet. Suspicious samples from Tier I and II assessments are then subjected to a colorimetric assay for active ingredients identification and quantification. In this article, we evaluate a novel colorimetric assay for the simultaneous assessment of both lumefantrine and artemether in co-formulated Coartem™ tablets, and integrate the method with two novel, low-cost, fluorescence and laser photometric devices. Image analysis software is used for the assessments. Although artemether-lumefantrine is used as an example, the strategy may be adapted to other medicines.

We report a large multicenter genome-wide association study of Plasmodium falciparum resistance to artemisinin, the frontline antimalarial drug. Across 15 locations in Southeast Asia, we identified at least 20 mutations in kelch13 (PF3D7_1343700) affecting the encoded propeller and BTB/POZ domains, which were associated with a slow parasite clearance rate after treatment with artemisinin derivatives. Nonsynonymous polymorphisms in fd (ferredoxin), arps10 (apicoplast ribosomal protein S10), mdr2 (multidrug resistance protein 2) and crt (chloroquine resistance transporter) also showed strong associations with artemisinin resistance. Analysis of the fine structure of the parasite population showed that the fd, arps10, mdr2 and crt polymorphisms are markers of a genetic background on which kelch13 mutations are particularly likely to arise and that they correlate with the contemporary geographical boundaries and population frequencies of artemisinin resistance. These findings indicate that the risk of new resistance-causing mutations emerging is determined by specific predisposing genetic factors in the underlying parasite population.

Because of reductions in the incidence of Plasmodium falciparum malaria in Laos, identification of the causes of fever in people without malaria, and discussion of the best empirical treatment options, are urgently needed. We aimed to identify the causes of non-malarial acute fever in patients in rural Laos.

Plasmodium knowlesi is the most common cause of malaria in Malaysia. However, microscopic diagnosis is inaccurate and rapid diagnostic tests (RDTs) are insufficiently sensitive. PCR is sensitive and specific but not feasible at a district level. Loop-mediated isothermal amplification (LAMP) shows potential with only basic requirements. A commercially available LAMP assay, the Eiken Loopamp™ MALARIA Pan Detection kit, is sensitive for Plasmodium falciparum and Plasmodium vivax, but has not previously been evaluated for P. knowlesi. This study aims to determine the sensitivity of this LAMP assay for detecting P. knowlesi infection.

Rapid diagnostic tests (RDTs) are widely used for malaria diagnosis, but lack of quality control at point of care restricts trust in test results. Prototype positive control wells (PCW) containing recombinant malaria antigens have been developed to identify poor-quality RDT lots. This study assessed community and facility health workers' (HW) ability to use PCWs to detect degraded RDTs, the impact of PCW availability on RDT use and prescribing, and preferred strategies for implementation in Lao People's Democratic Republic (Laos) and Uganda. A total of 557 HWs participated in Laos (267) and Uganda (290). After training, most (88% to ≥ 99%) participants correctly performed the six key individual PCW steps; performance was generally maintained during the 6-month study period. Nearly all (97%) reported a correct action based on PCW use at routine work sites. In Uganda, where data for 127,775 individual patients were available, PCW introduction in health facilities was followed by a decrease in antimalarial prescribing for RDT-negative patients ≥ 5 years of age (4.7-1.9%); among community-based HWs, the decrease was 12.2% (P < 0.05) for all patients. Qualitative data revealed PCWs as a way to confirm RDT quality and restore confidence in RDT results. HWs in malaria-endemic areas are able to use prototype PCWs for quality control of malaria RDTs. PCW availability can improve HWs' confidence in RDT results, and benefit malaria diagnostic programs. Lessons learned from this study may be valuable for introduction of other point-of-care diagnostic and quality-control tools. Future work should evaluate longer term impacts of PCWs on patient management.

Diagnosis of malaria relies on parasite detection by microscopy or antigen detection; both fail to detect low-density infections. New tests providing rapid, sensitive diagnosis with minimal need for training would enhance both malaria diagnosis and malaria control activities. We determined the diagnostic accuracy of a new loop-mediated amplification (LAMP) kit in febrile returned travelers.

The fixed dose combination of artemether-lumefantrine (AL) is the most widely used treatment for uncomplicated Plasmodium falciparum malaria. Relatively lower cure rates and lumefantrine levels have been reported in young children and in pregnant women during their second and third trimester. The aim of this study was to investigate the pharmacokinetic and pharmacodynamic properties of lumefantrine and the pharmacokinetic properties of its metabolite, desbutyl-lumefantrine, in order to inform optimal dosing regimens in all patient populations.

In 2012, a stratified random survey, using mystery shoppers, was conducted to investigate the availability and quality of antibiotics sold to patients in the private sector in five southern provinces of the Lao People's Democratic Republic (Laos).

The global prevalence of diabetes mellitus is increasing alarmingly. However, the quality of vital medicines and medical products used to treat and monitor diabetes remains uncertain but of potential great public health significance. Here, we review the available evidence on the quality of antidiabetic medicines and supplies for self-monitoring of blood glucose (SMBG) and discuss their potential impact for the patients and society.

Fever commonly leads to healthcare seeking and hospital admission in sub-Saharan Africa and Asia. There is only limited guidance for clinicians managing non-malarial fevers, which often results in inappropriate treatment for patients. Furthermore, there is little evidence for estimates of disease burden, or to guide empirical therapy, control measures, resource allocation, prioritisation of clinical diagnostics or antimicrobial stewardship. The Febrile Illness Evaluation in a Broad Range of Endemicities (FIEBRE) study seeks to address these information gaps.

Phenotypic testing for drug susceptibility of Mycobacterium tuberculosis is critical to basic research and managing the evolving problem of antimicrobial resistance in tuberculosis management, but it remains a specialized technique to which access is severely limited. Here, we report on the development and validation of an improved phage-mediated detection system for M. tuberculosis We incorporated a nanoluciferase (Nluc) reporter gene cassette into the TM4 mycobacteriophage genome to create phage TM4-nluc. We assessed the performance of this reporter phage in the context of cellular limit of detection and drug susceptibility testing using multiple biosafety level 2 drug-sensitive and -resistant auxotrophs as well as virulent M. tuberculosis strains. For both limit of detection and drug susceptibility testing, we developed a standardized method consisting of a 96-hour cell preculture followed by a 72-hour experimental window for M. tuberculosis detection with or without antibiotic exposure. The cellular limit of detection of M. tuberculosis in a 96-well plate batch culture was ≤102 CFU. Consistent with other phenotypic methods for drug susceptibility testing, we found TM4-nluc to be compatible with antibiotics representing multiple classes and mechanisms of action, including inhibition of core central dogma functions, cell wall homeostasis, metabolic inhibitors, compounds currently in clinical trials (SQ109 and Q203), and susceptibility testing for bedaquiline, pretomanid, and linezolid (components of the BPaL regimen for the treatment of multi- and extensively drug-resistant tuberculosis). Using the same method, we accurately identified rifampin-resistant and multidrug-resistant M. tuberculosis strains.IMPORTANCEMycobacterium tuberculosis, the causative agent of tuberculosis disease, remains a public health crisis on a global scale, and development of new interventions and identification of drug resistance are pillars in the World Health Organization End TB Strategy. Leveraging the tractability of the TM4 mycobacteriophage and the sensitivity of the nanoluciferase reporter enzyme, the present work describes an evolution of phage-mediated detection and drug susceptibility testing of M. tuberculosis, adding a valuable tool in drug discovery and basic biology research. With additional validation, this system may play a role as a quantitative phenotypic reference method and complement to genotypic methods for diagnosis and antibiotic susceptibility testing.

Turoctocog alfa pegol (N8-GP) is a glycoPEGylated human recombinant factor VIII for the treatment of hemophilia A. The safety profile of rFVIII, and polyethylene glycols (PEG) technology, is well-established. Conducting long-term toxicity studies in animals using human proteins can be complicated by anti-drug antibody (ADA) development. To evaluate long-term safety of N8-GP, 26- and 52-week toxicity studies were conducted in immune-deficient rats dosed intravenously every fourth day with 0, 50, 150, 500, or 1200 IU/kg N8-GP. Observations included clinical observations, body weight, ophthalmoscopy, hematology, chemistry, coagulation, urinalysis, toxicokinetics, antibody analysis, and macroscopic/microscopic organ examination. Immunohistochemical staining examined the distribution of PEG in the brain. No adverse test item-related findings were seen and PEG was not detected in the brain. Exposure was confirmed for ~75% of the animals dosed with 500 and 1200 IU/kg N8-GP; the high lower limit of quantification of the bioanalysis assay prevented confirmation of exposure in the lower doses. A small number of animals developed ADAs, and the proportion of animals surviving until scheduled termination was >80%. N8-GP was well tolerated, and the immune-deficient rat proved suitable for testing long-term toxicity of human proteins that are immunogenic in animals.

The emergence of infectious diseases pose major global health threats. Estimates of total in-country human pathogen diversity, and insights as to how and when species were described through history, could be used to estimate the probability of new pathogen discoveries. Data from the Lao People's Democratic Republic (Laos) were used in this proof-of-concept study to estimate national human pathogen diversity and to examine historical discovery rate drivers.

Recent expansions of vector-borne diseases highlight the need for improved surveillance, especially in resource-poor settings. Dengue virus (DENV), chikungunya virus (CHIKV), and Zika virus (ZIKV) share the same vectors as well as similar clinical presentations, suggesting that combined surveillance would be useful. We hypothesized that blood spotted on dengue rapid diagnostic tests (RDTs) could be harnessed for sample collection in remote areas for subsequent detection of DENV, CHIKV, and ZIKV by reverse transcription real-time polymerase chain reaction (RT-qPCR). CHIKV and ZIKV dilutions were spotted on dengue RDTs (SD BIOLINE Dengue DUO, Standard Diagnostics, Gyeonggi-do, Republic of Korea), dried, and extracted. As reference, aliquots of each viral dilution were directly extracted. Using specific RT-qPCR tests, both viruses were successfully detected from RDT extracts. However, the limit of detection was slightly lower in comparison to direct extracts, two logfold for CHIKV and one logfold for ZIKV. For analysis of temperature stability, DENV dilutions were spotted on RDTs and stored for up to 2 months at -80°C, 4°C, or 35°C before testing. Storage of RDTs for 2 months at 35°C did not compromise detection of RNA by RT-qPCR; only minimal degradation was observed. This proof-of-principle study demonstrates the potential of using dengue RDTs for DENV/CHIKV/ZIKV combined surveillance in areas without access to laboratory facilities. Further investigations are needed for evaluation of tri-viral surveillance under field conditions using patient samples. Large-scale implementation of surveillance for these viruses is of crucial public health importance for the early detection of epidemics. This method also has important implications for improving understanding of the molecular epidemiology of the three viruses.

Klebsiella pneumoniae is a leading cause of bloodstream infection (BSI). Strains producing extended-spectrum beta-lactamases (ESBLs) or carbapenemases are considered global priority pathogens for which new treatment and prevention strategies are urgently required, due to severely limited therapeutic options. South and Southeast Asia are major hubs for antimicrobial-resistant (AMR) K. pneumoniae and also for the characteristically antimicrobial-sensitive, community-acquired "hypervirulent" strains. The emergence of hypervirulent AMR strains and lack of data on exopolysaccharide diversity pose a challenge for K. pneumoniae BSI control strategies worldwide.

Conventional descriptions of central nervous system (CNS) infections are variably categorized into clinical syndromes for patient investigation, management and research. Aetiologies of the most commonly recognized syndromes, encephalitis and meningitis, tend to be attributed predominantly to viruses and bacteria, respectively.

Bloodstream infections cause substantial morbidity and mortality. However, despite clinical suspicion of such infections, blood cultures are often negative. We investigated blood cultures that were negative after 5 days of incubation for the presence of bacterial pathogens using specific (Rickettsia spp. and Leptospira spp.) and a broad-range 16S rRNA PCR. From 190 samples, 53 (27.9%) were positive for bacterial DNA. There was also a high background incidence of dengue (90/112 patient serum positive, 80.4%). Twelve samples (6.3%) were positive for Rickettsia spp., including two Rickettsia typhi. The 16S rRNA PCR gave 41 positives; Escherichia coli and Klebsiella pneumoniae were identified in 11 and eight samples, respectively, and one Leptospira species was detected. Molecular investigation of negative blood cultures can identify potential pathogens that will otherwise be missed by routine culture. Patient management would have been influenced in all 53 patients for whom a bacterial organism was identified, and 2.3-6.1% of patients would likely have had an altered final outcome. These findings warrant further study, particularly to determine the cost-benefit for routine use, ways of implementation, and timing of PCR for organisms such as Rickettsia and Leptospira, which are important pathogens in rural Asia.