Feasibility and limitations of acridine orange fluorescence technique using a Malaria Diagnosis Microscope in Myanmar.

We studied parasite detectability in thick films by an acridine orange fluorescence technique (AO) to test its applicability and the use of a Malaria Diagnosis Microscope (MDM)-ESL in the detection of parasites, compared to the conventional Giemsa staining method. This study was conducted on 1,390 clinically suspected malaria cases of Thaton township, Myanmar. We found sensitivities of 82.8% for Plasmodium falciparum (P. falciparum) and 100% for Plasmodium vivax (P. vivax) and specificities of 97.1% for P. falciparum and 98.6% for P. vivax. AO had a higher sensitivity than Giemsa-stained films at low levels of parasitemia (&#60; 1,000/microl). AO showed lower sensitivity and higher specificity than the Giemsa method at parasite levels of more than 1,000/microl. The results of using the AO method, achieved by both novice and experienced observers, showed no significant difference and required less practice to perform the test as well as to identify the parasite. The acridine orange fluorescence technique using a malaria diagnosis microscope MDM-ESL series is simple, rapid and cost effective. The microscope is conveniently operable using standard AC power or a 12-V DC car battery, and it is easily convertible to a conventional biological microscope. With the exception of species differentiation, which is not possible with this method, this method would be appropriate for both clinical and epidemiological studies.</p

An ultrasensitive reverse transcription polymerase chain reaction assay to detect asymptomatic low-density Plasmodium falciparum and Plasmodium vivax infections in small volume blood samples.

BackgroundHighly sensitive, scalable diagnostic methods are needed to guide malaria elimination interventions. While traditional microscopy and rapid diagnostic tests (RDTs) are suitable for the diagnosis of symptomatic malaria infection, more sensitive tests are needed to screen for low-density, asymptomatic infections that are targeted by interventions aiming to eliminate the entire reservoir of malaria infection in humans.MethodsA reverse transcription polymerase chain reaction (RT- PCR) was developed for multiplexed detection of the 18S ribosomal RNA gene and ribosomal RNA of Plasmodium falciparum and Plasmodium vivax. Simulated field samples stored for 14 days with sample preservation buffer were used to assess the analytical sensitivity and specificity. Additionally, 1750 field samples from Southeastern Myanmar were tested both by RDT and ultrasensitive RT-PCR.ResultsLimits of detection (LoD) were determined under simulated field conditions. When 0.3 mL blood samples were stored for 14 days at 28 °C and 80% humidity, the LoD was less than 16 parasites/mL for P. falciparum and 19.7 copies/µL for P. vivax (using a plasmid surrogate), about 10,000-fold lower than RDTs. Of the 1739 samples successfully evaluated by both ultrasensitive RT-PCR and RDT, only two were RDT positive while 24 were positive for P. falciparum, 108 were positive for P. vivax, and 127 were positive for either P. vivax and/or P. falciparum using ultrasensitive RT-PCR.ConclusionsThis ultrasensitive RT-PCR method is a robust, field-tested screening method that is vastly more sensitive than RDTs. Further optimization may result in a truly scalable tool suitable for widespread surveillance of low-level asymptomatic P. falciparum and P. vivax parasitaemia

Prevalence and seroprevalence of Plasmodium infection in Myanmar reveals highly heterogeneous transmission and a large hidden reservoir of infection.

Malaria incidence in Myanmar has significantly reduced over recent years, however, completeness and timeliness of incidence data remain a challenge. The first ever nationwide malaria infection and seroprevalence survey was conducted in Myanmar in 2015 to better understand malaria epidemiology and highlight gaps in Annual Parasite Index (API) data. The survey was a cross-sectional two-stage stratified cluster-randomised household survey conducted from July-October 2015. Blood samples were collected from household members for ultra-sensitive PCR and serology testing for P. falciparum and P. vivax. Data was gathered on demography and a priori risk factors of participants. Data was analysed nationally and within each of four domains defined by API data. Prevalence and seroprevalence of malaria were 0.74% and 16.01% nationwide, respectively. Prevalent infection was primarily asymptomatic P. vivax, while P. falciparum was predominant in serology. There was large heterogeneity between villages and by domain. At the township level, API showed moderate correlation with P. falciparum seroprevalence. Risk factors for infection included socioeconomic status, domain, and household ownership of nets. Three K13 P. falciparum mutants were found in highly prevalent villages. There results highlight high heterogeneity of both P. falciparum and P. vivax transmission between villages, accentuated by a large hidden reservoir of asymptomatic P. vivax infection not captured by incidence data, and representing challenges for malaria elimination. Village-level surveillance and stratification to guide interventions to suit local context and targeting of transmission foci with evidence of drug resistance would aid elimination efforts

Spread of artemisinin resistance in Plasmodium falciparum malaria.

BACKGROUND: Artemisinin resistance in Plasmodium falciparum has emerged in Southeast Asia and now poses a threat to the control and elimination of malaria. Mapping the geographic extent of resistance is essential for planning containment and elimination strategies. METHODS: Between May 2011 and April 2013, we enrolled 1241 adults and children with acute, uncomplicated falciparum malaria in an open-label trial at 15 sites in 10 countries (7 in Asia and 3 in Africa). Patients received artesunate, administered orally at a daily dose of either 2 mg per kilogram of body weight per day or 4 mg per kilogram, for 3 days, followed by a standard 3-day course of artemisinin-based combination therapy. Parasite counts in peripheral-blood samples were measured every 6 hours, and the parasite clearance half-lives were determined. RESULTS: The median parasite clearance half-lives ranged from 1.9 hours in the Democratic Republic of Congo to 7.0 hours at the Thailand-Cambodia border. Slowly clearing infections (parasite clearance half-life >5 hours), strongly associated with single point mutations in the "propeller" region of the P. falciparum kelch protein gene on chromosome 13 (kelch13), were detected throughout mainland Southeast Asia from southern Vietnam to central Myanmar. The incidence of pretreatment and post-treatment gametocytemia was higher among patients with slow parasite clearance, suggesting greater potential for transmission. In western Cambodia, where artemisinin-based combination therapies are failing, the 6-day course of antimalarial therapy was associated with a cure rate of 97.7% (95% confidence interval, 90.9 to 99.4) at 42 days. CONCLUSIONS: Artemisinin resistance to P. falciparum, which is now prevalent across mainland Southeast Asia, is associated with mutations in kelch13. Prolonged courses of artemisinin-based combination therapies are currently efficacious in areas where standard 3-day treatments are failing. (Funded by the U.K. Department of International Development and others; ClinicalTrials.gov number, NCT01350856.)

Pneumocystis jirovecii pneumonia in tropical and low and middle income countries: a systematic review and meta-regression

Objective: Pneumocystis jirovecii pneumonia (PCP), the commonest opportunistic infection in HIV-infected patients in the developed world, is less commonly described in tropical and low and middle income countries (LMIC). We sought to investigate predictors of PCP in these settings. Design Systematic review and meta-regression. METHODS: Meta-regression of predictors of PCP diagnosis (33 studies). Qualitative and quantitative assessment of recorded CD4 counts, receipt of prophylaxis and antiretrovirals, sensitivity and specificity of clinical signs and symptoms for PCP, co-infection with other pathogens, and case fatality (117 studies). RESULTS: The most significant predictor of PCP was per capita Gross Domestic Product, which showed strong linear association with odds of PCP diagnosis (p30%; treatment was largely appropriate. Prophylaxis appeared to reduce the risk for development of PCP, however 24% of children with PCP were receiving prophylaxis. CD4 counts at presentation with PCP were usually <200×10 3/ ml. CONCLUSIONS: There is a positive relationship between GDP and risk of PCP diagnosis. Although failure to diagnose infection in poorer countries may contribute to this, we also hypothesise that poverty exposes at-risk patients to a wide range of infections and that the relatively non-pathogenic P. jirovecii is therefore under-represented. As LMIC develop economically they eliminate the conditions underlying transmission of virulent infection: P. jirovecii , ubiquitous in all settings, then becomes a greater relative threat

光散乱モデルに基づく顕微鏡画像解析による眼内レンズの特性評価に関する研究

Supervisor:小谷　一孔先端科学技術研究科博

Intensifying responsiveness towards neglected intestinal helminth infections in a resource-constrained setting

Abstract Neglected intestinal helminth infections afflict the marginalized communities in Asia. Since 2004, growing body of evidence in Myanmar indicated high prevalence of soil-transmitted helminths (STH) infections (30–40%) among school children. Co-existence of STH (23%) with food-borne trematodes was noted among 383 pregnant women in a selected township in 2012–2014 followed by molecular verification of very low prevalence of schistosome infection (<5%) in the same study site in 2016. The success of transmission elimination plans may depend upon sensitive diagnostic tools to detect persistent infections and polyparasitism. Addressing the research gaps in vulnerable sites requires an increased investment in resource-constrained settings

Comparison of microscopy and PCR for the detection of human Plasmodium species and Plasmodium knowlesi in southern Myanmar

Objectives: To determine the distribution of Plasmodium (P) species including Plasmodium knowlesi and to compare the specificity and sensitivity of microscopy with nested PCR in malaria diagnosis. Methods: The study was conducted in Kawthaung, southern Myanmar. Ninety clinically suspected malaria patients were screened for malaria by Giemsa stained microscopy and confirmed by nested PCR. Results: Among the participants, 57 (63.3%) were positive and 33 (36.7%) were negative by microscopy. Of positive samples, 39 (68.4%) were Plasmodium falciparum, 17 (29.8%) Plasmodium vivax and 1 (1.8%) Plasmodium malariae, whereas 59-amplified by PCR were 40 (67.8%), 18 (30.5%) and 1 (1.7%) respectively. PCR amplified 2 microscopy negative samples. Two samples of P. falciparum detected by microscopy were amplified as P. vivax and vice versa. All samples were negative for Plasmodium ovale, P. knowlesi and mixed infections. Microscopy had a very good measure of agreement (κ = 0.95) compared to nested PCR. Sensitivity and specificity of microscopy for diagnosis of P. falciparum were 92.5% (95% CI: 79.6–98.4) and 96.0% (95% CI: 86.3–99.5) respectively, whereas for P. vivax were 83.3% (95% CI: 58.6–96.4) and 97.2% (95% CI: 90.3–99.7). Conclusions: P. knowlesi was not detected by both microscopy and PCR. Giemsa stained microscopy can still be applied as primary method for malaria diagnosis and is considered as gold standard. As to the lower sensitivity of microscopy for vivax malaria, those with previous history of malaria and relapse cases should be diagnosed by RDT or PCR combined with microscopy. Inaccuracy of species diagnosis highlighted the requirement of training and refresher courses for microscopists

Access to primaquine in the last mile: challenges at the service delivery points in pre-elimination era, Myanmar

Abstract Background Alongside monitoring of the disease burden, the successful move towards malaria elimination relies on the readiness of the health care delivery system. However, there is a lack of evidence in the gap of existing National Guidelines and access to low dose primaquine in real practice under varying degrees of antimalarial resistance in the pre-elimination phase in Myanmar. Therefore, this study addressed the essential information from the service delivery points (SDPs) of public and private sectors on the availability and the use of primaquine in both supply and demand side. Concomitantly, the study aimed to underscore challenges in health system infrastructure to promote the sustained flow in rolling out primaquine in line with National Guidelines for malaria elimination. Methods A cross-sectional study conducted from September 2017 to February 2018 included six townships of three states/regions. The team used an observation checklist for documenting primaquine supplies at SDPs. Semi-structured interviews, key informant, and in-depth interviews focused both public and private sectors including staff from the Vector-Borne Diseases Control (VBDC) teams in each state/region and rural health centers (n = 25), those from the non-governmental organizations (NGOs), general practitioners and drug sellers (n = 11), and recently infected malaria patients (n = 11). Triangulation of quantitative and qualitative data provided meaningful interpretations. Results Public sector staff reported an adequate stock of primaquine, but it was unavailable at the general practitioners’ clinics without any connection to NGOs and also at the unlicensed drug shops. Health care providers of the public sector experienced challenges in poor compliance of malaria patients to primaquine treatment in conjunction with an artemisinin-based combination therapy, loss-to-follow-ups especially in conflict areas, and delays in timely substitution of new batches of primaquine. Respondents from the private sector demanded for the refresher training course on updated antimalarial treatment guidelines. Conclusion Monitoring compliance and safety of primaquine treatment was found as a barrier especially among mobile migrant workers and those who were in conflict areas. An alternative strategy by the NMCP could enable to prevent the underutilization of primaquine in vivax malaria to reach the malaria elimination targets