Evaluation of Two Malaria Rapid Diagnostic Tests Quality Assurance (mRDT’s QA) Methods in Peripheral Health Facilities, Rural Tanzania.

\ud WHO recommends confirming suspected malaria cases before initiation of treatment. Due to the imited availability of quality microscopy services, this recommendation has been followed with increased use of antigen-detecting malaria rapid diagnostic tests (mRDTs) in many malaria endemic countries. With the increased use of mRDTs, the need for a thorough mRDT quality assurance (RDT QA) method has become more apparent. One of the WHO recommendations for RDT QA is to monitor the tests in field use monthly, by comparing mRDT results to reference microscopy. This study was carried out to monitor mRDT performance in selected health facilities using two quality assurance methods; first based on ference microscopy and second based on detection of parasite DNA by real time quantitative PCR (qPCR) on dried blood spots (DBS); as well as assessing the cost and timeliness of the two QA methods. Blood samples were collected from patients undergoing a rapid test for malaria for two to three consecutive days per month, for five months, in 12 health facilities in Iringa rural and Mufindi districts. The health workers were instructed to label RDT cassettes, blood smear slides, and filter papers for DBS with matching unique ID stickers. A sticker was also placed in the log book where RDT results were recorded. Blood smears (BS) were first read at the district hospital (BS1) and then transported to Bagamoyo for a reference reading at the IHI- Bagamoyo laboratory (BS2). A third BS reader (BS3) was consulted from Muhimbili University of Health and Allied Sciences (MUHAS) in case of discordant results between BS1 and BS2. Molecular analysis involved extraction of parasite DNA from DBS samples using a QIAamp DNA Mini Kit. Sample DNA aliquots were compared against standard solutions with parasite DNA diluted 10-fold to give a parasitemia ranging from 200,000/μL to 20/μL. About 20% of the study DNA aliquots were sent to the CDC laboratory in Atlanta in order to validate qPCR results performed at the Bagamoyo laboratory. Data were entered in Microsoft Access (Microsoft Corporation, 2006) and analyzed in STATA 10 (StataCorp, Texas USA). Because of the known limitations of mRDTs to detect parasitemia below 200 parasites/μL, BS and PCR results greater than or equal to 5 parasites/200 WBC or 200 parasites/μL were considered positive in comparisons with mRDT performance. In the univariate analysis, proportions of positive tests were compared among the three types of tests: mRDT, microscopy and qPCR. Microscopy readings were categorized into 3 groups; BS1, BS2 and /or BS summary which is an average of BS1 and BS2. In case of discordant results between BS1 and BS2, a third reader- BS3 was consulted. Chi-squared test was done to assess differences in proportion of positive tests per district; whereas McNemar’s test was Malaria RDT QA Final Report, March 2012 5 used to assess the difference in test positivity by type of test. Kappa statistic was used to quantify the strength of the agreement between tests results. In addition, we examined health workers performance of the testing procedure when attending patients at a health facility, using a predefined checklist. Towards the end of the study, an evaluation of health worker acceptability was carried out to assess preferences between the two RDT QA methods. We received 2369 samples and 2324 (98%) had complete information. mRDTs had the highest positivity rate (6.5%). The proportion of positive tests by all types of tests was slightly higher in Iringa DC, but only qPCR and BS2 showed statistically significant differences in positivity rate between the two districts, where Iringa DC had more positive tests than Mufindi DC (p<0.05). When qPCR was a gold standard, mRDTs had higher sensitivity (68.6%, 95%CI: 55.0-79.7) than microscopy (53.7%, 95%CI: 38.7- 68.0) but highest mRDT sensitivity was achieved with comparison to microscopy (85.3%, 95%CI: 70.0- 93.6). All tests had higher inter-observer agreement than would be expected by chance. Substantial high inter-observer agreement (kappa =0.75; p<0.001) was seen amongst the microscopists i.e. district’s quality assurance officers and the reference microscopy readings. Assessment of the time needed to process BS at the district level revealed that, smears at district level took on average 8 days (min 2 to max 33) to be processed and provide feedback; but up to an average of 44 days (min 19 to max 98) to get a second reading. Many health workers were aware that the use of mRDTs was due to changes in treatment policy (11/30), and patients who qualify for the test are those suspected to have malaria. Majority (16/30) related assessment of control line as a measure of test accuracy and suggested the use of microscopy for quality control of mRDT results (15/30). Their major concerns were mRDTs’ inability to give parasite count, stock-out of the tests kits in their working areas and the frequency of negative results. This evaluation encountered several challenges, among them were 1. Poor quality of blood smears made at health facilities, especially dispensaries, which do not have laboratory services. 2. About 3.5% of BS1 slides could not be processed for BS2 because they were damaged during transportation and/or poor quality of smears. This accounts for the small difference in the numbers of BS assessed between two readers. 3. We were not able to prepare standard concentration solutions for qPCR analysis in the country. 4. Problems with PCR machine and inability to repair it that necessitated shipment of the machine, to and from, the manufacturers in Europe (Germany). Malaria RDT QA Final Report, March 2012 6 Due to these challenges, qPCR results were not available until after specimen collection had ended. In this study malaria positivity was higher with mRDTs than microscopy and qPCR for the 200 parasites/μL lower boundary of positivity threshold. This could either be due to the strict lower cut-off point for microscopy and qPCR parasite density or higher false positivity of mRDTs due to persistent antigen in blood, errors in mRDTs performance or other patient’s characteristics. When qPCR was taken as gold standard, mRDTs showed better sensitivity than microscopy, but when microscopy was regarded as a gold standard, mRDTs showed higher sensitivity than with qPCR. However, results of qPCR demonstrated a better correlation (inter-observer agreement) with those of microscopy than with mRDTs. The challenges of performing qPCR, as observed in this evaluation, make it unsuitable for quality assurance of mRDTs in routine care, Tanzania. The high inter-observer agreement between districts’ and reference microscopists (K=0.75) and higher tests performances of BS1 when BS2 was a comparator, demonstrates the competence shown by district’s technicians/ technologists to suffice their involvement as reference microscopists for quality assurance of mRDTs in their respective districts. This is also complimented by a fact that, both BS1 and BS2 had more similar performance when qPCR was taken as a gold standard. In this setting, a microscopy-based quality assurance system to assess mRDT performance in routine use may be a practical and suitable method. However, long distance transportation of smears should be avoided.\u

Field Evaluation of the Photo-induced Electron Transfer Fluorogenic Primers (PET) Real-time PCR for the Detection of Plasmodium falciparum in Tanzania.

Accurate diagnosis of malaria infections remains challenging, especially in the identification of submicroscopic infections. New molecular diagnostic tools that are inexpensive, sensitive enough to detect low-level infections and suitable in laboratory settings of resource-limited countries are required for malaria control and elimination programmes. Here the diagnostic potential of a recently developed photo-induced electron transfer fluorogenic primer (PET) real-time polymerase chain reaction (PCR) called PET-PCR was investigated. This study aimed to (i) evaluate the use of this assay as a method for the detection of both Plasmodium falciparum and other Plasmodium species infections in a developing country's diagnostic laboratory; and, (ii) determine the assay's sensitivity and specificity compared to a nested 18S rRNA PCR. Samples used in this study were obtained from a previous study conducted in the region of Iringa, Tanzania. A total of 303 samples from eight health facilities in Tanzania were utilized for this evaluation. All samples were screened using the multiplex PET-PCR assay designed to detect Plasmodium genus and P. falciparum initially in laboratory in Tanzania and then repeated at a reference laboratory at the CDC in the USA. Microscopy data was available for all the 303 samples. A subset of the samples were tested in a blinded fashion to find the sensitivity and specificity of the PET-PCR compared to the nested 18S rRNA PCR. Compared to microscopy, the PET-PCR assay was 59% more sensitive in detecting P. falciparum infections. The observed sensitivity and specificity were 100% (95% confidence interval (CI0.95) = 94-100%) and (CI0.95 = 96-100%), respectively, for the PET-PCR assay when compared to nested 18S rRNA PCR. When compared to 18S rRNA PCR, microscopy had a low sensitivity of 40% (CI0.95 = 23-61%) and specificity of 100% (CI0.95 = 96-100%). The PET-PCR results performed in the field laboratory in Tanzania were in 100% concordance with the results obtained at the reference laboratory in the USA. The PET-PCR is a new molecular diagnostic tool with similar performance characteristics as commonly used PCR methods that is less expensive, easy to use, and amiable to large scale-surveillance studies in developing country settings

A historical perspective on malaria control in Brazil

Malaria has always been an important public health problem in Brazil. The early history of Brazilian malaria and its control was powered by colonisation by Europeans and the forced relocation of Africans as slaves. Internal migration brought malaria to many regions in Brazil where, given suitableAnopheles mosquito vectors, it thrived. Almost from the start, officials recognised the problem malaria presented to economic development, but early control efforts were hampered by still developing public health control and ignorance of the underlying biology and ecology of malaria. Multiple regional and national malaria control efforts have been attempted with varying success. At present, the Amazon Basin accounts for 99% of Brazil’s reported malaria cases with regional increases in incidence often associated with large scale public works or migration. Here, we provide an exhaustive summary of primary literature in English, Spanish and Portuguese regarding Brazilian malaria control. Our goal was not to interpret the history of Brazilian malaria control from a particular political or theoretical perspective, but rather to provide a straightforward, chronological narrative of the events that have transpired in Brazil over the past 200 years and identify common themes

Prospective Study of Plasmodium vivax Malaria Recurrence after Radical Treatment with a Chloroquine-Primaquine Standard Regimen in Turbo, Colombia.

Plasmodium vivax recurrences help maintain malaria transmission. They are caused by recrudescence, reinfection, or relapse, which are not easily differentiated. A longitudinal observational study took place in Turbo municipality, Colombia. Participants with uncomplicated P. vivax infection received supervised treatment concomitantly with 25 mg/kg chloroquine and 0.25 mg/kg/day primaquine for 14 days. Incidence of recurrence was assessed over 180 days. Samples were genotyped, and origins of recurrences were established. A total of 134 participants were enrolled between February 2012 and July 2013, and 87 were followed for 180 days, during which 29 recurrences were detected. The cumulative incidence of first recurrence was 24.1% (21/87) (95% confidence interval [CI], 14.6 to 33.7%), and 86% (18/21) of these events occurred between days 51 and 110. High genetic diversity of P. vivax strains was found, and 12.5% (16/128) of the infections were polyclonal. Among detected recurrences, 93.1% (27/29) of strains were genotyped as genetically identical to the strain from the previous infection episode, and 65.5% (19/29) of infections were classified as relapses. Our results indicate that there is a high incidence of P. vivax malaria recurrence after treatment in Turbo municipality, Colombia, and that a large majority of these episodes are likely relapses from the previous infection. We attribute this to the primaquine regimen currently used in Colombia, which may be insufficient to eliminate hypnozoites

Availability and Utilization of Malaria Prevention Strategies in Pregnancy in Eastern India

BACKGROUND. Malaria in pregnancy in India, as elsewhere, is responsible for maternal anemia and adverse pregnancy outcomes such as low birth weight and preterm birth. It is not known whether prevention and treatment strategies for malaria in pregnancy (case management, insecticide-treated bednets, intermittent preventive therapy) are widely utilized in India. METHODS. This cross-sectional study was conducted during 2006-2008 in two states of India, Jharkhand and Chhattisgarh, at 7 facilities representing a range of rural and urban populations and areas of more versus less stable malaria transmission. 280 antenatal visits (40/site) were observed by study personnel coupled with exit interviews of pregnant women to assess emphasis upon, availability and utilization of malaria prevention practices by health workers and pregnant women. The facilities were assessed for the availability of antimalarials, lab supplies and bednets. RESULTS. All participating facilities were equipped to perform malaria blood smears; none used rapid diagnostic tests. Chloroquine, endorsed for chemoprophylaxis during pregnancy by the government at the time of the study, was stocked regularly at all facilities although the quantity stocked varied. Availability of alternative antimalarials for use in pregnancy was less consistent. In Jharkhand, no health worker recommended bednet use during the antenatal visit yet over 90% of pregnant women had bednets in their household. In Chhattisgarh, bednets were available at all facilities but only 14.4% of health workers recommended their use. 40% of the pregnant women interviewed had bednets in their household. Only 1.4% of all households owned an insecticide-treated bednet; yet 40% of all women reported their households had been sprayed with insecticide. Antimalarial chemoprophylaxis with chloroquine was prescribed in only 2 (0.7%) and intermittent preventive therapy prescribed in only one (0.4%) of the 280 observed visits. CONCLUSIONS. A disconnect remains between routine antenatal practices in India and known strategies to prevent and treat malaria in pregnancy. Prevention strategies, in particular the use of insecticide-treated bednets, are underutilized. Gaps highlighted by this study combined with recent estimates of the prevalence of malaria during pregnancy in these areas should be used to revise governmental policy and target increased educational efforts among health care workers and pregnant women.United States Agency for International Development/India mission (cooperative agreement GHS-A-00-03-00020-00); National Institute of Allergy and Infectious Disease (R03 HD52167-01); Indian National Institute of Malaria Research; Indo-US Program for Contraception and Reproductive Health Researc

Plasma levels of angiopoietin-1 and -2 predict cerebral malaria outcome in Central India

<p>Abstract</p> <p>Background</p> <p>The mechanisms underlying the pathogenesis of cerebral malaria (CM) syndrome are not well understood. Previous studies have shown a strong association of inflammatory chemokines, apoptotic markers and angiogenic molecules with CM associated mortality. Recognizing the importance of angiopoietins (ANG) in the pathogenesis of CM, a retrospective investigation was carried out in a hospital cohort of malaria patients with <it>Plasmodium </it>infection in central India to determine if these factors could be suitable markers of CM associated severity.</p> <p>Methods</p> <p>Patients enrolled in the study were clinically characterized as healthy controls (HC), mild malaria (MM), CM survivors (CMS) and CM non-survivors (CMNS) based on their malaria status and hospital treatment outcome. Plasma ANG-1 and ANG-2 levels were assessed using sandwich ELISA. Receiver operating characteristic (ROC) curve analysis was used to calculate area under the curve (AUC) for each biomarker in order to assess predictive accuracy of individual biomarkers.</p> <p>Results</p> <p>The plasma levels of ANG-1 were lower in CMS and CMNS compared to control groups (mild malaria and healthy controls) at the time of hospital admission. On the contrary, ANG-2 levels positively correlated with malaria severity and were significantly higher in CMNS. The ratio of ANG-2/ANG-1 was highest in CMNS compared to other groups. Receiver operating characteristic curves revealed that compared to ANG-1 (AUC = 0.35), ANG-2 (AUC = 0.95) and ratio of ANG-2/ANG-1 (AUC = 0.90) were better markers to discriminate CMNS from MM cases. However, they were less specific in predicting fatal outcome amongst CM cases at the time of hospital admission.</p> <p>Conclusion</p> <p>These results suggest that at the time of admission plasma levels of ANG-2 and ratio of ANG-2/ANG-1 are clinically informative biomarkers to predict fatal CM from MM cases while they have limited usefulness in discriminating fatal CM outcomes in a pool of CM cases in endemic settings of Central India.</p

Antibody responses to the merozoite surface protein-1 complex in cerebral malaria patients in India

<p>Abstract</p> <p>Background</p> <p><it>Plasmodium falciparum </it>infection causes cerebral malaria (CM) in a subset of patients with anti-malarial treatment protecting only about 70% to 80% of patients. Why a subset of malaria patients develops CM complications, including neurological sequelae or death, is still not well understood. It is believed that host immune factors may modulate CM outcomes and there is substantial evidence that cellular immune factors, such as cytokines, play an important role in this process. In this study, the potential relationship between the antibody responses to the merozoite surface protein (MSP)-1 complex (which consists of four fragments namely: MSP-1₈₃, MSP-1₃₀, MSP-1₃₈and MSP-1₄₂), MSP-6₃₆and MSP-7₂₂and CM was investigated.</p> <p>Methods</p> <p>Peripheral blood antibody responses to recombinant antigens of the two major allelic forms of MSP-1 complex, MSP-6₃₆and MSP-7₂₂were compared between healthy subjects, mild malaria patients (MM) and CM patients residing in a malaria endemic region of central India. Total IgG and IgG subclass antibody responses were determined using ELISA method.</p> <p>Results</p> <p>The prevalence and levels of IgG and its subclasses in the plasma varied for each antigen. In general, the prevalence of total IgG, IgG1 and IgG3 was higher in the MM patients and lower in CM patients compared to healthy controls. Significantly lower levels of total IgG antibodies to the MSP-1_f38, IgG1 levels to MSP-1_d83, MSP-1₁₉and MSP-6₃₆and IgG3 levels to MSP-1_f42and MSP-7₂₂were observed in CM patients as compared to MM patients.</p> <p>Conclusion</p> <p>These results suggest that there may be some dysregulation in the generation of antibody responses to some MSP antigens in CM patients and it is worth investigating further whether perturbations of antibody responses in CM patients contribute to pathogenesis.</p

Differences in selective pressure on dhps and dhfr drug resistant mutations in western Kenya

<p>Abstract</p> <p>Background</p> <p>Understanding the origin and spread of mutations associated with drug resistance, especially in the context of combination therapy, will help guide strategies to halt and prevent the emergence of resistance. Unfortunately, studies have assessed these complex processes when resistance is already highly prevalent. Even further, information on the evolutionary dynamics leading to multidrug-resistant parasites is scattered and limited to areas with low or seasonal malaria transmission. This study describes the dynamics of strong selection for mutations conferring resistance against sulphadoxine-pyrimethamine (SP), a combination therapy, in western Kenya between 1992 and 1999, just before SP became first-line therapy (1999). Importantly, the study is based on longitudinal data, which allows for a comprehensive analysis that contrasts with previous cross-sectional studies carried out in other endemic regions.</p> <p>Methods</p> <p>This study used 236 blood samples collected between 1992 and 1999 in the Asembo Bay area of Kenya. Pyrosequencing was used to determine the alleles of dihydrofolate reductase (<it>dhfr</it>) and dihydropterote synthase <it>(dhps) </it>genes. Microsatellite alleles spanning 138 kb around <it>dhfr </it>and <it>dhps</it>, as well as, neutral markers spanning approximately 100 kb on chromosomes 2 and 3 were characterized.</p> <p>Results</p> <p>By 1992, the South-Asian <it>dhfr </it>triple mutant was already spreading, albeit in low frequency, in this holoendemic Kenyan population, prior to the use of SP as a first-line therapy. Additionally, <it>dhfr </it>triple mutant alleles that originated independently from the predominant Southeast Asian lineage were present in the sample set. Likewise, <it>dhps </it>double mutants were already present as early as 1992. There is evidence for soft selective sweeps of two <it>dhfr </it>mutant alleles and the possible emergence of a selective sweep of double mutant <it>dhps </it>alleles between 1992 and 1997. The longitudinal structure of the dataset allowed estimation of selection pressures on various <it>dhfr </it>and <it>dhps </it>mutants relative to each other based on a theoretical model tailored to <it>P. falciparum</it>. The data indicate that drug selection acted differently on the resistant alleles of <it>dhfr </it>and <it>dhps</it>, as evidenced by fitness differences. Thus a combination drug therapy such as SP, by itself, does not appear to select for "multidrug"-resistant parasites in areas with high recombination rate.</p> <p>Conclusions</p> <p>The complexity of these observations emphasizes the importance of population-based studies to evaluate the effects of strong drug selection on <it>Plasmodium falciparum </it>populations.</p