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

Exploring the Impact of Targeted Distribution of Free Bed Nets on Households Bed Net Ownership, Socio-Economic Disparities and Childhood Malaria Infection Rates: Analysis of National Malaria Survey Data from three Sub-Saharan Africa countries.

The last decade has witnessed increased funding for malaria control. Malaria experts have used the opportunity to advocate for rollout of such interventions as free bed nets. A free bed net distribution strategy is seen as the quickest way to improve coverage of effective malaria control tools especially among poorest communities. Evidence to support this claim is however, sparse. This study explored the effectiveness of targeted free bed net distribution strategy in achieving equity in terms of ownership and use of bed nets and also reduction of malaria prevalence among children under-five years of age. National malaria indicator survey (MIS) data from Angola, Tanzania and Uganda was used in the analysis. Hierarchical multilevel logistic regression models were used to analyse the relationship between variables of interest. Outcome variables were defined as: childhood test-confirmed malaria infections, household ownership of any mosquito net and children's use of any mosquito nets. Marginal effects of having free bed net distribution on households with different wealth status were calculated. Angolan children from wealthier households were 6.4 percentage points less likely to be parasitaemic than those in poorest households, whereas those from Tanzania and Uganda were less likely to test malaria positive by 7 and 11.6 percentage points respectively (p < 0.001). The study estimates and present results on the marginal effects based on the impact of free bed net distribution on children's malaria status given their socio-economic background. Poorest households were less likely to own a net by 21.4% in Tanzania, and 2.8% in Uganda, whereas both poorer and wealthier Angolan households almost achieved parity in bed net ownership (p < 0.001). Wealthier households had a higher margin of using nets than poorest people in both Tanzania and Uganda by 11.4% and 3.9% respectively. However, the poorest household in Angola had a 6.1% net use advantage over children in wealthier households (p < 0.001). This is the first study to use nationally representative data to explore inequalities in bed net ownership and related consequences on childhood malaria infection rates across different countries. While targeted distribution of free bed nets improved overall bed net ownership, it did not overcome ownership inequalities as measured by household socioeconomic status. Use of bed nets was disproportionately lower among poorest children, except for Angola where bed net use was higher among poorest households when compared to children in wealthier households. The study highlights the need for malaria control world governing bodies and policy makers to continue working on finding appropriate strategies to improve access to effective malaria control tools especially by the poorest who often times bears the brunt of malaria burden than their wealthier counterparts

How Patients Take Malaria Treatment: A Systematic Review of the Literature on Adherence to Antimalarial Drugs

High levels of patient adherence to antimalarial treatment are important in ensuring drug effectiveness. To achieve this goal, it is important to understand levels of patient adherence, and the range of study designs and methodological challenges involved in measuring adherence and interpreting results. Since antimalarial adherence was reviewed in 2004, there has been a major expansion in the use of artemisinin-based combination therapies (ACTs) in the public sector, as well as initiatives to make them more widely accessible through community health workers and private retailers. These changes and the large number of recent adherence studies raise the need for an updated review on this topic. We conducted a systematic review of studies reporting quantitative results on patient adherence to antimalarials obtained for treatment. The 55 studies identified reported extensive variation in patient adherence to antimalarials, with many studies reporting very high adherence (90–100%) and others finding adherence of less than 50%. We identified five overarching approaches to assessing adherence based on the definition of adherence and the methods used to measure it. Overall, there was no clear pattern in adherence results by approach. However, adherence tended to be higher among studies where informed consent was collected at the time of obtaining the drug, where patient consultations were directly observed by research staff, and where a diagnostic test was obtained. Variations in reported adherence may reflect factors related to patient characteristics and the nature of their consultation with the provider, as well as methodological variations such as interaction between the research team and patients before and during the treatment. Future studies can benefit from an awareness of the impact of study procedures on adherence outcomes, and the identification of improved measurement methods less dependent on self-report

Putting the genie back in the bottle? Availability and presentation of oral artemisinin compounds at retail pharmacies in urban Dar-es-Salaam

BACKGROUND: Recently global health advocates have called for the introduction of artemisinin-containing antimalarial combination therapies to help curb the impact of drug-resistant malaria in Africa. Retail trade in artemisinin monotherapies could undermine efforts to restrict this class of medicines to more theoretically sound combination treatments. METHODS: This paper describes a systematic search for artemisinin-containing products at a random sample of licensed pharmacies in Dar-es-Salaam, Tanzania in July 2005. RESULTS: Nineteen different artemisinin-containing oral pharmaceutical products, including one co-formulated product, one co-packaged product, and 17 monotherapies were identified. All but one of the products were legally registered and samples of each product were obtained without a prescription. Packaging and labeling of the products seldom included local language or illustrated instructions for low-literate clients. Packaging and inserts compared reasonably well with standards recommended by the national regulatory authority with some important exceptions. Dosing instructions were inconsistent, and most recommended inadequate doses based on international standards. None of the monotherapy products mentioned potential benefits of combining the treatment with another antimalarial drug. CONCLUSION: The findings confirm the widespread availability of artemisinin monotherapies that led the World Health Organization to call for the voluntary withdrawal of these drugs in malaria-endemic countries. As the global public health community gathers resources to deploy artemisinin-containing combination therapies in Africa, planners should be mindful that these drugs will coexist with artemisinin monotherapies in an already well-established market place. In particular, regulatory authorities should be incorporated urgently into the process of planning for rational deployment of artemisinin-containing antimalarial combination therapies

Getting antimalarials on target: impact of national roll-out of malaria rapid diagnostic tests on health facility treatment in three regions of Tanzania.

OBJECTIVES: Parasitological confirmation of malaria prior to treatment is recommended for patients of all ages, with malaria rapid diagnostic tests (mRDTs) an important tool to target artemisinin-based combination therapies (ACTs) to patients with malaria. To evaluate the impact on case management practices of routine government implementation of mRDTs, we conducted large-scale health facility surveys in three regions of Tanzania before and after mRDT roll-out. METHODS: Febrile patients at randomly selected health facilities were interviewed about care received at the facility, and blood samples were collected for reference blood smears. Health facility staff were interviewed about their qualifications and availability of malaria diagnostics and drugs. RESULTS: The percentage of febrile patients tested for malaria at the facility increased from 15.8% in 2010 to 54.9% in 2012. ACTs were obtained by 65.8% of patients positive by reference blood smear in 2010 and by 50.2% in 2012 (P = 0.0675); no antimalarial was obtained by 57.8% of malaria-negative patients in 2010 and by 82.3% in 2012 (P < 0.0001). Overall, ACT use decreased (39.9-21.3%, P < 0.0001) and antibiotic use increased (31.2-48.5%, P < 0.0001). CONCLUSION: Roll-out of mRDTs in Tanzania dramatically improved diagnostic testing for malaria and reduced overuse of ACTs for patients without parasitemia. However, post-roll-out almost 50% of febrile patients did not receive a diagnostic test, and almost 50% of patients testing positive did not receive ACTs. Stock-outs of ACTs and mRDTs were important problems. Further investigation is needed to determine reasons for not providing ACTs to patients with malaria and potential for inappropriate antibiotic use

The genetic change in P. falciparum populations of rural Tanzania resulting from national policy on firstline malaria treatment and pilot Sulfadoxine/pyrimethamine and Artesunate combination

Malaria Journal 2010, 9(Suppl 2):P20Theory predicts that we can protect the efficacy of future antimalarials by changing treatment practice or drug formulation, but the potential success of such interventions rests upon their impact on drug pressure in the field. So far, gathering field data on the relationship between policy, drug pressure, recombination and the evolution of resistance has been entirely challenging. To test these predictions, dhfr and dhps frequency changes were measured in two rural districts of Rufiji and Kilombero/Ulanga during 2000-2006, and the frequencies of the two genes compared prior, during and after antimalarial policy change from first line CQ to first line SP in 2001. Furthermore, while SP first line was maintained in Kilombero/Ulanga, pilot combination therapy of SP+Artesunate (ART) was introduced in Rufiji in 2002 to replace SP and dhfr and dhps frequency changes compared between the two districts. Size polymorphisms at three sets of microsatellite loci linked to dhfr and three other sets of unlinked microsatellite loci were analysed. Genetic analysis of SP resistance genes was carried out on 9,662 Plasmodium falciparum infections identified in a series of annual cross sectional surveys conducted in the two districts between 2000-2006. The frequency of dhfr and dhps resistance alleles did not change significantly while SP was the recommended second-line treatment, but highly significant changes occurred during the subsequent year after the switch to first line SP. The frequency of the triple mutant dhfr allele increased by 37% -63% and that of double mutant dhps allele increased 200%-300%. A strong association between these unlinked alleles also emerged; confirming that they are co-selected by SP. Distribution of major lineages indicates that there is extensive genetic exchange among the geographic regions. Combination therapy had visible effect on the frequencies of dhfr and dhps resistance alleles. The findings of this study provide insight on the interplay between policy, drug pressure, recombination and the evolution of resistance

Increased proportions of outdoor feeding among residual malaria vector populations following increased use of insecticide-treated nets in rural Tanzania

Abstract Background: Insecticide-treated nets (ITNs) and indoor residual spraying (IRS) represent the front-line tools for malaria vector control globally, but are optimally effective where the majority of baseline transmission occurs indoors. In the surveyed area of rural southern Tanzania, bed net use steadily increased over the last decade, reducing malaria transmission intensity by 94%. Methods: Starting before bed nets were introduced (1997), and then after two milestones of net use had been reached-75% community-wide use of untreated nets (2004) and then 47% use of ITNs (2009)-hourly biting rates of malaria vectors from the Anopheles gambiae complex and Anopheles funestus group were surveyed. Results: In 1997, An. gambiae s.l. and An. funestus mosquitoes exhibited a tendency to bite humans inside houses late at night. For An. gambiae s.l., by 2009, nocturnal activity was less (p = 0.0018). At this time, the sibling species composition of the complex had shifted from predominantly An. gambiae s.s. to predominantly An. arabiensis. For An.funestus, by 2009, nocturnal activity was less (p = 0.0054) as well as the proportion biting indoors (p < 0.0001). At this time, An. funestus s.s. remained the predominant species within this group. As a consequence of these altered feeding patterns, the proportion (mean ± standard error) of human contact with mosquitoes (bites per person per night)occurring indoors dropped from 0.99 ± 0.002 in 1997 to 0.82 ± 0.008 in 2009 for the An. gambiae complex (p = 0.0143)and from 1.00 ± <0.001 to only 0.50 ± 0.048 for the An. funestus complex (p = 0.0004) over the same time period. Conclusions: High usage of ITNs can dramatically alter African vector populations so that intense, predominantly indoor transmission is replaced by greatly lowered residual transmission, a greater proportion of which occurs outdoors. Regardless of the underlying mechanism, the residual, self-sustaining transmission will respond poorly to further insecticidal measures within houses. Additional vector control tools which target outdoor biting mosquitoes at the adult or immature stages are required to complement ITNs and IRS

Caution is required when using health facility-based data to evaluate the health impact of malaria control efforts in Africa

The global health community is interested in the health impact of the billions of dollars invested to fight malaria in Africa. A recent publication used trends in malaria cases and deaths based on health facility records to evaluate the impact of malaria control efforts in Rwanda and Ethiopia. Although the authors demonstrate the use of facility-based data to estimate the impact of malaria control efforts, they also illustrate several pitfalls of such analyses that should be avoided, minimized, or actively acknowledged. A critique of this analysis is presented because many country programmes and donors are interested in evaluating programmatic impact with facility-based data. Key concerns related to: 1) clarifying the objective of the analysis; 2) data validity; 3) data representativeness; 4) the exploration of trends in factors that could influence malaria rates and thus confound the relationship between intervention scale-up and the observed changes in malaria outcomes; 5) the analytic approaches, including small numbers of patient outcomes, selective reporting of results, and choice of statistical and modeling methods; and 6) internal inconsistency on the strength and interpretation of the data. In conclusion, evaluations of malaria burden reduction using facility-based data could be very helpful, but those data should be collected, analysed, and interpreted with care, transparency, and a full recognition of their limitations

Media, Health Workers, and Policy Makers' Relationship and Their Impact on Antimalarial Policy Adoption: A Population Genetics Perspective

Drug resistance negatively impacts malaria treatments, making treatment policy revision unavoidable. So far, studies relating sociopolitical and technical issues on policy change with malaria parasite genetic change are lacking. We have quantified the effect of malaria treatment policy on drug pressure and the influence of the media, policy makers, and health worker relationship on parasite population genetic change in Kilombro/Ulanga district. Cross-sectional surveys of asymptomatic infections conducted before, during and after the switch from chloroquine to sulphadoxine/pyrimethamine were used for genetic analysis of SP resistance genes in 4,513 asymptomatic infections identified, and their frequency change was compared with retrospective study of the documented process of policy change. Highly significant changes of dhfr and dhps resistance alleles occurred within one year of switch to SP first line, followed by a decline of their rate of selection caused by reduction of SP usage, as a result of negative media reports on SP usage and lack of adequate preparations

Quantification of markers of antimalarial drug resistance from an area of high malaria transmission: Comparing frequency with prevalence

Molecular monitoring of markers of antimalarial drug resistance offers an affordable alternative to the in vivo method for the detection of resistance, and has the potential to guide public health policy in a timely manner. However, the optimal way of analyzing and reporting these data, particularly those emanating from areas of moderate to high malaria transmission, has never been fully explored or agreed upon, given the potential of being confounded by coinfections. By using large number of real field samples, we quantified the difference between prevalence and frequency when reporting field data on antimalarial drug resistance obtained by direct counting of haplotypes. Polymerase chain reaction (PCR) and sequence specific oligonucleotide probing was used to generate point mutations which were used to construct haplotypes. Results indicate that frequency underestimates haplotypes present at low levels while also amplifying haplotypes present at high levels; prevalence on the other hand behaved in a vice versa manner. Both prevalence and frequency are therefore essential, as each may have relevance in different contexts in high malaria transmission settings. Frequency is essential to gauge the impact of intervention on antimalarial drug resistance while prevalence may be more relevant when the aim is to determine parasite clearance. Key words: Molecular markers, polymerase chain reaction (PCR) - sequence specific oligonucleotide probing (SSOP), prevalence, frequency