A case of dual chloroquine and halofantrine treatment failure in Zimbabwe

A CAJM article on malaria treatment and health policy considerations.A case of malaria treatment failure with chloroquine and halofantrine is reported. The likely determinants and policy considerations are addressed

Selection for Chloroquine-Sensitive Plasmodium Falciparum by Wild Anopheles Arabiensis in Southern Zambia

Background: The emergence of parasite drug resistance, especially Plasmodium falciparum, persists as a major obstacle for malaria control and elimination. To develop effective public health containment strategies, a clear understanding of factors that govern the emergence and spread of resistant parasites in the field is important. The current study documents selection for chloroquine-sensitive malaria parasites by wild Anopheles arabiensis in southern Zambia. Methods. In a 2,000-sq km region, mosquitoes were collected from human sleeping rooms using pyrethrum spray catches during the 2006 malaria transmission season. After morphological examination and molecular confirmation, vector mosquitoes were dissected to separate head and thorax from the abdominal section, followed by PCR screening for P. falciparum infection. Human residents of all ages were tested for P. falciparum parasitaemia by microscopy and PCR. Plasmodium falciparum infections were genotyped at the chloroquine resistance-conferring amino acid codon 76 of the PfCRT gene, using PCR and restriction enzyme digestion. Results: In the human population there was nearly 90% prevalence of the chloroquine-resistant PfCRT K76T mutant, with no significant differences in polymorphism among smear-positive and smear-negative (submicroscopic) infections (p = 0.323, n = 128). However, infections in both abdominal and salivary gland phases of the An. arabiensis vector exhibited wild type K76-bearing parasites with up to 9X higher odds (OR (95% CI): 9 (3.7-20.2), p \u3c 0.0005, n = 125), despite having been acquired from humans within a few weeks. Conclusions: Anopheles arabiensis selects for wild-type K76-bearing P. falciparum during both abdominal and salivary gland phases of parasite development. The rapid vectorial selection, also recently seen with antifolate resistance, is evidence for parasite fitness cost in the mosquito, and may underpin regional heterogeneity in the emergence, spread and waning of drug resistance. Understanding the nature and direction of vector selection could be instrumental for rational curtailment of the spread of drug resistance in integrated malaria control and elimination programmes

A comparative study of the schizontocidal efficacy and safety of artemether versus chloroquine in uncomplicated malaria

A comparative study of medications being used for malaria treatment in Zimbabwe

High Prevalence of DHFR and DHPS Molecular Markers in Plasmodium Falciparum in Pregnant Women of Nchelenge District, Northern Zambia

Background: Sulphadoxine-pyrimethamine (SP) is the recommended drug for intermittent preventive treatment in pregnancy (IPTp) in most African countries, including Zambia. However, malaria is still one of the leading causes of morbidity and mortality in pregnant women despite reports of greater than 50% of women taking at least two doses of SP in IPTp. Studies have shown that resistance to SP is associated with mutations in the dhfr and dhps gene of Plasmodium falciparum. This study examined the prevalence of dhfr and dhps polymorphisms in P. falciparum found in pregnant women of Nchelenge district. Method: This cross-sectional study was conducted in 2013 in Nchelenge, a holoendemic area with malaria prevalence estimated at 50% throughout the year. Three rural health centres were randomly selected and a census survey carried out at each health centre. A questionnaire was administered and malaria testing done using RDT and microscopy, with collection of a dried blood spot. A chelex extraction was done to extract parasite DNA from dried blood spots followed by nested PCR and enzyme restriction digestion. Results: Of the enrolled participants (n∈=∈375), the median age of the women was 23. The prevalence of malaria by PCR was 22%. The PCR positive samples examined (n∈=∈72) showed a high prevalence of dhfr triple (Asn-108∈+∈Arg-59∈+∈Ile-59) mutant (68%) and dhps double (Gly -437∈+∈Glu-540) mutant (21%). The quintuple haplotype was found in 17% with 2 samples with an additional Gly-581mutation. In addition 6% mutations at Val-16 were found and none found at Thr-108 respectively, these both confer resistance to cycloguanil. Multivariate analysis showed that there was an association between malaria and women aged 30-34 years old p∈\u3c∈0.05(AOR: 0.36) at 95% CI. Conclusion: This study showed a high number of mutations in the dhfr and dhps genes. The high malaria endemicity in the general population of this area may have contributed to the high prevalence of resistant parasites in pregnant women, suggesting a need to examine the efficacy of SP given that it is the only approved drug for IPTp in Zambia. © 2015 Siame et al.; licensee BioMed Central

A Simple Chelex Protocol for DNA Extraction From Anopheles Spp

Endemic countries are increasingly adopting molecular tools for efficient typing, identification and surveillance against malaria parasites and vector mosquitoes, as an integral part of their control programs. For sustainable establishment of these accurate approaches in operations research to strengthen malaria control and elimination efforts, simple and affordable methods, with parsimonious reagent and equipment requirements are essential. Here we present a simple Chelex-based technique for extracting malaria parasite and vector DNA from field collected mosquito specimens. We morphologically identified 72 Anopheles gambiae sl. from 156 mosquitoes captured by pyrethrum spray catches in sleeping rooms of households within a 2,000 km(2) vicinity of the Malaria Institute at Macha. After dissection to separate the head and thorax from the abdomen for all 72 Anopheles gambiae sl. mosquitoes, the two sections were individually placed in 1.5 ml microcentrifuge tubes and submerged in 20 μl of deionized water. Using a sterile pipette tip, each mosquito section was separately homogenized to a uniform suspension in the deionized water. Of the ensuing homogenate from each mosquito section, 10 μl was retained while the other 10 μl was transferred to a separate autoclaved 1.5 ml tube. The separate aliquots were subjected to DNA extraction by either the simplified Chelex or the standard salting out extraction protocol(9,10). The salting out protocol is so-called and widely used because it employs high salt concentrations in lieu of hazardous organic solvents (such as phenol and chloroform) for the protein precipitation step during DNA extraction(9). Extracts were used as templates for PCR amplification using primers targeting arthropod mitochondrial nicotinamide adenine dinucleotide dehydrogenase (NADH) subunit 4 gene (ND4) to check DNA quality, a PCR for identification of Anopheles gambiae sibling species(10) and a nested PCR for typing of Plasmodium falciparum infection. Comparison using DNA quality (ND4) PCR showed 93% sensitivity and 82% specificity for the Chelex approach relative to the established salting out protocol. Corresponding values of sensitivity and specificity were 100% and 78%, respectively, using sibling species identification PCR and 92% and 80%, respectively for P. falciparum detection PCR. There were no significant differences in proportion of samples giving amplicon signal with the Chelex or the regular salting out protocol across all three PCR applications. The Chelex approach required three simple reagents and 37 min to complete, while the salting out protocol entailed 10 different reagents and 2 hr and 47 min\u27 processing time, including an overnight step. Our results show that the Chelex method is comparable to the existing salting out extraction and can be substituted as a simple and sustainable approach in resource-limited settings where a constant reagent supply chain is often difficult to maintain

Malaria Research Challenges in Low Prevalence Settings

The prevalence of malaria has reduced significantly in some areas over the past decade. These reductions have made local elimination possible and the research agenda has shifted to this new priority. However, there are critical issues that arise when studying malaria in low transmission settings, particularly identifying asymptomatic infections, accurate detection of individuals with microparasitaemic infections, and achieving a sufficient sample size to have an adequately powered study. These challenges could adversely impact the study of malaria elimination if they remain unanswered

Temporal and Spatial Patterns of Serologic Responses to Plasmodium Falciparum Antigens in a Region of Declining Malaria Transmission in Southern Zambia

Background: Critical to sustaining progress in malaria control is comprehensive surveillance to identify outbreaks and prevent resurgence. Serologic responses to Plasmodium falciparum antigens can serve as a marker of recent transmission and serosurveillance may be feasible on a large scale. Methods. Satellite images were used to construct a sampling frame for the random selection of households enrolled in prospective longitudinal and cross-sectional surveys in two study areas in Southern Province, Zambia, one in 2007 and the other in 2008 and 2009. Blood was collected and stored as dried spots from participating household members. A malaria rapid diagnostic test (RDT) was used to diagnose malaria. An enzyme immunoassay (EIA) was used to detect IgG antibodies to asexual stage P. falciparum whole parasite lysate using serum eluted from dried blood spots. The expected mean annual increase in optical density (OD) value for individuals with a documented prior history of recent malaria was determined using mixed models. SatScan was used to determine the spatial clustering of households with individuals with serological evidence of recent malaria, and these households were plotted on a malaria risk map. Results: RDT positivity differed markedly between the study areas and years: 28% of participants for whom serologic data were available were RDT positive in the 2007 study area, compared to 8.1% and 1.4% in the 2008 and 2009 study area, respectively. Baseline antibody levels were measured in 234 participants between April and July 2007, 435 participants between February and December 2008, and 855 participants between January and December 2009. As expected, the proportion of seropositive individuals increased with age in each year. In a subset of participants followed longitudinally, RDT positivity at the prior visit was positively correlated with an increase in EIA OD values after adjusting for age in 2007 (0.261, p = 0.003) and in 2008 (0.116, p = 0.03). RDT positivity at the concurrent visit also was associated with an increase in EIA OD value in 2007 (mean increase 0.177, p = 0.002) but not in 2008 (-0.063, p =0.50). Households comprised of individuals with serologic evidence of recent malaria overlapped areas of high malaria risk for serologic data from 2009, when parasite prevalence was lowest. Conclusions: Serological surveys to whole asexual P. falciparum antigens using blood collected as dried blood spots can be used to detect temporal and spatial patterns of malaria transmission in a region of declining malaria burden, and have the potential to identify focal areas of recent transmission

Malaria Research Challenges in Low Prevalence Settings

The prevalence of malaria has reduced significantly in some areas over the past decade. These reductions have made local elimination possible and the research agenda has shifted to this new priority. However, there are critical issues that arise when studying malaria in low transmission settings, particularly identifying asymptomatic infections, accurate detection of individuals with microparasitaemic infections, and achieving a sufficient sample size to have an adequately powered study. These challenges could adversely impact the study of malaria elimination if they remain unanswered

Comparison of a PfHRP2-Based Rapid Diagnostic Test and Pcr for Malaria in a Low Prevalence Setting in Rural Southern Zambia: Implications for Elimination

Background: Rapid diagnostic tests (RDTs) detecting histidine-rich protein 2 (PfHRP2) antigen are used to identify individuals with Plasmodium falciparum infection even in low transmission settings seeking to achieve elimination. However, these RDTs lack sensitivity to detect low-density infections, produce false negatives for P. falciparum strains lacking pfhrp2 gene and do not detect species other than P. falciparum. Methods: Results of a PfHRP2-based RDT and Plasmodium nested PCR were compared in a region of declining malaria transmission in southern Zambia using samples from community-based, cross-sectional surveys from 2008 to 2012. Participants were tested with a PfHRP2-based RDT and a finger prick blood sample was spotted onto filter paper for PCR analysis and used to prepare blood smears for microscopy. Species-specific, real-time, quantitative PCR (q-PCR) was performed on samples that tested positive either by microscopy, RDT or nested PCR. Results: Of 3,292 total participants enrolled, 12 (0.4%) tested positive by microscopy and 42 (1.3%) by RDT. Of 3,213 (98%) samples tested by nested PCR, 57 (1.8%) were positive, resulting in 87 participants positive by at least one of the three tests. Of these, 61 tested positive for P. falciparum by q-PCR with copy numbers ≤ 2 × 103 copies/μL, 5 were positive for both P. falciparum and Plasmodium malariae and 2 were positive for P. malariae alone. RDT detected 32 (53%) of P. falciparum positives, failing to detect three of the dual infections with P. malariae. Among 2,975 participants enrolled during a low transmission period between 2009 and 2012, sensitivity of the PfHRP2-based RDT compared to nested PCR was only 17%, with specificity of \u3e99%. The pfhrp gene was detected in 80% of P. falciparum positives; however, comparison of copy number between RDT negative and RDT positive samples suggested that RDT negatives resulted from low parasitaemia and not pfhrp2 gene deletion. Conclusions: Low-density P. falciparum infections not identified by currently used PfHRP2-based RDTs and the inability to detect non-falciparum malaria will hinder progress to further reduce malaria in low transmission settings of Zambia. More sensitive and specific diagnostic tests will likely be necessary to identify parasite reservoirs and achieve malaria elimination

Malaria Antifolate Resistance With Contrasting Plasmodium Falciparum Dihydrofolate Reductase (DHFR) Polymorphisms in Humans and Anopheles Mosquitoes

Surveillance for drug-resistant parasites in human blood is a major effort in malaria control. Here we report contrasting antifolate resistance polymorphisms in Plasmodium falciparum when parasites in human blood were compared with parasites in Anopheles vector mosquitoes from sleeping huts in rural Zambia. DNA encoding P. falciparum dihydrofolate reductase (EC 1.5.1.3) was amplified by PCR with allele-specific restriction enzyme digestions. Markedly prevalent pyrimethamine-resistant mutants were evident in human P. falciparum infections - S108N (\u3e90%), with N51I, C59R, and 108N+51I+59R triple mutants (30-80%). This resistance level may be from selection pressure due to decades of sulfadoxine/pyrimethamine use in the region. In contrast, cycloguanil-resistant mutants were detected in very low frequency in parasites from human blood samples - S108T (13%), with A16V and 108T+16V double mutants (∼4%). Surprisingly, pyrimethamine-resistant mutants were of very low prevalence (2-12%) in the midguts of Anopheles arabiensis vector mosquitoes, but cycloguanil-resistant mutants were highly prevalent - S108T (90%), with A16V and the 108T+16V double mutant (49-57%). Structural analysis of the dihydrofolate reductase by in silico modeling revealed a key difference in the enzyme within the NADPH binding pocket, predicting the S108N enzyme to have reduced stability but the S108T enzyme to have increased stability. We conclude that P. falciparum can bear highly host-specific drug-resistant polymorphisms, most likely reflecting different selective pressures found in humans and mosquitoes. Thus, it may be useful to sample both human and mosquito vector infections to accurately ascertain the epidemiological status of drug-resistant alleles