Markers of anti-malarial drug resistance in Plasmodium falciparum isolates from Swaziland: identification of pfmdr1-86F in natural parasite isolates

BACKGROUND: The development of Plasmodium falciparum resistance to chloroquine (CQ) has limited its use in many malaria endemic areas of the world. However, despite recent drug policy changes to adopt the more effective artemisinin-based combination (ACT) in Africa and in the Southern African region, in 2007 Swaziland still relied on CQ as first-line anti-malarial drug. METHODS: Parasite DNA was amplified from P. falciparum isolates from Swaziland collected in 1999 (thick smear blood slides) and 2007 (filter paper blood spots). Markers of CQ and sulphadoxine-pyrimethamine (SP) resistance were identified by probe-based qPCR and DNA sequencing. RESULTS: Retrospective microscopy, confirmed by PCR amplification, found that only six of 252 patients treated for uncomplicated malaria in 2007 carried detectable P. falciparum. The pfcrt haplotype 72C/73V/74I/75E/76T occurred at a prevalence of 70% (n = 64) in 1999 and 83% (n = 6) in 2007. Prevalence of the pfmdr1-86N allele was 24% in 1999 and 67% in 2007. A novel substitution of phenylalanine for asparagine at codon 86 of pfmdr1 (N86F) occurred in two of 51 isolates successfully amplified from 1999. The pfmdr1-1246Y allele was common in 1999, with a prevalence of 49%, but was absent among isolates collected in 2007. The 86N/184F/1246D pfmdr1 haplotype, associated with enhanced parasite survival in patients treated with artemether-lumefantrine, comprised 8% of 1999 isolates, and 67% among 2007 isolates. The pfdhfr triple-mutant 16C/51I/59R/108N/164I haplotype associated with pyrimethamine resistance was common in both 1999 (82%, n = 34) and 2007 (50%, n = 6), as was the wild-type 431I/436S/437A/540K/581A/613A haplotype of pfdhps (100% and 93% respectively in 1999 and 2007). The quintuple-mutant haplotype pfdhfr/pfdhps-CIRNI/ISGEAA, associated with high-level resistance to SP, was rare (9%) among 1999 isolates and absent among 2007 isolates. CONCLUSIONS: The prevalence of pfcrt and pfmdr1 alleles reported in this study is consistent with a parasite population under sustained CQ drug pressure. The low prevalence of dhps-437G and dhps-540E mutations (ISGEAA) and the rarity of quintuple-mutant haplotype pfdhfr/pfdhps-CIRNI/ISGEAA suggest that SP retains some efficacy in Swaziland. Anti-malarial policy changes in neighbouring countries may have had an impact on the prevalence of molecular markers of anti-malarial resistance in Swaziland, and it is hoped that this new information will add to understanding of the regional anti-malarial resistance map

ARCHIVED GIEMSA-STAINED THICK-SMEAR BLOOD SAMPLES AS A SOURCE OF DNA FOR PLASMODIUM FALCIPARUM DETECTION BY THE POLYMERASE CHAIN REACTION (PCR): AN IDEA FOR LOW TRANSMISSION SETTINGS

The absence of a reasonable number of cases of malaria in low transmission settings often presents problems for epidemiologic studies of antimalarial drug resistance. Whole blood from filter paper spots is normally used as a source of deoxyribonucleic acid (DNA). The number of samples available to derive statistical power is usually limited due to low transmission rates. This study suggests an alternative source of DNA from Giemsa-stained thick smears (GSTS) for epidemiological studies of Plasmodium falciparum drug resistance. A total of 73 archived GSTS and 6 whole blood filter paper samples were available for this analysis. DNA obtained from GSTS was successfully extracted, genotyped and sequenced for 64 (88%) samples for the Plasmodium falciparum chloroquine resistance transporter (pfcrt) gene, 51 (70%) samples for the Plasmodium falciparum multi-drug resistance 1 (pfmdr1) gene, 34 (47%) samples for the Plasmodium falciparum dihydrofolate reductase (pfdhfr) gene and 27 (37%) samples for the Plasmodium falciparum dihydropteroate synthase (pfdhps) gene. Whole blood from the 6 filter paper samples that were also run for comparison were all (100%) successfully genotyped and sequenced in a single attempt for all the four genes. The ease of the analysis of the filter paper DNA samples and the quality of the gel electrophoresis pictures suggests that filter paper DNA sources are much more sensitive than archived GSTS samples. Nonetheless, the results of this study suggest that safely stored and clearly labelled GSTS can provide a cheap and somewhat reliable alternative source of DNA for retrospective epidemiologic studies using the polymerase chain reaction (PCR) analyses where filter paper sources are either not available or insufficient. It is, therefore, strongly recommended that laboratories in malaria low transmission settings develop guidelines for safe storage of GSTS for future use in genotyping and other experiments. Protocols to refine extraction and PCR methods as well as the design of appropriate primer pairs may improve the sensitivity of current PCR methods to improve the results of GSTS

Knowledge of human social and behavioral factors essential for the success of community malaria control intervention programs: The case of Lomahasha in Swaziland

Although malaria control programs have made rapid progress recently, they neglect important social and behavioral factors associated with the disease. Social, political, and cultural factors are involved in malaria control, and individuals in a community may be comfortable in behaving in ways that, to an outsider, may seem contrary to commonly held perceptions. Malaria control efforts can no longer afford to overlook the multidimensional human contexts that create and support varying notions of malaria and its prevention, treatment, and control. This study aimed to assess the knowledge and perceptions of malaria issues in the community, and to identify practices that support or hinder the progress of malaria control programs. Methods: A triangulation study involving individual interviews, focus group discussions, and observatory analysis between 2003 and 2010 at Lomahasha, a malarious community on the eastern border of Swaziland and Mozambique, was conducted. Results: Results indicated that a high knowledge level and good perception of the disease were observed in the age group of < 40 years, contrary to those in higher age groups, among the Lomahasha community members. However, behavior of certain community groups includes practices that are not supportive of the national control program's aspirations, such as delay in seeking medical attention, staying outdoors until late, maintaining stagnant water in roadside excavations, and seeking medical assistance from wrong sources. Malpractices are more commonly observed among men, boys, and those who drink alcohol. Conclusion: This study suggests a thorough community diagnosis before all intervention programs for malaria control are instituted

Schistosomiasis knowledge, attitude, practices, and associated factors among primary school children in the Siphofaneni area in the Lowveld of Swaziland

Background: Schistosomiasis, a worldwide concern, has received attention in Swaziland through control programs such as deworming programs, education programs, and school health programs; however, these programs neglect the importance of monitoring and evaluation strategies such as assessing children's knowledge, attitudes and practices (KAPs) and the prevalence of the disease. Children are a high-risk group because of their water contact practices, and need to be informed about schistosomiasis to influence their attitudes and practices. Social and cultural factors are involved in schistosomiasis control because they instill myths and misconceptions about the disease. As a result, children in the community may be comfortable with bad practices. This study aimed to assess the KAPs of schoolchildren on schistosomiasis, and to identify practices that support or hinder the progress of schistosomiasis control. Methods: In 2014, a descriptive quantitative cross-sectional survey was conducted through questionnaires among Siphofaneni primary schools, an area hit by schistosomiasis in the Lowveld of Swaziland. A logistic regression model was applied to analyze the data. Results: Moderate knowledge, good attitudes, and fairly good practices were observed in the children. However, practices of certain children were risky and they still had some misconceptions. Knowledge was correlated with practice and with predictors of good and bad practices such as male sex, always urinating in water, and always using river water for domestic practices. Conclusion: This study suggests that empowering children with knowledge and attempting to modify their water contact, and improved human waste disposal practices are necessary for schistosomiasis control

Schistosomiasis knowledge, attitude, practices, and associated factors among primary school children in the Siphofaneni area in the Lowveld of Swaziland

AbstractBackgroundSchistosomiasis, a worldwide concern, has received attention in Swaziland through control programs such as deworming programs, education programs, and school health programs; however, these programs neglect the importance of monitoring and evaluation strategies such as assessing children's knowledge, attitudes and practices (KAPs) and the prevalence of the disease. Children are a high-risk group because of their water contact practices, and need to be informed about schistosomiasis to influence their attitudes and practices. Social and cultural factors are involved in schistosomiasis control because they instill myths and misconceptions about the disease. As a result, children in the community may be comfortable with bad practices. This study aimed to assess the KAPs of schoolchildren on schistosomiasis, and to identify practices that support or hinder the progress of schistosomiasis control.MethodsIn 2014, a descriptive quantitative cross-sectional survey was conducted through questionnaires among Siphofaneni primary schools, an area hit by schistosomiasis in the Lowveld of Swaziland. A logistic regression model was applied to analyze the data.ResultsModerate knowledge, good attitudes, and fairly good practices were observed in the children. However, practices of certain children were risky and they still had some misconceptions. Knowledge was correlated with practice and with predictors of good and bad practices such as male sex, always urinating in water, and always using river water for domestic practices.ConclusionThis study suggests that empowering children with knowledge and attempting to modify their water contact, and improved human waste disposal practices are necessary for schistosomiasis control