Spread and distribution of drug resistance and compensatory mutations in Plasmodium falciparum [Elektronisk resurs]

In 2010 there were an estimated 216 million cases of malaria worldwide. In Honduras there were ~9000 cases of which 88% were due to Plasmodium vivax mono-infection. Chloroquine (CQ) resistant Plasmodium falciparum have spread throughout the world curtailing its use. The only exception appears to be north of Panama where CQ reportedly remains efficacious and the drug of choice for treating both P. falciparum and P. vivax. Resistance to antimalarials is associated with specific genetic polymorphisms and recently a putative H+ pump (pfvp2) has been suggested to be linked to CQ resistant P. falciparum. The aim of this thesis was to identify resistance associated genetic polymorphisms in P. falciparum and P.vivax from Honduras and to describe the worldwide distribution of pfvp2 polymorphisms and their correlation to CQ resistance. Resistance associated genetic polymorphisms in P. falciparum and P. vivax multidrug resistance gene (pfmdr1 and pvmdr1), dihydrofolate reductase (pfdhfr and pvdhfr), P. falciparum chloroquine resistance transporter (pfcrt), dihydropteroate synthase (pfdhps) and V-type H+ pyrophosphatase (pfvp2) were identified in field samples using PCR based methods. From Honduras, 37 P. falciparum and 64 P. vivax samples, collected from symptomatic patients were used. In addition, 50 samples from each of Colombia, Liberia, Guinea-Bissau, Tanzania, Iran, Thailand and Vanuatu were used. The samples represented a time period from 1978 to 2009 and areas with different prevalence of CQ resistant P. falciparum. In samples from Honduras no genetic polymorphisms associated with CQ or sulphadoxinepyrimethamine (SP) resistance were found in P. falciparum. In P. vivax, the CQ resistance associated pvmdr1 976F allele was found in 7/37 samples and the SP resistance associated pvdhfr 57L+58R alleles were found in 2/57 samples. When analysing the worldwide collection of samples, the pfvp2 405V, 582K and 711P haplotype was associated with the for CQ resistance essential allele, pfcrt 76T (P=0.007). Samples with pfvp2 405I and/or 582R and/or 711S were significantly more common in Liberia in 1978-1980 (P=0.01), all African countries (P=0.004) and all African countries + Honduras (P=0.01) compared to the rest of the world. Our results suggest that P. falciparum and P. vivax in Honduras are sensitive to CQ and SP. However, small numbers of P. vivax had genetic polymorphisms suggesting a degree of tolerance to CQ and SP. The association between pfcrt 76T and the pfvp2 405V, 582K and 711P haplotype suggest that this haplotype is associated with CQ resistance. This is in line with previous research that has described increased expression of pfvp2 during CQ exposure. The higher frequency of pfvp2 405I and/or 582R and/or 711S in CQ sensitive settings in Africa and Honduras suggests a larger variation in the pfvp2 genome prior to the spread of CQ resistance further supporting the association between pfvp2 and CQ resistance

Plasmodium vivax mdr1 genotypes in isolates from successfully cured patients living in endemic and non-endemic Brazilian areas

BACKGROUND: Plasmodium vivax is the most widely distributed species causing the highest number of malaria cases in the world. In Brazil, P. vivax is responsible for approximately 84 % of reported cases. In the absence of a vaccine, control strategies are based on the management of cases through rapid diagnosis and adequate treatment, in addition to vector control measures. The approaches used to investigate P. vivax resistance to chloroquine (CQ) were exclusively in vivo studies because of the difficulty in keeping parasites in continuous in vitro culture. In view of the limitations related to follow-up of patients and to assessing the plasma dosage of CQ and its metabolites, an alternative approach to monitor chemo-resistance (QR) is to use molecular markers. Single nucleotide polymorphisms (SNPs) in the multidrug resistance gene pvmdr1 are putative determinants of CQ resistance (CQR), but such SNPs in P. vivax isolates from patients with good response to treatment should be further explored. The aim of this study is to investigate the mutations in the gene, supposedly associated to QR, in P. vivax isolates from successfully cured patients, living in Brazilian endemic and non-endemic areas. METHODS: Blood samples were collected from 49 vivax malaria patients from endemic (Amazon Basin: 45) and non-endemic (Atlantic Forest: four) Brazilian regions and analysed for SNPs in the CQR-related P. vivax gene (pvmdr1), using PCR-based methods. RESULTS: Among the 49 isolates genetically characterized for the gene pvmdr1, 34 (70 %) presented at least one mutation. T958M mutant alleles were the most frequent (73 %) followed Y976F (15 %) and F1076L (12 %). Single mutation was detected in 24 (70.5 %) isolates and double mutations in ten (29.5 %). The most common single mutant genotype was the 958M/Y976/F1076 (79 %), followed by 976F/F1076 (21 %) whereas 958M/Y976/1076L (60 %) and 976F/1076L (40 %) double mutant genotypes were detected. Single mutant profile was observed only in isolates from Amazon Basin, although double mutants were found both in the Amazon and Atlantic Forest regions. Interestingly, the genotype 958M/Y976/1076L was present in all isolates from the Atlantic Forest in the Rio de Janeiro State. CONCLUSIONS: Considering that primaquine (PQ) efficacy is highly dependent on concurrent administration of a blood schizontocidal agent and that PQ could not circumvent CQR, together with the fact that no pvmdr1 mutation should be expected in successfully cured patients, these findings seem to indicate that the pvmdr1 gene is not a reliable marker of CQR. Further investigations are needed to define a reliable molecular marker for monitoring P. vivax CQR in P. vivax populations

Prevalence of Plasmodium falciparum infection among pregnant women at first antenatal visit in post-Ebola Monrovia, Liberia

Abstract Background Disruption of malaria control strategies during the West African 2014–2016 Ebola epidemic led to an increase in malaria-attributable mortality. However, recent data on malaria infection in vulnerable groups, such as pregnant women, are lacking in this post-Ebola scenario. This cross-sectional study aimed to assess the prevalence of Plasmodium falciparum infection and of molecular markers of drug resistance among pregnant women attending antenatal care in Monrovia, capital of Liberia. Methods From October 2016 to June 2017, all pregnant women attending their first antenatal care visit at the Saint Joseph’s Catholic Hospital, Monrovia, were invited to participate in the study. In addition to their routine antenatal care tests, capillary blood spotted onto filter papers were collected from all consenting participants to determine presence of P. falciparum by real-time quantitative PCR. Molecular markers of anti-malarial drug resistance were assessed through Sanger sequencing and quantitative PCR in specimens positive for P. falciparum analysis. Results Of the 195 women participants, 24 (12.3%) were P. falciparum-positive by qPCR. Infected women tended to be more commonly primigravidae and younger than uninfected ones. Parasite densities were higher in primigravidae. Fever was more frequently detected among the infected women. No statistically significant association between P. falciparum infection and haemoglobin levels or insecticide-treated net use was found. While high prevalence of genetic polymorphisms associated with chloroquine and amodiaquine resistance were detected, no molecular markers of artemisinin resistance were observed. Conclusion Plasmodium falciparum infections are expected to occur in at least one in every eight women attending first ANC at private clinics in Monrovia and outside the peak of the rainy season. Young primigravidae are at increased risk of P. falciparum infection. Molecular analyses did not provide evidence of resistance to artemisinins among the P. falciparum isolates tested. Further epidemiological studies involving pregnant women are necessary to describe the risk of malaria in this highly susceptible group outside Monrovia, as well as to closely monitor the emergence of resistance to anti-malarials, as recommended by the Liberian National Malaria Control Programme