Subpatent Plasmodium with mutant pfmdr1, pfcrt, and pvmdr1 alleles from endemic provinces in Mindanao, the Philippines: implications for local malaria elimination.

OBJECTIVES: This study was performed to identify and characterize circulating Plasmodium species in three provinces of Mindanao approaching malaria elimination. METHODS: Rapid diagnostic tests (RDT), microscopic examination, and PCR were used to detect malaria parasites. PCR-positive isolates were genotyped for polymorphisms in loci of interest. RESULTS: A total of 2639 participants were surveyed in Mindanao between 2010 and 2013. Malaria prevalence by PCR was 3.8% (95% confidence interval (CI): 2.7-5.2%) in Sarangani, 10% (95% CI: 7.7-12.7%) in South Cotabato, and 4.2% (95% CI: 3.2-5.6%) in Tawi-Tawi. P. falciparum and P. vivax were identified in all three provinces, and there was one case of P. malariae in South Cotabato. RDT was inferior to PCR for detecting asymptomatic P. falciparum and P. vivax. In Tawi-Tawi, microscopy failed to identify 46 PCR-positive malaria infections. The presence of pfcrt haplotypes CVMNK, CVIET, and SMNT (codons 72-76), pfmdr1 haplotype NFSND (codons 86, 184, 1034, 1042, 1246), and pvmdr1 haplotype NFL (codons 91, 976, 1076) was confirmed in Mindanao. CONCLUSIONS: Asymptomatic Plasmodium infections persisted in local communities between 2010 and 2013. PCR successfully identified subpatent malaria infections, and can better characterize malaria epidemiology in communities seeking malaria control and elimination at the local level.

Antimalarial drug resistance markers in human immunodeficiency virus (HIV)-positive and HIV-negative adults with asymptomatic malaria infections in Port Harcourt, Nigeria.

BACKGROUND: In Nigeria, indiscriminate use of antimalarial drugs may contribute to the threat of drug resistance, but this has not been evaluated among people living with human immunodeficiency virus (HIV). METHODS: HIV-positive adults attending a university hospital HIV clinic and HIV-negative adult volunteers from the university hospital community with a positive blood film were treated with artemether-lumefantrine. Parasite DNA from before and after treatment was polymerase chain reaction amplified to identify molecular markers of drug susceptibility. RESULTS: The pfcrt76T genotype was prevalent among both HIV-positive and HIV-negative participants (78.6% and 68.2%, respectively). Three new mutations in the pfmdr1 gene-F73S, S97L and G165R-and the uncommon pfdhps S436F variant were detected, whereas pfdhps K540E and pfdhfr I164L were absent. The A437G allele of pfdhps predominated (62/66 [94%]). The I431 V mutation was found in 19 of 66 pretreatment pfdhps sequences (28.8%). The pfmdr1 86N allele was significantly more common at day 3 post-treatment than at baseline (odds ratio 8.77 [95% confidence interval 1.21 to 380]). CONCLUSIONS: We found evidence of continued chloroquine use among HIV-positive individuals. Selection for the pfmdr1 86N after artemether-lumefantrine treatment was observed, indicating a possible threat to antimalarial efficacy in the study area. The complexity of pfdhps haplotypes emphasises the need for careful monitoring of anti-folate susceptibility in Nigeria.

Mosquito and human hepatocyte infections with Plasmodium ovale curtisi and Plasmodium ovale wallikeri.

BACKGROUND: Human ovale malaria is caused by the two closely related species, Plasmodium ovale curtisi and P. ovale wallikeri. Both species are known to relapse from quiescent hepatic forms months or years after the primary infection occurred. Although some studies have succeeded in establishing mosquito transmission for ovale malaria, none have specifically described transmission and human hepatocyte infection of both sibling species. METHODS: Here we describe a simplified protocol for successful transmission of both P. ovale curtisi and P. ovale wallikeri to Anopheles coluzzii mosquitoes and streamlined monitoring of infection using sensitive parasite DNA detection, by loop-activated amplification, in blood-fed mosquitoes. RESULTS: In one experimental infection with P. ovale curtisi and one with P. ovale wallikeri, viable sporozoites were isolated from mosquito salivary glands and used to successfully infect cultured human hepatocytes. CONCLUSIONS: This protocol provides a method for the utilisation of pretreatment clinical blood samples from ovale malaria patients, collected in EDTA, for mosquito infection studies and generation of the hepatic life cycle stages of P. ovale curtisi and P. ovale wallikeri. We also demonstrate the utility of loop-activated amplification as a rapid and sensitive alternative to dissection for estimating the prevalence of infection in Anopheles mosquitoes fed with Plasmodium-infected blood.

Plasmodium malariae and Plasmodium ovale infections and their association with common red blood cell polymorphisms in a highly endemic area of Uganda.

BACKGROUND: Plasmodium ovale and Plasmodium malariae infections are scarcely studied in sub-Saharan Africa, where the Plasmodium falciparum species predominates. The objective of this study is to investigate the prevalence of P. ovale and P. malariae infections and their relationship with common red blood cell polymorphisms in a cohort of 509 individuals from Uganda. METHODS: Three cross-sectional surveys were conducted in individuals of 1-10 and >20 y of age from the Apac district at baseline and 6 and 16 weeks after drug treatment. Malaria infections were assessed by polymerase chain reaction and genotyping was performed for the sickle-cell allele, α-thalassaemia and glucose-6-phosphate dehydrogenase. RESULTS: At baseline, the prevalence of infection was 7.5%, 12.6% and 57.4% for P. ovale, P. malariae and P. falciparum species, respectively. Co-infections were present in 14.1% of individuals, all including P. falciparum parasites. In children 1-5 y of age, the prevalence of P. ovale mono-infections increased significantly from 1.7% to 7.3% over time (p=0.004) while the prevalence of P. malariae and P. falciparum infections declined significantly during this study. After adjusting for confounding and multiple testing, only α-thalassaemia had a statistically significant increase in the odds of P. falciparum infections (odds ratio 1.93 [95% confidence interval 1.26 to 2.94]). CONCLUSIONS: Common red blood cell polymorphisms do not show strong effects on mild Plasmodium infections in this Ugandan population. To understand the extent of this result, similar studies should be carried out in other populations using larger cohorts.

Molecular determinants of sulfadoxine-pyrimethamine resistance in Plasmodium falciparum in Nigeria and the regional emergence of dhps 431V.

There are few published reports of mutations in dihydropteroate synthetase (dhps) and dihydrofolate reductase (dhfr) genes in P. falciparum populations in Nigeria, but one previous study has recorded a novel dhps mutation at codon 431 among infections imported to the United Kingdom from Nigeria. To assess how widespread this mutation is among parasites in different parts of the country and consequently fill the gap in sulfadoxine-pyrimethamine (SP) resistance data in Nigeria, we retrospectively analysed 1000 filter paper blood spots collected in surveys of pregnant women and children with uncomplicated falciparum malaria between 2003 and 2015 from four sites in the south and north. Genomic DNA was extracted from filter paper blood spots and placental impressions. Point mutations at codons 16, 50, 51, 59, 108, 140 and 164 of the dhfr gene and codons 431, 436, 437, 540, 581 and 613 of the dhps gene were evaluated by nested PCR amplification followed by direct sequencing. The distribution of the dhps-431V mutation was widespread throughout Nigeria with the highest prevalence in Enugu (46%). In Ibadan where we had sequential sampling, its prevalence increased from 0% to 6.5% between 2003 and 2008. Although there were various combinations of dhps mutations with 431V, the combination 431V + 436A + 437G+581G+613S was the most common. All these observations support the view that dhps-431V is on the increase. In addition, P. falciparum DHPS crystal structure modelling shows that the change from Isoleucine to Valine (dhps-431V) could alter the effects of both S436A/F and A437G, which closely follow the 2nd ß-strand. Consequently, it is now a research priority to assess the implications of dhps-VAGKGS mutant haplotype on continuing use of SP in seasonal malaria chemoprevention (SMC) and intermittent preventive treatment in pregnancy (IPTp). Our data also provides surveillance data for SP resistance markers in Nigeria between 2003 and 2015.

Paper-Origami-Based Multiplexed Malaria Diagnostics from Whole Blood.

We demonstrate, for the first time, the multiplexed determination of microbial species from whole blood using the paper-folding technique of origami to enable the sequential steps of DNA extraction, loop-mediated isothermal amplification (LAMP), and array-based fluorescence detection. A low-cost handheld flashlight reveals the presence of the final DNA amplicon to the naked eye, providing a "sample-to-answer" diagnosis from a finger-prick volume of human blood, within 45 min, with minimal user intervention. To demonstrate the method, we showed the identification of three species of Plasmodium, analyzing 80 patient samples benchmarked against the gold-standard polymerase chain reaction (PCR) assay in an operator-blinded study. We also show that the test retains its diagnostic accuracy when using stored or fixed reference samples.

Genome-scale comparison of expanded gene families in Plasmodium ovale wallikeri and Plasmodium ovale curtisi with Plasmodium malariae and with other Plasmodium species.

Malaria in humans is caused by six species of Plasmodium parasites, of which the nuclear genome sequences for the two Plasmodium ovale spp., P. ovale curtisi and P. ovale wallikeri, and Plasmodium malariae have not yet been analyzed. Here we present an analysis of the nuclear genome sequences of these three parasites, and describe gene family expansions therein. Plasmodium ovale curtisi and P. ovale wallikeri are genetically distinct but morphologically indistinguishable and have sympatric ranges through the tropics of Africa, Asia and Oceania. Both P. ovale spp. show expansion of the surfin variant gene family, and an amplification of the Plasmodium interspersed repeat (pir) superfamily which results in an approximately 30% increase in genome size. For comparison, we have also analyzed the draft nuclear genome of P. malariae, a malaria parasite causing mild malaria symptoms with a quartan life cycle, long-term chronic infections, and wide geographic distribution. Plasmodium malariae shows only a moderate level of expansion of pir genes, and unique expansions of a highly diverged transmembrane protein family with over 550 members and the gamete P25/27 gene family. The observed diversity in the P. ovale wallikeri and P. ovale curtisi surface antigens, combined with their phylogenetic separation, supports consideration that the two parasites be given species status.

Assessment of Markers of Antimalarial Drug Resistance in Plasmodium falciparum Isolates from Pregnant Women in Lagos, Nigeria.

BACKGROUND: The use of antimalarial drugs for prevention and treatment is a major strategy in the prevention of malaria in pregnancy. Although sulphadoxine-pyrimethamine (SP) is currently recommended for intermittent preventive treatment of malaria during pregnancy in Nigeria, previously used drugs for prophylaxis such as chloroquine (CQ) and pyrimethamine are accessible as they are purchased over the counter. This study describes the markers of absence or presence of resistance to quinoline (Pfcrt and Pfmdr 1) and type 1 antifolate antimalarial medicines (Pfdhfr). METHODS: Plasmodium falciparum-positive dried blood spots from pregnant women attending antenatal clinics for the first time during current pregnancy were investigated for the presence of mutations at codons 72-76 of Plasmodium falciparum chloroquine resistance transporter (Pfcrt) gene by real time polymerase chain reaction (PCR) using haplotype-specific probes. PCR followed by sequence analysis was used to identify mutations at codons 86, 184, 1034, 1042 and 1246 of P. falciparum multi-drug resistance-1 (Pfmdr1) gene; and codons 16, 50, 51, 59, 108, 140 and 164 of Pfdhfr gene. RESULTS: Two haplotypes of Pfcrt (n = 54) were observed: CVMNK 13(24.2%) and CVIET 41 (75.9%) of the samples. The SVMNT haplotype was absent in this population. The Pfmdr1 (n = 28) haplotypes were NYSND 15(53.6%), YYSND 5(17.9%), NFSND 6(21.4%) and YFSND 2(7.1%). The Pfdhfr (n = 15) were ACNCSVI 4(26.7%), and ACICNSVI 1(6.7%) and ACIRNVI 10 (66.7%). The rate of occurrence of Pfcrt 76T, Pfdhfr108N, Pfmdr186Y and 184F were 75.9%, 73.3%, 25% and 28.1% respectively. The Pfmdr1 86Y was associated with low parasitaemia (median = 71 parasites/µl, P = 0.024) while Pfcrt 76T was associated with young maternal age (mean 24.1 ± 4.5 years; P = 0.006). The median parasitaemia were similar (P>0.05) in wild and mutant strains of Pfcrt 76, Pfmdr1 184 and Pfdhfr 108. There was no association between gravidity or gestational age of the women and presence of mutations in the Pfcrt, Pfmdr1 or Pfdhfr genes (P>0.05). CONCLUSION: Markers of resistance to chloroquine and pyrimethamine were high, whereas cycloguanil-resistance marker was not present in the studied population. The low level of mutations in the Pfmdr1gene indicates likely efficacy of amodiaquine against malaria in pregnancy.

Dimorphism in genes encoding sexual-stage proteins of Plasmodium ovale curtisi and Plasmodium ovale wallikeri.

Plasmodium ovale curtisi and Plasmodium ovale wallikeri are distinct species of malaria parasite which are sympatric throughout the tropics, except for the Americas. Despite this complete overlap in geographic range, these two species do not recombine. Although morphologically very similar, the two taxa must possess distinct characters which prevent recombination between them. We hypothesised that proteins required for sexual reproduction have sufficiently diverged between the two species to prevent recombination in any mosquito blood meal in which gametocytes of both species are ingested. In order to investigate possible barriers to inter-species mating between P. ovale curtisi and P. ovale wallikeri, homologues of genes encoding sexual stage proteins in other plasmodia were identified and compared between the two species. Database searches with motifs for 6-cysteine, Limulus Coagulation factor C domain-containing proteins and other relevant sexual stage proteins in the genus Plasmodium were performed in the available P. ovale curtisi partial genome database (Wellcome Trust Sanger Institute, UK). Sequence fragments obtained were used as the basis for PCR walking along each gene of interest in reference isolates of both P. ovale curtisi and P. ovale wallikeri. Sequence alignment of the homologues of each gene in each species showed complete dimorphism across all isolates. In conclusion, substantial divergence between sexual stage proteins in the two P. ovale spp. was observed, providing further evidence that these do not recombine in nature. Incompatibility of proteins involved in sexual development and fertilisation thus remains a plausible explanation for the observed lack of natural recombination between P. ovale curtisi and P. ovale wallikeri.

Alternatively spliced transcripts and novel pseudogenes of the Plasmodium falciparum resistance-associated locus pfcrt detected in East African malaria patients.

OBJECTIVES: Polymorphisms in the lysosomal transporter encoded by the pfcrt gene directly impact on Plasmodium falciparum susceptibility to aminoquinolines. The Lys76Thr mutation is the critical change conferring chloroquine resistance in vitro and in vivo, but always occurs with additional non-synonymous changes in the pfcrt coding sequence. We sought to better describe pfcrt polymorphisms distal to codon 76. METHODS: Small-volume samples (≤ 500 µL) of parasite-infected blood collected directly from malaria patients presenting for treatment in Sudan and Tanzania were immediately preserved for RNA extraction. The pfcrt locus was amplified from cDNA preparations by nested PCR and sequenced directly to derive full-length mRNA sequences. RESULTS: In one of two sites in Sudan, two patients were found with an unorthodox spliced form of pfcrt mRNA in which two exons were skipped, but it was not possible to test for the presence of the putative protein products of these aberrant transcripts. Genomic DNA sequencing from dried blood spots collected in parallel confirmed the presence of spliced pfcrt pseudogenes in a minority of parasite isolates. Full-length cDNA from conventionally spliced mRNA molecules in all study sites demonstrated the existence of a variety of pfcrt haplotypes in East Africa, and thus provides evidence of intragenic recombination. CONCLUSIONS: The presence of pseudogenes, although unlikely to have any direct public health impact, may confound results obtained from simple genotyping methods that consider only codon 76 and the adjacent residues of pfcrt.

Polymorphisms in Plasmodium falciparum chloroquine resistance transporter and multidrug resistance 1 genes: parasite risk factors that affect treatment outcomes for P. falciparum malaria after artemether-lumefantrine and artesunate-amodiaquine.

Adequate clinical and parasitologic cure by artemisinin combination therapies relies on the artemisinin component and the partner drug. Polymorphisms in the Plasmodium falciparum chloroquine resistance transporter (pfcrt) and P. falciparum multidrug resistance 1 (pfmdr1) genes are associated with decreased sensitivity to amodiaquine and lumefantrine, but effects of these polymorphisms on therapeutic responses to artesunate-amodiaquine (ASAQ) and artemether-lumefantrine (AL) have not been clearly defined. Individual patient data from 31 clinical trials were harmonized and pooled by using standardized methods from the WorldWide Antimalarial Resistance Network. Data for more than 7,000 patients were analyzed to assess relationships between parasite polymorphisms in pfcrt and pfmdr1 and clinically relevant outcomes after treatment with AL or ASAQ. Presence of the pfmdr1 gene N86 (adjusted hazards ratio = 4.74, 95% confidence interval = 2.29 - 9.78, P < 0.001) and increased pfmdr1 copy number (adjusted hazards ratio = 6.52, 95% confidence interval = 2.36-17.97, P < 0.001 : were significant independent risk factors for recrudescence in patients treated with AL. AL and ASAQ exerted opposing selective effects on single-nucleotide polymorphisms in pfcrt and pfmdr1. Monitoring selection and responding to emerging signs of drug resistance are critical tools for preserving efficacy of artemisinin combination therapies; determination of the prevalence of at least pfcrt K76T and pfmdr1 N86Y should now be routine.

An observational study of malaria in British travellers: Plasmodium ovale wallikeri and Plasmodium ovale curtisi differ significantly in the duration of latency.

OBJECTIVES: Ovale malaria is caused by two closely related species of protozoan parasite: Plasmodium ovale curtisi and Plasmodium ovale wallikeri Although clearly distinct genetically, there have been no studies comparing the morphology, life cycle or epidemiology of these parasites. We tested the hypothesis that the two species differ in the duration of latency prior to presentation with symptoms of blood-stage infection. DESIGN: PCR was used to identify P ovale curtisi and P ovale wallikeri infections among archived blood from UK malaria patients. Latency periods, estimated as the time between entry into the UK and diagnosis of malaria, were compared between the two groups. SETTING: UK National Reference Laboratory. PARTICIPANTS: None. Archived parasite material and surveillance data for 74 P ovale curtisi and 60 P ovale wallikeri infections were analysed. Additional epidemiological data were taken from a database of 1045 imported cases. OUTCOMES: None. RESULTS: No differences between the two species were identified by a detailed comparison of parasite morphology (N=9, N=8, respectively) and sex ratio (N=5, N=4) in archived blood films. The geometric mean latency period in P ovale wallikeri was 40.6 days (95% CI 28.9 to 57.0), whereas that for P ovale curtisi was more than twice as long at 85.7 days (95% CI 66.1 to 111.1; p=0.002). Further, the proportion of ovale malaria sensu lato which occurred in patients reporting chemoprophylaxis use was higher than for Plasmodium falciparum (OR 7.56; p<0.0001) or P vivax (OR 1.82; p<0.0001). CONCLUSIONS: These findings provide the first difference of epidemiological significance observed between the two parasites which cause ovale malaria, and suggest that control measures aimed at P falciparum may not be adequate for reducing the burden of malaria caused by P ovale curtisi and P ovale wallikeri.

Persistent detection of Plasmodium falciparum, P. malariae, P. ovale curtisi and P. ovale wallikeri after ACT treatment of asymptomatic Ghanaian school-children.

Two hundred and seventy four asymptomatic Ghanaian school-children aged 5 to 17 years were screened for malaria parasites by examination of blood films. One hundred and fifty five microscopically-positive individuals were treated with dihydroartemisinin-piperaquine and followed for 3 weeks. Retrospective species-specific PCR of all 274 screened samples identified an additional 60 children with sub-patent parasitaemia, and a substantial proportion of co-infections with Plasmodium malariae, Plasmodium ovale curtisi and Plasmodium ovale wallikeri. One hundred individuals harboured at least one non-falciparum parasite species. Using standard double-read microscopy, the 21-day efficacy of treatment against Plasmodium falciparum was 91.4% among the 117 children seen at all 5 visits. Using nested PCR to test 152 visit 5 blood samples, 22 were found to be parasite-positive. Twenty individuals harboured P. falciparum, four harboured P. ovale spp. and two P. malariae, with four of these 22 isolates being mixed species infections. The persistent detection of low density Plasmodium sp. infections following antimalarial treatment suggests these may be a hitherto unrecognised obstacle to malaria elimination.

Increased pfmdr1 copy number and sequence polymorphisms in Plasmodium falciparum isolates from Sudanese malaria patients treated with artemether-lumefantrine.

Molecular markers for surveillance of Plasmodium falciparum resistance to current antimalarials are sorely needed. A 28-day efficacy study of artemether-lumefantrine in eastern Sudan identified 5 treatment failures among 100 evaluable patients; 9 further individuals were parasite positive by PCR during follow-up. Polymorphisms in pfatpase6 and pfmdr1 were evaluated by DNA sequencing. One individual carried parasites with a novel pfmdr1 polymorphism (F1044L). pfmdr1 gene amplification in parasites prior to treatment occurred in three individuals who had recurrent infection during follow-up.

Plasmodium ovale curtisi and Plasmodium ovale wallikeri circulate simultaneously in African communities.

It has been proposed that ovale malaria in humans is caused by two closely related but distinct species of malaria parasite, Plasmodium ovale curtisi and Plasmodium ovale wallikeri. It was recently shown that these two parasite types are sympatric at the country level. However, it remains possible that localised geographic, temporal or ecological barriers exist within endemic countries which prevent recombination between the genomes of the two species. Here, using conventional and real-time quantitative PCR (qPCR) methods specifically designed to discriminate P. o. curtisi and P. o. wallikeri, it is shown that both species are present among clinic attendees in Congo-Brazzaville, and occur simultaneously both in lake-side and inland districts in Uganda and on Bioko Island, Equatorial Guinea. Thus P. o. curtisi and P. o. wallikeri in these localities are exactly sympatric in both time and space. These findings are consistent with the existence of a biological barrier, rather than geographical or ecological factors, preventing recombination between P. o. curtisi and P. o. wallikeri. In cross-sectional surveys carried out in Uganda and Bioko, our results show that infections with P. ovale spp. are more common than previously thought, occurring at a frequency of 1-6% in population samples, with both proposed species contributing to ovale malaria in six sites. Malaria elimination programmes in Africa need to include strategies for control of P. o. curtisi and P. o. wallikeri.

Two nonrecombining sympatric forms of the human malaria parasite Plasmodium ovale occur globally.

BACKGROUND: Malaria in humans is caused by apicomplexan parasites belonging to 5 species of the genus Plasmodium. Infections with Plasmodium ovale are widely distributed but rarely investigated, and the resulting burden of disease is not known. Dimorphism in defined genes has led to P. ovale parasites being divided into classic and variant types. We hypothesized that these dimorphs represent distinct parasite species. METHODS: Multilocus sequence analysis of 6 genetic characters was carried out among 55 isolates from 12 African and 3 Asia-Pacific countries. RESULTS: Each genetic character displayed complete dimorphism and segregated perfectly between the 2 types. Both types were identified in samples from Ghana, Nigeria, São Tomé, Sierra Leone, and Uganda and have been described previously in Myanmar. Splitting of the 2 lineages is estimated to have occurred between 1.0 and 3.5 million years ago in hominid hosts. CONCLUSIONS: We propose that P. ovale comprises 2 nonrecombining species that are sympatric in Africa and Asia. We speculate on possible scenarios that could have led to this speciation. Furthermore, the relatively high frequency of imported cases of symptomatic P. ovale infection in the United Kingdom suggests that the morbidity caused by ovale malaria has been underestimated.