Molecular detection of human Plasmodium species using a multiplex real time PCR.

Molecular detection methods have revealed higher sensitivity and specificity than conventional microscopy or rapid diagnostic tests for malaria diagnosis. In this study, we implemented, evaluated and validated according to the ISO 15,189 requirements, a multiplex real-time PCR assay to detect and identify the five human malaria parasites. DNA samples were extracted from whole blood or dried blood spots drawn from patients. Based on the External Quality Assessment (whole blood), this method shows 100% sensitivity and specificity. This PCR detected P. vivax up to 0.25 p/µl, P. falciparum and P. knowlesi up to 0.5 p/µl, P. ovale up to 1 p/µl and P. malariae up to 5 p/µl of blood. From blood spots (extraction from four punches), it detected P. vivax at 5 p/µl, P. falciparum, P. ovale and P. knowlesi at 20 p/µl and P. malariae at 125 p/µl. In conclusion, this quantitative PCR shows excellent performance, is easy to use and DNA saver. It is especially useful to actively screen large population groups and identify the five human malaria parasites in a context of low malaria transmission.

Declines in prevalence alter the optimal level of sexual investment for the malaria parasite Plasmodium falciparum.

Successful infectious disease interventions can result in large reductions in parasite prevalence. Such demographic change has fitness implications for individual parasites and may shift the parasite's optimal life history strategy. Here, we explore whether declining infection rates can alter Plasmodium falciparum's investment in sexual versus asexual growth. Using a multiscale mathematical model, we demonstrate how the proportion of polyclonal infections, which decreases as parasite prevalence declines, affects the optimal sexual development strategy: Within-host competition in multiclone infections favors a greater investment in asexual growth whereas single-clone infections benefit from higher conversion to sexual forms. At the same time, drug treatment also imposes selection pressure on sexual development by shortening infection length and reducing within-host competition. We assess these models using 148 P. falciparum parasite genomes sampled in French Guiana over an 18-y period of intensive intervention (1998 to 2015). During this time frame, multiple public health measures, including the introduction of new drugs and expanded rapid diagnostic testing, were implemented, reducing P. falciparum malaria cases by an order of magnitude. Consistent with this prevalence decline, we see an increase in the relatedness among parasites, but no single clonal background grew to dominate the population. Analyzing individual allele frequency trajectories, we identify genes that likely experienced selective sweeps. Supporting our model predictions, genes showing the strongest signatures of selection include transcription factors involved in the development of P. falciparum's sexual gametocyte form. These results highlight how public health interventions impose wide-ranging selection pressures that affect basic parasite life history traits.

Emergence of Plasmodium vivax Resistance to Chloroquine in French Guiana.

In South America, Plasmodium vivax resistance to chloroquine was recently reported in Brazil and Bolivia. The objective of this study was to collect data on chloroquine resistance in French Guiana by associating a retrospective evaluation of therapeutic efficacy with an analysis of recurrent parasitemia from any patients. Patients with P. vivax infection, confirmed by microscopy and a body temperature of ≥37.5°C, were retrospectively identified at Cayenne Hospital between 2009 and 2015. Follow-up and treatment responses were performed according to the World Health Organization protocol. Parasite resistance was confirmed after dosage of a plasma concentration of chloroquine and microsatellite characterization. The pvmdr1 and pvcrt-o genes were analyzed for sequence and gene copy number variation. Among the 172 patients followed for 28 days, 164 presented adequate clinical and parasitological responses. Eight cases of treatment failures were identified (4.7%; n = 8/172), all after 14 days. The therapeutic efficacy of chloroquine was estimated at 95.3% (95% confidence interval [CI], 92.5 to 98.1%; n = 164/172). Among the eight failures, five were characterized: two cases were true P. vivax chloroquine resistance (1.2%; 95% CI, 0 to 2.6%; n = 2/172), and three cases were found with subtherapeutic concentrations of chloroquine. No particular polymorphism in the Plasmodium vivaxpvmdr1 and pvcrt-o genes was identified in the resistant parasites. This identified level of resistance of P. vivax to chloroquine in French Guiana does not require a change in therapeutic recommendations. However, primaquine should be administered more frequently to limit the spread of resistance, and there is still a need for a reliable molecular marker to facilitate the monitoring of P. vivax resistance to chloroquine.

Plasmodium vivax multidrug resistance-1 gene polymorphism in French Guiana.

BACKGROUND: Plasmodium vivax malaria is a major public health problem in French Guiana. Some cases of resistance to chloroquine, the first-line treatment used against P. vivax malaria, have been described in the Brazilian Amazon region. The aim of this study is to investigate a possible dispersion of chloroquine-resistant P. vivax isolates in French Guiana. The genotype, polymorphism and copy number variation, of the P. vivax multidrug resistance gene-1 (pvmdr1) have been previously associated with modification of the susceptibility to chloroquine. METHODS: The pvmdr1 gene polymorphism was evaluated by sequencing and copy number variation was assessed by real-time PCR, in P. vivax isolates obtained from 591 symptomatic patients from 1997 to 2013. RESULTS: The results reveal that 1.0% [95% CI 0.4-2.2] of French Guiana isolates carry the mutations Y976F and F1076L, and that the proportion of isolates with multiple copies of pvmdr1 has significantly decreased over time, from 71.3% (OR = 6.2 [95% CI 62.9-78.7], p < 0.0001) in 1997-2004 to 12.8% (OR = 0.03 [95% CI 9.4-16.9], p < 0.0001) in 2009-2013. A statistically significant relationship was found between Guf-A (harboring the single mutation T958M) and Sal-1 (wild type) alleles and pvmdr1 copy number. CONCLUSIONS: Few P. vivax isolates harboring chloroquine-resistant mutations in the pvmdr1 gene are circulating in French Guiana. However, the decrease in the prevalence of isolates carrying multiple copies of pvmdr1 might indicate that the P. vivax population in French Guiana is evolving towards a decreased susceptibility to chloroquine.

Adaptive evolution of malaria parasites in French Guiana: Reversal of chloroquine resistance by acquisition of a mutation in pfcrt.

In regions with high malaria endemicity, the withdrawal of chloroquine (CQ) as first-line treatment of Plasmodium falciparum infections has typically led to the restoration of CQ susceptibility through the reexpansion of the wild-type (WT) allele K76 of the chloroquine resistance transporter gene (pfcrt) at the expense of less fit mutant alleles carrying the CQ resistance (CQR) marker K76T. In low-transmission settings, such as South America, drug resistance mutations can attain 100% prevalence, thereby precluding the return of WT parasites after the complete removal of drug pressure. In French Guiana, despite the fixation of the K76T allele, the prevalence of CQR isolates progressively dropped from >90% to <30% during 17 y after CQ withdrawal in 1995. Using a genome-wide association study with CQ-sensitive (CQS) and CQR isolates, we have identified a single mutation in pfcrt encoding a C350R substitution that is associated with the restoration of CQ susceptibility. Genome editing of the CQR reference strain 7G8 to incorporate PfCRT C350R caused a complete loss of CQR. A retrospective molecular survey on 580 isolates collected from 1997 to 2012 identified all C350R mutant parasites as being CQS. This mutation emerged in 2002 and rapidly spread throughout the P. falciparum population. The C350R allele is also associated with a significant decrease in piperaquine susceptibility in vitro, suggesting that piperaquine pressure in addition to potential fitness costs associated with the 7G8-type CQR pfcrt allele may have selected for this mutation. These findings have important implications for understanding the evolutionary dynamics of antimalarial drug resistance.

Absence of correlation between ex vivo susceptibility to doxycycline and pfteQ-pfmdt gene polymorphism in French Guiana.

BACKGROUND: In French Guiana, doxycycline is used for both chemoprophylaxis and the treatment of malaria. The presence of isolates with reduced ex vivo susceptibility to doxycycline in French Guiana makes it critical to identify any genetic determinants contributing to the chemosusceptibility level of Plasmodium falciparum to doxycycline, such as pfmdt and pftetQ, which were recently identified as potential molecular markers in African isolates. METHODS: A Bayesian statistical approach was used to define different ex vivo doxycycline phenotypes. The pfmdt and pftetQ gene copy numbers were quantified by quantitative real-time polymerase chain reaction in 129 P. falciparum isolates collected between 2000 and 2010, and pftetQ, pfrps7, pfssurRNA, and pflsurRNA sequences were analysed after amplification by polymerase chain reaction. RESULTS: PftetQ and pfmdt copy numbers were not associated with reduced susceptibility to doxycycline in P. falciparum within French Guiana. Sequence analysis of the genes revealed five known single nucleotide polymorphisms. Three new SNPs were identified in the apicoplast ribosomal RNA long sub-unit (pflsurRNA): C740T, A1875C and A1875T. These polymorphisms were not associated with reduced chemosusceptibility to doxycycline. CONCLUSIONS: The present study does not validate pfmdt and pftetQ genes as molecular markers of decreased susceptibility to doxycycline in P. falciparum isolates in French Guiana.

High performance of histidine-rich protein 2 based rapid diagnostic tests in French Guiana are explained by the absence of pfhrp2 gene deletion in P. falciparum.

BACKGROUND: Care for malaria patients in endemic areas has been improved through the increasing use of Rapid Diagnostic Tests (RDTs). Most RDTs target the histidine-rich protein-2 antigen (PfHRP2) to detect P. falciparum, as it is abundant and shows great heat stability. However, their use in South America has been widely questioned following a recent publication that pinpoints the high prevalence of Peruvian field isolates lacking the gene encoding this protein. In the remote rural health centers of French Guiana, RDTs are the main diagnosis tools. Therefore, a study of PfHRP2 RDT performances and pfhrp2 genotyping was conducted to determine whether a replacement of the current pLDH-based kit could be considered. METHODS: The performance study compared the SD Malaria Ag test P.f/Pan® kit with the current gold standard diagnosis by microscopy. The prevalence of pfhrp2 and pfhrp3 deletions were evaluated from 221 P. falciparum isolates collected between 2009 and 2011 in French Guiana. RESULTS: Between January 2010 and August 2011, 960 suspected cases of malaria were analyzed using microscopy and RDTs. The sensitivity of the SD Malaria Ag test P.f/Pan® for detection of P. falciparum was 96.8% (95% CI: 90.9-99.3), and 86.0% (95% CI: 78.9-91.5) for the detection of P. vivax. No isolates (95% CI: 0-4.5) lacking either exon of the pfhrp2 gene were identified among the 221 P. falciparum isolates analyzed, but 7.4% (95% CI: 2.8-15.4) lacked the exon 2 part of the pfhrp3 gene. CONCLUSIONS: Field isolates lacking either exon of the pfhrp2 gene are absent in this western part of South America. Despite its sensibility to detect P. vivax, the SD Malaria Ag test P.f/Pan® kit is a satisfying alternative to microscopy in remote health centers, where it is difficult to provide highly skilled microscopists and to maintain the necessary equipment.

Discordant temporal evolution of Pfcrt and Pfmdr1 genotypes and Plasmodium falciparum in vitro drug susceptibility to 4-aminoquinolines after drug policy change in French Guiana.

Analysis of the evolution of drug target genes under changing drug policy is needed to assist monitoring of Plasmodium falciparum drug resistance in the field. Here we genotype Pfcrt and Pfdmr1 of 700 isolates collected in French Guiana from 2000 (5 years after withdrawal of chloroquine) to 2008, i.e., the period when the artemether-lumefantrine combination was progressively introduced and mefloquine was abandoned. Gene sequencing showed fixation of the 7G8-type Pfcrt SMVNT resistance haplotype and near fixation of the NYCDY Pfdmr1 haplotype. Pfdmr1 gene copy number correlated with 50% inhibitory concentrations of mefloquine and halofantrine (r = 0.64 and 0.47, respectively, n = 547); its temporal changes paralleled changes in in vitro mefloquine susceptibility. However, the molecular parameters studied did not account for the regained in vitro susceptibility to chloroquine and showed a poor correlation with susceptibility to artemether, lumefantrine, or quinine. Identification of novel markers of resistance to these antimalarials is needed in this South American area.

Genetic diversity of msp3alpha and msp1_b5 markers of Plasmodium vivax in French Guiana.

BACKGROUND: Reliable molecular typing tools are required for a better understanding of the molecular epidemiology of Plasmodium vivax. The genes msp3a and msp1_block5 are highly polymorphic and have been used as markers in many P. vivax population studies. These markers were used to assess the genetic diversity of P. vivax strains from French Guiana (South America) and to develop a molecular typing protocol. METHODS: A total of 120 blood samples from 109 patients (including 10 patients suffered from more than one malaria episode, samples were collected during each episode) with P. vivax infection were genotyped. All samples were analysed by msp3a PCR-RFLP and msp1_b5 gene sequencing was performed on 57 samples. Genotyping protocol applied to distinguish between new infection or relapse from heterologus hypnozoites and treatment failure or relapse from homologus hypnozoites was based on analysing first msp3a by PCR-RFLP and secondly, only if the genotypes of the two samples are identical, on sequencing the msp1_b5 gene. RESULTS: msp3a alleles of three sizes were amplified by PCR: types A, B and C. Eleven different genotypes were identified among the 109 samples analysed by msp3a PCR-RFLP. In 13.8% of cases, a mixed genotype infection was observed. The sequence of msp1_b5 gene revealed 22 unique genotypes and 12.3% of cases with mixed infection. In the 57 samples analysed by both methods, 45 genotypes were found and 21% were mixed. Among ten patients with two or three malaria episodes, the protocol allowed to identify five new infections or relapses from heterologous hypnozoites and six treatment failures of relapses from homologous hypnozoites. CONCLUSION: The study showed a high diversity of msp3a and msp1_b5 genetic markers among P. vivax strains in French Guiana with a low polyclonal infection rate. These results indicated that the P. vivax genotyping protocol presented has a good discrimination power and can be used in clinical drug trials or epidemiological studies.

In vitro monitoring of Plasmodium falciparum drug resistance in French Guiana: a synopsis of continuous assessment from 1994 to 2005.

Implemented as one arm of the malaria control program in French Guiana in the early 1990s, our laboratory has since established in vitro profiles for parasite drug susceptibility to a panel of eight antimalarials for more than 1,000 Plasmodium falciparum isolates from infected patients. The quinine-doxycycline combination was introduced in 1995 as the first-line drug treatment against uncomplicated P. falciparum malaria, replacing chloroquine, and the first-line drug combination was changed to the artemether-lumefantrine combination in 2002. Resistance to chloroquine declined 5 years after it was dropped in 1995 as the first-line drug, but unlike similar situations in Africa, there was a rapid halt to this decline. Doxycycline susceptibility substantially decreased from 2002 to 2005, suggesting parasite selection under quinine-doxycycline drug pressure. Susceptibility to mefloquine decreased from 1997 onward. Throughout the period from 1994 to 2005, most isolates were sensitive in vitro to quinine, amodiaquine, and atovaquone. Susceptibility to amodiaquine was strongly correlated with that to chloroquine and to a lesser extent with that to mefloquine and halofantrine. Susceptibilities to mefloquine and to halofantrine were also strongly correlated. There were two alerts issued for in vitro artemether resistance in the period from 2002 to 2003 and again in 2005, both of which could be associated with the presence of an S769N polymorphism in the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA)-type P. falciparum ATPase6 (PfATPase6) gene. Analysis of susceptibility to lumefantrine, conducted for the first time in 2005, indicates an alarming rate of elevated 50% inhibitory concentrations. In vitro monitoring of parasite drug susceptibility should be pursued to further document the consequences of specific drug policies on the local parasite population and, in particular, to establish profiles of susceptibility to individual components of drug combinations to provide early warning signs of emerging parasite resistance.

Resistance of Plasmodium falciparum field isolates to in-vitro artemether and point mutations of the SERCA-type PfATPase6.

Artemisinin derivatives are an essential component of treatment against multidrug-resistant Plasmodium falciparum malaria. We aimed to investigate in-vitro resistance to artemisinin derivatives in field isolates. In-vitro susceptibility of 530 P falciparum isolates from three countries (Cambodia, French Guiana, and Senegal) with different artemisinin use was assessed with an isotopic microtest. Artemether IC50 up to 117 and 45 nmol/L was seen in French Guiana and Senegal, respectively. DNA sequencing in a subsample of 60 isolates lends support to SERCA-PfATPase6 as the target for artemisinins. The S769N PfATPase6 mutation, noted exclusively in French Guiana, was associated with raised (>30 nmol/L) artemether IC50s (p<0.0001, Mann-Whitney). All resistant isolates came from areas with uncontrolled use of artemisinin derivatives. This rise in resistance indicates the need for increased vigilance and a coordinated and rapid deployment of drug combinations.

Molecular analysis of two local falciparum malaria outbreaks on the French Guiana coast confirms the msp1 B-K1/varD genotype association with severe malaria.

BACKGROUND: Plasmodium falciparum outbreaks can occur in the coastal area of French Guiana, where the population is essentially non-immune. Two sporadic outbreaks were observed, including one with severe malaria cases. To characterize these outbreaks and verify previous observations of specific genotype characteristics in severe malaria in this area, all cases from each outbreak were studied. METHODS: P. falciparum genotypes for six genetic loci were determined by PCR amplification from peripheral blood parasites. The msp1/block2 and msp2 genotypes were determined by DNA sequencing. Microsatellite and varD genotyping was based on size polymorphism and locus-specific amplification. RESULTS: The outbreak including severe malaria cases was associated with a single genotype. The other mild malaria outbreak was due to at least five distinct genotypes. CONCLUSION: Two distinct types of outbreak occurred despite systematic and sustained deployment of malaria control measures, indicating a need for reinforced vigilance. The varD/B-K1 msp1 linkage and its association with severe malaria in this area was confirmed.

A sero-epidemiological study of malaria in human and monkey populations in French Guiana.

This paper describes a sero-epidemiological study of malaria prevalence in French Guiana. An immunofluorescence assay and an enzyme-linked immunosorbent assay were used to detect antibodies against blood-stage antigens and synthetic peptides mimicking the repetitive epitope of the sporozoites of Plasmodium falciparum, Plasmodium vivax and Plasmodium malariae/brasilianum, in 218 human sera and 113 non-human primate sera collected in French Guiana. Almost all the monkey sera tested had antibodies against malaria blood-stages (98%) and a large majority (73%) also tested positive with the P. malariae/brasilianum circumsporozoite peptide. A number of primate samples also reacted positively with P. falciparum NANP repeats in a very specific manner, suggesting that monkeys in the rainforest are bitten by mosquitoes infected with human malaria parasites. Seroprevalences were lower in the humans tested but Indian tribes on the borders with Suriname and Brazil were clearly more exposed to malaria than other ethnic groups, with a prevalence of nearly 70% seropositivity. P. vivax infections accounted for much of the observed pattern of reactivity, but there was also a high frequency of positive reactions to the P. brasilianum/malariae peptide. Similarly, a large proportion of the sera obtained from Bush Negro populations tested positive for P. malariae/brasilianum repeats. These data add to the emerging evidence that non-human primates might constitute a natural reservoir, not only for simian, but also for human malaria, and therefore suggest that they might be responsible for the maintenance of foci of P. malariae, and possibly of other malaria species, in isolated areas of the Amazonian rainforest.