Nonradioactive heteroduplex tracking assay for the detection of minority-variant chloroquine-resistant Plasmodium falciparum in Madagascar

<p>Abstract</p> <p>Background</p> <p>Strains of <it>Plasmodium falciparum </it>genetically resistant to chloroquine (CQ) due to the presence of <it>pfcrt </it>76T appear to have been recently introduced to the island of Madagascar. The prevalence of such resistant genotypes is reported to be low (< 3%) when evaluated by conventional PCR. However, these methods are insensitive to low levels of mutant parasites present in patients with polyclonal infections. Thus, the current estimates may be an under representation of the prevalence of the CQ-resistant <it>P. falciparum </it>isolates on the island. Previously, minority variant chloroquine resistant parasites were described in Malawian patients using an isotopic heteroduplex tracking assay (HTA), which can detect <it>pfcrt </it>76T-bearing <it>P. falciparum </it>minority variants in individual patients that were undetectable by conventional PCR. However, as this assay required a radiolabeled probe, it could not be used in many resource-limited settings.</p> <p>Methods</p> <p>This study describes a digoxigenin (DIG)-labeled chemiluminescent heteroduplex tracking assay (DIG-HTA) to detect <it>pfcrt </it>76T-bearing minority variant <it>P. falciparum</it>. This assay was compared to restriction fragment length polymorphism (RFLP) analysis and to the isotopic HTA for detection of genetically CQ-resistant parasites in clinical samples.</p> <p>Results</p> <p>Thirty one clinical <it>P. falciparum </it>isolates (15 primary isolates and 16 recurrent isolates) from 17 Malagasy children treated with CQ for uncomplicated malaria were genotyped for the <it>pfcrt </it>K76T mutation. Two (11.7%) of 17 patients harboured genetically CQ-resistant <it>P. falciparum </it>strains after therapy as detected by HTA. RFLP analysis failed to detect any <it>pfcrt </it>K76T-bearing isolates.</p> <p>Conclusion</p> <p>These findings indicate that genetically CQ-resistant <it>P. falciparum </it>are more common than previously thought in Madagascar even though the fitness of the minority variant <it>pfcrt </it>76T parasites remains unclear. In addition, HTAs for malaria drug resistance alleles are promising tools for the surveillance of anti-malarial resistance. The use of a non-radioactive label allows for the use of HTAs in malaria endemic countries.</p

Plasmodium vivax dhfr and dhps mutations in isolates from Madagascar and therapeutic response to sulphadoxine-pyrimethamine

<p>Abstract</p> <p>Background</p> <p>Four of five <it>Plasmodium </it>species infecting humans are present in Madagascar. <it>Plasmodium vivax </it>remains the second most prevalent species, but is understudied. No data is available on its susceptibility to sulphadoxine-pyrimethamine, the drug recommended for intermittent preventive treatment during pregnancy. In this study, the prevalence of <it>P. vivax </it>infection and the polymorphisms in the <it>pvdhfr </it>and <it>pvdhps </it>genes were investigated. The correlation between these polymorphisms and clinical and parasitological responses was also investigated in <it>P. vivax</it>-infected patients.</p> <p>Methods</p> <p><it>Plasmodium vivax </it>clinical isolates were collected in eight sentinel sites from the four major epidemiological areas for malaria across Madagascar in 2006/2007. <it>Pvdhfr </it>and <it>pvdhps </it>genes were sequenced for polymorphism analysis. The therapeutic efficacy of SP in <it>P. vivax </it>infections was assessed in Tsiroanomandidy, in the foothill of the central highlands. An intention-to-treat analysis of treatment outcome was carried out.</p> <p>Results</p> <p>A total of 159 <it>P. vivax </it>samples were sequenced in the <it>pvdhfr/pvdhps </it>genes. Mutant-types in <it>pvdhfr </it>gene were found in 71% of samples, and in <it>pvdhps </it>gene in 16% of samples. Six non-synonymous mutations were identified in <it>pvdhfr</it>, including two novel mutations at codons 21 and 130. For <it>pvdhps</it>, beside the known mutation at codon 383, a new one was found at codon 422. For the two genes, different combinations were ranged from wild-type to quadruple mutant-type. Among the 16 patients enrolled in the sulphadoxine-pyrimethamine clinical trial (28 days of follow-up) and after adjustment by genotyping, 3 (19%, 95% CI: 5%–43%) of them were classified as treatment failure and were <it>pvdhfr </it>58R/117N double mutant carriers with or without the <it>pvdhps </it>383G mutation.</p> <p>Conclusion</p> <p>This study highlights (i) that genotyping in the <it>pvdhfr </it>and <it>pvdhps </it>genes remains a useful tool to monitor the emergence and the spread of <it>P. vivax </it>sulphadoxine-pyrimethamine resistant in order to improve the national antimalarial drug policy, (ii) the issue of using sulphadoxine-pyrimethamine as a monotherapy for intermittent preventive treatment of pregnant women or children.</p

Susceptibility of Colombian Plasmodium falciparum isolates to 4-aminoquinolines and the definition of amodiaquine resistance in vitro

There are wide variations in the threshold used to define in vitro resistance of Plasmodium falciparum to amodiaquine (AQ), probably due to differences in methodology and interpretation. In vitro susceptibility data of Colombian P. falciparum strains to AQ and N-desethylamodiaquine is used to illustrate the need to standardized methodologies and compare inhibitory concentrations, instead of resistant/susceptible phenotypes, when studying the mechanisms of resistance to AQ and monitoring drug susceptibility trends in the field

FlexiChip package: an universal microarray with a dedicated analysis software for high-thoughput SNPs detection linked to anti-malarial drug resistance

BACKGROUND: A number of molecular tools have been developed to monitor the emergence and spread of anti-malarial drug resistance to Plasmodium falciparum. One of the major obstacles to the wider implementation of these tools is the absence of practical methods enabling high throughput analysis. Here a new Zip-code array is described, called FlexiChip, linked to a dedicated software program, which largely overcomes this problem. METHODS: Previously published microarray probes detecting single-nucleotide polymorphisms (SNP) associated with parasite resistance to anti-malarial drugs (ResMalChip) were adapted for a universal microarray FlexiChip format. To evaluate the overall sensitivity of the FlexiChip package (microarray + software), the results of FlexiChip were compared to ResMalChip microarray, using the same extension probes and with the same PCR products. In both cases, sequence results were used as gold standard to calculate sensitivity and specificity. FlexiChip results obtained with a set of field isolates were then compared to those assessed in an independent reference laboratory. RESULTS: The FlexiChip package gave results identical to the ResMalChip results in 92.7% of samples (kappa coefficient 0.8491, with a standard error 0.021) and had a sensitivity of 95.88% and a specificity of 97.68% compared to the sequencing as the reference method. Moreover the method performed well compared to the results obtained in the reference laboratories, with 99.7% of identical results (kappa coefficient 0.9923, S.E. 0.0523). CONCLUSION: Microarrays could be employed to monitor P. falciparum drug resistance markers with greater cost effectiveness and the possibility for high throughput analysis. The FlexiChip package is a promising tool for use in poor resource settings of malaria endemic countrie