Molecular markers for detection and differentiation of Plasmodium falciparum and Plasmodium vivax in human blood samples by pyrosequencing.
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ABSTRACT:
SUBMITTER: Lulitanond V
PROVIDER: S-EPMC3318504 | biostudies-literature | 2012 Apr
REPOSITORIES: biostudies-literature
Publications
Molecular markers for detection and differentiation of Plasmodium falciparum and Plasmodium vivax in human blood samples by pyrosequencing.
Lulitanond Viraphong V Intapan Pewpan M PM Tantrawatpan Chairat C Sankuntaw Nipaporn N Sanpool Oranuch O Janwan Penchom P Maleewong Wanchai W
Journal of clinical microbiology 20120201 4
PCR amplification coupled with pyrosequencing was used to measure molecular markers that could be used to detect and differentiate Plasmodium falciparum and Plasmodium vivax in human blood samples. The detection rates were in agreement with the results of Giemsa-stained film microscopy, which is the current gold standard for detection. This method provides an exciting alternative for malaria diagnosis. ...[more]
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Project description:INTRODUCTION:Malaria is a human disease of which causes high morbidity and mortality. In Plasmodium falciparum malaria, the resistance to antimalarial drugs, especially chloroquine (CQ) is one of the paramount factors contributing to the global increase in morbidity and mortality, due to malaria. Hence, there is a need for detection of chloroquine drug resistance genes i.e., pfcrt-o (Plasmodium falciparum chloroquine resistance transporter-o) and pfmdr-1 (Plasmodium falciparum multidrug resistance-1) of P. falciparum and pvcrt-o (Plasmodium vivax chloroquine resistance transporter-o) and pvmdr-1 (Plasmodium vivax multidrug resistance-1) of P. vivax by using molecular methods to prevent mortality in malarial cases. AIM:To standardize chloroquine drug sensitivity testing by molecular method so as to provide reports of chloroquine within 6-8 hours to physicians for better treatment. MATERIALS AND METHODS:This study was conducted over a period of one year from January to December 2014. A Total of 300 blood samples were collected from malaria suspected patient attending MGM Hospital, Kamothe, Navi Mumbai, India. Out of 300 blood samples, 44 were malaria positive as assessed by Thick and Thin blood smear stained, by Leishman's method and examination with light microscope. Chloroquine drug sensitivity testing was performed using WHO III plate method (micro test). Nested PCR was done for detection of pfcrt-o and pfmdr-1 for P. falciparum and pvcrt-o, pvmdr-1 genes for P. vivax. RESULTS:Total 44 samples were included in this study, out of which 22 samples confirmed for Plasmodium falciparum and 22 samples confirmed for Plasmodium vivax. Out of 22 P. falciparum 15 (68.18%) samples were chloroquine resistant. P. vivax showed chloroquine resistance to 5 samples (22.73%) by method similar to WHO III plate method (micro test) and nested PCR. CONCLUSION:Drug resistance testing by molecular methods is useful for early detection of antimalarial drug resistance. pfmdr-1 along with pfcrt-o can be used as biomarker for chloroquine drug resistance in P. falciparum and pvmdr-1 along with pvcrt-o for P. vivax.
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Project description:BackgroundLoop-mediated isothermal amplification (LAMP) for malaria diagnosis at the point of care (POC) depends on the detection capacity of synthesized nucleic acids and the specificity of the amplification target. To improve malaria diagnosis, new colorimetric LAMP tests were developed using multicopy targets for Plasmodium vivax and Plasmodium falciparum detection.MethodsThe cytochrome oxidase I (COX1) mitochondrial gene and the non-coding sequence Pvr47 for P. vivax, and the sub-telomeric sequence of erythrocyte membrane protein 1 (EMP1) and the non-coding sequence Pfr364 for P. falciparum were targeted to design new LAMP primers. The limit of detection (LOD) of each colorimetric LAMP was established and assessed with DNA extracted by mini spin column kit and the Boil & Spin method from 28 microscopy infections, 101 malaria submicroscopic infections detected by real-time PCR only, and 183 negatives infections by both microscopy and PCR.ResultsThe LODs for the colorimetric LAMPs were estimated between 2.4 to 3.7 parasites/µL of whole blood. For P. vivax detection, the colorimetric LAMP using the COX1 target showed a better performance than the Pvr47 target, whereas the Pfr364 target was the most specific for P. falciparum detection. All microscopic infections of P. vivax were detected by PvCOX1-LAMP using the mini spin column kit DNA extraction method and 81% (17/21) were detected using Boil & Spin sample preparation. Moreover, all microscopic infections of P. falciparum were detected by Pfr364-LAMP using both sample preparation methods. In total, PvCOX1-LAMP and Pfr364-LAMP detected 80.2% (81 samples) of the submicroscopic infections using the DNA extraction method by mini spin column kit, while 36.6% (37 samples) were detected using the Boil & Spin sample preparation method.ConclusionThe colorimetric LAMPs with multicopy targets using the COX1 target for P. vivax and the Pfr364 for P. falciparum have a high potential to improve POC malaria diagnosis detecting a greater number of submicroscopic Plasmodium infections.
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Project description:BackgroundIn Brazil, malaria is endemic in the Amazon River basin and non-endemic in the extra-Amazon region, which includes areas of São Paulo state. In this state, a number of autochthonous cases of malaria occur annually, and the prevalence of subclinical infection is unknown. Asymptomatic infections may remain undetected, maintaining transmission of the pathogen, including by blood transfusion. In these report it has been described subclinical Plasmodium infection in blood donors from a blood transfusion centre in São Paulo, Brazil.MethodsIn this cross-sectional study, representative samples of blood were obtained from 1,108 healthy blood donors at the Fundação Pró-Sangue Hemocentro de São Paulo, the main blood transfusion centre in São Paulo. Malaria exposure was defined by the home region (exposed: forest region; non-exposed: non-forest region). Real-time PCR was used to detect Plasmodium falciparum and Plasmodium vivax. Subclinical malaria cases were geo-referenced.ResultsEighty-four (7.41%) blood donors tested positive for Plasmodium; 57 of these were infected by P. falciparum, 25 by P. vivax, and 2 by both. The prevalence of P. falciparum and P. vivax was 5.14 and 2.26, respectively. The overall prevalence ratio (PR) was 3.23 (95% confidence interval (CI) 2.03, 5.13); P. falciparum PR was 16.11 (95% CI 5.87, 44.21) and P. vivax PR was 0.47 (95% CI 0.2, 1.12). Plasmodium falciparum subclinical malaria infection in the Atlantic Forest domain was present in the mountain regions while P. vivax infection was observed in cities from forest-surrounded areas.ConclusionsThe presence of Plasmodium in healthy blood donors from a region known as non-endemic, which is important in the context of transfusion biosafety, was described. Infected recipients may become asymptomatic carriers and a reservoir for parasites, maintaining their transmission. Furthermore, P. falciparum PR was positively associated with the forest environment, and P. vivax was associated with forest fragmentation.
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Project description:This molecular epidemiological study was designed to determine the antimalarial drug resistance pattern, and the genetic diversity of malaria isolates collected from a war-altered Federally Administered Tribal Area (FATA), in Pakistan. Clinical isolates were collected from Bajaur, Mohmand, Khyber, Orakzai and Kurram agencies of FATA region between May 2017 and May 2018, and they underwent DNA extraction and amplification. The investigation of gene polymorphisms in drug resistance genes (dhfr, dhps, crt, and mdr1) of Plasmodium falciparum and Plasmodium vivax was carried out by pyrosequencing and Sanger sequencing, respectively. Out of 679 PCR-confirmed malaria samples, 523 (77%) were P. vivax, 121 (18%) P. falciparum, and 35 (5%) had mixed-species infections. All P. falciparum isolates had pfdhfr double mutants (C59R+S108N), while pfdhfr/pfdhps triple mutants (C59R+S108N+A437G) were detected in 11.5% of the samples. About 97.4% of P. falciparum isolates contained pfcrt K76T mutation, while pfmdr1 N86Y and Y184F mutations were present in 18.2% and 10.2% of the samples. P. vivax pvdhfr S58R mutation was present in 24.9% of isolates and the S117N mutation in 36.2%, while no mutation in the pvdhps gene was found. Pvmdr1 F1076L mutation was found in nearly all samples, as it was observed in 98.9% of isolates. No significant anti-folate and chloroquine resistance was observed in P. vivax; however, mutations associated with antifolate-resistance were found, and the chloroquine-resistant gene has been observed in 100% of P. falciparum isolates. Chloroquine and sulphadoxine-pyrimethamine resistance were found to be high in P. falciparum and low in P. vivax. Chloroquine could still be used for P. vivax infection but need to be tested in vivo, whereas a replacement of the artemisinin combination therapy for P. falciparum appears to be justified.
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Project description:BACKGROUND:Parasite genetic diversity and multiplicity of infection (MOI) affect clinical outcomes, response to drug treatment and naturally-acquired or vaccine-induced immunity. Traditional methods often underestimate the frequency and diversity of multiclonal infections due to technical sensitivity and specificity. Next-generation sequencing techniques provide a novel opportunity to study complexity of parasite populations and molecular epidemiology. METHODS:Symptomatic and asymptomatic Plasmodium vivax samples were collected from health centres/hospitals and schools, respectively, from 2011 to 2015 in Ethiopia. Similarly, both symptomatic and asymptomatic Plasmodium falciparum samples were collected, respectively, from hospitals and schools in 2005 and 2015 in Kenya. Finger-pricked blood samples were collected and dried on filter paper. Long amplicon (> 400 bp) deep sequencing of merozoite surface protein 1 (msp1) gene was conducted to determine multiplicity and molecular epidemiology of P. vivax and P. falciparum infections. The results were compared with those based on short amplicon (117 bp) deep sequencing. RESULTS:A total of 139 P. vivax and 222 P. falciparum samples were pyro-sequenced for pvmsp1 and pfmsp1, yielding a total of 21 P. vivax and 99 P. falciparum predominant haplotypes. The average MOI for P. vivax and P. falciparum were 2.16 and 2.68, respectively, which were significantly higher than that of microsatellite markers and short amplicon (117 bp) deep sequencing. Multiclonal infections were detected in 62.2% of the samples for P. vivax and 74.8% of the samples for P. falciparum. Four out of the five subjects with recurrent P. vivax malaria were found to be a relapse 44-65 days after clearance of parasites. No difference was observed in MOI among P. vivax patients of different symptoms, ages and genders. Similar patterns were also observed in P. falciparum except for one study site in Kenyan lowland areas with significantly higher MOI. CONCLUSIONS:The study used a novel method to evaluate Plasmodium MOI and molecular epidemiological patterns by long amplicon ultra-deep sequencing. The complexity of infections were similar among age groups, symptoms, genders, transmission settings (spatial heterogeneity), as well as over years (pre- vs. post-scale-up interventions). This study demonstrated that long amplicon deep sequencing is a useful tool to investigate multiplicity and molecular epidemiology of Plasmodium parasite infections.
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