scholarly journals Fixation of pfcrt chloroquine resistance alleles in Plasmodium falciparum clinical isolates collected from unrest tribal agencies of Pakistan

2023 ◽  
Vol 83 ◽  
Author(s):  
M. F. Nadeem ◽  
N. Zeeshan ◽  
A. A. Khattak ◽  
U. A. Awan ◽  
A. Yaqoob
Keyword(s):  
Plasmodium Falciparum ◽  
Nested Pcr ◽  
Resistant Mutant ◽  
Small Subunit ◽  
Clinical Isolates ◽  
Chloroquine Resistance ◽  
Mortality And Morbidity ◽  
Blood Smears ◽  
Tribal Areas ◽  
Pcr Targeting

Abstract Plasmodium falciparum resistance to Chloroquine (CQ) is a significant cause of mortality and morbidity worldwide. There is a paucity of documented data on the prevalence of CQ-resistant mutant haplotypes of Pfcrt and Pfmdr1 genes from malaria-endemic war effected Federally Administered Tribal Areas of Pakistan. The objective of this study was to investigate the prevalence of P. falciparum CQ-resistance in this area. Clinical isolates were collected between May 2017 and May 2018 from North Waziristan and South Waziristan agencies of Federally Administrated Trial Area. Subsequently, Giemsa-stained blood smears were examined to detect Plasmodium falciparum. Extraction of malarial DNA was done from microscopy positive P. falciparum samples, and P. falciparum infections were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes). All PCR confirmed P. falciparum samples were sequenced by pyrosequencing to find out mutation in Pfcrt gene at codon K76T and in pfmdr1 at codons N86Y, Y184F, N1042D, and D1246Y. Out of 121 microscopies positive P. falciparum cases, 109 samples were positive for P. falciparum by nested PCR. Pfcrt K76T mutation was found in 96% of isolates, Pfmdr1 N86Y mutation was observed in 20%, and 11% harboured Y184F mutation. All samples were wild type for Pfmdr1 codon N1042D and D1246Y. In the FATA, Pakistan, the frequency of resistant allele 76T remained high despite the removal of CQ. However, current findings of the study suggest complete fixation of P. falciparum CQ-resistant genotype in the study area.

scholarly journals Genetic Polymorphisms of Pfcrt K76T and Pfmdr1 N86Y among Asymptomatic School Children in Forest Communities of Ekondo Titi Subdivision along the Cameroon-Nigeria Border Area

2019 ◽  
pp. 1-16
Author(s):  
Bonaventure Tientche ◽  
Jerome Fru-Cho ◽  
Damian Nota Anong ◽  
Theresa K. Nkuo-Akenji
Keyword(s):  
Plasmodium Falciparum ◽  
School Children ◽  
Nested Pcr ◽  
Primary Schools ◽  
Point Mutations ◽  
Plasmodium Species ◽  
Small Subunit ◽  
Chloroquine Resistance ◽  
Rrna Gene ◽  
Cross Sectional

Aims: The study sought to quantify Plasmodium infection and molecular markers for chloroquine resistance among asymptomatic school children. Study Design: The study was cross-sectional. Place and Duration of Study: The study was carried out in Ekondo Titi Subdivision near Cameroon's south-western border with Nigeria from March to May and from September to October 2014. Methodology: The prevalence of human Plasmodium species was determined by nested PCR (Polymerase Chain Reaction) using DNA from dried blood spot in six primary schools. A PCR/RFLP analysis (Restriction Fragment Length Polymorphism) was used to determine the prevalence of chloroquine resistance (CQR) associated pfcrt 76T and pfmdr 1 56Y point mutations in Plasmodium falciparum asymptomatic school children. Results: A nested PCR amplifying the 18S small-subunit ribosomal RNA (SSU rRNA) gene of Plasmodium in 205 samples confirmed 76.1% of the isolates as asymptomatic P. falciparum infections, with a substantial proportion 22% of P. malariae infection. Among these, 3.6% were single P. malariae infections and 15.1% were P. falciparum and P. malariae mixed infections. Mixed P. falciparum and   P. ovale infections were 2.0%. Of the 156 Plasmodium falciparum, positive samples by species-specific PCR, 107 samples with P. falciparum mono-infection were analyzed for the presence of drug resistant alleles pfcrt 76T and pfmdr1- Y 86. The prevalence of pfcrt 76T mutation (74.6%) was higher than that of the pfmdr1-Y86 mutation (25.4%). Logistic regression analysis of socio-demographic factors predicted no significant association between pfcrt 76T mutation with gender and communities. Conclusions: The results indicated a high prevalence of P. malariae and mixed infection in the area under study. The high-level distribution of the pfcrtT76 observed in the study could be possibly attributed to the fact that CQ remained widely used at the community level more than 14 years after  withdrawal.

scholarly journals DNA recovery from archived RDTs for genetic characterization of Plasmodium falciparum in a routine setting in Lambaréné, Gabon

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
The Trong Nguyen ◽  
Brice Nzigou Mombo ◽  
Albert Lalremruata ◽  
Erik Koehne ◽  
Rella Zoleko Manego ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Real Time ◽  
Real Time Pcr ◽  
Rural Areas ◽  
Large Scale ◽  
Genetic Material ◽  
Chloroquine Resistance ◽  
Dna Recovery ◽  
Parasite Populations ◽  
Pcr Targeting

Abstract Background Rapid diagnostic tests (RDTs) have been described as a source of genetic material to analyse malaria parasites in proof-of-concept studies. The increasing use of RDTs (e.g., in focal or mass screening and treatment campaigns) makes this approach particularly attractive for large-scale investigations of parasite populations. In this study, the complexity of Plasmodium falciparum infections, parasite load and chloroquine resistance transporter gene mutations were investigated in DNA samples extracted from positive RDTs, obtained in a routine setting and archived at ambient temperature. Methods A total of 669 archived RDTs collected from malaria cases in urban, semi-urban and rural areas of central Gabon were used for P. falciparum DNA extraction. Performance of RDTs as a source of DNA for PCR was determined using: (i) amplification of a single copy merozoite surface protein 1 (msp1) gene followed by highly sensitive and automated capillary electrophoresis; (ii) genotyping of the pfcrt gene locus 72–76 using haplotype-specific-probe-based real-time PCR to characterize chloroquine resistance; and, (iii) real-time PCR targeting 18S genes to detect and quantify Plasmodium parasites. Results Out of the 669 archived RDTs, amplification of P. falciparum nucleic materials had a success rate of 97% for 18S real-time PCR, and 88% for the msp1 gene. The multiplicity of infections (MOI) of the whole population was 2.6 (95% CI 2.5–2.8). The highest number of alleles detected in one infection was 11. The MOI decreased with increasing age (β = − 0.0046, p = 0.02) and residence in Lambaréné was associated with smaller MOIs (p < 0.001). The overall prevalence of mutations associated with chloroquine resistance was 78.5% and was not associated with age. In Lambaréné, prevalence of chloroquine resistance was lower compared to rural Moyen-Ogooué (β = − 0.809, p-value = 0.011). Conclusion RDT is a reliable source of DNA for P. falciparum detection and genotyping assays. Furthermore, the increasing use of RDTs allows them to be an alternative source of DNA for large-scale genetic epidemiological studies. Parasite populations in the study area are highly diverse and prevalence of chloroquine-resistant P. falciparum remains high, especially in rural areas.

scholarly journals Assessment of Microscopic detection of Malaria with Nested Polymerase Chain Reaction in War-torn Federally Administered Tribal Areas of Pakistan

2021 ◽  
Author(s):  
Muhammad Faisal Nadeem ◽  
Aamer Ali Khattak ◽  
Adnan Yaqoob ◽  
Usman Ayub Awan ◽  
Nadia Zeeshan
Keyword(s):  
Diagnostic Accuracy ◽  
Sensitivity And Specificity ◽  
Mixed Infection ◽  
Microscopic Examination ◽  
Nested Pcr ◽  
Venous Blood ◽  
Pcr Analysis ◽  
Microscopic Detection ◽  
Tribal Areas ◽  
Pcr Targeting

Abstract Background: Diagnostic accuracy of malaria is critical for early treatment, control, and elimination of malaria, especially in war-affected malaria endemic areas. Microscopic detection of Plasmodium species has been the gold standard in remote malaria-endemic regions. However, the diagnostic accuracy is still questioned, especially in discriminating mixed and submicroscopic parasitic levels. This study was designed to evaluate the diagnostic performance of microscopic examination against nested PCR analysis in war-torn malaria-endemic Federally Administered Tribal Areas (FATA) of Pakistan. Methods: Venous blood samples were collected from symptomatic patients for microscopic examination and nested PCR analysis from January 2016 - December 2016 from five Agencies (Bajaur, Mohmand, Khyber, Orakzai and Kurram Agency) and four Frontier Regions (Peshawar, Kohat, Bannu, and Dera Ismail Khan Frontier Region) of FATA. Malaria-positive isolates were confirmed by nested PCR (targeting Plasmodium small subunit ribosomal ribonucleic acid (ssrRNA) genes) for speciation. Results: Among enrolled participants, 762 were found positive for malaria parasite on microscopic examination of the blood film. P. vivax was found in 623, P. falciparum in 132 and 7 were diagnosed with mixed infection (P. vivax and P. falciparum coinfection). Nested PCR detected Plasmodium infection in 679 samples (523 P. vivax, 121 P. falciparum, and 35 mixed infections). Compared with microscopy, the sensitivity of nested PCR was 98.94%, and specificity was 98.27%, while the sensitivity and specificity of slide microscopy 89.34% and 87.99% respectively. Conclusion: The conventional microscopy method has low sensitivity to detect mixed infection as compared to nested PCR. High sensitivity and specificity observed in nested PCR makes this molecular tool a useful technique for monitoring, controlling, and eliminating malaria-endemic regions.

scholarly journals Genetic diversity of Plasmodium falciparum populations in three malaria transmission settings in Madagascar

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Fanomezantsoa Ralinoro ◽  
Tovonahary Angelo Rakotomanga ◽  
Rianasoambolanoro Rakotosaona ◽  
Danielle A. Doll Rakoto ◽  
Didier Menard ◽  
...  
Keyword(s):  
Genetic Diversity ◽  
Plasmodium Falciparum ◽  
Malaria Transmission ◽  
Nested Pcr ◽  
Drug Efficacy ◽  
Control Measures ◽  
Epidemiological Strata ◽  
Allele Specific ◽  
The Impact ◽  
Pcr Targeting

Abstract Background Assessment of the genetic diversity of Plasmodium falciparum parasites from various malaria transmission settings could help to define tailored local strategies for malaria control and elimination. Such assessments are currently scarce in Madagascar. The study presented here aimed to bridge this gap by investigating the genetic diversity of P. falciparum populations in three epidemiological strata (Equatorial, Tropical and Fringes) in Madagascar. Methods Two-hundred and sixty-six P. falciparum isolates were obtained from patients with uncomplicated malaria enrolled in clinical drug efficacy studies conducted at health centres in Tsaratanana (Equatorial stratum), Antanimbary (Tropical stratum) and Anjoma Ramartina (Fringes) in 2013 and 2016. Parasite DNA was extracted from blood samples collected before anti-malarial treatment. Plasmodium species were identified by nested PCR targeting the 18 S rRNA gene. The genetic profiles of P. falciparum parasites were defined by allele-specific nested PCR on the polymorphic regions of the msp-1 and msp-2 genes. Results Fifty-eight alleles were detected in the P. falciparum samples tested: 18 alleles for msp-1 and 40 for msp-2. K1 (62.9%, 139/221) and FC27 (69.5%, 114/164) were the principal msp-1 and msp-2 allele families detected, although the proportions of the msp-1 and msp-2 alleles varied significantly between sites. Polyclonal infections were more frequent at sites in the Equatorial stratum (69.8%) than at sites in the Tropical stratum (60.5%) or Fringes (58.1%). Population genetics analyses showed that genetic diversity was similar between sites and that parasite flow within sites was limited. Conclusions This study provides recent information about the genetic diversity of P. falciparum populations in three transmission strata in Madagascar, and valuable baseline data for further evaluation of the impact of the control measures implemented in Madagascar.

scholarly journals Rare mutations in Pfmdr1 gene of Plasmodium falciparum detected in clinical isolates from patients treated with anti-malarial drug in Nigeria

2019 ◽  
Vol 18 (1) ◽  
Author(s):  
Abel O. Idowu ◽  
Wellington A. Oyibo ◽  
Sanjib Bhattacharyya ◽  
Manjeet Khubbar ◽  
Udoma E. Mendie ◽  
...  
Keyword(s):  
Plasmodium Falciparum ◽  
Clinical Isolates ◽  
Chloroquine Resistance ◽  
Malaria Therapy ◽  
Chloroquine Resistance Transporter ◽  
Rare Mutations ◽  
High Prevalence ◽  
Resistance Markers ◽  
Next Generation Sequencing Ngs ◽  
Effective Drugs

Abstract Background Plasmodium falciparum, the deadliest causative agent of malaria, has high prevalence in Nigeria. Drug resistance causing failure of previously effective drugs has compromised anti-malarial treatment. On this basis, there is need for a proactive surveillance for resistance markers to the currently recommended artemisinin-based combination therapy (ACT), for early detection of resistance before it become widespread. Methods This study assessed anti-malarial resistance genes polymorphism in patients with uncomplicated P. falciparum malaria in Lagos, Nigeria. Sanger and Next Generation Sequencing (NGS) methods were used to screen for mutations in thirty-seven malaria positive blood samples targeting the P. falciparum chloroquine-resistance transporter (Pfcrt), P. falciparum multidrug-resistance 1 (Pfmdr1), and P. falciparum kelch 13 (Pfk13) genes, which have been previously associated with anti-malarial resistance. Results Expectedly, the NGS method was more proficient, detecting six Pfmdr1, seven Pfcrt and three Pfk13 mutations in the studied clinical isolates from Nigeria, a malaria endemic area. These mutations included rare Pfmdr1 mutations, N504K, N649D, F938Y and S967N, which were previously unreported. In addition, there was moderate prevalence of the K76T mutation (34.6%) associated with chloroquine and amodiaquine resistance, and high prevalence of the N86 wild type allele (92.3%) associated with lumefantrine resistance. Conclusion Widespread circulation of mutations associated with resistance to current anti-malarial drugs could potentially limit effective malaria therapy in endemic populations.

Molecular Pattern of Anti-malarial Drug Resistance of Plasmodium falciparum in Bangladeshi Troops Working in Endemic Areas of Bangladesh and Africa

2020 ◽  
Vol 37 (1) ◽  
pp. 1-6
Author(s):  
Md Nurul Amin ◽  
Mahmuda Yasmin ◽  
Marufa Zerin Akhtar ◽  
Chowdhury Rafiqul Ahsan
Keyword(s):  
Plasmodium Falciparum ◽  
Nested Pcr ◽  
Plasmodium Species ◽  
Armed Forces ◽  
Chloroquine Resistance ◽  
Chittagong Hill Tracts ◽  
Resistance Marker ◽  
African Countries ◽  
Endemic Areas ◽  
Sub Saharan

Members of Bangladesh Armed Forces work in two different malaria endemic area, Chittagong Hill Tracts (CHT) in Bangladesh and Sub-Saharan countries in Africa. This under-recognized group remained unexplored for long in respect to drug resistant falciparum malaria they usually suffer from. In this study, a total of 252 ‘dried blood samples on filter paper’ were collected between November 2014 and February 2016, from Plasmodium falciparum positive Bangladeshi troops working in Chittagong Hill Tracts (CHT), Bangladesh and five Sub Saharan African Countries namely, Central African Republic (CAR), Democratic Republic of Congo (DRC), Liberia, Mali and Ivory Coast. After DNA extraction from all these samples (94 from Bangladesh and 138 from African countries), plasmodium species was confirmed by a nested PCR following standard protocol with minor modifications. Thereafter, a multiplex nested PCR followed by restriction fragment length polymorphism (RFLP) method was employed to investigate the presence of chloroquine resistance marker ‘K76T mutation’ in P. falciparum chloroquine resistance transporters (pfcrt) gene and lumifantrine and mefloquine resistance marker ‘N86Y mutation’ in P. falciparum multidrug resistance1 (pfmdr1) gene. The P. falciparum DNA was confirmed in 35 (37.23%) Bangladeshi and 45 (28.48%) African samples. The ‘pfcrt (K76T) mutation’ that confers resistance to chloroquine, was detected in 93.10% Bangladeshi and 29.27% African samples. The ‘pfmdr1 (N86Y) mutation’ that confers resistance to lumifantrine and mefloquine, was detected in 20.69% Bangladeshi and only 2.44% African samples. The higher prevalence of chloroquine resistance of P. falciparum in Bangladesh than in African countries revealed that possible withdrawal of chloroquine from endemic areas and also periodic molecular survey to monitor pf resistance to chloroquine, mefloquine, lumefantrine and artemisinin among these troops working in both endemic areas. Bangladesh J Microbiol, Volume 37 Number 1 June 2020, pp 1-6

scholarly journals The impact of HIV-associated immunosuppression on the Plasmodium falciparum chloroquine resistance transporter gene (PfCRT) of HIV patients in Akure, Nigeria

2020 ◽  
Vol 44 (1) ◽  
Author(s):  
Iyabo Adepeju Simon-Oke ◽  
Adeola Olanireti Ade-Alao ◽  
Foluso Ologundudu
Keyword(s):  
Plasmodium Falciparum ◽  
Malaria Prevalence ◽  
Cd4 Count ◽  
Chloroquine Resistance ◽  
Hiv Care ◽  
Transporter Gene ◽  
Hiv Patients ◽  
Chloroquine Resistance Transporter ◽  
Hiv Test ◽  
Low Prevalence

Abstract Background The study evaluated the prevalence of malaria and Plasmodium falciparum chloroquine resistance transporter gene (PfCRT) in HIV patients attending Specialist Hospital, Akure. This study was carried out between April and June 2019. Three hundred and seventeen (317) patients attending the antiretroviral clinic (ART) were involved, out of which 89 (28.08%) were males and 228 (71.92%) were females. HIV test was done using the Unigold® HIV test kit, malaria test was done using thick and thin blood smear, CD4 test was done using the Partec® CD4 counter and PCR was used to detect the presence of plasmodium falciparum mutant gene. The data obtained from this analysis was subjected to Pearson’s Chi-square test. Results The overall result showed low prevalence of malaria (23.03%) in the sampled patients. Highest malaria prevalence (31.0%) was recorded in HIV patients with CD4 count between 200–500 cells/μl of blood, with the males recording 24.7% malaria prevalence. The age group 20–29 years recorded the highest prevalence of 27.3%. A higher prevalence 91.1% of PfCRT gene in HIV-positive and (40.0%) in HIV-negative patients was recorded with 100% prevalence in patients with CD4 count ≤ 200. This shows that the low prevalence of malaria recorded in this study could be credited to good health-seeking attitude of HIV patients and the upscale of HIV care and treatment centres. Conclusion The high prevalence of PfCRT gene shows that treatment of malaria with chloroquine is still being practised despite the availability of artemisinin-based combination therapy (ACTs) as the recommended regimen for malaria treatment.

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