Anti-malarial drug resistance and associated genetic polymorphism of Plasmodium falciparum in members of Armed Forces of Bangladesh working in endemic areas at home and abroad

Abstract

Despite achieving a trend of reduction in global morbidity and mortality from malaria since beginning of 21st century the parasitic disease continues to have a devastating impact on people’s health and livelihoods in Asia and Africa. Plasmodium falciparum (pf), responsible for most malaria-related deaths among all 5 parasite species, has a long tradition of acquiring resistance against anti-malarial drugs. Development of pf resistance to artemisinin, the world’s most efficient antimalarial drug, is now threatening the achievements of global malaria control and elimination programmes. Armed forces’ personnel in South East Asia and Sub-Saharan Africa traditionally remain exposed to this volatile malarial situation as they usually need to operate in malaria endemic areas. Members of Bangladesh Armed Forces operate in endemic areas of both home and Africa because of their deployment in hilly areas at home and peace keeping missions in Africa. Not surprisingly, they need to travel between different endemic regions. Unfortunately, there is no data available on the genetic types of falciparum malaria they suffer from or probable resistant strains they might transmit between different endemic areas they travel. The present study is designed to investigate and compare molecular pattern and epidemiological aspects of anti-malarial drug resistance of P. falciparum in members of Armed Forces of Bangladesh working in endemic areas at home and Africa. A total of 252 ‘dried blood samples on filter paper’ were collected between November 2014 and February 2016, from P. falciparum positive Bangladeshi soldiers working in Chittagong Hill Tracts (CHT), Bangladesh and 5 Sub Saharan African Countries namely, Central African Republic (CAR), Democratic Republic of Congo (DRC), Liberia, Mali and Ivory Coast. These samples (94 from Bangladesh and 138 from African countries) were then transported to Microbiology Laboratories of University of Dhaka, where pf DNA extraction was done from all of them using QIAamp® DNA Mini Kit (Qiagen GmbH, Germany), following manufacturer’s protocol. Plasmodium species was confirmed by a nested PCR following standard protocol with minor modifications. Thereafter, a multiplexix 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 propeller region of the kelch 13 (pfk13) gene in 29 Bangladeshi and 40 African samples was amplified by a nested PCR following a protocol developed by Pasteur Institute, Paris and Cambodia and then sequenced to see markers of artemisinin resistance. Some data on demography, clinical features, epidemiological aspects and Knowledge attitude and practice (KAP) were also collected from the respondent soldiers using a pretested structured questionnaire and a check list. All molecular and epidemiological data were entered and analyzed in statistical software IBM SPSS version 19. 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. None of the Bangladeshi samples had mutation in k13 propeller domain. On the other hand, 9 (22.50%) African samples exhibited pfk13 mutations including 5 non-synonymous and 3 synonymous mutations, reported for the first time. All of these new non-synonymous mutations namely A617P, Y616F, S491F, N458k and Y616F and synonymous mutations namely F614F, I616I and K503K were found in samples from D R Congo. Mutations F614F and A617P were detected in a single isolate and S491F was detected in two of the isolates. The most common African mutation A578S was detected in a sample from Ivory Coast. None of the pfk13 mutations, so far recognized to be associated/candidate or validated as artemisinin resistance marker by World Health Organization (WHO), was detected in this study. While analyzing clinical data it was found that, duration of fever, number of fever episodes, failure of drug, referral and length of hospital stay were associated with the 1st line antimalarial drug used (p ˂ 0.05). Epidemiological data in this study revealed a yearlyx incidence of 180 cases of malaria per thousand population under mefloquine prophylaxis in Liberia, although variables like duration of fever, drug failure and mutation in pfmdr1 were not associated with this prophylaxis in all the study areas. While analyzing data on knowledge attitude and practice (KAP) it is found that troops under mefloquine prophylaxis were more confident on the efficacy of this prophylactic drug (P=0.00). Majority (92%) of the troops could identify mosquito bite as the route of malaria transmission while 62.30% chose bed net as the most important preventive measure against malaria. The correctness of their understanding about malaria transmission, cause of resistance and preventive priorities, was associated with their source of information (p ˂ 0.05). More than 90% of the troops used bed net as a protective measure although only 19% of them used insecticide treated net. Regular use of protective clothing to prevent mosquito bite by the soldiers in different study areas varied between 70% and 95%. A routine practice of outdoor mosquito-cidal spray was reported by more than 80% troops working in different countries. Preventive behavior of the troops like use of protective clothing, insecticide treated bed net and mosquito repellent were found to be associated with their source of information (p ˂ 0.05). This study, first of its kind to be conducted in members of Bangladesh Armed Forces deployed in malaria endemic areas of both Bangladesh and Africa, attempted to investigate and compare molecular and epidemiological pattern of antimalarial drug resistance. It revealed 5 new non-synonymous and 3 synonymous mutations in pfk13 gene found in its African samples. Further study is required to see the relationship of these new mutations with delayed parasite clearance and eventually artemisinin resistance. Further study is also required to examine the potential transportability of drug resistant strains of pf malaria between endemic areas.