Introduction Malaria, a terrifying earthly disease, is sometimes regarded as the ''King of diseases" which is majorly caused by a protozoan of genus Plasmodium. 1 It is often regarded as one of the most important parasitic infection especially in tropical and subtropical regions of the World. 2,3 Because of the suitable temperature, rainfall required for the development of the Anopheles mosquitos and unhygienic conditions, poor population of these regions are at a greater risk of malaria. 4 The world's most malaria endemic regions comprised of Asia, Africa, Central and South America. 5 The four Plasmodium species responsible for causing malaria in humans include Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae and Plasmodiumovale. The occurrence of these species depends on the geographical location and climatic conditions of the area. 6 Pakistan is highly endemic to plasmodiumfalciparum and plasmodiumvivax. Being a tropical country, Pakistan holds a vast irrigation system, large number of stagnant water bodies which provide the mosquitoes standard climatic conditions for their breeding. 7 The aptitude of malaria has also risen due to monsoon rains in the country. In Pakistan, lower incidence of malaria was restricted to Punjab and Azad Kashmir (AJK) areas while Balochistan and Federally Administered Tribal Areas (FATA) were found with higher malarial incidences during 2004. 8 P.falciparum is thought to be the cause of most serious and sometime the fatal kind of disease and causes most of the mortality cases. P.vivax can also cause severe illness and morbidity but the mortality tolls are lower than P.falciparum whereas P.malariae and P.ovale infections are rare across the world. 2,3 On a global level, Plasmodiumvivax threatens approximately 2.8 billion people and its eradication could be more difficult than P.falciparum due to its complicated biological characteristics such as presence of liver hypnozoites which causes numerous relapses. 9-13 Accurate treatment of malaria is solely dependent upon the early and accurate diagnosis. 8 Microscopic screening of Giemse-stained thick and thin blood smears is considered to be the gold standard and is used as a conventional method to diagnose malaria in various industrialized countries. 14,15 Despite the fact that this technique is economical, quick and J Pak Med Assoc 677 ORIGINAL ARTICLE Amplification of Mitochondrial DNA for detection of Plasmodiumvivax in Balochistan Muhammad Naeem Shahwani, 1 Samia Nisar, 2 Abdul Aleem, 3 Marina Panezai, 4 Sarwat Afridi, 5 Shaukat Iqbal Malik 6 Abstract Objective: To access a new step using PCR to amplify the targeted mtDNA sequence for detecting specifically Plasmodium vivax and its co-infections, false positive and false negative results with Plasmodium falciparum. Methods: In this study we have standardized a new technical approach in which the target mitochondrial DNA sequence (mtDNA) was amplified by using a PCR technique as a tool to detect Plasmodium spp. Species specific primers were designed to hybridize with cytochrome c oxidase gene of P. vivax (cox I) and P. falciparum (cox III). Two hundred blood samples were collected on the basis of clinical symptoms which were initially examined through microscopic analysis after preparing Giemsa stained thick and thin blood smears. Afterwards genomic DNA was extracted from all samples and was then subjected to PCR amplification by using species specific primers and amplified segments were sequenced for confirmation of results. Results: One-hundred and thirty-two blood samples were detected as positive for malaria by PCR, out of which 64 were found to be positive by PCR and 53 by both microscopy and PCR for P.vivax infection. Nine samples were found to be false negative, one P.vivax mono infection was declared as co infection by PCR and 3 samples identified as having P.falciparum gametes were confirmed as P.vivax by PCR amplification. Sensitivity and specificity were found to be 85% and 92% respectively. Conclusion: Results obtained through PCR method were comparatively better and reliable than microscopy. Keywords: Malaria diagnosis, Plasmodium vivax, Polymerase chain reaction, mtDNA, cox I gene. (JPMA 67: 677; 2017) 1-5 Department of Biotechnology, Faculty of Life Sciences and Informatics (FLS&I), Balochistan University of Information Technology, Engineering and Management Sciences (BUITEMS), Quetta, Balochistan, 6 Department of Bioinformatics & Biological Sciences, Capital University of Science & Technology, Expressway, Islamabad, Pakistan. Correspondence: Muhammad Naeem Shahwani. Email: muhammad.naeem@buitms.edu.pk