Tehran University of Medical Sciences Iranian Journal of Parasitology 1735-7020 17 1 2022 03 14 43 52 Muhammad Aurongzeb 1. Jamil-ur-Rahman Center for Genome Research, Dr. Panjwani Center for Molecular Medicine and Drug Research, International Center for Chemical and Biological Sciences, University of Karachi, Karachi, 75270, Pakistan 2. Institute of Biotechnology & Genetic Engineering, University of Sindh, Jamshoro, Pakistan Yasmeen Rashid Department of Biochemistry, University of Karachi, Karachi, 75270, Pakistan Syed Naqvi Institute of Biotechnology & Genetic Engineering, University of Sindh, Jamshoro, Pakistan Ambrina Khatoon Department of Research, Ziauddin University, 4/B, Saharah-e-Ghalib, Block-6, Clifton, Karachi, 7500, Pakistan Sadia Abdul Haq Department of Biochemistry, University of Karachi, Karachi, 75270, Pakistan Muhammad Kamran Azim Department of Biosciences, Mohammad Ali Jinnah University, Karachi, Pakistan Imdad Kaleem Department of Bioinformatics and Bioscience, COMSATS University (CUI), Islamabad, Pakistan Shahid Bashir Neuroscience Center, King Fahad Specialist Hospital, Dammam, Saudi Arabia 2021 02 03 2021 06 15 Background: Primary amoebic meningoencephalitis (PAM) is an acute and fulminant CNS infection caused by Naegleria fowleri. Recreational activities and ritual ablution with contaminated warm fresh water are the main reason of PAM. Pakistan ranked the second most affected country, where most of the PAM incidences were reported from Karachi, Pakistan. Methods: In May, 2019, a 28-yr-old suspected PAM patient came to the Imam Zain-Ul-Abdin Hospital, Karachi. Biochemical and cytological investigations of patient`s CSF were carried out at Karachi Diagnostic Center and Molecular Biology Lab. Sequencing of Naegleria sp. specific (ITS) primer-based amplicons was performed from both patient`s CSF and water samples followed by multiple sequence alignment and phylogenetic studies. Results: Biochemical and cytological investigations of patient`s CSF showed 5 mg/dl glucose, 240 mg/dl total protein and 2260/mm3 TLC suggesting acute meningoencephalitis. PCR-based analyses of patient`s CSF and his residential tap water samples using Naegleria sp. specific (ITS) and N. fowleri specific primers revealed the presence of N. fowleri DNA. Nucleotide sequences of ITS primer-based amplicons from both patient`s CSF and water samples were submitted in GenBank under the accession numbers MT726981.1 and MT726226.1, respectively. According to phylogenetic analysis, N. fowleri isolate from Pakistan has shown the least node age of seven. Conclusion: Here, for the very first time in Pakistan, N. fowleri genotype has been identified as type-2. Phylogenetic analysis showed that N. fowleri isolate from Pakistan is among the latest descendants, i.e., evolved later in life. https://ijpa.tums.ac.ir/index.php/ijpa/article/view/3255 https://ijpa.tums.ac.ir/index.php/ijpa/article/download/3255/1194