Original Article

Naegleria fowleri from Pakistan Has Type-2 Genotype


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.

1. Shakoor S, Beg MA, Mahmood SF, et al. Primary amebic meningoencephalitis caused by Naegleria fowleri, Karachi, Pakistan. Emerg Infect Dis. 2011; 17(2):258-61.
2. De Jonckheere JF. The impact of man on the occurrence of the pathogenic free-living amoeboflagellate Naegleria fowleri. Future Microbiol. 2012; 7(1):5-7
3. Marciano-Cabral F, MacLean R, Mensah A, et al . Identification of Naegleria fowleri in domestic water sources by nested PCR. Appl Environ Microbiol. 2003; 69(10):5864-9.
4. Martinez AJ, Visvesvara GS. Free-living, amphizoic and opportunistic amebas. Brain Pathol. 1997; 7(1):583-98.
5. Visvesvara GS. Free-living amebae as opportunistic agents of human disease. J Neuroparasitol. 2010; 1:1-3.
6. Griffin JL. Temperature tolerance of pathogenic and nonpathogenic free-living amoebas. Science. 1972 ;178(4063):869-70
7. Martínez-Castillo M, Cárdenas-Zúñiga R, Coronado-Velázquez D, et al. Naegleria fowleri after 50 years: is it a neglected pathogen? J Med Microbiol. 2016 ;65(9):885-896.
8. Mishra BB, Gundra UM, Teale JM. Toll-like receptors in CNS parasitic infections. Curr Top Microbiol Immunol. 2009; 336:83-104.
9. De Jonckheere JF. Origin and evolution of the worldwide distributed pathogenic amoeboflagellate Naegleria fowleri. Infect Genet Evol. 2011 ;11(7):1520-8.
10. Wang Q, Li J, Ji J, et al. A case of Naegleria fowleri related primary amoebic meningoencephalitis in China diagnosed by next-generation sequencing. BMC Infect Dis. 2018; 18(1):349.
11. Chomba M, Mucheleng'anga LA, Fwoloshi S, et al. A case report: primary amoebic meningoencephalitis in a young Zambian adult. BMC Infect Dis. 2017; 17:532.
12. Panda A, Khalil S, Mirdha BR, et al. Prevalence of Naegleria fowleri in Environmental Samples from Northern Part of India. PLoS One. 2015; 10(10):e0137736.
13. Petit F, Vilchez V, Torres G, Molina O, et al. [Primary amebic meningoencephalitis: two new cases report from Venezuela]. Arq Neuropsiquiatr. 2006; 64:1043-6.
14. Craun GF, Calderon RL, Craun MF. Outbreaks associated with recreational water in the United States. Int J Environ Health Res. 2005; 15(4):243-62.
15. Tung MC, Hsu BM, Tao CW, et al. Identification and significance of Naegleria fowleri isolated from the hot spring which related to the first primary amebic meningoencephalitis (PAM) patient in Taiwan. Int J Parasitol. 2013; 43(9):691-6.
16. Quist-Paulsen E, Kran AB, Lindland ES, et al. To what extent can clinical characteristics be used to distinguish encephalitis from encephalopathy of other causes? Results from a prospective observational study. BMC Infect Dis. 2019; 19(1):80.
17. Naqvi AA, Yazdani N, Ahmad R, et al. Epidemiology of primary amoebic meningoencephalitis-related deaths due to Naegleria fowleri infections from freshwater in Pakistan: An analysis of 8-year dataset. Arch Pharm Pract. 2016; 7(4):119-29.
18. Ali M, Jamal SB, Farhat SM. Naegleria fowleri in Pakistan. Lancet Infect Dis. 2020; 20(1):27-8.
19. Khalid M, Saif UR, Shahzain K. Suspected Case of Naegleria fowleri (Primary Amebic Meningo-encephalitis). Pak Pediatr J. 2014; 38(3):196-8.
20. De Jonckheere JF. Sequence Variation in the Ribosomal Internal Transcribed Spacers, Including the 5.8S rDNA, of Naegleria spp. Protist. 1998 ;149(3):221-8.
21. De Jonckheere JF. What do we know by now about the genus Naegleria? Exp Parasitol. 2014; 145 Suppl:S2-9.
22. Escrig JI, Hahn HJ, Debnath A. Activity of Auranofin against Multiple Genotypes of Naegleria fowleri and Its Synergistic Effect with Amphotericin B In Vitro. ACS Chem Neurosci. 2020 ;11(16):2464-2471.
23. Zein NN. Clinical significance of hepatitis C virus genotypes. Clin Microbiol Rev. 2000 ;13(2):223-35.
24. Zhang LL, Wu M, Hu BC, et al. Identification and molecular typing of Naegleria fowleri from a patient with primary amebic meningoencephal-itis in China. Int J Infect Dis. 2018 ;72:28-33.
25. Sazzad HMS, Luby SP, Sejvar J, et al. A case of primary amebic meningoencephalitis caused by Naegleria fowleri in Bangladesh. Parasitol Res. 2020; 119(1):339-344.
26. Deisenhammer F, Bartos A, Egg R, et al. Guidelines on routine cerebrospinal fluid analysis. Report from an EFNS task force. Eur J Neurol. 2006; 13(9):913-22.
27. Teunissen CE, Petzold A, Bennett JL, et al. A consensus protocol for the standardization of cerebrospinal fluid collection and biobanking. Neurology. 2009; 73(22):1914-22.
28. Ithoi I, Ahmad AF, Nissapatorn V, et al. Detection of Naegleria species in environmental samples from Peninsular Malaysia. PLoS One. 2011; 6(9):e24327.
29. Barnett ND, Kaplan AM, Hopkin RJ, et al. Primary amoebic meningoencephalitis with Naegleria fowleri: clinical review. Pediatr Neurol. 1996; 15(3):230-4.
30. Moussa M, De Jonckheere JF, Guerlotté J, et al. Survey of Naegleria fowleri in geothermal recreational waters of Guadeloupe (French West Indies). PLoS One. 2013; 8(1):e54414.
31. Kang H, Seong GS, Sohn HJ, et al. Effective PCR-based detection of Naegleria fowleri from cultured sample and PAM-developed mouse. Eur J Protistol. 2015; 51(5):401-8.
32. Wittwer CT, Ririe KM, Andrew RV, et al. The Light Cycler: a microvolume multisample fluorimeter with rapid temperature control. Biotechniques. 1997; 22(1):176-81.
33. Fotedar R, Stark D, Beebe N, et al. Laboratory diagnostic techniques for Entamoeba species. Clin Microbiol Rev. 2007; 20(3):511-32.
34. Behera HS, Satpathy G, Tripathi M. Isolation and genotyping of Acanthamoeba spp. from Acanthamoeba meningitis/ meningoencephalitis (AME) patients in India. Parasit Vectors. 2016; 9:442.
35. Pélandakis M, Serre S, Pernin P. Analysis of the 5.8S rRNA gene and the internal transcribed spacers in Naegleria spp. and in N. fowleri. J Eukaryot Microbiol. 2000 ;47(2):116-21.
36. Dobrowsky PH, Khan S, Cloete TE, et al. Molecular detection of Acanthamoeba spp., Naegleria fowleri and Vermamoeba (Hartmannella) vermiformis as vectors for Legionella spp. in untreated and solar pasteurized harvested rainwater. Parasit Vectors. 2016 ;9(1):539.
37. Kumar S, Stecher G, Li M, et al. MEGA X: Molecular Evolutionary Genetics Analysis across Computing Platforms. Mol Biol Evol. 2018; 35(6):1547-1549.
38. Tamura K, Nei M, Kumar S. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proc Natl Acad Sci U S A. 2004; 101(30):11030-5.
39. U.o.E. Institute of Evolutionary Biology, Edinburgh, FigTree v1.3.1, (2010).
40. De Jonckheere JF. Century of research on the amoeboflagellate genus Naegleria. Acta Protozool. 2002;41:309-42.
41. Phu NH, Hoang Mai NT, Nghia HD, et al. Fatal consequences of freshwater pearl diving. Lancet. 2013;381(9861):176.
42. Nicolas M, De Jonckheere JF, Pernin P, et al.[Molecular diagnosis of a fatal primary amoebic meningoencephalitis in Guadeloupe (French West Indies)]. Bull Soc Pathol Exot. 2010;103(1):14-8.
IssueVol 17 No 1 (2022) QRcode
SectionOriginal Article(s)
DOI https://doi.org/10.18502/ijpa.v17i1.9015
Naegleria fowleri Genotyping Primary amoebic meningoencephalitis

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Aurongzeb M, Rashid Y, Naqvi S, Khatoon A, Abdul Haq S, Azim MK, Kaleem I, Bashir S. Naegleria fowleri from Pakistan Has Type-2 Genotype. Iran J Parasitol. 2022;17(1):43-52.