Nested PCR assay for the rapid detection of Naegleria fowleri from swimming pools in Egypt W.M. Hikal a,b, ⁎, M.A. Dkhil c,d a Parasitology Laboratory, Water Pollution Research Department, National Research Centre, 33 El Bohouth St. (former El Tahrir St.), Dokki, 12622 Giza, Egypt b Department of Biology, Faculty of Science, University of Tabuk, P.O. Box 741, Tabuk 71491, Saudi Arabia c Department of Zoology, College of Science, King Saud University, P.O. Box 2455, Riyadh 11451, Saudi Arabia d Department of Zoology and Entomology, Faculty of Science, Helwan University, Helwan 11795, Egypt abstract article info Article history: Received 30 April 2016 Received in revised form 13 June 2017 Accepted 15 June 2017 Available online xxxx The free-living amoeboflagellate Naegleria fowleri is the only species infects humans world widely distributed. N. fowleri is the causative agent of very rare but severe brain infection called primary amoebic meningo-encephalitis (PAM), a rapidly fatal disease of the central nervous system mainly in immuno-compromised individuals. N. fowleri infects human through the entry of the nose, and it happens when human swimming or diving in warm freshwater, such as lakes, rivers and swimming pools. The disease is acute, and patients often die within 5–10 days and before the infectious agent can be diagnosed. Limited information is available about the existence of pathogenic N. fowleri, in Egypt, so the present of N. fowleri is an important public health. In the present study, we examined hundred water, dust and swap samples collected from 5 swimming pools in Cairo, Egypt. Based on morphological characteristics of trophozoite and cyst, flagellation test 56% of thermo-tolerant Naegleria like amoeba was detected. The incidence of thermo-tolerant free-living amoebae reached 84, 80and 70% from water, cotton swap and dust samples, respectively at cultivation temperature of 45 °C. The highest occurrence of thermo-tolerant amoebae were recorded in summer (100 & 87.5%) while the lowest one were recorded in winter (58 & 37.5%) in both water and dust samples, respectively. In swap samples, the highest occurrence of thermo-tolerant free-living amoeba was recorded in both summer and spring (100%), while the lowest one was recorded in winter (40%). N. fowleri was performed on 24 samples from a total of 56 (42.2%) samples which are positive by culture. Nested PCR using Mp2Cl5 gene primers that is unique to N. fowleri was carried out. The N. fowleri specific primer showed band at 166 bp against 24 of 56 (42.2%) samples. The majority of pos- itive samples unique to N. fowleri was detected in water samples followed by swap samples and finally dust sam- ples 14 of 24 (58%), 7 of 24 (29%), 3 of 24 (13%), respectively. In conclusion, swimming pools water may be the source of Naegleria invasion. The use of molecular methods to identify free-living amoebae N. fowleri could pro- vide a more rapid means to diagnose infections caused by those amoebae. © 2017 Ecological Society of China. Published by Elsevier B.V. All rights reserved. Keywords: Naegleria fowleri Swimming pools PCR Culture Egypt 1. Introduction Free-living amoebae (FLA) are a very diverse and comprehensive- ness organisms that have been isolated from soil and water environ- ments [19,36,39]. Naegleria is a free-living amoeba that is distributed ubiquitously in the environment all over the world. N. fowleri is a ther- mo-tolerant, free-living amoebo-flagellate characterized by a life-cycle of trophozoite, flagellate and cyst stage [8,10,20,25,26,33]. Only N. fowleri was found to cause human disease, that result in primary amoe- bic meningo-encephalitis (PAM), which is quick and fatal infection of the central nervous system (CNS) [42]. The most frequently reported PAM in healthy young men with the recent history of swimming or other water activities in warm water [11,38,44]. Also, PAM was associat- ed with pipe and storage ponds used by Muslims for routine ritual ablu- tion, which involves the inhalation of water through the nostrils [23,34]. It was documented that, N. fowleri infects the CNS via inhalation through the nasal passages of the trophozoite, flagellate and cyst forms that pol- lute water or dust. Inside the nasal passage, amoebae travel up the olfac- tory mucosa, along olfactory nerve fibers, and through the cribriform plate into the brain. Inside the brain, N. fowleri amoebae feed on red blood cells, white blood cells, and brain tissue causing hemorrhagic ne- crosis and edema. Disease progresses vary rapidly and symptoms occur in a few days, followed by a dramatic clinical course and death within 3– 7 days following the onset of symptoms. Most victims of PAM are healthy children and young adults with a relatively recent history of Acta Ecologica Sinica xxx (2017) xxx–xxx ⁎ Corresponding author at: Parasitology Laboratory, Water Pollution Research Department, National Research Centre, 33 El Bohouth St. (former El Tahrir St.), Dokki, 12622 Giza, Egypt. E-mail address: whikal@ut.edu.sa (W.M. Hikal). CHNAES-00508; No of Pages 6 http://dx.doi.org/10.1016/j.chnaes.2017.06.013 1872-2032/© 2017 Ecological Society of China. Published by Elsevier B.V. All rights reserved. Contents lists available at ScienceDirect Acta Ecologica Sinica journal homepage: www.elsevier.com/locate/chnaes Please cite this article as: W.M. Hikal, M.A. Dkhil, Nested PCR assay for the rapid detection of Naegleria fowleri from swimming pools in Egypt, Acta Ecologica Sinica (2017), http://dx.doi.org/10.1016/j.chnaes.2017.06.013