Potentially pathogenic free-living amoebae (FLA) are unique protozoa that present a healthcare challenge. The appropriate choice of staining technique is crucial to improving the efficiency of microscopic diagnosis of FLA isolated from environmental as well as clinical samples. This study aimed to evaluate different staining techniques for morphological identification of Acanthamoeba and Naegleria isolated from tap water samples in different localities in Assiut City, Egypt. A multi-attribute evaluation was performed to compare these techniques considering staining quality, ease of performance, cost and time of each procedure. Seven staining techniques were used including Lugol's iodine and lactophenol cotton blue as temporary stains, with Giemsa, trichrome, iron hematoxylin, acid-fast and Gram stains as permanent stains. Both lactophenol cotton blue and Lugol's iodine showed good structural details of different stages of FLA and good contrast at a very short time and reasonable cost, followed by Giemsa. Both trichrome and iron hematoxylin stains gave comparable staining quality but at a longer time with more complex staining procedures and higher cost. Both modified acid-fast and Gram stains didn't provide sufficient staining quality to identify morphological details of FLA. According to the multi-attribute evaluation, both Lugol's iodine and lactophenol cotton blue achieved the highest rank (92.85%) followed by Giemsa and Gram stains (64.28 %), modified acid-fast stain (57.14%), while iron hematoxylin and Masson's trichrome had the last rank (46.42%).

Free-living amoebae (FLA) are gaining attention due to the increasing number of related grave central nervous system (CNS) and sight-threatening eye infections and their role as Trojan horses for many bacteria and viruses. This study was conducted in Assiut City, Egypt to detect the presence of FLA in different water sources using morphological and molecular approaches and determine their potential pathogenicity. A total of 188 water samples (100 tap, 80 tank, and 8 swimming pool samples) were collected, cultivated on non-nutrient agar seeded with Escherichia coli, and inspected for FLA. Thermo- and osmo-tolerance assays were performed to determine their pathogenicity. Polymerase chain reaction and sequence analysis were performed to confirm the identification and analyze the genotype. Overall, 52 samples (27.7%) were positive for FLA. Of these, 20.7% were identified as Acanthamoeba, 1.6% as Vahlkampfiidae, and 5.3% as mixed Acanthamoeba and Vahlkampfiidae. Seven species of Acanthamoeba were recognized, of which A. triangularis, A. polyphaga, A. lenticulata, and A. culbertsoni are thermo- and osmo-tolerant, and A. astronyxis, A. comandoni, and A. echinulata are non-thermo- and non-osmo-tolerant. The phylogeny analysis revealed T4 and T7 genotypes. Among Vahlkampfiids, 61.5% were identified as thermo- and osmo-tolerant Vahlkampfia, and 30.8% were identified as non-pathogenic Naegleria. One isolate (7.7%) was identified as potentially pathogenic Allovahlkampfia, as confirmed by sequencing. This is the first report documenting the occurrence and phylogeny of waterborne FLA (Acanthamoeba/Vahlkampfiidae) in Assiut, Egypt. The presence of potentially pathogenic FLA highlights the possible health hazards and the need for preventive measures.