ABSTRACT
BACKGROUND: Pathogenic free-living amoeba (FLA) such as Acanthamoeba spp., Naegleria fowleri, and Balamuthia mandrillaris are causative agents of fatal amoebic encephalitis/meningoencephalitis. The diagnosis of such infections is challenging due to a lack of clinical suspicion and expertise in microscopic identification. We evaluated the performance of molecular assays for the timely and accurate detection of FLA-causing central nervous system (CNS) afflictions. METHODS: This study included samples from 156 patients with suspected encephalitis/meningoencephalitis, including 149 cerebrospinal fluid (CSF) samples, 5 brain tissue biopsies, and 2 brain abscess samples. All the samples were subjected to PCR-based detection of Acanthamoeba spp., N. fowleri, and B. mandrillaris. The diagnostic characteristics and the inter-rater reliability scores were evaluated for parasite-specific polymerase chain reaction (PCR) using culture on non-nutrient agar (NNA)/microscopy or histopathological examination as a confirmatory test for Acanthamoeba spp. and N. fowleri and histopathology for B. mandrillaris. RESULTS: We detected 11 samples positive for FLA, including 6 Acanthamoeba spp., 3 B. mandrillaris, and 2 N. fowleri. Furthermore, all 11 samples were positive according to the confirmatory tests, i.e., culture on NNA/microscopy/histopathology in the case of Acanthamoeba spp. and N. fowleri and histopathology of tissue biopsies for B. mandrillaris. The inter-rater reliability between the PCRs and the confirmatory tests for the detection of Acanthamoeba spp., N. fowleri, and B. mandrillaris was 100%. CONCLUSIONS: The PCR-based detection of FLA in patients suspected of encephalitis/meningoencephalitis was found to be fast, efficient, and reliable in our study. We suggest the use of these PCRs in laboratories to obtain additional data on their efficiency in diagnosing FLA infections of the CNS. The present study was conducted with a small sample size of 156 patient samples, and we found only six Acanthamoeba spp., three B. mandrillaris, and two N. fowleri. The present study should be conducted on a larger sample size for better evaluation of the primer pairs.
ABSTRACT
BACKGROUND: The clinical features of Acanthamoeba keratitis (AK) are non-specific and closely resemble bacterial, viral and fungal keratitis. MATERIALS AND METHODS: We compared loop-mediated isothermal amplification (LAMP) with microscopy, non-nutrient agar (NNA) culture and polymerase chain reaction (PCR) in clinical suspects of AK. RESULTS: Of 52 clinical samples (42 AK suspects and 10 proven bacterial, viral or fungal keratitis), 3 were positive by direct microscopy (sensitivity 60%, confidence interval [CI]: 17%-92.7%), and 5 by NNA culture, 18S rDNA PCR and LAMP (sensitivity 100%, CI: 46.3%-100%). The limit of detection of Acanthamoeba DNA was 1 pg/µl by both LAMP and PCR. CONCLUSION: PCR and LAMP assays targeting 18S rDNA gene were found particularly suitable for a rapid and accurate diagnosis of AK. LAMP assay takes 2-3 h lesser than PCR, and thus offers a rapid, highly sensitive and specific, simple and affordable diagnostic modality for patients suspected of AK, especially in resource limited settings.
