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Acanthamoebae can be easily grown in bacterised cultures, but their growth in axenic media is tedious and many times unsuccessful. We thus experimented with some additives in the conventional axenic medium for growth of various isolates of Acanthamoeba. Addition of Torula yeast RNA was found to significantly enhance the growth of Acanthamoebae in the axenic culture medium.

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.

Background: Isolation of free-living amoebae (FLA) is reported sparsely from water taps, ventilators, air conditioners, haemodialysis units and dental irrigation systems of hospitals worldwide. Their prevalence in hospital environment especially in wards having immunocompromised patients may pose a risk to this group of susceptible population as they may cause disease themselves or may carry pathogens inside them. No study from India has performed such surveillance., Objective: To evaluate extent of FLA contamination in water sources of bone marrow transplant (BMT) intensive care unit (ICU), transplant ICU, haemodialysis unit and high dependency unit in a tertiary care hospital in India., Materials and Methods: A total of hundred samples including fifty each of tap water samples and swabs from mouth of taps used for drinking, bathing and hand washing purposes in these units were collected according to standard procedure. Samples were inoculated onto non-nutrient agar plates at room temperature followed by morphological confirmation. Molecular identification including polymerase chain reaction (PCR) and sequencing was performed in culture positive samples., Results: Four tap water samples and ten swab samples showed growth of trophozoites and cyst formation. Morphologically, four amoebae resembled Acanthamoeba spp. which was further confirmed by PCR and sequencing showed them to be of T3 and T4 genotypes., Conclusion: The presence of these FLA in hospital water sources emphasises the urgent need of implementing effective preventive measures. Further studies are required to estimate the true prevalence of FLA in Indian hospitals by taking larger number of samples.