Assessment of pathogenic potential of Acanthamoeba isolates by in vitro and in vivo tests.

Acanthamoeba are free-living protozoa present ubiquitously in numerous environmental reservoirs that exist as an actively feeding trophozoite or a dormant cyst stage. The pathogenic Acanthamoeba are known to cause Acanthamoeba keratitis (AK) and granulomatous amoebic encephalitis (GAE). Despite their omnipresence, the number of infections is quite low. The reason behind this low frequency of Acanthamoeba infections could be the existence of many non-pathogenic strains or a successful host immune response to these infections. Studies in the past have proposed a few physiological parameters for the differentiation of pathogenic and non-pathogenic strains. Additionally, in vivo experiments are known to play an essential role in understanding the virulence of parasites, immunological aspects, and disease pathogenesis. The thermotolerance (30 °C, 37 °C, and 40 °C) and osmotolerance (0.5 M, 1 M, and 1.5 M) tests were performed on 43 Acanthamoeba isolates from patients with keratitis (n = 22), encephalitis (n = 5), and water samples (n = 16). In addition, the genotype of 10 Acanthamoeba isolates (keratitis (n = 2), encephalitis (n = 2), water (n = 6)) was determined and were then evaluated for pathogenicity on mouse model by inducing Acanthamoeba keratitis and amoebic encephalitis. The results of the thermotolerance and osmotolerance assays categorized 29/43 (67.4%) isolates as pathogenic, 8 as low pathogenic (18.6%), and the remaining 6 (13.9%) as non-pathogenic. The 10 Acanthamoeba isolates were categorized as T11 (5 isolates), T5 (2 isolates), T4 (2 isolates), and T10 (1 isolate) genotypes. Out of 10 Acanthamoeba isolates, 9 were successful in establishing AK, amoebic encephalitis, or both in the mice model, and a single isolate was found non-pathogenic. Two isolates from water samples were non-pathogenic in the physiological tests but successfully established Acanthamoeba infection in the mice model. The results of the physiological assays and in vivo experiments were analogous for 7 isolates while 1 isolate from the water was low pathogenic in the physiological assays but failed to produce pathogenicity during in vivo experiments. The physiological parameters are not very dependable to test the pathogenic potential of Acanthamoeba isolates, and thus results must always be validated by in vivo experiments. There is no infallible approach for determining the potential pathogenicity of environmental isolates of Acanthamoeba because several parameters regulate the pathogenic potential.

Evaluation of in vitro activity of five antimicrobial agents on Acanthamoeba isolates and their toxicity on human corneal epithelium.

BACKGROUND: Acanthamoeba keratitis (AK) is an important cause of ocular morbidity in both contact lens wearers and non wearers. Medical management comprises prolonged empiric treatment with multiple drugs, leading to adverse effects and suboptimal cure. The present study evaluated the efficiency and safety of common antimicrobial agents used in treatment of AK. METHODS: Six Acanthamoeba isolates (four AK, two water samples) were axenized and subjected to in vitro susceptibility testing against chlorhexidine, pentamidine isethionate, polymyxin B, miltefosine, and fluconazole to check for trophocidal and cysticidal activity. The safety profile was analysed by observing the cytotoxicity of the highest cidal concentration toward human corneal epithelial cell (HCEC) line. RESULTS: Chlorhexidine had the lowest cidal concentration against both cysts and trophozoites (range 4.16-25 µg/ml) followed by pentamidine isethionate (range 25-166.7 µg/ml). Both agents were nontoxic to HCEC. Polymyxin B (range 25-200 µg/ml) and fluconazole (range 64-512 µg/ml) had relatively higher minimum inhibitory concentrations (MIC); fluconazole was nontoxic even at 1024 µg/ml, but cytotoxicity was observed at 400 µg/ml with polymyxin B. Miltefosine was not effective against cysts at tested concentrations. A. castellanii were more susceptible to all agents (except pentamidine isethionate) than A. lenticulata. Clinical isolates were less susceptible to polymyxin B and fluconazole than environmental isolates, reverse was true for miltefosine. CONCLUSION: Chlorhexidine and pentamidine isethionate were the most effective and safe agents against both trophozoites and cysts forms of our Acanthamoeba isolates. Fluconazole had higher MIC but was nontoxic. Polymyxin B was effective at high MIC but therapeutic dose was found toxic. Miltefosine, at tested concentrations, could not inhibit cysts of Acanthamoeba. Clinical isolates had higher MICs for polymyxin B and fluconazole.

Microbiological diagnosis of Acanthamoebic keratitis: experience from tertiary care center of North India.

Acanthamoeba keratitis (AK) is a painful vision-threatening infection caused by pathogenic free-living Acanthamoeba. Due to the non-specific clinical presentation, this condition tends to be misdiagnosed by clinicians. A timely diagnosis is crucial for favorable visual outcome. Three hundred patients with suspected microbial keratitis presenting to the Advanced Eye Center at our tertiary care center in North India during the period from 2014 to 2018 were included. Patient's corneal scrapings, contact lens, lens solution, lens case, and tears were processed for microscopic examination by Giemsa and Calcofluor staining, non-nutrient agar (NNA) culture and molecular diagnosis by conventional PCR (cPCR) and Real-time PCR (qPCR). 18S rDNA gene sequencing was done to assess phylogenetic relationship. AK was found in 3.6% (11/300) of non-bacterial non-fungal keratitis patients. Among microbiological techniques, microscopy for Acanthamoeba was positive in 7 cases, NNA culture was positive in 9 cases and 11 cases were detected both by cPCR and qPCR. The sensitivity of microscopy, culture, cPCR and qPCR was 63.64%, 81.82 %, 100%, and 100% respectively whereas specificity was 100% for all the tests. 18S rDNA sequencing revealed that A. castellanii was the predominant species and isolates were genetically distinct. AK should be considered in the differential diagnosis of infectious keratitis. Molecular tests are useful for rapid, sensitive and specific diagnosis and must be included in workup of keratitis.

DrugRepV: a compendium of repurposed drugs and chemicals targeting epidemic and pandemic viruses.

Viruses are responsible for causing various epidemics and pandemics with a high mortality rate e.g. ongoing SARS-CoronaVirus-2 crisis. The discovery of novel antivirals remains a challenge but drug repurposing is emerging as a potential solution to develop antivirals in a cost-effective manner. In this regard, we collated the information of repurposed drugs tested for antiviral activity from literature and presented it in the form of a user-friendly web server named 'DrugRepV'. The database contains 8485 entries (3448 unique) with biological, chemical, clinical and structural information of 23 viruses responsible to cause epidemics/pandemics. The database harbors browse and search options to explore the repurposed drug entries. The data can be explored by some important fields like drugs, viruses, drug targets, clinical trials, assays, etc. For summarizing the data, we provide overall statistics of the repurposed candidates. To make the database more informative, it is hyperlinked to various external repositories like DrugBank, PubChem, NCBI-Taxonomy, Clinicaltrials.gov, World Health Organization and many more. 'DrugRepV' database (https://bioinfo.imtech.res.in/manojk/drugrepv/) would be highly useful to the research community working to develop antivirals.

Acanthamoeba keratitis: A 4-year review from a tertiary care hospital in North India.

INTRODUCTION: Acanthamoeba keratitis (AK) is a blinding condition reported from both developed and developing countries. Limited knowledge on the clinical characteristics of AK and scarce laboratory diagnostic facilities in such countries poses difficulties in the accurate diagnosis. OBJECTIVE: To describe the epidemiological and clinical characteristics as well as management of Acanthamoeba keratitis in a tertiary care hospital in North India. METHODS: All clinically suspicious cases of Acanthamoeba keratitis (AK) presenting to our centre were screened for Acanthamoeba. All patients diagnosed as Acanthamoeba on microscopic examination, culture and polymerase chain reaction (PCR) were given Polyhexamethylene biguanide (PHMB) eye drops 0.02% half hourly for 1 week, then hourly for 1 week and then gradually tapered according to the response. Out of 300 consecutive patients evaluated, Acanthamoeba was detected in 11(3.6%) patients. A history of trauma was elicited in majority of the patients, 6 (55%). The most common complaints were eye pain, redness and watering in all of the patients, diminution of vision (8, 72.7%), photophobia (7, 63.6%) and foreign body sensation (2, 18.2%). Complete healing with vascularization and scarring was observed in 7 patients (63.6%) patients whereas progression to perforation of corneal ulcer and corneal melt was seen in 3 (27.3%) cases and these patients underwent therapeutic keratoplasty later. One patient did not come for follow up examination. CONCLUSION: The most common risk factor for the occurrence of Acanthamoeba Keratitis is trauma followed by contact lens use.

NipahVR: a resource of multi-targeted putative therapeutics and epitopes for the Nipah virus.

Nipah virus (NiV) is an emerging and priority pathogen from the Paramyxoviridae family with a high fatality rate. It causes various diseases such as respiratory ailments and encephalitis and poses a great threat to humans and livestock. Despite various efforts, there is no approved antiviral treatment available. Therefore, to expedite and assist the research, we have developed an integrative resource NipahVR (http://bioinfo.imtech.res.in/manojk/nipahvr/) for the multi-targeted putative therapeutics and epitopes for NiV. It is structured into different sections, i.e. genomes, codon usage, phylogenomics, molecular diagnostic primers, therapeutics (siRNAs, sgRNAs, miRNAs) and vaccine epitopes (B-cell, CTL, MHC-I and -II binders). Most decisively, potentially efficient therapeutic regimens targeting different NiV proteins and genes were anticipated and projected. We hope this computational resource would be helpful in developing combating strategies against this deadly pathogen. Database URL: http://bioinfo.imtech.res.in/manojk/nipahvr/.

Demographic and clinical profile of microspodial keratitis in North India: an underreported entity.

Ocular microsporidiosis was first described in immunocompromised subjects but recent reports have also shown cases in immunocompetent hosts. The database of existing clinical studies is very limited. The aim of present study was to analyse demographic profile, clinical features, microbiological profile, treatment and outcome of ocular microsporidiosis in northern India. The present study was carried out in the Department of Medical Parasitology, Advanced Eye Center, Postgraduate Institute of Medical Education and Research, and Bharat Vikas Parishad Charitable trust, Chandigarh. A total of 250 patients during year 2013-17 and suspected of microsporidial keratitis (either clinically or after exclusion of bacterial, viral or fungal agents). Corneal scraping were taken and subjected for various staining methods and PCR. 8 patients of microsporidial keratitis were confirmed, based on positivity by at least any two of the above mentioned techniques. None of the patients had history of contact lens wear or exposure to swimming pool. All these patients were systemically healthy and HIV serology was negative except one had history of diabetes mellitus. This study is a reminder to clinicians that when multifocal diffuse epithelial keratitis and culture-negative keratoconjunctivitis not responding to medical therapy, regardless of immune status are found in patients with symptoms suggesting an infectious keratitis, microsporidial keratoconjunctivitis should be included in the differential diagnosis.

Protein profiling of Acanthamoeba species using MALDI-TOF MS for specific identification of Acanthamoeba genotype.

Acanthamoeba spp. are ubiquitous in the environment and have the potential to cause severe infections. The different genotypes of Acanthamoeba have been shown to influence the severity of the disease and response to therapy. Characterizing Acanthamoeba spp. upto genotype can aid in infection control practices. Twenty-five Acanthamoeba isolates, characterized by 18S rDNA sequencing, were subjected to MALDI-TOF MS analysis by creating a database for the individual genotypes. The differentiating features of the various spectra were observed; the coded samples were then tested against the created database. The results of identification were compared with sequencing. Five different genotypes were obtained-T3, T4, T5, T10, and T11. Spectral analysis revealed genus-specific and genotype-specific peaks. The peak patterns for individual genotype were discrete and reproducible. Clinical isolates produced different peaks from the environmental isolate of the same genotype. A concordance of 92% was obtained with MALDI-TOF MS in comparison with 18sDNA sequencing. MALDI-TOF MS, once optimized, has the potential to reliably identify the genotype of Acanthamoeba spp. and to differentiate clinical isolate from mere contaminant.

Osteo-cutaneous acanthamoebiasis in a non-immunocompromised patient with a favorable outcome.

Osteo-cutaneous form is a rare presentation of acanthamoebiasis. We present the first such case from India in an apparently healthy male who developed cutaneous lesion with bone involvement after traumatic inoculation of Acanthamoeba cysts. The diagnosis was established by routine microbiological techniques and confirmed by 18SrRNA gene sequencing. Aggressive therapy with terbinafine, chlorhexidine, rifampicin and co-trimoxazole was successful in clearing the lesion and preventing encephalitis.

Evaluation of loop-mediated isothermal amplification assay for rapid diagnosis of Acanthamoeba keratitis.

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.

An improvised medium for axenic cultivation of Acanthamoeba spp.

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.

Excessive zinc in diet induces leptin resistance in Wistar rat through increased uptake of nutrients at intestinal level.

PROJECT: The ob gene has either been found to be mutant defective resulting in a deficiency of its product leptin or leptin has been found to be resistant to its receptors in obese human and rodents. The factors inducing leptin resistance have not been identified. Since excessive bioavailability of Zn has been implicated in obesity, we investigated if its excess in diet induces leptin resistance. PROCEDURE: For the investigations, three groups of Wistar rats were included in this study and they were fed on equicalories semi synthetic basal diet containing 20 mg, 40 mg or 80 mg Zn/kg diet for 120 days. There after they were sacrificed for hormonal status and intestinal investigations. RESULTS: The data of this study revealed that the food intake, gain in body weight, serum leptin, glucose, insulin, cortisol increased with increased Zn concentration in diet. TEM study showed a positive correlation between Zn concentration in diet and number of microvilli/unit surface area of the mucosal epithelial cells of the intestine. CONCLUSION: The results of this study suggest that excessive bioavailability of Zn induces leptin resistance through increased uptake of nutrients at intestinal level, leading to the growth of the fat cells which aggravated the leptin synthesis and its release in the blood stream. In spite of its higher circulating level, it was unable to reduce the food intake and gain in body weight in Zn treated rats equivalent to the control group.