Safeguarding health on every plate: Intestinal parasitic infection survey of food handlers in a tertiary care centre.

Food borne infections pose significant public health problem, especially in developing countries of the world. A continuous surveillance to ensure the health of the personnel involved in preparation of the hospital food is important as they can be a source of spreading the infections and possible outbreaks. We analysed the data of the prevalence of intestinal parasitic infections in food handlers in our tertiary care centre from 2018 to 2022 and 6.8% were observed to harbour intestinal parasites during the period. This signifies the importance of routine screening, and the need of awareness and education of the food handlers in hospitals.

Modified trichrome stain for faster and improved detection of intestinal protozoan parasites.

Permanent stains such as trichrome have better sensitivity but are time-consuming and the fixative includes toxic mercuric chloride. Thus, a newer modification was tested and found to be a superior, faster and safer staining technique for intestinal parasitic detection. Our study lasted 9 months and a single stool sample was collected from each enrolled patient. We evaluated classical trichrome (T1 - using Schaudinn fixative) with newer modifications, which involved different fixatives with mordant combinations (T2 - acetic acid + hydrated aluminium sulphate, T3 - citric acid + copper sulphate hydrate). Conventional PCR targeting Entamoeba histolytica, Giardia lamblia and Cryptosporidium spp. was taken as the reference. Out of 175 stool samples, 25.1% protozoa were identified by wet mount, 24% by each T1 and T2, 25.7% by T3. Statistically, T3 and T2 had higher sensitivity as compared to T1 and wet mount when PCR was used as reference.

Case Report: A Series of Three Meningoencephalitis Cases Caused by Acanthamoeba spp. from Eastern India.

Acanthamoeba spp. are rare etiological agents of meningoencephalitis with high mortality. We present three cases of Acanthamoeba meningoencephalitis in immunocompetent individuals from Eastern India. The first patient presented with fever and headache; the second with headache, visual disturbance, and squint; and the third presented in a drowsy state. The cases presented on March 3, 18, and 21, 2023 respectively. The first two patients had concomitant tubercular meningitis for which they received antitubercular therapy and steroid. Their cerebrospinal fluid showed slight lymphocytic pleocytosis and increased protein. The diagnosis was done by microscopy, culture, and polymerase chain reaction. They received a combination therapy comprising rifampicin, fluconazole, and trimethoprim-sulfamethoxazole. The first patient additionally received miltefosine. She responded well to therapy and survived, but the other two patients died despite intensive care. Detection of three cases within a period of 1 month from Eastern India is unusual. It is imperative to sensitize healthcare providers about Acanthamoeba meningoencephalitis to facilitate timely diagnosis and treatment of the disease.

Opportunistic microsporidiosis unveiled by fine-needle aspiration cytology of cervical lymph node with literature review.

Microsporidia are highly specialized obligate intracellular organisms closely related to fungi, traditionally linked to diarrheal diseases in acquired immunodeficiency syndrome patients. Over the past two decades, an increasing incidence of extraintestinal infections affecting various organ systems, especially in immunocompromised individuals, has been observed. The report presents a unique case of lymph node microsporidiosis in a 38-year-old male, positive for human immunodeficiency virus, with coinfections of hepatitis B and C. Fine-needle aspiration cytology (FNAC) from cervical lymph node yielded pus-like, necrotic material with periodic acid-Schiff stained smear uncovering small round to oval spores on microscopy suspicious for microsporidia. Based on polymerase chain reaction and sequencing done with aspiration material, the causative agent was identified as Vittaforma corneae. This rare encounter highlights the significance of recognizing unique morphological characteristics of infectious organisms and employing appropriate ancillary techniques for precise identification. The case underscores the crucial role of FNAC in diagnosing opportunistic infections involving the lymph nodes and the growing significance of molecular tests for specific pathogen confirmation.

Comparison of B1 and RE 529 gene targets by real time PCR and LAMP assay for diagnosis of toxoplasmosis in pregnant females.

PURPOSE: The aim of this study is to accurately diagnose the presence of toxoplasmosis in pregnant women. In this study we evaluated two gene targets B1 and RE-529 using two different molecular methods i.e., real-time PCR and LAMP. PROCEDURE: A total of 150 blood samples were collected from pregnant women attending the PGIMER outpatient clinic. The serum and Buffy layer were extracted and various serological (ELISA) and molecular tests (qPCR and LAMP) targeting B1 and RE-529 were carried out. FINDING: Out of 150 patients, 32 were seropositive. Amongst which for the RE-529 gene, 18 were LAMP positive and 16 were qPCR positive, while for the B1 gene, 14 were LAMP positive and 13 were qPCR positive. CONCLUSIONS: Molecular methods were more sensitive than serological tests to diagnose congenital toxoplasmosis in antenatal females. Few seronegative patients were reported positive using molecular methods. In addition, LAMP targeting the RE-529 gene is more sensitive than qPCR, and LAMP targets the B1 gene.

Real-time loop-mediated isothermal amplification for the detection of Giardia duodenalis in fecal specimens.

INTRODUCTION: Giardiasis is a leading cause of subacute or chronic diarrhoea and is frequently associated with impaired physical, cognitive and psychosocial development, especially in children. The diagnosis relies mainly on the microscopic evaluation of stool specimens that have a low sensitivity. In contrast, molecular advancements like the polymerase chain reaction and Real-time loop-mediated isothermal amplification (Real-time LAMP) are promising techniques and reportedly have better diagnostic characteristics. METHODS: We have evaluated the performance of Real-time LAMP for detecting Giardia in ninety stool specimens compared to microscopy and nested PCR. RESULTS: A total of 35 fecal samples were detected positive by microscopy, 41 by nested PCR and 43 by real-time LAMP. Microscopy and nested PCR detected 33, microscopy and real-time LAMP detected 35, and nested PCR and real-time LAMP detected 41 positive samples. CONCLUSION: The real-time LAMP assay was found suitable for the rapid and accurate detection of G. duodenalis with a better sensitivity in comparison to nested PCR and microscopy. Furthermore, besides being sensitive and rapid, LAMP had the advantage of an adequate rapid turn-around time of eleven to 15 â€‹min as compared to 5 â€‹h of nested PCR.

Parafilm as an efficient transport matrix for corneal scrapings.

Introduction: Acanthamoeba spp. are free-living parasites increasingly implicated in causing Acanthamoeba keratitis (AK). AK is diagnosed by demonstration of parasites in corneal samples by direct microscopy, culture, and nucleic acid amplification. Most commonly, corneal scrapings are sent to the laboratory smeared between two glass slides. These scrapings are suitable for direct microscopy but less suitable for culture and polymerase chain reaction (PCR) which, in turn, are more sensitive for the diagnosis of AK. Aim: The aim of the study was to explore better alternatives for transporting corneal scrapings from the point-of-care eye center to the concerned laboratories. Materials and Methods: The study used small Parafilm (Bemis Company Inc., USA) squares (PSs) of 1 cm each prepared by cutting Parafilm using a surgical blade under sterile conditions. Each of the four different dilutions of Acanthamoeba suspension (15, 30, 60, and 120 cells) was used in this study. Each dilution was added onto the surface of 36 PSs and kept at room temperature for 24-h, 48-h, and 72-h incubation. The PSs for one particular time point and dilution were used for calcofluor white staining, its inoculation onto the surface of nonnutrient agar having a lawn of Escherichia coli, and Acanthamoeba-specific PCR amplification. In addition, two PSs inoculated with 30 cells and incubated for 24 h and 72 h were used for scanning electron microscopy (SEM). Results and Conclusion: All three diagnostic techniques, i.e. microscopy, culture, and PCR, detected the presence of Acanthamoeba at all the tested concentrations and time points. However, the growth pattern on culture changed directly in proportion to increased incubation periods and increased concentration of inoculum. In addition, the adherence of Acanthamoeba to the Parafilm was confirmed by SEM; these results suggest the use of these PSs as a suitable matrix for the transport of corneal scrapings.

Primate malaria of human importance.

Nonhuman primate (NHP) malaria poses a major threat to the malaria control programs. The last two decades have witnessed a paradigm shift in our understanding of the malaria caused by species other than the traditionally known human Plasmodium species - Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, and Plasmodium ovale. The emergence of the malaria parasite of long-tailed macaque monkeys, Plasmodium knowlesi, as the fifth malaria species of humans has made the scientific community consider the risk of other zoonotic malaria, such as Plasmodium cynomolgi, Plasmodium simium, Plasmodium inui, and others, to humans. The development of knowledge about P. knowlesi as a pathogen which was earlier only known to experimentally cause malaria in humans and rarely cause natural infection, toward its acknowledgment as a significant cause of human malaria and a threat of malaria control programs has been made possible by the use of advanced molecular techniques such as polymerase chain reaction and gene sequencing. This review explores the various aspects of NHP malaria, and the association of various factors with their emergence and potential to cause human malaria which are important to understand to be able to control these emerging infections.

The gene expression and proteomic profiling of Acanthamoeba isolates.

INTRODUCTION: The free-living protozoan Acanthamoeba can cause severe keratitis known as Acanthamoeba Keratitis (AK) and granulomatous amoebic encephalitis (GAE). The pathogenesis of Acanthamoeba includes intricate interactions between the organism and the host's immune system. The downstream analysis of a well-annotated genome assembly along with proteomic analysis can unravel several biological processes and aid in the identification of potential genes involved in pathogenicity. METHODS: Based on the next-generation sequencing data analysis, genes including lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein were selected as probable pathogenic targets that were validated by conventional PCR in a total of 30 Acanthamoeba isolates. This was followed by real-time PCR for the evaluation of relative gene expression in the keratitis and amoebic encephalitis animal model induced using keratitis (CHA5), encephalitis (CHA24) and non-pathogenic environmental isolate (CHA36). In addition, liquid chromatography-mass spectrometry (LC-MS/MS) was performed for keratitis, encephalitis, and non-pathogenic environmental isolate before and after treatment with polyhexamethylene biguanide (PHMB). RESULTS: The conventional PCR demonstrated the successful amplification of lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein genes in clinical and environmental isolates. The expression analysis revealed phospholipase, lysophospholipase, and mannose-binding genes to be significantly upregulated in the keratitis isolate (CHA 5) during AK in the animal model. In the case of the amoebic encephalitis model, phospholipase, lysophospholipase, S8/S53 peptidase, and carboxylesterase were significantly upregulated in the encephalitis isolate compared to the keratitis isolate. The proteomic data revealed differential protein expression in pathogenic versus non-pathogenic isolates in the pre and post-treatment with PHMB. CONCLUSION: The gene expression data suggests that lysophospholipase, phospholipase, S8/S53 peptidase, carboxylesterase, and mannose-binding protein (MBP) could play a role in the contact-dependent and independent mechanisms of Acanthamoeba pathogenesis. In addition, the proteomic profiling of the 3 isolates revealed differential protein expression crucial for parasite growth, survival, and virulence. Our results provide baseline data for selecting possible pathogenic targets that could be utilized for designing knockout experiments in the future.

A rapid multiplex loop-mediated isothermal amplification (mLAMP) assay for detection of Entamoeba histolytica and Giardia duodenalis.

We developed a rapid multiplex loop-mediated isothermal amplification (mLAMP) assay for two common intestinal parasites-Entamoeba histolytica and Giardia duodenalis, where early detection may be helpful. The mLAMP assay was optimized for the detection of DNA of E. histolytica (18S rRNA gene) and G. duodenalis (Elongation factor 1 alpha gene) from standard strains by using six specific primers FIP (forward inner primer), BIP (backward inner primer), F3 (forward outer primer), B3 (backward outer primer), loopF (forward loop primer), and loopB (backward loop primer) for each gene target. The amplification time was 16-26 min for E. histolytica and 10-15 min for G. duodenalis, and the parasites could be distinguished based on melting-curve analysis for specific annealing temperatures (Tm) of 84°C-86°C and 88°C-90°C for E. histolytica and G. duodenalis, respectively. The analytical sensitivity was one fg, and no cross-reactivity with other intestinal pathogens was observed. Thus, the mLAMP assay could detect and clearly distinguish E. histolytica and G. duodenalis with a rapid turnaround time and excellent analytical sensitivity and specificity.

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.

Prevalence of Trichomonas vaginalis by polymerase chain reaction-based molecular method among symptomatic women from Northern India.

Introduction: Trichomoniasis remains one of the most common sexually transmitted infections, which is curable. To prevent complications and transmission, prompt and correct diagnosis is essential to treat Trichomonas vaginalis. The present study was done to evaluate polymerase chain reaction (PCR) with other conventional techniques for the diagnosis of T. vaginalis infection and determine the prevalence of T. vaginalis in women with vaginal discharge based on PCR assay. Methods: Vaginal swabs were collected by the trained health-care professional using FLOQSwabs™ (Copan, Italy) during routine pelvic examinations among 1974 symptomatic females. The wet microscopy, culture, and PCR were performed. Results: The sensitivity of wet mount and culture in comparison to PCR was 60.87% and 56.52%, respectively. The kappa inter-rater agreement of T. vaginalis PCR showed substantial agreement with wet mount microscopy (κ = 0.742) and culture (κ = 0.707). The PCR detected an additional 17 cases that were missed by conventional techniques. Discussion: The study highlights the importance of PCR for T. vaginalis screening among symptomatic females.

Seroprevalence of human cystic echinococcosis: A study from a tertiary care center of North India.

Present study retrospectively analysed the serological data of patients suspected of cystic echinococcosis (CE) attending the outpatient clinics or admitted in our hospital. An enzyme-linked immunoassay was used to analyse anti-CE antibodies in serum samples of 3680 patients. Microscopy of aspirated cystic fluid was performed on 170 cases only. CE seropositive cases were 595 (16.2%), of which 293 (49.2%) were males and 302 (50.8%) were females. A higher percentage of seropositivity was found in adults within age range of 21-40 years of age. There has been a decrease in seropositivity in the study years (2016-2021) in comparison to previous years (1999-2015).

Molecular Diagnosis of Encephalitis/Meningoencephalitis Caused by Free-Living Amoebae from a Tertiary Center in India.

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.

An Insight into the Genome of Pathogenic and Non-Pathogenic Acanthamoeba.

BACKGROUND: Acanthamoeba are amphizoic amoeba majorly responsible for causing Acanthamoeba keratitis (AK) and Granulomatous amoebic encephalitis (GAE). Despite its ubiquitous nature, the frequency of infections is not high, probably due to the existence of non-pathogenic isolates. The whole-genome sequencing and an annotated genome assembly can unravel the biological functions and help in identifying probable genes related to pathogenicity. METHODS: Illumina and Nanopore sequencing were performed for keratitis, encephalitis, and non-pathogenic environmental isolates. Hybrid assembly was prepared for the AK and GAE isolates, while only the Illumina reads were utilized for a non-pathogenic environmental isolate. Protein coding genes were identified using the GeneMark-ES program and BLASTx module of Diamond used for gene prediction. Additionally, the Kyoto Encyclopedia of Genes and Genomes annotation and cluster of orthologous group's annotation using RPS-blast against the CDD database was performed. The subsequent data analysis and validation will help identify probable pathogenic genes. RESULTS: The genome assemblies of 9.67, 8.34, and 8.89 GBs were reported for GAE, AK, and non-pathogenic isolate, respectively. KEGG reported 22,946 in GAE, 24,231 in keratitis, and 9367 genes in the environmental isolate. The COG annotation revealed 3232 in GAE, 3403 in keratitis, and 1314 genes in the non-pathogenic isolate. CONCLUSION: The present study has attempted to generate de novo hybrid genome assemblies of Acanthamoeba that would help decode the genome of free-living amoeba and will provide genomic data for a better understanding of virulence-related factors.

Bilateral Pulmonary Hydatid Cyst in a Young Child: A Rare Case Report from North India.

Echinococcosis or hydatid disease is caused by the larval stage of the dog tapeworm, that is, Echinococcus granulosus , E. multilocularis , E. vogeli , or E. oligarthrus . Echinococcus granulosus causes cystic echinococcosis, which has a worldwide distribution. Liver is the most common site, affecting approximately two-third of the patients, whereas lung involvement is seen in approximately 25% of cases. This case is a very rare scenario of bilateral pulmonary hydatid cysts in a young child having exposure to a pet dog with negative hydatid serology and normal eosinophil count.

Environmental water surveillance for free-living amoeba in North India.

PURPOSE: Free-living amoebae (FLA) exist in various environmental sources and are the causative agents of many serious human diseases. We surveyed various natural and artificial water bodies in areas of north India to detect the presence of FLA species. MATERIALS AND METHODS: Surface water samples were collected from 29 water bodies and subjected to microscopy, non-nutrient agar (NNA) culture, and various polymerase chain reaction (PCR) assays for the detection of FLA. RESULTS: FLA were found in 62.1% (18/29) of the natural and artificial water bodies by NNA culture. Based on PCR based detection, Acanthamoeba spp. was identified in 41.4% (12/29), Vannella spp./Vahlkampfia ovis in 17.2% (5/29) and Naegleria spp. in 20.7% (6/29) of the samples from different locations. PCR-sequencing of Naegleria positive samples identified them as Naegleria australiensis and Naegleria species. CONCLUSIONS: The present study reports widespread occurrence of medically important FLA species in various natural and artificial water bodies, and highlights the need for regular molecular surveillance of FLA in the environment for better control and prevention of the diseases caused by FLA.

Local cytokine/chemokine profiles in BALB/c and C57BL/6 mice in response to T. vaginalis infection.

Trichomonas vaginalis is the causative agent of Trichomoniasis (a sexually transmitted infection). Recent reports have shown that stimulation of cellular immunity can reduce trichomoniasis infection. Animal studies are essential to understanding the pathogenesis of infection and developing new potential drugs and vaccines to treat the infection. Therefore, we have tried to understand the pathogenesis of T. vaginalis infection by investigating the differences in the expression of chemokine/cytokine levels in vaginal and cervical tissues of BALB/c and C57BL/6 mice. Different pathological symptoms, like desquamation, neutrophil infiltration, and hemorrhage, were recorded in BALB/c and C57BL/6 in response to T. vaginalis infection. Vaginal and cervical tissues of BALB/c showed these symptoms on 2nd dpi, which became severe on 7th dpi and turned to mild or normal till 14th dpi compared to C57BL/6 strain. Immunohistochemistry in the vagina and cervical tissues of BALB/c and C57BL/6 mice was done to assess cytokines at different time intervals post-infection. Significant expression of Interleukin-1ß (IL-1ß) (a pro-inflammatory cytokine) was found in BALB/c compared to the C57BL/6 mice, on 7th dpi and 2nd dpi in vaginal and cervical tissues, respectively. Higher expression of MIP-2 (neutrophil chemoattractant) was observed in the vaginal tissues of BALB/c mice on 7th dpi compared to the C57BL/6 group. In addition, higher expression of TGF-ß (immune-suppressor) was observed on 7th dpi in the vaginal tissue of BALB/c mice. The present study demonstrates that more pathological signs of T. vaginalis infection developed in BALB/c mice than C57BL/6 mice. Also, significant levels of IL-1ß and MIP-2 were measured in BALB/c mice in response to T. vaginalis compared to C57BL/6.

Cyclosporiasis in an immunocompromised patient who had undergone renal allograft transplant-A rare case report.

Cyclospora spp. is an important cause of traveler's diarrhea or water and food-borne diarrhoeal diseases. We present an interesting but rare case report of cyclosporiasis in a 51-year-old male who had undergone renal allograft transplant six years ago. He also had a past history of tuberculosis, cytomegalovirus, severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and hepatitis C infection and was being treated with immunosuppressants. The patient had a prolonged history of gastrointestinal manifestations with recent acute onset of watery diarrhea associated with abdominal cramps. Stool examination after modified Ziehl-Neelsen staining revealed oocysts of Cyclospora spp. The patient was successfully treated with cotrimoxazole.

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.