The diagnostic conundrum of hyper eosinophilia-Sheer tenacity of a parasite.

We present an interesting and rare case of Capillaria hepatica infection in a 2-year-old boy, who presented with fever, rash, hepatomegaly and peripheral eosinophilia. FNAC of hepatic lesion showed parasitic eggs and PCR from the aspirate confirmed the diagnosis. We describe the cytomorphological features and provide educational multiple-choice questions related to the topic.

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.

Latent Upregulation of Nlrp3, Nlrc4 and Aim2 Differentiates between Asymptomatic and Symptomatic Trichomonas vaginalis Infection.

Trichomonas vaginalis is a parasitic protozoan that causes trichomoniasis. The involvement of NLRP3 inflammasome in trichomoniasis has been discussed in recent studies. The present study aimed to find out the involvement of Nlrp3, Nlrc4, and Aim2 in the BALB/c mouse model infected with symptomatic and asymptomatic isolates of T. vaginalis by quantitative real-time PCR and immunohistochemistry. Our results showed a significantly increased expression of Nlrp3 in the vaginal tissue of the symptomatic group on the 2nd dpi and 14th dpi in the asymptomatic group, respectively. The cervical tissue of asymptomatic groups expressed higher Nlrp3 on 14th dpi than the symptomatic group. The Nlrc4 was expressed on 14th dpi in the vaginal and cervical tissues of mice infected with asymptomatic group as compared to the symptomatic group. Aim2 expression in vaginal tissue was highest at early time points in both the infected groups as compared to controls. However, in cervical tissues, a significant increase of Aim2 expression was observed on 14th dpi in asymptomatic as compared to the symptomatic group. The significantly higher expression of caspase-1 and caspase-4 was observed in cervical tissues of the asymptomatic group on 14th dpi as compared to the symptomatic group, respectively. All NLRs together resulted in higher IL-1ß expression in the vaginal tissues of the symptomatic and asymptomatic groups. We conclude from this study that early expression of Nlrp3, Nlrc4, and Aim2 was seen in the symptomatic group as compared to the late-onset asymptomatic in the vaginal and cervical tissues.

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.

Role of Polymerase Chain Reaction in Stool and Duodenal Biopsy for Diagnosis of Giardiasis in Patients with Persistent/Chronic Diarrhea.

BACKGROUND: Giardia duodenalis is a common cause of chronic diarrhea especially in tropical countries. Diagnosis is based on microscopy (three stool samples) for trophozoites/cysts. Role of stool or duodenal biopsy PCR as a diagnostic method needs to be defined. We conducted a prospective study to determine the diagnostic characteristics of G. duodenalis stool and duodenal biopsy PCR in comparison to stool microscopy (reference standard). Later, we compared other techniques with stool PCR, considering it as new reference standard and characterized the type of Giardia assemblage. METHODS: G. duodenalis stool nested PCR was first evaluated using 40 positive controls and 50 negative controls considering stool microscopy as reference standard. Patients with chronic diarrhea (n = 100) were evaluated by stool microscopy and nested PCR. In 30 patients in whom upper gastrointestinal endoscopy was performed, duodenal biopsy samples were obtained and evaluated by histopathology, imprint cytology, and nested PCR. The type of Giardia assemblage was detected by assemblage-specific PCR. RESULTS: Stool nested PCR was found to have sensitivity and specificity of 100% and 94%, respectively, compared to stool microscopy. In patients with chronic diarrhea, 48% had evidence of Giardia infection. Stool microscopy detected 65%, stool PCR detected an additional 27%, and duodenal biopsy PCR detected an additional 8% of cases. The commonest assemblage found was assemblage B. Clinical and demographic characteristics were similar in patients harboring either assemblage A or B. CONCLUSION: Stool PCR is more sensitive than stool microscopy. By utilizing stool microscopy, stool nested PCR, and duodenal biopsy PCR in sequential manner, diagnostic yield can be increased.

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.

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.

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.

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.

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.

Multiplex PCR for gastrointestinal parasites in stool: Benchmarking against direct microscopy and simplex PCR.

PURPOSE: To develop and validate a multiplex conventional PCR assay to simultaneously detect Cryptosporidium spp., Entamoeba histolytica, and Giardia lamblia in diarrheal samples as a rapid, cost-effective, and sensitive diagnostic tool for prevalent co-infections for improved diagnostic accuracy and efficiency in resource-limited settings. METHODS: Stool samples collected from patients with gastrointestinal symptoms after taking written consent, processed via wet mount, iodine mount, and PCR assays. Cohen's kappa statistical analysis was done to test agreement. RESULT: Among 240 patients, 28.75% showed intestinal protozoa via Microscopy; Single-plex and multiplex PCR demonstrated 100% concordance, detecting 27.9%; confirmed by sequencing. Highest parasite positivity was observed in transplant and immunocompromised patients, with moderate to almost perfect agreement between microscopy and molecular methods. CONCLUSION: Multiplex-conventional PCR offers superior sensitivity and specificity over microscopy and 100% concordance with single-plex PCR, enabling rapid, cost-effective diagnosis of multiple parasites from single stool sample. Its adoption could revolutionize parasitic infection management in routine diagnostics.

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.

Immunogenicity and protective efficacy of heparan sulphate binding proteins of Entamoeba histolytica in a guinea pig model of intestinal amoebiasis.

Entamoeba histolytica infection is associated with considerable morbidity and mortality in the form of intestinal and extraintestinal amoebiasis. No vaccine is yet available for amoebiasis. Heparan Sulphate Binding Proteins (HSBPs) from E. histolytica were evaluated for immunogenicity and protective efficacy in a Guinea pig model. Animals were immunized subcutaneously with 30µg of HSBP by three weekly inoculations. The immunogenicity of HSBP was determined by antibody response (IgG, IgM and IgA), splenocyte proliferation assay and in vitro direct amoebicidal assay with splenic lymphocytes and monocytes from vaccinated and control animals. The efficacy of the vaccine was evaluated by challenge infection to vaccinated and control animals by intra-caecal inoculation of E. histolytica trophozoites and comparing gross and histopathological findings in caeca of these animals. HSBP was found to induce specific anti-amoebic response as seen by specific antibody production and direct amoebicidal activity of splenocytes. The vaccine also showed partial protection against challenge infection in vaccinated animals as shown by mild/absent lesions and histopathological findings.

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.

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).