Central nervous system infections caused by pathogenic free-living amoebae: An Indian perspective

@#Pathogenic free-living amoebae (FLA), namely Acanthamoeba sp., Naegleria fowleri and Balamuthia mandrillaris are distributed worldwide. These neurotropic amoebae can cause fatal central nervous system (CNS) infections in humans. This review deals with the demographic characteristics, symptoms, diagnosis, and treatment outcomes of patients with CNS infections caused by FLA documented in India. There have been 42, 25, and 4 case reports of Acanthamoeba granulomatous amoebic encephalitis (GAE), N. fowleri primary amoebic meningoencephalitis (PAM), and B. mandrillaris meningoencephalitis (BAE), respectively. Overall, 17% of Acanthamoeba GAE patients and one of the four BAE patients had some form of immunosuppression, and more than half of the N. fowleri PAM cases had history of exposure to freshwater. Acanthamoeba GAE, PAM, and BAE were most commonly seen in males. Fever, headache, vomiting, seizures, and altered sensorium appear to be common symptoms in these patients. Some patients showed multiple lesions with edema, exudates or hydrocephalus in their brain CT/MRI. The cerebrospinal fluid (CSF) of these patients showed elevated protein and WBC levels. Direct microscopy of CSF was positive for amoebic trophozoites in 69% of Acanthamoeba GAE and 96% of PAM patients. One-fourth of the Acanthamoeba GAE and all the BAE patients were diagnosed only by histopathology following autopsy/biopsy samples. Twenty-one Acanthamoeba GAE survivors were treated with cotrimoxazole, rifampicin, and ketoconazole/amphotericin B, and all eleven PAM survivors were treated with amphotericin B alongside other drugs. A thorough search for these organisms in CNS samples is necessary to develop optimum treatment strategies.

Oral metronomic chemotherapy for recurrent & refractory epithelial ovarian cancer: A retrospective analysis

Background & objectives: Advanced epithelial ovarian cancer (EOC) is associated with dismal outcome and progression-free survival (PFS) shortens with each subsequent relapse. For patients with recurrent and platinum refractory disease, therapeutic options are limited. Oral metronomic therapy (OMT) is associated with symptomatic relief and stable response in a significant proportion of patients. We retrospectively evaluated the outcome of patients with EOC treated with OMT at a tertiary care hospital in north India. Methods: Between January 2011 to December 2017, 36 EOC patients received OMT. Patients' median age was 50 yr (range, 38-81 yr) and they had received a median of two lines of prior chemotherapy. OMT regimen included a combination of cyclophosphamide, etoposide (VP-16) and celecoxib with or without pazopanib along with supportive care. Response rates and outcomes were ascertained using the Gynecological Cancer Intergroup Guidelines. The toxicity was graded according to the Common Terminology Criteria for Adverse Events v.4.03. Results: The median CA-125 before initiating OMT was 160 U/ml (range, 42.23-5330 U/ml). The median interval between last chemotherapy and starting OMT regimen was 159 days (range, 1-1211 days). The overall response rate was 50 per cent. The median progression-free survival (PFS) was 8.2 months [95% confidence interval (CI): 5.03-10.33], and the median overall survival was 38 months (95% CI: 25.6-NR). Patients who received two lines of chemotherapy before OMT (P=0.052) and those who received pazopanib-based OMT (P=0.0513) had better PFS. Interpretation & conclusions: For patients with relapse and refractory EOC, OMT could be a reasonable option. A combination of oral etoposide (VP-16) and pazopanib needs further evaluation in a large number of patients in a randomized trial.

Seroprevalence of Toxoplasmosis at a Tertiary Care Centre in North India from 2004 to 2014

Purpose: This study was carried out to determine the seroprevalence of anti-Toxoplasma gondii antibodies in different groups of patients at a tertiary care hospital in North India. Materials and Methods: Clinical and demographic data such as age and gender of patients who had undergone testing for the presence of anti-T. gondii IgG and IgM antibodies between January 2004 and October 2014 were retrospectively analysed. Results: Amongst the 8397 serum samples, an overall seropositivity of 21% (n = 1763) and IgG and IgM seropositivity of 5.7% (n = 481) and 15.3% (n = 1282) were respectively observed. Compared to the period of 2004–2012 (median seroprevalence: 23.6%), a decline in seropositivity to 9.7% in 2013 and 8.1% in 2014 was noted. A rising seroprevalence with age and a higher seroprevalence in females versus males (29.5%, n = 1179 vs. 13.3%, n = 584) were recorded. The highest seroprevalence was observed in suspected ocular toxoplasmosis (47.2%, n = 47), followed by neurological (26.8%, n = 77), human immunodeficiency virus/acquired immunodeficiency syndrome (18.9%, n = 267), post-transplant (17.1%, n = 12) and congenital (7.2%, n = 144) toxoplasmosis. In patients screened for Toxoplasma exposure, the seropositivity was 47.8% (n = 11) in transplant screening and 44.9% (n = 781) in antenatal screening. Conclusion: Toxoplasma infection is highly prevalent in the population of North India across various clinical categories of patients. Future studies focusing on continuous monitoring of seroprevalence trends and elucidation of the risk factors associated with seropositivity in more defined groups of patients are needed.

Protocol for developing a surveillance system for surgical site infections

Purpose: Healthcare-associated infections (HCAIs/ HAIs) are the most common adverse occurrences during health care delivery. Across the globe, millions of patients are affected by HAIs annually, with a higher burden and impact in developing nations. a major lacuna in planning preventing protocols is the absence of National Surveillance Systems in most low-middle income countries, which also prevents allocation of resources to the high-priority areas. Among all the HAIs, there is a huge global burden of SSIs, in terms of morbidity, prolonged hospital stays, increased antimicrobial treatment as well as attributable mortality. Method: This manuscript details the process of establishment of an SSI surveillance protocol at a level-1 trauma centre in North India. Result and Conclusion: Surveillance is an essential tool to reduce this burden. It is also an important primary step in recognizing problems and priorities, and it plays a crucial role in identifying risk factors for SSI and to be able to target modifiable risk factors. Therefore, it is imperative to establish reliable systems for surveillance of HAIs, to regularly estimate the actual burden of HAIs, and to use these data for developing indigenous preventive measures, tailored to the country's priorities.

Diagnostic performance of mini parasep® solvent-free foecal parasite concentrator for the diagnosis of intestinal parasitic infections

Introduction: Various stool concentration techniques have been used to increase the microscopic detection of parasites. We assessed the enclosed, single-vial, Mini Parasep® technique in comparison to the currently used coprodiagnosis procedures. Materials and Methods: A total of 150 stool samples were collected. Samples concentrated by formol–ether method (FEM) and Mini Parasep® SF foecal concentrator and unconcentrated samples were subjected to wet mount, iodine mount microscopy and smear examination by modified acid-fast staining. Results: Direct wet mount detected 72 positive samples (48.6%), whereas 77 (51.3%) and 80 (53.3%) samples were detected by FEM and Mini Parasep® SF methods, respectively. The sensitivity of detection of parasites was 98.7%, 95% and 90.1% with Mini Parasep®, FEM and direct microscopy, respectively. A clearer background with less foecal debris and a better yield of Hymenolepis nana, Trichuris trichiura, Entamoeba coli and Giardia lamblia were noted with Mini Parasep®. Conclusion: Mini Parasep® SF technique is simple, rapid and less cumbersome than conventional diagnostics, making it suitable for routine use. In addition, it offers higher sensitivity and better background clearance than both direct stool examination and FEM.

A clinicopathological study of persistent fetal vasculature

Purpose: To study the clinicopathological findings of Persistent Fetal Vasculature (PFV) in patients with congenital cataract and PFV. Methods: Six eyes with anterior or combined PFV with cataract underwent phacoaspiration with primary posterior capsulotomy with anterior vitrectomy with intraocular lens implantation followed by histopathological evaluation of the PFV stalk and membrane. Results: Four and two patients had combined and anterior PFV respectively. There was no postoperative hyphema, vitreous haemorrhage, glaucoma or retinal detachment in six months. Haematoxylin and eosin staining showed inflammatory cells predominantly with extramedullary hematopoeisis and vascularisation. Conclusion: We recommend IOL implantation in PFV, with early and aggressive amblyopia therapy.

Primary amoebic meningoencephalitis in an infant

Primary amoebic meningoencephalitis is rare but fatal disease encountered in immunocompetent individuals. Here, we present a case of a previously healthy 8-month-old female child, who presented with features of meningoencephalitis of 2 days' duration. Rapidly moving trophozoites of amoeba were observed in cerebrospinal fluid, which were confirmed to be Naegleria fowleri on polymerase chain reaction. Broad-spectrum antimicrobial therapy with ceftriaxone, vancomycin, amphotericin B and acyclovir was initiated. However, the patient deteriorated and left the hospital against medical advice. The isolation of N. fowleri in this case demands for increased awareness for prompt diagnosis and management in view of its high mortality.

Pathogen burden & associated antibiogram of Pseudomonas spp. in a tertiary care hospital of India

Antimicrobial resistance particularly in Gram-negative bacilli is an increasing problem worldwide. Pseudomonas spp. is one the most common Gram-negative bacteria associated with nosocomial infections and therefore, its trend of antimicrobial resistance needs to be studied. The aim of this study was to evaluate the rate of antimicrobial resistance and changes in resistance pattern over a period of five years (2012-2016) in Pseudomonas spp. isolated from trauma patients attending a tertiary care hospital in north India. During the study, a total of 2444 Pseudomonas spp. were isolated from the various clinical sample. The most common species isolated was P. aeruginosa (2331, 95%). The highest level of resistance was observed against levofloxacin (1678, 69%) and the lowest level of resistance was observed against tobramycin (1254, 51%). Irrational and inappropriate use of antibiotics was found to be responsible for multidrug resistance in Pseudomonas spp. Hence, there is an urgent need to emphasize strict antibiotic policy to minimize the misuse of antimicrobials.

Phenotypic & genotypic profile of antimicrobial resistance in Pseudomonas species in hospitalized patients

Background & objectives: Nosocomial infections caused by multidrug-resistant, Pseudomonas species have become a major clinical and public health concern. The aim of this study was to characterize phenotypic and genotypic profile of antimicrobial resistance (AMR) in Pseudomonas spp. isolated from hospitalized patients. Methods: A total of 126 consecutive, non-duplicate isolates of Pseudomonas spp. isolated from various clinical samples were included in the study over a period of two years. Identification and antimicrobial sensitivity was performed using automated culture system according to the Clinical and Laboratory Standards Institute (CLSI) recommendations. Phenotypic detection of extended-spectrum ?-lactamases (ESBLs), Amp-C ?-lactamase (AmpC) and metallo-?-lactamases (MBLs) were done by various combinations of disc-diffusion and E-test methods, followed by polymerase chain reaction-based detection of ?-lactamase-encoding genes. Results: Among 126 clinical isolates, 121 (96.1%) isolates were identified as Pseudomonas aeruginosa. Most of the isolates were recovered from pus sample, 35 (27.8%) followed by urine, 25 (19.84%); endotracheal aspirate, 24 (19.04%); blood, 14 (11.11%) and sputum, four (3.17%). The highest rate of resistance was against ticarcillin-clavulanic acid, 113 (89.7%) followed by meropenem, 92 (72.5%) and ceftazidime, 91 (72.3%). Overall, ESBLs, AmpC and carbapenemase production was detected in 109 (96.4%), 64 (50.8%) and 105 (94.6%) isolates by phenotypic methods. The most prevalent ESBL gene was blaTEMin 72 (57.1%) and the least prevalent was blaSHVin 19 (15.1%) isolates. AmpC gene was seen less compared to ESBL gene. The most prevalent carbapenemases gene was blaNDM-141 (46.06%) followed by blaVIM and blaOXA-1. Interpretation & conclusions: Our findings suggested that a high rate of ESBLs and carbapenemases production was observed in Pseudomonas spp. Therefore, phenotypic and genotypic detection of AMR needs to be combined for better characterization of resistance patterns in Pseudomonas spp.

Clinical and laboratory characteristics of patients with toxocariasis encountered at a tertiary care centre in North India

Toxocariasis is a neglected soil-transmitted helminthic infection caused by either Toxocara canis or Toxocara cati where humans get infected by accidental ingestion of embryonated eggs, and the definitive hosts are dogs and cats. This study aims to analyse the clinical and laboratory characteristics of Toxocara-infected patients and assessment of response to standard treatment with review of literature. The clinical details of patients with Toxocara serology positive for IgG antibodies by ELISA in 5 years (2013–2017) were retrospectively analysed. A total of 29 patients with clinical features and serology suggestive of Toxocara infection were evaluated. A complete history of 14 patients was available for the analysis. Majority (13/14; 96.5%) of cases were children <15 years, males (79%) and belonged to lower socioeconomic status (64.3%). The most common clinical presentation was fever (78.5%) with respiratory symptoms (57%) followed by gastrointestinal features (35.7%). All the patients had eosinophilia (range 8.3%–85%) and raised total IgE levels. Toxocariasis is more common in children, and the true prevalence may be underestimated. In endemic areas, Toxocara workup should be considered in patients with eosinophilia presenting with respiratory and gastrointestinal features.

An ocular cysticercosis case: Caused by Asian genotype of Taenia solium.

An ocular cysticercosis case of a 42-year-old male, who presented with anterior uveitis is being reported. Microscopical examination of the cyst revealed presence of only one hooklet suggestive of T. solium cysticercus. Mitochondrial DNA analysis confirmed it to be T. solium cysticercus of Asian genotype. This is the first report on molecular typing of cysticercus isolate from ocular cysticercosis patient in India. The study suggests that the molecular analysis of cox1 gene may be a useful diagnostic tool in cases where microscopic examination is not confirmatory.

Free living amoebae in water sources of critical units in a tertiary care hospital in India.

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.

Emergence of Balamuthia mandrillaris meningoencephalitis in India.

We report two cases of fatal chronic meningoencephalitis caused by Balamuthia mandrillaris in immunocompetent men. Diagnosis of amoebic meningoencephalitis was made ante‑mortem in one case and postmortem in another by histopathological examination and confirmed by demonstration of B. mandrillaris deoxyribonucleic acid (DNA) by polymerase chain reaction (PCR).

Development of touch down‑multiplex PCR for the diagnosis of toxoplasmosis.

Purpose: The diagnosis of toxoplasmosis is challenging since conventional methods like culture and immunofluorescence are not universally available. Serology, which is used regularly might be negative during early phase of infection and in immunosuppressed patients or may remain positive for a long time. Several molecular tests have been used for the diagnosis of toxoplasmosis, but none of them have an internal control which would inform us regarding the presence of polymerase chain reaction (PCR) inhibitors thus, undermining the confidence of a laboratory physician. Materials and Methods: We designed a multiplex PCR containing primers targeting human beta globin gene which would act as internal control and two primers against the B1 gene and 5s gene which aid in sensitive detection of T. gondii. Results: Multiplex PCR had a sensitivity of 83.3% and specificity of 100%. Conclusion: Multiplex PCR may provide a sensitive and specific tool for diagnosis of human toxoplasmosis.

Isolation, Identification and Optimization of Culture Conditions of Bacillus sp. Strain PM1 for Alkalo-thermostable Amylase Production.

Aims: To isolate and optimize the culture conditions for thermo stable and alkaline amylase production from bacteria. Study Design: Optimization of different physiological and nutritional parameters for amylase production and kinetic studies of amylase. Place and Duration of Study: Soil Samples: Herbal garden of Amity University Haryana, Gurgaon (Manesar), India, between April 2012 and September 2012. Methodology: Amylolytic isolates were selected by flooding the nutrient agar plates containing 2% starch with Lugol solution. Isolates were selected on the basis of higher ratio of clear zone to colony size and grown in nutrient broth containing 2% starch. The level of amylase was detected in the culture filtrate. The selected isolate showing maximum amylase production was identified on the basis of 16S rDNA amplification. Results: An Alkalo-thermostable amylase producing bacterial isolate from soil was identified as Bacillus sp. strain PM1 on the basis of 16S rRNA. It yielded 3.5 U/ml of amylase in medium containing (%) starch 2.0, beef extract 0.5, NaCl 0.5 at 50ºC, pH 7.0 at 180 rpm after 72 h. The optimum pH and temperature for amylase activity was 8.0 and 50°C, respectively. The enzyme exhibited 67% activity after 60 minute incubation at 50ºC. At pH 8.0, the enzyme retained 78% activity after 4 h. Conclusion: The properties of the isolated enzyme are adequate for its use in starch processing and baking industry.

Isolation and Identification of Xylanolytic Enzyme from an Effective Strain Bacillus Licheniformis Isolated from the Decaying Wood.

A new species of Bacillus licheniformis produced extracellular xylanase under submerged fermentation when wheat bran is used as carbon source. The xylan is the most common hemicellulosic polysaccharide in food industry and agricultural wastes, comprising a backbone of xylose residues linked by β-1,4 glycosidic bonds. Bacillus licheniformis has been shown to be a promising organism for enhanced production of xylanases & β-xylosidase under submerged fermentation (SmF). The optimization of cultural conditions and carbon, nitrogen sources for enzymes production. The bacterial strain Bacillus licheniformis was cultivated using as substrate xylan, wheat bran, corn straw, corncob, and sugarcane bagasse. Wheat bran has been a good xylanase (16.8U/ml) & β xylosidase (5.6U/ml) activity after 48h of fermentation. Maximum enzyme activity was observed in xylan as carbon source and peptone as nitrogen source. Both crude enzymes were characterized and a bacterial xylanase shows optimum pH for xylanase activity at 6.5 & β xylosidase were found to be 6.0. The optimum temperatures were 450C for both and they were thermally stable up to 500C. The parameters of Vmax and Km obtained using Line weaver-Burk plot method were 277.7μmol / min/mg and 5.26 mg /L correspondingly.

Differences in consumption of food items between obese and normal-weight people in India.

Background. There is a rising prevalence of obesity in India, and diet may be a major determinant of this. We aimed to assess differences in types and quantities of food items consumed by obese and normal-weight people in India. Methods. Cross-sectional data of 7067 factory workers and their families were used from the Indian Migration Study, conducted in four cities across northern, central and southern India. Food frequency questionnaire data were used to compare the quantities of consumption of 184 food items between 287 obese (body mass index >30 kg/m2) and 1871 normalweight (body mass index 18.50–22.99 kg/m2) individuals, using t tests and ANCOVAs. Individuals with diabetes, hypertension and cardio-vascular disease were excluded. SPSS 16.0 was used for analysis. Results. After adjusting for age, sex, location and socioeconomic status, obese individuals were found to eat significantly larger quantities of 11 food items compared with normalweight individuals. These included phulkas, chapatis/parathas/ naan, plain dosa, mutton/chicken pulao/biryani, chicken fried/ grilled, rasam, mixed vegetable sagu, vegetable raitha, honey, beetroot and bottlegourd (p<0.01). Consumption of plain milk was higher among normal-weight than among obese individuals (p<0.05). Consumption of some of these food items was also found to increase by socioeconomic status, decrease by age, and be higher among men relative to women. Conclusion. Obese individuals were found to consume larger quantities of certain food items compared with normal-weight individuals. Interventions should aim at limiting overall food consumption among obese individuals.