Characterization and pro-inflammatory responses of spore and hyphae samples from various mold species.

Mold particles from Aspergillus fumigatus, Penicillium chrysogenum, Aspergillus versicolor, and Stachybotrys chartarum have been linked to respiratory-related diseases. We characterized X-ray-inactivated spores and hyphae fragments from these species by number of particles, morphology, and mycotoxin, ß-glucan and protease content/activity. The pro-inflammatory properties of mold particles were examined in human bronchial epithelial cells (BEAS-2B) and THP-1 monocytes and phorbol 12-myristate 13-acetate (PMA)-differentiated THP-1. Spores from P. chrysogenum and S. chartarum contained some hyphae fragments, whereas the other preparations contained either spores or hyphae. Each mold species produced mainly one gelatin-degrading protease that was either of the metallo- or serine type, while one remains unclassified. Mycotoxin levels were generally low. Detectable levels of ß-glucans were found mainly in hyphae particle preparations. PMA-differentiated THP-1 macrophages were by far the most sensitive model with effects in the order of 10 ng/cm2 . Hyphae preparations of A. fumigatus and P. chrysogenum were more potent than respective spore preparations, whereas the opposite seems to be true for A. versicolor and S. chartarum. Hyphae fragments of A. fumigatus, P. chrysogenum, and A. versicolor enhanced the release of metalloprotease (proMMP-9) most markedly. In conclusion, species, growth stage, and characteristics are all important factors for pro-inflammatory potential.

Humoral and T cell-mediated immune response against trichomoniasis.

Trichomonas vaginalis (T. vaginalis) infection leads to the synthesis of specific antibodies in the serum and local secretions. The profile of T. vaginalis-specific antibodies and T cell-mediated immune responses may influence the outcome of infection, towards parasite elimination, persistence or pathological reactions. Studies have indicated that Th1-, Th17- and Th22 cell-related cytokines may be protective or pathogenic, whereas Th2- and Treg cell-related cytokines can exert anti-inflammatory effects during T. vaginalis infection. A number of T. vaginalis-related components such as lipophosphoglycan (TvLPG), α-actinin, migration inhibitory factor (TvMIF), pyruvate:ferredoxin oxidoreductase (PFO), legumain-1 (TvLEGU-1), adhesins and cysteine proteases lead to the induction of specific antibodies. T. vaginalis has acquired several strategies to evade the humoral immune responses such as degradation of immunoglobulins by cysteine proteases, antigenic variation and killing of antibody-producing B cells. The characterization of the T. vaginalis-specific antibodies to significant immunogenic molecules and formulation of strategies to promote their induction in vaginal mucosa may reveal their potential protective effects against trichomoniasis. In this review, we discuss the current understanding of antibody and T cell-mediated immune responses to T. vaginalis and highlight novel insights into the possible role of immune responses in protection against parasite.

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.

Immunity in urogenital protozoa.

Innate and adaptive immunity play a significant role in urogenital infections. Innate immunity is provided by the epithelial cells and mucus lining along with acidic pH, which forms a strong physical barrier against the pathogens in female reproductive tract. Cells of innate immune system, antimicrobial peptides, cytokines, chemokines and adaptive immunity in the reproductive tract are evolved during infection, and a pro-inflammatory response is generated to fight against the invading pathogen Trichomonas vaginalis, a primary urogenital protozoa, the etiological agent of human trichomoniasis, a curable sexually transmitted infection. The involvement of the urogenital tract by other protozoal infections such as P. falciparum, Trypanosoma, Leishmania, Toxoplasma, Entamoeba histolytica and Acanthamoeba infection is rarely reported. Trichomonas induce pro-inflammatory and immunosuppressive responses in infected subjects. Multifactorial pathogenic mechanisms including parasite adherence, cysteine proteases, lipophosphoglycan, free radical, cytokine generation and Toll-like receptors appear to interplay with the induction of local and systemic immune responses that ultimately determine the outcome of the infection. However, the involvement of urogenital pathogen-specific immune mechanisms and effect of normal local resident flora on the outcome (symptomatic vs. asymptomatic) of infection are poorly understood. Moreover, immune interactions in trichomoniasis subjects co-infected with bacterial and viral pathogens need to be elucidated.

The impact of hearing loss in older adults: a tertiary care hospital based study.

Hearing loss is the most common sensory deficit in the elderly, and is becoming a severe social and health problem. Presbycusis is the result of aging which can lead to communication problems compromising the quality of life (QoL).Since the elderly population is increasing worldwide, presbycusis is showing a similar trend. This study intended to identify the impact of hearing loss in the social life of the elderly. A total of 70 elderly patients attending Nepal Medical College Teaching Hospital who were found to have sensorineural hearing loss (SNHL) on pure tone audiometry (PTA) were recruited for this study.None of the patients had used hearing aids in the past. To assess their handicap due to hearing impairment a Hearing Handicap Inventory for the Elderly (HHIE) questionnaire was used and patients were graded as: no handicap, mild to moderate handicap and significant handicap. Pure-tone averages (PTA) were calculated for the thresholds at 0.5, 1 and 2 kHz in each ear. Patients with their PTA values between 26 to 40 dBHL were interpreted as having mild SNHL, those between 41 to 55dBHL as moderate SNHL and those above 55 dBHL as severe SNHL. Out of 70 patients, 65 had some degree of handicap ranging from mild to severe. The severity of handicap was significantly associated with the degree of hearing loss in both ears.

The presence of dsRNA virus in Trichomonas vaginalis isolates from symptomatic and asymptomatic Indian women and its correlation with in vitro metronidazole sensitivity.

PURPOSE: Trichomonas vaginalis, a protozoan parasite, is the causative agent of human trichomoniasis, the most common non-viral sexually transmitted disease. The infection encompasses from a complete asymptomatic presentation to severe sequelae; yet, the virulence markers have been poorly understood. It is suggested that the presence of Trichomonas vaginalis virus (TVV) in T. vaginalis may have an impact on its virulence, and its relatedness to in vitro metronidazole resistance has been reported. The aim of the study was to assess the presence of TVV in fresh and Long -Term Cultivated ( LTC) maintained T. vaginalis isolates from symptomatic (S) and asymptomatic (AS) Indian women and its relatedness, if any, with symptomatology and in vitro drug sensitivity. MATERIALS AND METHODS: One thousand women (537 S and 463 AS) were screened for the presence of T. vaginalis by wet smear and culture examination of vaginal swab and urine sample. Fresh and LTC (6 months-2 years) maintained 15 isolates each from 15 S and 15 AS women were subjected to agarose gel electrophoresis following total cellular RNA extraction to evaluate the presence of double stranded (ds) RNA viral infection. The susceptibility of isolates to metronidazole was determined in vitro. RESULTS: On agarose gel electrophoresis, three bands (5.5, 2.5 and 1.5 kb) were observed in all the 30 fresh isolates from 15 S and 15 AS women and only in 7 LTC isolates from 3 S and 4 AS women. All the fresh isolates harbouring TVV were found to be sensitive to metronidazole in vitro irrespective of the symptomatology of subjects, and out of seven LTC isolates harbouring TVV, six were sensitive to metronidazole and one showed borderline resistance. CONCLUSIONS: The results suggest that the presence of TVV alone may not be a virulence marker and loss of TVV on LTC appears to be related to drug resistance. The T. vaginalis Indian isolates are sensitive to metronidazole.

Ear diseases in children presenting at Nepal Medical College Teaching Hospital.

Ear diseases are a common presentation in the ENT out-patient department. These diseases can have sequelae if not treated early which may cause increased morbidity, hearing disability and even mortality. Identifying these conditions early and treating them can reduce these unwanted sequelae. A prospective study over a period of three years was undertaken in a tertiary care hospital in Kathmandu. All children presenting with ear diseases consecutively in the ENT out-patients were included and their data was recorded in a pro forma. The data was analysed for frequency and cross-tabulated. The study included 2218 children with ear diseases among which 868 (39.1%) were pre-school and 1350 (60.9%) were school going children. The male to female ratio was 1.5:1. The most common otological problem was wax impaction (40.2%), followed by acute otitis media (AOM) with 24.3%, chronic suppurative otitis media (CSOM) with 17.7% and acute otitis externa (AOE) with 7.5% of the total cases. The other conditions were otitis media with effusion (2.8%), foreign bodies in the ear (2.3%), otomycosis (1.7%), preauricular sinus (1.1%), sensorineural hearing loss (0.8%), trauma to the ear (0.7%), keloids (0.3%), microtia (0.2%) and perichondritis (0.2%).The three year olds had the highest number of otological diseases with 9.2% of the total among the ages studied. It is felt that proper education of health care providers regarding ear diseases and also awareness in the community can prevent and reduce the disability and complications of these conditions.

Human subcutaneous dirofilariasis in India: a report of three cases with brief review of literature.

Human subcutaneous dirofilariasis is a rare infection caused by filarial worms of the genus Dirofilaria. The parasites are transmitted to man by mosquitoes and the infection is manifested as subcutaneous nodules. Excision of the lesion is both diagnostic and therapeutic. Hereby we report three cases of human subcutaneous dirofilariasis. The worms were sent to our department for identification over a period of four years (2006-2009). Of these three patients, two men and one woman were between 15 and 45 years of age. In two cases, the infection manifested as a nodule on face, in one case near lower eyelid and in the other on the cheek, while in the third case as an itchy nodule on the abdomen. It is emphasized that both clinicians and microbiologists should have an increased awareness of this entity and include dirofilariasis in the differential diagnosis of patients presenting with subcutaneous nodules.

Ophthalmomyiasis: three cases from North India.

Three cases of external ophthalmomyiasis are reported here. The larvae were identified to be Oestrus ovis in two cases and Cochliomyia hominivorax in one. Two of the patients were immunocompetent while one was undergoing treatment for squamous cell carcinoma of eyelid. In the latter myiasis led to complete destruction of the eye.

Serological screening for antenatal toxoplasma infection in India.

PURPOSE: Detection of infection caused by Toxoplasma gondii during pregnancy to prevent congenital infection. MATERIALS AND METHODS: This study was carried out from January 2005 to 2006 in 300 pregnant women. Antitoxoplasma IgG, IgM, IgA antibody and IgG avidity were assessed using ELISA. At least two samples were taken at least 3 weeks apart preferably one in each trimester. RESULT: Of these 300 pregnant women, anti toxoplasma IgG antibodies were detected in 46 (15.33%) cases, while 9 (3%) had positive anti toxoplasma IgM with IgA and /low IgG avidity antibodies suggestive of acute infection during or just before pregnancy. CONCLUSION: The results indicate that about 85% of female population of Chandigarh is susceptible to toxoplasma infection and thus should be specifically educated about prevention of this infection during pregnancy.

Concomitant intestinal parasitism and non-cholera vibrio infection.

Concomitant parasitism is not uncommon especially in tropical countries with low socioeconomic status. Here we report an unusual combination of intestinal infection due to Strongyloides stercoralis, Blastomyces hominis and non-cholera Vibrio in a patient suffering from acute gastroenteritis and hypoalbuminemia. Early recognition and accurate treatment of gastrointestinal infections and infestations before the patient develops complications is important.

Genetic polymorphism in msp-2, ama-1 and csp genes in Plasmodium falciparum field isolates from north and north-western India.

BACKGROUND & OBJECTIVES: Malaria is a major public health problem in tropical and sub-tropical countries. Malaria vaccine is highly desirable as an adjunct to existing malaria control measures. The polymorphism in vaccine candidate antigens might be a hurdle in developing an effective vaccine. Merozoite surface protein-2, apical membrane antigen-1 and circumsporozoite protein of Plasmodium falciparum are vaccine candidate antigens. The aim of this study was to detect extent of genetic polymorphism in potential vaccine candidate antigen genes, i.e. msp-2, ama-1 and csp of P. falciparum isolates prevalent in northern and north-western parts of India. METHODS: Overall 88 parasite isolates of P. falciparum were collected during July 1998-March 2002 from different parts of northern and north-western India. DNA was extracted and analyzed for genetic polymorphism by PCR-RFLP method. For msp-2 gene, family-specific (FC-27 and 3D7) nested PCR was also performed. RESULTS: PCR showed size polymorphism in all the target genes. Three alleles were observed in msp-2 and ama-1, while only two in csp. RFLP of ama-1 and csp with Dra-1 and Ssp-1 endonucleases respectively, failed to differentiate isolates in sub-allelic types, while Hinf-I digestion of msp-2 amplicons differentiated three alleles into two distinct allelic families, i.e. FC-27 and 3D7. The allelic family-specific PCR generally confirmed the results of PCR-RFLP except in a few isolates, which showed mixed (two) clones of msp-2 gene. INTERPRETATION & CONCLUSION: There was extensive polymorphism in msp-2 gene while ama-1 and csp genes showed low polymorphism which may be due to the functional constraints of these proteins. The low level transmission of malaria in the study area may also be a factor for low polymorphism.

Evaluation of excretory secretory and 10-30 kDa antigens of Taenia solium Cysticerci by EITB assay for the diagnosis of neurocysticercosis.

Neurocysticercosis (NCC), caused by the presence of Taenia solium Cysticerci in the Central Nervous System is the most common neurological disease of parasite aetiology. The serodiagnostic methods available at present have variable sensitivity and specificity depending upon the antigen and technique used. The present study was aimed to assess the efficacy of T. solium Cysticerci excretory secretory (ES) and lower molecular mass (LMM) 10-30 kDa antigenic fractions for antibody detection in serum and urine samples by enzyme-linked immunoelectrotransfer blot (EITB) for the diagnosis of NCC. Serum and urine samples were collected from 125 clinically suspected and radiologically proven NCC children (111 patients with single lesion and 14 with multiple lesions) and 125 control subjects. With the use of ES and LMM antigenic fractions, the sensitivity of the EITB assay was 85.6% and 80.8% with serum and 76.8% and 50.4% with urine, respectively. The specificity was 64% and 61.6% with serum and 48% and 33.6% with urine samples, respectively. The study suggests that antibody detection to ES antigen in serum by EITB assay may serve better purpose for the serodiagnosis of human NCC.

Neurocysticercosis immunodiagnosis using Taenia solium cysticerci crude soluble extract, excretory secretory and lower molecular mass antigens in serum and urine samples of Indian children.

Neurocysticercosis (NCC), the most common neurological disorder of parasite etiology, results from lodgement of Taenia solium cysticerci in the central nervous system and is now increasingly being recognized in children. The confirmed diagnosis is based collectively on radiological findings and serodiagnostic techniques. The serodiagnostic techniques have variable sensitivity and specificity depending upon the technique, antigens used, location and number of cysts. Crude soluble extract (CSE), excretory secretory (ES) and lower molecular mass (LMM) (10-30 kDa) antigenic fraction of T. solium cysticerci were evaluated for antibody detection in serum and urine samples by ELISA. Serum and urine samples were collected each from 125 clinically suspected and radiologically proven NCC (111 with single Computed Tomography (CT) lesions and 14 with multiple CT lesions) and 125 control subjects (60 with neurological disorders other than NCC, 40 with other parasitic diseases and 25 apparently healthy subjects). The sensitivity of the ELISA with the use of CSE, ES and LMM antigenic fractions was 38.4%, 63.2% and 30.4% with serum (cut off dilution 400), 46.4%, 44% and 47.2% with neat urine and the specificity was 88%, 76.8% and 85.6% with serum (cut off dilution 400), 66.4%, 65.2% and 58.4% with neat urine samples, respectively. The study suggests that detection of antibody to ES antigen in serum samples may serve useful purpose for the serodiagnosis of human NCC.

Polymorphism in merozoite surface protein-1 gene in north & northwest Indian field isolates of Plasmodium vivax.

BACKGROUND & OBJECTIVE: Merozoite surface protein-1 of Plasmodium vivax (Pvmsp-1) is a strong vaccine candidate against asexual blood stages. Extensive polymorphism in msp-1 gene has been reported in P. vivax isolates from different geographical regions which is necessary before a field trial of any malaria vaccine based on msp-1 is undertaken. There are only a few reports available on polymorphism in msp-1 gene in Indian field isolates of P. vivax. The aim of the present study was therefore to investigate the polymorphism in Pvmsp-1 gene in 25 isolates of P. vivax collected from malaria patients from regions of north and northwest India. METHODS: DNA was extracted from whole blood samples collected in citrated anticoagulant. The polymorphic region-5, the most variable region of the Pvmsp-1 gene was amplified by PCR. The PCR products were further analyzed by restriction fragment length polymorphism (RFLP) using Mva-1 restriction enzyme. The DNA fragments obtained on PCR and RFLP were analyzed by agarose gel electrophoresis. RESULTS: On the basis of PCR, significant size polymorphism was seen and 4 allelic types were observed amongst the 25 isolates. Further analysis by RFLP discriminated these 4 allelic types into 9 sub-allelic types indicating that PCR-RFLP can be a good tool to study polymorphism in msp-1 gene of Plasmodium. INTERPRETATION & CONCLUSION: Marked genetic polymorphism was observed in msp-1 gene among the isolates of P. vivax. These observations stress the need to study larger numbers of isolates from different regions of India. The findings could have important implications on the vaccine development strategies for P. vivax.

Cysteine proteinase 30 (CP30) and antibody response to CP30 in serum and vaginal washes of symptomatic and asymptomatic Trichomonas vaginalis-infected women.

Infection with Trichomonas vaginalis may be asymptomatic or with symptoms suggestive of vaginitis. Because cysteine proteinase 30 (CP30) of T. vaginalis is known to be a virulence marker that plays a role in cytoadherence, the aim of this study was to analyse the presence of CP30 and antibody to CP30 in clinical samples of symptomatic and asymptomatic infected women. CP30 was detected in all the serum and vaginal washes (VWs) of symptomatic women and in 65% of the serum and 80% of the VWs of asymptomatic women. This suggested that the majority of asymptomatic women also exhibit CP30 in the serum and VWs. Antibody to CP30 was detected in all the serum samples of symptomatic and asymptomatic women and in the VWs of only 54.5% of the symptomatic and 35% of the asymptomatic women. Antibody to CP30 was also detected in 3/20 of the serum samples and in none of the VWs from uninfected women. Significantly higher amounts of antibody (mean OD values) were observed in serum and VWs of symptomatic as compared to asymptomatic and healthy women (P<0.001). These results indicate that besides CP30, other factors may also be playing a role in leading to symptomatic infection, because CP30 was detected in clinical samples from all the symptomatic and the majority of the asymptomatic women. Although anti-CP30 antibodies do not appear to be protective, detection of antibody to CP30 antigen in serum samples may be used as a diagnostic tool.

Kinetics of serum and local cytokine profile in experimental intravaginal trichomoniasis induced with Trichomonas vaginalis isolates from symptomatic and asymptomatic women.

Trichomoniasis, the most common non-viral sexually transmitted disease, is caused by infection with the protist Trichomonas vaginalis. The clinical spectrum varies from an asymptomatic to a severe symptomatic state. However, the exact factors leading to varied symptomatology have not been well elucidated. The role of cytokines in the pathogenesis of many microbial diseases has been reported. The present study reports the cytokine levels (IL-2, IL-4, IFN-gamma) on different days post infection (3rd, 7th, 14th, 21st and 28th d.p.i.) in serum and vaginal washes (VWs) of mice infected intravaginally with T. vaginalis isolates from 15 symptomatic and 15 asymptomatic women. Significantly higher production of IL-2 and IFN-gamma was observed on the 3rd to 28th d.p.i., and IL-4 on the 7th to 21st d.p.i., in infected as compared to uninfected mice. A significant increase in cytokine IL-2 and IFN-gamma was observed on the 3rd to 28th d.p.i. in serum and VWs of mice infected with T. vaginalis isolates from asymptomatic as compared to symptomatic women. IL-2 (P < 0.001) and IFN-gamma (P < 0.05) concentrations were significantly higher on the 14th d.p.i. in serum samples as compared to VWs of mice infected with T. vaginalis isolates from asymptomatic and symptomatic women, while no significant difference was observed in IL-4 concentration between the two groups of mice. The study indicates the involvement of a Th-1 (IL-2 and IFN-gamma) like response in mice infected with isolates from asymptomatic women as compared to symptomatic women and suggests that Th-1 type cytokines might be playing a role in maintaining low levels of infection.

Nitric oxide radicals in leucocytes and vaginal washes of Trichomonas vaginalis -infected symptomatic and asymptomatic women.

The clinical spectrum of Trichomonas vaginalis infection varies from asymptomatic to mild, moderate or severe vaginitis. Nitric oxide and other reactive nitrogen radicals produced by immune effector cells are important cytotoxic and cytostatic mediators against several microorganisms including parasites. In the present study, inducible nitric oxide synthase (iNOS) and reactive nitrogen intermediates (RNI) were determined in leucocyte cultures (stimulated with T. vaginalis in vitro) and vaginal washes (VWs) of 22 symptomatic and 20 asymptomatic T. vaginalis-infected and 20 healthy women by immunoblotting and Griess method respectively. The iNOS protein was detected in leucocytes and VWs of all the symptomatic and asymptomatic women, but was not detected in any of the samples from healthy women. Mean iNOS protein band intensity was significantly higher in leucocytes as compared to VWs (P<0.001) of both symptomatic and asymptomatic women and was also higher in leucocytes of asymptomatic as compared to symptomatic women (P<0.05). Mean RNI concentration was also significantly higher in leucocytes (P<0.01) and VWs (P<0.05) of asymptomatic as compared to symptomatic women, and was also higher in samples of infected as compared to healthy women (P<0.001). These results suggest that reactive nitrogen radicals may have a role in limiting T. vaginalis infection in asymptomatic women.

Evaluation of ELISA and dot blots for the serodiagnosis of neurocysticercosis, in children found to have single or multiple enhancing lesions in computerized tomographic scans of the brain.

Although human neurocysticercosis (NCC) is being increasingly recognized in children, diagnosis of the disease can be difficult, and the 'gold standard' criteria that indicate an unambiguous case have still to be established. In the present study, the performances of an ELISA and dot-blot assay, for the detection of antibodies against antigens from larval Taenia solium, were investigated and compared, using sera, from children aged 5-12 years, that were diluted to at least 1:400. Eighty of the subjects (20 aged 5-<8 years and 60 older children) each had the signs and symptoms of NCC, including one brain lesion (N=69) or multiple brain lesions (N=11) that were visible by computed tomography. Another 100 sera, from children who had tubercular meningitis (N=20) or a parasitic disease other than taeniasis/cysticercosis (N=20) or, apart from a minor respiratory-tract infection, appeared healthy (N=60), were also investigated. Most (86%) of the cases of NCC had presented with focal seizures. Analysis of antibody response indicated that the optimum threshold titres for seropositivity were 1:800 for the ELISA and 1:6400 for the dot-blot assays. When used with these thresholds, the ELISA gave a sensitivity, specificity, positive and negative predictive values and diagnostic efficacy of 89%, 81%, 79%, 90%, 85%, respectively. The corresponding values for the dot-blot assay were similar, at 89%, 73%, 72.5%, 89%, 82%, respectively. Both assays were more sensitive, in the detection of the specific antibody response, when used among the paediatric cases of NCC who had multiple brain lesions (100%) than when used among the single-lesion cases (87%). As the ELISA gave higher specificity and diagnostic efficacy than the dot-blot assay, it should be considered the better method for the serological confirmation of NCC in children.