Cloning, expression & evaluation of potential immunogenic recombinant capsid premembrane protein of West Nile virus

Background & objectives: West Nile virus (WNV) is a neurotropic flavivirus that has emerged globally as a significant cause of viral encephalitis. The early confirmatory diagnosis of WNV infections is important for timely clinical management and in areas where multiple flaviviruses are endemic. Diagnosis of WNV infection is primarily based on serodiagnosis, followed by virus isolation and identification. The aim of this study was to develop and evaluate a highly sensitive and specific immunoglobulin M (IgM) ELISA using the recombinant CprM protein (rWNV-CprM) for rapid, early and accurate diagnosis of WNV. Methods: The gene coding for the CprM protein of WNV was cloned and expressed in pET 28a vector followed by purification. An indirect IgM microplate ELISA using purified rWNV-CprM protein was optimized having no cross-reactivity with healthy human serum and serum samples obtained from patients with dengue and Japanese encephalitis viruses infection. Results: The comparative evaluation of this rWNV-CprM protein-specific IgM ELISA with plaque reduction neutralization test using 105 blood samples collected from patients suspected to have acute WNV infection revealed 98 per cent concordance with sensitivity and specificity of 100 and 97 per cent, respectively. Interpretation & conclusions: The recombinant CprM protein-based WNV-specific ELISA reported in this study may be useful for rapid screening of large numbers of blood samples in endemic areas during outbreaks.

Evaluation of real-time reverse-transcription loop-mediated isothermal amplification assay for clinical diagnosis of West Nile virus in patients

Background & objectives: West Nile virus (WNV) is a mosquito-borne flavivirus. The disease can be diagnosed by isolation followed by fluorescent antibody tests, enzyme-linked immunosorbent assay and polymerase chain reaction (PCR) assay. These diagnostic methods are laborious and time-consuming. The present study was aimed to evaluate the real-time reverse-transcription loop-mediated isothermal amplification (RT-LAMP) method for rapid, early and accurate diagnosis of WNV. Methods: A one-step single tube accelerated quantitative RT-LAMP assay was evaluated by targeting the Env gene of WNV. The gene amplification was accomplished by incubating the reaction mixture at 63°C for 60 min in both real time turbidimeter as well as routine laboratory water bath/dry heating bath. To rule out contamination issues, proper negative controls, including no template, no primer; and no enzyme, were always kept alongside each run. The RT-LAMP assay was evaluated on 105 clinical samples from individuals having ocular infection. Results: Of the 105 samples tested, 27 were positive for WNV by RT-LAMP assay. The comparative evaluation with conventional RT-PCR revealed 100 per cent accordance with sensitivity and specificity of 100 and 95 per cent, respectively. The specificity of this assay was confirmed with serum samples obtained from patients with dengue and chikungunya. Interpretation & conclusions: The RT-LAMP test seemed to be a sensitive and specific method for rapid detection of WNV infection and would be useful for rapid screening of a large number of clinical samples in endemic areas during outbreaks.

Ocular toxoplasmosis associated with scleritis.

We report an atypical presentation of Toxoplasma retinochoroiditis with associated scleritis in a young and immunocompetent patient. The diagnosis was done on the basis of Polymerase chain reaction of vitreous sample, and the clinical response to specific treatment. This case highlights the unusual presentation of ocular toxoplasmosis as scleritis.

Algorithmic approach in the diagnosis of uveitis.

Uveitis is caused by disorders of diverse etiologies including wide spectrum of infectious and non-infectious causes. Often clinical signs are less specific and shared by different diseases. On several occasions, uveitis represents diseases that are developing elsewhere in the body and ocular signs may be the first evidence of such systemic diseases. Uveitis specialists need to have a thorough knowledge of all entities and their work up has to be systematic and complete including systemic and ocular examinations. Creating an algorithmic approach on critical steps to be taken would help the ophthalmologist in arriving at the etiological diagnosis.

Efficiency of two commercial kits in serodiagnosis of leptospiral uveitis.

Purpose: Uveitis is an important complication of systemic leptospirosis that can occur months to years after systemic infection. The gold standard technique Microscopic Agglutination Test (MAT) is less sensitive and more complicated. All the commercial kits currently available are for early detection of acute systemic leptospiral infection. The purpose of this study is to evaluate the efficiency of two commercial kits in serodiagnosis of leptospiral uveitis, which is a late manifestation. Materials and Methods: Serum samples from leptospiral uveitis patients 20 MAT positive, 20 MAT negative, 15 non-leptospiral uveitis patients, 20 systemic leptospiral infected patients and 21 controls were selected. These samples were tested for the presence of leptospiral IgM antibodies by (i) MAT using a panel of 20 serovars, (ii) LEPTO IgM MICROLISA (J.Mitra & Co.Pvt. Ltd, India) and (iii) Leptocheck (Zephyr Biomedicals, India). The statistical analysis was carried out using stata 11.0. Results: Total of 96 samples were tested with two commercial kits, Lepto IgM MICROLISA and Leptocheck. The sensitivity and specificity of Lepto IgM MICROLISA was 60% and 55% and Leptocheck was 80% and 59% respectively in comparison to MAT. In comparison to clinical diagnosis the sensitivity of IgM Microlisa was 55%, Leptocheck 70% and specificity of IgM MICROLISA was 58.33% and leptocheck was 69.44%. Conclusion: Commercial kits though sensitive and specific for systemic leptospirosis, have limited diagnostic capacity for leptospiral uveitis. Therefore it is essential to develop an inhouse serodiagnostic method specific for leptospiral uveitis patients using local leptospiral isolates.

Intermediate uveitis.

Intermediate uveitis (IU) is described as inflammation in the anterior vitreous, ciliary body and the peripheral retina. In the Standardization of Uveitis Nomenclature (SUN) working group's international workshop for reporting clinical data the consensus reached was that the term IU should be used for that subset of uveitis where the vitreous is the major site of the inflammation and if there is an associated infection (for example, Lyme disease) or systemic disease (for example, sarcoidosis). The diagnostic term pars planitis should be used only for that subset of IU where there is snow bank or snowball formation occurring in the absence of an associated infection or systemic disease (that is, “idiopathic”). This article discusses the clinical features, etiology, pathogenesis, investigations and treatment of IU.

Fine needle aspiration cytology in childhood TB lymphadenitis.

Objective. To evaluate the efficacy of Fine Needle Aspiration Cytology (FNAC) to diagnose Tuberculous (TB) lymphadenitis with compare to excision biopsy and to correlate TB lymphadenitis with clinical, cytological, radiological and mantoux test features. Methods. This was a prospective correlational study. FNAC was done by a pediatrician for 135 children with persisting lymphadenitis after two weeks of antibiotic therapy in the period of January 2005 to June 2006 and compared with excision biopsy in a tertiary care hospital. Results. Forty Six cases (34.07%) were TB lymphadenitis diagnosed by FNAC. Excision biopsy and cytological correlation was done in 100 cases. Sensitivity, specificity and diagnostic accuracy for TB lymphadenitis were found to be 98%, 100% and 99% respectively. Positive and negative predictive values were 100 and 98 respectively. Large (>2cm) (86.9%), multiple (52.1%), matted (47.8%), posterior cervical and submandibular group nodes with history of contact (P=0.0016), positive mantoux test (P=0.0001) and Grade III and IV Protein Energy Malnutrition (PEM) (P=0.0041) were significantly seen in TB lymphadenitis. Ziehl Neelson staining for Acid Fast Bacilli (AFB) was positive in 32.5% cases of TB Lymphadenitis. Conclusion. Pediatrician himself can do FNAC which is an excellent first line method to diagnose TB lymphadenitis and it has equal accuracy to excision biopsy.

Rapid progression of HIV infection in infancy.

Transmission of HIV from mother to child can occur in utero, during labor or after delivery via breast feeding. Data on the fate of babies born with HIV in India are scarce. We present details of 25 infants with perinatally acquired HIV infection (virologically confirmed) to highlight the observed high rate of morbidity and mortality within the first 18 months of life. Our findings of rapid disease progression among perinatally infected HIV positive children underline the importance of early diagnosis and treatment.

Histopathological study of ocular erythema nodosum leprosum and post-therapeutic scleral perforation: a case report.

Leprosy is a chronic granulomatous disease caused by Mycobacterium leprae , clinically present either as tuberculoid, borderline or lepromatous type. Erythema nodosum leprosum (ENL) is an acute humoral response in the chronic course of lepromatous leprosy. Although very severe ENL reactions are known in systemic leprosy, such severity is rare in ocular tissues. A leprosy uveitis patient suffered from a severe form of post-therapeutic ENL reaction which resulted in perforation of the globe at the site of preexisting subconjunctival leproma. Painful blind eye was enucleated. Histopathological study revealed infiltration of numerous polymorphs and macrophages packed with acid-fast bacilli in the conjunctiva, cornea, ciliary body, ora serrata and sclera. A profuse influx of neutrophils on a background of macrophages packed with M. leprae confirmed the ocular ENL reaction. This case is reported to alert the ophthalmologists to a rare ocular complication of ENL.

A pre- and post-treatment evaluation of vision-related quality of life in uveitis.

AIM: To study the effect of treatment on vision-related quality of life (VR-QOL) in uveitis patients. MATERIALS AND METHODS: Interviewer-administered questionnaire-based evaluation of visual function and VR-QOL in Tamil-speaking adult patients with active uveitis at presentation and follow-up by the same interviewer. RESULTS: Ninety-eight patients participated in this study. There was a statistically significant improvement in VR-QOL in all the scales following treatment ( P < 0.001). Patients with chronic uveitis showed better improvement upon treatment than patients with acute uveitis. The visual symptoms scale showed moderate gains following treatment (effect size 0.56). Persons with bilateral disease had poorer mean scores compared to those with unilateral disease. Visual acuity was closely correlated with VR-QOL scores. CONCLUSION: The VR-QOL measurement has shown that it is sensitive to demonstrate the problems of patients with uveitis irrespective of their demographic profile. The scores improved significantly in patients with uveitis following treatment and have shown close correlation to visual acuity thus demonstrating that VR-QOL is effective in assessing the response to treatment.

Field rats form a major infection source of leptospirosis in and around Madurai, India.

AIMS: To determine the seroprevalence of leptospires and to isolate Leptospira spp. from field rats and bandicoots in and around Madurai. MATERIALS AND METHODS: Thirteen rats and five bandicoots were trapped alive from fields in and around Madurai. Blood samples were tested for anti-leptospiral antibodies by microscopic agglutination test while the urine and kidney samples were used for isolation of leptospires. The isolated leptospires were tested for pathogenic status (13 degrees C test and PCR) followed by serological and genetic characterization. RESULTS: Serology revealed the presence of anti-leptospiral antibodies in 58% (7/12) of field rats and leptospires were isolated from two urine and six kidney samples. The bandicoots were negative in both serology and culture. Analysis of the isolates from field rats revealed that all the isolates were pathogenic except for one, which was further confirmed by serological and genetic characterization. Six of the seven pathogenic isolates were identified as L. interrogans serogroup Autumnalis serovar Akiyami A and one as L. borgpetersenii serogroup Javanica serovar Veldrat Batavia 46. CONCLUSIONS: Serology and isolation reveals that field rats are major natural carriers and shedders of leptospires in and around Madurai.

Recurrent herpes zoster in early childhood.

Herpes Zoster is produced by reactivation of latent Varicella Zoster Virus from the dorsal root ganglion of sensory nerves. It is common in older individuals and rarely described in the pediatric age group. We report a case of recurrent herpes zoster in a 3-year-old HIV positive child involving T4 dermatome on the first occasion and subsequently involving T10 dermatome. The child responded well to oral acyclovir.

Global variation and pattern changes in epidemiology of uveitis.

Uveitis, a complex intraocular inflammatory disease results from several etiological entities. Causes of uveitis are known to vary in different populations depending upon the ecological, racial and socioeconomic variations of the population studied. Tropical countries are unique in their climate, prevailing pathogens and in the existing diseases, which further influence the epidemiological and geographical distribution of specific entities. We provide an overview of the pattern of uveitis of 15221 cases in 24 case series reported from several countries over 35 years (1972-2007) and we integrate it with our experience of an additional 8759 cases seen over six years (1996-2001) at a large community-based eye hospital. Uveitis accounted for 0.8% of our hospital-based outpatient visits. The uveitis was idiopathic in 44.6%, the most commonly identified entities in the cohort included leptospiral uveitis (9.7%), tuberculous uveitis (5.6%) and herpetic uveitis (4.9%). The most common uveitis in children below 16 years (616 patients; 7.0% of the total cohort) was pediatric parasitic anterior uveitis, (182 children, 29.5% of the pediatric cohort), whereas the most common uveitis in patients above 60 years (642 patients; 7.3% of the total cohort) was herpetic anterior uveitis, (78 patients, 12.1% of the elderly cohort). Etiologies varied with the age group of the patients. As in other tropical countries, a high prevalence of infectious uveitis was seen in this population.

Ocular manifestations of leptospirosis.

Leptospiral uveitis is a common entity in tropical countries. Ocular manifestations are noted in the second phase of illness, but these remain under-diagnosed mainly because of the prolonged symptom-free period that separates the systemic manifestations from detection of ocular manifestations.Varying ophthalmic presentations and the intrinsic nature of different types of uveitis to mimic one another also challenge the accuracy of the diagnosis. Of the individual ocular signs, the combination of acute, non-granulomatous, panuveitis, hypopyon, vasculitis, optic disc edema, membranous vitreous opacities and absence of choroiditis or retinitis have high predictive value for the clinical diagnosis of leptospiral uveitis. Geographic location of the patient, occupation, socio-economic status, risk factors related to exposure, past history of fever or jaundice also aid in diagnosis.Steroids are the mainstay of treatment for leptospiral uveitis. Depending upon the severity and anatomical location of inflammatory lesion, topical, peri-ocular and/or systemic steroids are given. The prognosis is generally good, even when the inflammation is severe.

Ocular infections due to non-tuberculous mycobacteria.

PURPOSE: To investigate the types and causes of non-tuberculous ocular infections and study their response to topical antibiotic therapy. METHOD: A single center, retrospective review of 18 patients with non-tuberculous mycobacterial ocular infections, seen over a 3 year period was done. Laboratory diagnosis was established by growth on blood agar, LJ medium and Ziehl-Nielsen acid fast stain. RESULTS: Out of 18 patients, six had post corneal graft infection, six had corneal ulcers, three had endogenous endophthalmitis, one had post operative endophthalmitis and two cases were of post surgical wound infection. History of trauma was reported in two cases and surgery in nine cases. M.chelonae was grown in blood agar for all patients. For corneal infections fortified genatmicin and fortified amikacin topical eye drops were given while the cases of endophthalmitis received intravitreal amikacin. Response to treatment was poor in 16 cases (88.9%). Only two cases of corneal ulcer improved after prolonged treatment. There was a misdiagnosis of Corynebacterium spp. on Gram stain in the initial cases. Majority of the isolates were sensitive to gentamicin (72.2%) followed by amikacin (44.4%). CONCLUSIONS: Early clinical recognition and prompt laboratory diagnosis together with aggressive topical antibiotic therapy may shorten morbidity and improve the clinical outcome of non-tuberculous mycobacterial ocular infection.