Genotypic Detection of Epstein-Barr Virus in Pediatric Transplant Recipients From India.

Objective: To determine the rate of occurrence and genotypes of Epstein-Barr Virus (EBV) among pediatric renal and liver transplants recipients. Design: Observational study. Setting: Vision Research Foundation referral center and Institute of Liver Disease and Transplantation, Chennai, India. Participants: 70 pediatric solid organ transplant recipients and 60 voluntary healthy donors. Methods: Polymerase chain reaction (PCR) for detection and genotyping of EBV were carried out using genes targeting Viral capsid antigen, Nuclear antigen 1, 2 and 3, followed by real time PCR for viral load determination and further confirmed by phylogenetic analysis. Results: EBV was detected in 35 (51.4%) samples (32 liver and 4 renal transplants) with high viral load. Type A was detected in 33 samples, Type B in 2 liver transplant patients, and co-infection in one liver transplant patient who developed Post-transplant Lymphoproliferative Disorder (PTLD). Real time PCR results correlated with conventional PCR. The mean viral load for patients who did not develop PTLD was 50,424 copies/mL. Overall EBV load in patient with PTLD ranged from 1,40,392 copies/mL prior to PTLD diagnosis to 62,124 copies /mL post treatment. Conclusion: EBV infection is the high risk factor for PTLD after liver transplantation. PCR targeting of EBV can be applied to diagnose EBV infections and monitor treatment for EBV in pediatric solid organ transplant recipients.

Application of real time polymerase chain reaction targeting kex 1 gene & its comparison with the conventional methods for rapid detection of Pneumocystis jirovecii in clinical specimens.

Background & objectives: As there are no standard laboratory techniques for the rapid detection of Pneumocystis jirovecii in India, this study was undertaken to evaluate and establish an optimal and rapid technique for the detection of P. jirovecii by comparing three different techniques - staining technique, application of a real time polymerase chain reaction (RT-PCR) targeting kex 1 gene and application of nested PCR targeting mitochondrial large subunit (mtLSU) gene for rapid detection of P. jirovecii in HIV positive patients. Methods: One hundred and fifty sputum specimens from HIV positive (n = 75) and HIV negative (n = 75) patients were subjected to three different techniques -KOH/Calcoflour and Grocott methanamine silver staining (GMS), RT-PCR targeting kex1 gene, PCR targeting mtLSU region followed by DNA sequencing and BLAST analysis. Results: Among the 75 HIV positive patients, P. jirovecii was detected in 19 (25.33%) patients by the staining techniques, and in 23 (30.65%) patients each by PCR targeting mtLSU region and by RT- PCR targeting kex1 gene of P. jirovecii. PCR based DNA sequencing targeting mtLSU region revealed 97-100 per cent sequence homology with P. jirovecii sequences in GenBank. Interpretation & conclusions: Of the three techniques for detection of P. jirovecii evaluated in this study, false negativity was found to be more in staining technique and it also required high technical expertise to interpret the result. Both nested PCR and RT-PCR were reliable and equally sensitive, in rapid detection of P. jirovecii, but RT-PCR technique also generated the copy numbers for knowing the severity of infection.

A study on the incidence, microbiological analysis and investigations on the source of infection of postoperative infectious endophthalmitis in a tertiary care ophthalmic hospital: An 8-year study.

Background: The objective of the study was the determination of the incidence of culture-proven postoperative endophthalmitis and probable sources of infection. Materials and Methods: It was a prospective study on the microbiology, incidence and probable sources of infection in patients with postoperative infectious endophthalmitis carried out in a tertiary care eye hospital. Consecutive patients diagnosed with postoperative infectious endophthalmitis during the years 2000-2007 were investigated for the causative infective agent and possible sources of infection. The surgical data and microbiological data including the investigations performed to trace the source were recorded in a specific formatted form and were gathered and compiled for analysis. Results: Data of analysis showed that 98 (0.042%) out of 2,31,259 patients who underwent intra-ocular surgery developed infectious endophthalmitis. Among these, 70 (0.053%) occurred after cataract, 10 (0.5%) after penetrating keratoplasty (PK) and 18 (0.018%) following other types of intra-ocular surgeries. The predominant infectious agents isolated were bacteria (89.7%), with equal proportions of Gram-positive and Gram-negative bacteria. Polymicrobial infection was noted in four and fungi in seven patients. Occurrence of postoperative endophthalmitis was sporadic and not related to any specific part of period in a year. Sources of infection were donor corneal rim in six post-PK patients and phaco probe in one who had postphacoemulsification endophthalmitis Conclusions: Overall incidence of postoperative endophthalmitis over an 8-year period was quite low. The sources of infection could be established in six post-PK endophthalmitis patients and in a postcataract surgery.

Seroprevalence of human immunodeficiency virus, hepatitis B virus and hepatitis C virus among eye donors.

Blood specimens collected at the time of enucleation of the eyes from 483 consecutive eye donors were tested for sero-markers of Human Immunodeficiency Virus (HIV), Hepatitis B Virus (HBV) and Hepatitis C Virus (HCV). Antibodies to HIV1 were detected in 3 (0.62%), HBsAg in 17 (3.52%) and antibodies to HCV in 7 (1.45%).

Endophthalmitis caused by Acinetobacter calcoaceticus. A profile.

PURPOSE: To report the clinical and microbiological profile of endophthalmitis caused by Acinetobacter calcoaceticus. METHODS: A retrospective study of case series of Acinetobacter calcoaceticus endophthalmitis. Outcome measures included ability to sterilise the eye, anatomical result (clear media and attached retina) and visual recovery (visual acuity > 6/60). RESULTS: Of the 20 cases studied, 10 were cases of postoperative endophthalmitis, 3 were posttraumatic, 6 were endogenous and one was bleb-related endophthalmitis. Specific features of interest observed were relative chronicity of presentation and absence of any obvious predisposing factor in endogenous endophthalmitis cases. All cases could be sterilised except one, which needed evisceration. Cases with postoperative endophthalmitis had better anatomical outcome (7/10 with attached retina and clear media) and visual outcome (4/10 regained vision > 6/18). Higher smear positivity was seen in vitreous samples (72.2%) compared to aqueous samples (37.5%). Culture positivity was higher from the vitreous cavity compared to aqueous. The organism was sensitive to ciprofloxacin in a high percentage (88.9%) of cases. CONCLUSIONS: Visual recovery in Acinetobacter calcoaceticus endophthalmitis is modest. Ciprofloxacin is the antibiotic of choice.

The diagnostic significance of enzyme linked immuno-sorbent assay for herpes simplex, varicella zoster and cytomegalovirus retinitis.

PURPOSE: To evaluate the diagnostic usefulness of enzyme linked immuno-sorbent assay (ELISA) in single serum samples to associate herpes simplex virus (HSV), varicella zoster virus (VZV) or cytomegalovirus (CMV) with viral retinitis as against polymerase chain reaction (PCR) on intraocular specimens. It was also designed to study the seroprevalence in normal healthy individuals, and the genomic prevalence of HSV, VZV and CMV in patients without an active viral inflammatory process. METHODS: PCR for the detection of HSV, VZV and CMV genomes was done on 33 and 90 intraocular fluids from viral retinal patients and non-viral controls respectively. ELISA was done on 30 and 100 serum samples from viral retinitis patients and normal healthy controls respectively. RESULTS: PCR did not detect HSV, VZV and CMV genomes except one, in which VZV-DNA was detected. ELISA showed prevalence rates of 28%, 83% and 90% for antibodies against HSV, VZV and CMV respectively in the normal population. In the 30 viral retinitis patients, PCR detected HSV-DNA in 2 (6.7%), VZV-DNA in 7 (23.3%) and CMV-DNA in 6 (20.0%) patients, while ELISA detected antibodies against HSV, VZV and CMV in 13 (43.3%), 24 (80.0%) and 23 (76.7%) patients respectively. ELISA was of value in indirect diagnosis only in 6 (20.0%) as compared to 15 (50.0%) of 30 patients by PCR, this difference was statistically significant (McNemar test, P value = 0.005). CONCLUSION: Serology by ELISA is no longer a useful diagnostic tool to associate HSV, VZV and CMV viruses with viral retinitis.

Preparation of amniotic membrane for ocular surface reconstruction.

We describe the preparation and preservation of human amniotic membrane required for transplantation in the management of ocular surface diseases. Informed consent is obtained and the donor is screened to exclude risk of transmissible infections such as human immunodeficiency virus (HIV), hepatitis B virus, hepatitis C virus, and Treponema pallidum infections. Ideally, the media and washing solutions needed for the preparation of amniotic membrane are prepared only a week to 10 days prior to use and not stored in the freezer weeks ahead. The AM obtained under sterile conditions after elective caesarian section is washed free of blood clots and chorion. With the epithelial surface up, amniotic membrane is spread uniformly without folds or tears on individually sterilized 0.22 micron nitrocellulose membranes of the required sizes. The prepared filter membrane with the adherent amniotic membrane is placed in the preservative medium and stored at -80 degrees C. The membranes are released when the repeat serology for HIV after the window period has excluded virus infection in the donor. Depending on consumption they may be used up to 6 months after preparation, though many have recommended storage for an indefinite period. Since the amniotic membrane has only incomplete expression of HLA antigens and amniotic epithelial cells do not express them, it is not rejected after transplantation. The presence of several cytokines in the amniotic membrane promotes epithelialization with reduction of fibrosis during healing.

Clinical profile and immunological status of cytomegalovirus retinitis in organ transplant recipients.

PURPOSE: Cytomegalovirus retinitis (CMV) is the most common ocular opportunistic infection in transplant recipients. This retrospective study attempts to report the differences in occurrence of cytomegalovirus retinetis in transplant recipients from those reported in patients with acquired immunodeficiency syndrome (AIDS). METHODS: 25 eyes of 15 transplant recipients (14 renal and one cardiac) with cytomegalovirus retinitis were retrospectively reviewed. Immunological profile included CD4+ and CD8+ T lymphocyte counts, CD4+/CD8+ cell ratio (5 cases) and serology for the viral antibodies (8 cases). RESULTS: A predominantly bilateral presentation (60%) was noted. Active cytomegalovirus retinitis (72%) in zone 2 (92%) of the inferotemporal quadrant (68%) was noted. The average cell counts were within normal limits (mean CD4 cell count-711/microliter), unlike in late stages of AIDS with cytomegalovirus retinitis (CD4 count < 50/microliter). Serology revealed an IgM positivity of 53%. Retinal detachment (52%) was the most common complication occurring after an average of 5.4 months. CONCLUSION: CMV retinitis in organ transplant recipients appears to differ from that in AIDS patients. CMV retinitis presents early and has different immunological profile, probably owing to differences in pathogenesis.

Current approaches to diagnosis and management of ocular lesions in human immunodeficiency virus positive patients.

Human immunovirus infection in India is rapidly increasing. Ocular lesions due to highly active antiretroviral therapy have been well recognized. Acquired immunodeficiency syndrome can affect all parts of the eye. However, posterior segment lesions are the most common and of these, Human immunodeficiency virus retinopathy and cytomegalovirus retinitis predominate. Often clinical examination can establish the diagnosis of many ocular lesions in acquired immunodeficiency syndrome; therefore, ophthalmologists need to be aware of the more common ones. Various drugs in different routes can used to treat cytomegalovirus retinitis. Highly active antiretroviral therapy has remarkably reduced systemic and ocular morbidity among acquired immunodeficiency syndrome patients. To facilitate care of these patients aseptic precautions for ophthalmic care personnel are now well established and therefore ophthalmologist should not hesitate to provide ophthalmic care to acquired immunodeficiency syndrome patients.

Further investigations on the association of Mycobacterium tuberculosis with Eales' disease.

PURPOSE: To apply polymerase chain reaction (PCR) on vitreous fluid (VF) from Eales' disease to further confirm its association with Mycobacterium tuberculosis. METHODS: Sixty nine VF samples from 69 patients (24 Eales' disease and 45 Non-Eales' as controls) were processed by conventional methods for detection of mycobacteria. Polymerase chain reaction (PCR) specific for IS 6110 and nested PCR (nPCR) using primers coding for MPB 64 gene were applied on all 69 VF. PCR based dot-blot hybridisation was applied on the IS 6110 amplified products of n PCR-positive VFs. RESULTS: Conventional methods (direct smear and culture) did not detect mycobacteria in any of the 69 VF samples. Five (20.8%) of 24 VF from Eales' and 2 (4.2%) of 45 VF from control patients tested positive for M. tuberculosis DNA by nPCR. This difference was statistically significant (P < 0.05). All 69 VF were negative by PCR for IS 6110. Two VF of Eales' patients positive by nPCR were also positive by DNA probe dot-blot hybridisation for IS 6110. CONCLUSION: Detection of M. tuberculosis DNA by PCR in a significant number of VF of Eales' disease patients reemphasizes the association of this bacterium with Eales' disease.