Serum-derived hepatitis C virus 1a infection of human astrocyte cell line SVG.

Neuroinvasion of hepatitis C virus (HCV) is evidenced by recent clinical studies. In this study, serum-derived HCV infection of astrocytes was analysed. Astrocytes were infected with HCV-positive serum, and viral replication was assessed on different days postinfection. RT-PCR was positive for HCV-negative strand on 5th and 7th day postinfection in the HCV-positive serum-infected astrocytes. Real-time RNA count in the cell culture supernatant was steadily increasing from day 3 to day 7. To reconfirm the viral replication, astrocytes were treated with an antiviral before the serum infection, and the antiviral treatment significantly reduced the viral RNA count. Further, the virus-infected cells stained positive for the presence of viral core protein. Electron microscopy revealed the presence of HCV-like particles in the astrocyte cell culture supernatant. In conclusion, serum-derived HCV replicates in human astrocyte cell line SVG.

A pilot study on expression of toll like receptors (TLRs) in response to herpes simplex virus (HSV) infection in acute retinal pigment epithelial cells (ARPE) cells.

INTRODUCTION: Toll like receptors (TLRs) have been proven to play an important role in mounting the innate immune response in an infected host. The expression of TLRs against herpes simplex virus (HSV) have not been studied in retinitis. Therefore, the current study was undertaken to determine the same using the retinal pigment epithelial (ARPE-19) cell line. MATERIALS AND METHODS: APRE cells cultured in vitro were challenged with HSV 1 and 2 standard strains and 20 other clinical isolates. The cells were observed for cytopathic changes. The cell culture harvest was subjected to RNA extraction using a Total RNA mini kit. The RNA was subjected to reverse transcriptase polymerase chain reaction (PCR) for the amplification of TLRs 3, 4 and 9 and GAPDH housekeeping gene. The amplified products were subjected to electrophoresis on a 2% agarose gel and viewed under a transilluminator. RESULTS: TLR 3 and 4 were expressed by ARPE treated with all the 22 isolates. TLR 9 expression was seen in 16 of the 22 isolates. Bacterial contamination was ruled out by subjecting the harvests to PCR amplification of 16sRNA gene amplification of the eubacterial genome. CONCLUSIONS: The expression of TLR 4 has been reported for the first time in HSV infection. TLR 4 along with TLR 3 and 9 is responsible for the antiviral response in HSV infections.

Isolation of a variant human adenovirus identified based on phylogenetic analysis during an outbreak of acute keratoconjunctivitis in Chennai.

BACKGROUND & OBJECTIVES: Though several viruses are responsible for conjunctivitis, but human adenovirus (HAdV) is by far the most common cause. Epidemic conjunctivitis causes morbidity and early detection of aetiological agent is essential in preventing spread of disease as some of serotypes of adenoviruses cause a severe form of conjunctivitis. This study was undertaken to identify the causative agent of conjunctivitis outbreak in Chennai in 2010. METHODS: Conjunctival samples collected from 17 patients with conjunctivitis were subjected to virological investigations. Culture and PCR for detection of adenovirus and enterovirus were carried out. PCR positive products were further subjected for DNA sequencing. The nucleotide sequences of the hexons of isolates were analyzed by comparison with all 51 human adenovirus strains. Phylogenetic tree was constructed using DAMBE software. RESULTS: Among 17 patients, seven were positive for adenovirus by PCR on the direct specimen, none was positive for enterovirus. Eleven of 30 conjunctival swabs showed cytopathic effect in HEp-2 cell line and were confirmed as HAdV by PCR. The DNA sequence data of the 11 isolates had equal percentage of homology with HAdV 6 and 2 on blast analysis. On phylogenetic analysis with GeneBank data of 51 adenovirus strains, 11 isolates from patients during the outbreak of conjunctivitis formed a separate clade indicating a new variant strain. INTERPRETATION & CONCLUSIONS: Based on phylogenetic analysis it was concluded that the recent conjunctivitis outbreak that occurred in Chennai was caused by a variant adenovirus strain.

Detection of Mycobacterium tuberculosis directly from sputum specimens & phenotypic drug resistance pattern of M. tuberculosis isolates from suspected tuberculosis patients in Chennai.

BACKGROUND & OBJECTIVES: mRNA is more rapidly destroyed in cells than rRNA or genomic DNA, an assay targeting bacterial mRNA would provide a better guide to mycobacterial viability than amplification tests directed at DNA or rRNA targets. This study was carried out to standardize reverse transcriptase PCR (RT-PCR) targeting 85B gene for the rapid detection of viable Mycobacterium tuberculosis from sputum specimens of suspected TB patients at Chennai, South India and to detect MDR-TB circulating in this population. METHODS: Sputum samples from clinically suspected tuberculosis patients (n=301) and 78 controls were included in the study. The sputum samples were collected in sterile diethyl pyrocarbonate (DEPC) treated containers and transported in ice to the laboratory within 2 h to prevent degradation of RNA. RT-PCR targeting 85B gene, mycobacterial culture and phenotypic drug susceptibility testing for the first line drugs streptomycin (S), isoniazid (H), rifampicin (R), ethambutol (E) and pyrazinamide (Z) were performed by BACTEC microMGIT culture system for all the sputum specimens. RESULTS: All the 78 controls were negative for culture and RT-PCR. Among the 301 sputum specimens from patients, 231 (76.8%) were RT-PCR positive and 70 (23.2%) were negative. There were 166 M. tuberculosis isolates, of which 11 (2.9%) were MDR-TB, 33 (8.7%) were polyresistant, 31 (8.2%) were monoresistant and 91 (30.2%) were sensitive to all five first line anti-tuberculous drugs by phenotypic drug susceptibility testing. Monoresistance was higher with Z [20 (20.8%)], followed by S [6 (3%)]. INTERPRETATION & CONCLUSIONS: RT-PCR targeting 85B gene of M. tuberculosis was a specific, rapid, reliable technique to detect the M. tuberculosis directly from sputum specimens. Our results showed that 2.9 per cent of M. tuberculosis isolates in the study population of Chennai were MDR.

Utility of QuantiFERON®-TB Gold test in diagnosis and management of suspected tubercular uveitis in India.

The tuberculin skin test, used to detect latent systemic tuberculosis (TB), has its limitations. The utility of interferon-gamma assays, found useful in the diagnosis of latent TB, is still unestablished in tubercular uveitis. We present the results of QuantiFERON(®)-TB Gold (QFT-G) test and its relevance in the diagnosis and management of suspected tubercular uveitis in India. All suspected tubercular uveitis patients seen at our uveitis clinic between October 2006 and June 2008 who underwent relevant blood investigations, chest X-rays, Mantoux tests and QFT-G tests were included. Clinical profile, systemic correlation and outcome with treatment were analysed. Fifty suspected tubercular uveitis patients underwent QFT-G testing. The age range of the patients was 6-55 years (mean 32.66 years). Seven patients presented with active and three with a past history of systemic TB. The QFT-G test was positive in 29 patients. Radiological findings of TB were seen in four patients with a positive QFT-G and one patient with a negative QFT-G test. In 11 patients both QFT-G and Mantoux tests were positive. Eighteen Mantoux-negative patients were QFT-G-positive. Significantly, no patient with a positive Mantoux had a negative QFT-G test. Of the 32 patients with posterior uveitis, 17 patients had serpiginous choroiditis, four patients had a choroidal granuloma, six patients had multifocal choroiditis, four patients had retinal vasculitis, and one patient had a subretinal abscess. All QFT-G-positive patients were treated with anti-tuberculosis therapy as well as systemic steroids with a favorable clinical outcome. Our study shows that the QFT-G test is very useful in the diagnosis and management of suspected ocular TB. It was found to be very sensitive in identifying latent TB patients who, upon treatment, had a significantly reduced frequency of recurrences. It was more sensitive than the Mantoux test and is not significantly affected by previous treatment with systemic steroids or immunosuppressives. A negative QFT-G test can also be used as an adjunct before initiation of systemic steroids or immunosuppressives in uveitic patients particularly in an endemic setting like India.

Infectious aetiology of congenital cataract based on TORCHES screening in a tertiary eye hospital in Chennai, Tamil Nadu, India.

BACKGROUND & OBJECTIVES: We undertook this study to determine the infectious aetiology of congenital cataract based on the presence of IgM antibodies to TORCHES [(Toxoplasma gondii (T. gondii), Rubella virus (RV), Cytomegalovirus (CMV), Herpes simplex virus (HSV) and Syphilis (caused by Treponema pallidum)] in the serum samples of congenital cataract patients. METHODS: Serum samples collected from 593 infants and children (10 days to 12 months old) with clinically diagnosed congenital cataract at Sankara Nethralaya, a referral eye hospital in Chennai, were tested for the presence of specific IgG and IgM antibodies to T. gondii, RV, CMV, HSV by ELISA and specific treponemal antibodies by T. pallidum haemagglutination test (TPHA). RESULTS: IgM antibodies were detected against T. gondii in 1.7 per cent, RV in 8.4 per cent, CMV in 17.8 per cent and HSV in 5.1 per cent, and that of specific IgG in 8.9, 25.0, 66.1 and 2.6 per cent respectively. Presence of IgM antibodies to T. Gondii in the study group was significantly lower when compared to IgM antibodies to RV, CMV and HSV. All serum samples were negative for the presence of anti treponemal antibodies by TPHA. Overall, IgM antibodies to one or more of the four infectious agents were detected in 20.2 per cent of the study population, and among these co-infections to more than one infectious agents were detected in 12.5 per cent. INTERPRETATION & CONCLUSION: The results of the present retrospective analysis showed association of RV, CMV, HSV and T. gondii with congenital cataract based on the presence of specific IgM antibodies.

Antibiotic susceptibility pattern of rapidly growing mycobacteria.

BACKGROUND: The rapidly growing mycobacteria (RGM) causing human infections primarily consist of the Mycobacterium fortuitum group, Mycobacterium abscessus and Mycobacterium chelonae. The antibiotic susceptibility testing is important to determine the appropriate therapy as the antibiotics used to treat RGM are different from those used for treating infections caused by slow growers of mycobacteria. AIM: To determine antibiotic susceptibility of RGM using Kirby Bauer method and following Clinical and Laboratory Standards Institute (CLSI) guidelines. SETTINGS AND DESIGN: Larsen and Toubro Microbiology Research Centre, Vision Research Foundation, Sankara Nethralaya, Chennai, Retrospective study. MATERIALS AND METHODS: The antibiotic susceptibility testing was performed following CLSI method for the drugs Amikacin, Azithromycin, Tobramycin, Ceftazidime, Cephotaxime, Cefuroxime, Cefaperazone, Ceftriaxone, Ciprofloxacin, Ofloxacin, Norfloxacin, Gatifloxacin and Moxifloxacin. RESULTS AND CONCLUSIONS: Out of the 148 RGM isolates 146 (98%) were susceptible to amikacin, 138 (91%) to gatifloxacin, 132 (87%) to moxifloxacin, 122 (76%) to ciprofloxacin and 116 (74%) to Norfloxacin. Majority of the RGM were resistant to Ceftazidime, Cephotaxime and Cefaperazone. All the M. abscessus isolates were resistant to tobramycin. The in vitro antibiotic susceptibility testing by disc diffusion method showed that majority of the RGM were sensitive to Amikacin followed by Gatifloxacin, Moxifloxacin and Ciprofloxacin.

Unfolding COVID-19: Lessons-in-Learning in Ophthalmology.

IMPORTANCE: An observant Chinese doctor Li Wenliang became the first physician to alert the world about COVID-19. Being an ophthalmologist himself, he has put the additional onus on us. The fact that the ocular manifestation could be the first presenting feature of novel coronavirus pneumonia should not be ignored and the possibility of spread of SARS-CoV-2 through the ocular secretions cannot be ruled out. However, with breakthroughs still evolving about this disease, the calls are now louder for closer examination on the pathogenesis of conjunctivitis associated with it. Hence, we conducted a scoping review of all available literature till date to fill in the "potential" gaps in currently available knowledge on ocular manifestations of SARS-CoV-2 infection in an attempt to establish continuity in the "chain of information" from December 2019 till April 2020. We also summarize a possible hypothesis on much less understood and highly debated topics on regard to the etiopathogenesis of ocular involvement in SARS-CoV-2 based on either presence or absence of ACE2 receptor in the ocular surface. METHODS: We conducted a scoping review search of published and unpublished SARS-CoV-2-related English language articles from December 2019 till mid of April 2020 from the online databases. The findings were summarized using text, tables, diagrams, and flowcharts. RESULTS: The commonest ocular manifestation in SARS-CoV-2 infection is follicular conjunctivitis and has been the first manifestation of SARS-CoV-2 infection in 3 reported cases till date. The ocular surface inoculated with the SARS-CoV-2 leads to the facilitation of the virus to the respiratory system via the lacrimal passage. RT-PCR analysis of the ocular secretions has shown the presence of the SARS-CoV-2 nucleotides indicating the possibility of infection of ocular secretions. ACE2 receptors and its expression on the ocular mucosal surface are linked behind the etiopathogenesis of conjunctivitis. CONCLUSION: Conjunctivitis can be the presenting manifestation but may go unnoticed due to its mild nature. The ocular surface could serve as the entry gateway for the virus and ocular secretions could play a role in virus shed. The eye care personnel, as well as the general people, need to be more vigilant and adopt protective eye measures.

Analysis of mixed infections by multiple genotypes of human cytomegalovirus in immunocompromised patients.

Human cytomegalovirus (HCMV) is a significant cause of morbidity and mortality in immunocompromised patients. The present study was carried out to determine the frequency of occurrence of multiple genotypes of HCMV in immunocompromised patients, to determine if there is any discrepancy in identification of mixed infections by multiple genotypes in paired clinical specimens obtained from patients and to determine the significance of viral load differences between patients infected with single and multiple genotypes. One hundred clinical specimens from 75 patients were included in the study. Real-time PCR; Multiplex PCR and PCR-based RFLP were applied for the determination of viral load and genotyping of HCMV, respectively. Out of the 75 patients, 36 (48%) carried multiple genotypes. Discrepancy with regard to detection of genotypes were found in 17/25 patients whose paired clinical specimens were analyzed. Mixed genotypes were found more often in peripheral blood than urine or intraocular fluids collected from the same patient. There was a statistically significant difference between the median viral loads of clinical specimens carrying single genotypes and multiple genotypes. Mixed infections with multiple genotypes were found predominantly in the leukocyte fraction of peripheral blood specimens. The detection of mixed infections by multiple genotypes in the hypervariable regions of HCMV can be a surrogate marker of an increase in viral load.

Application of PCR based-RFLP for species identification of ocular isolates of methicillin resistant staphylococci (MRS).

BACKGROUND & OBJECTIVE: Early detection of methicillin resistant staphylococci (MRS) from clinical specimens enables institution of appropriate antimicrobial therapy. Limited information is available on speciation of MRS. This study was undertaken to compare results of conventional and molecular methods in detection of methicillin resistance (MR) and application of PCR-restriction fragment length polymorphism (RFLP) and DNA sequencing for speciation of ocular isolates of MRS. METHODS: A total of 110 consecutive ocular staphylococcal isolates were screened for MR. MRS was speciated by PCR-RFLP of gap gene and results were confirmed by DNA sequencing. All isolates were processed within 48 h of isolation. A single colony of bacterium, stocked as stab cultures in Hyer's and Johnson agar, was stored at 4 degrees C and sub-cultured at every 15 days interval. RESULTS: Seventy (63.6%) of 110 isolates were identified as MRS and 40 (36.4%) were MSS by conventional and molecular method (100% correlation). Of the 70 MRS, 18 (25.7%) were Staphylococcus aureus, remaining 52 (74.3%) were CNS by conventional and molecular method (100% correlation). PCR-RFLP of gap gene identified 18 (25.71%) MRS as S. aureus, 11 (15.71%) S. epidermidis, 27 (38.57%) S. haemolyticus, 6 (8.57%) S. cohnii subsp. urealyticum, 6 (8.57%) S. equorum, 1 (1.42%) S. xylosus and 1 (1.42%) S. hominis. INTERPRETATION & CONCLUSION: Overall rate of isolation MRS was 63.6 per cent and were predominantly isolated from conjunctival swab (23.6%) and donor corneal scleral rim (23.6%) of non hospitalized patients indicating their community origin. Detection of MR by mecA gene was easier and less time consuming compared to conventional methods. Speciation of MRS was possible by gap gene PCR - RFLP and the predominant MRS in our study was S. haemolyticus.

DNA sequencing by Microseq kit targeting 16S rRNA gene for species level identification of mycobacteria.

BACKGROUND & OBJECTIVES: Identification of mycobacteria to the species level is of therapeutic significance. Conventional methods are laborious and time consuming so we did 16S rRNA sequencing using a commercial MicroSeq sequencing kit, which includes DNA sequencing with software package for identification and phylogenetic analysis of clinical mycobacterial isolates. METHODS: A total of 47 mycobacteria were tested by both conventional and genotypic method using commercially available MicroSeq 500 amplification kit assay. The identification was determined by comparing the 500 bp amplified product of 16S rDNA sequence to the MicroSeq database. RESULTS: The phenotypic identification was concordant with genotypic identification in 33 (70.2%) isolates of 14 Mycobacterium tuberculosis, 11 M. fortuitum, 7 M. abscessus and 1 M. duvalii. For the discrepant isolates, identification was possible only by DNA sequencing in 14 (29.7%) isolates. The 14 discrepant isolates were 5 M. farcinogenes, 3 M. genavense, 2 M. species. nov and 1 each of M. fortuitum, M. immuogenum, M. simiae and M. wolinskyi. Of these, five were uncommon species that were difficult to identify by phenotypic method. INTERPRETATION & CONCLUSION: The MicroSeq DNA sequencing is an excellent tool for species identification of mycobacteria, which reduces the turn around time, makes repeat analysis easy as compared to phenotypic identification specially for mycobacterial isolates with ambiguous biochemical profiles.

Newer emerging pathogens of ocular non-sporulating molds (NSM) identified by polymerase chain reaction (PCR)-based DNA sequencing technique targeting internal transcribed spacer (ITS) region.

PURPOSE: To apply Polymerase Chain Reaction (PCR)-based DNA sequencing targeting Internal Transcribed Spacer (ITS) region for identification of non-sporulating molds (NSM) to species level which formed 12% of ocular isolates of fungi in a tertiary eye hospital in South India. MATERIALS AND METHODS: Fifty ocular filamentous fungal NSM isolates recovered from 45 patients were included in the study. PCR-based DNA sequencing technique targeting ITS region was applied to identify NSM. RESULTS: PCR-based DNA sequencing revealed 23 established pathogens involving 8 genera (Aspergillus, Fusarium, Bipolaris, Pythium, Cochilobolus, Exserohilum, Pseudoallescheria, and Scedosporiumspecies) and 27 emerging pathogens involving 7 genera (Botryosphaeria Lasiodiplodia species, Thielavia tortuosa, Glomerulla singulata, Macrophomina phaseolina, Rhizoctonia bataticola, and Podosporaspecies) reported for the first time in literature related to ocular infections. Fifteen (30%) patients with fungal keratitis caused by NSM failed to respond to standard antifungal therapy. CONCLUSION: PCR-based DNA sequencing technique is a rapid, reliable, and valuable tool to identify 54% of NSM as newer potential pathogens of fungi causing ocular mycoses.

Influence of feeder layer on the expression of stem cell markers in cultured limbal corneal epithelial cells.

BACKGROUND & OBJECTIVE: The limbus is enriched with the stem cells of corneal epithelium. Auto- and allograft limbal transplantations are effective in restoring the corneal epithelium and inhibiting inflammation and neovascularization. Preserved human amniotic membrane (AM) is now widely used as a substrate for ocular surface reconstruction. The combination of limbal and AM transplantation has been shown to improve the surgical outcome in patients with total limbal stem cell deficiency (LSCD). The purpose of this study was to compare the expression of putative stem cell markers ATP binding cassette protein (ABCG2) and keratinocyte stem cell marker: p63 and differentiation markers. (connexin 43 and keratin 3 / keratin 12) on the limbal epithelial cells cultured over the denuded AM with and without the 3T3 murine fibroblast cells as feeder layer. METHODS: Human limbal tissues obtained from the cadaveric donor eyes were cultured over the denuded human amniotic membrane in the presence of mitomycin C treated 3T3 fibroblasts and the cultured cells studied for the expression of ABCG2 and p63 by immunohistochemistry and Western blot. Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR) was done on the cultured cells at varying intervals of time for expression of ABCG2, p63, connexin43 (Cnx43), and keratin 3 (K3) and keratin 12 (K12). RESULTS: The growth rates were similar in both denuded AM and denuded AM + 3T3. The cells cultured over AM + 3T3 showed the expression of p63 and ABCG2 till 21 days of incubation by immunohistochemistry and Western blot. The expression of p63 and ABCG2 were retained till 21 days of incubation on the cells cultured over denuded AM + 3T3, whereas it was expressed only till day 8 on the cells cultured over the denuded membrane by semi quantitative RT-PCR. Cnx43 and K3/K12 were observed in both the conditions. INTERPRETATION & CONCLUSION: The limbal epithelial cells cultured in the presence of mitomycin C treated 3T3 feeder layer were able to maintain the expression of putative stem cell markers. Further in vitro studies using feeder layer will enable us to understand the factors, which play a role in maintaining the limbal stem cell niche.

Relative efficiency of polymerase chain reaction and enzyme-linked immunosorbant assay in determination of viral etiology in congenital cataract in infants.

BACKGROUND: Perinatal viral infections of fetus are among the leading causes of congenital cataract and identifying the viral etiology is important. OBJECTIVES: To detect the presence of Rubella virus (RV), herpes simplex virus (HSV) and cytomegalovirus (CMV) in lens aspirate specimens obtained from patients with congenital cataract and relate the results with serology. SETTING AND DESIGN: Prospective study carried out in tertiary care hospital. MATERIALS AND METHODS: Fifty lens aspirates from 50 infants with congenital cataract were subjected to HSV, RV isolation and polymerase chain reaction (PCR) for detection of HSV and CMV. Reverse transcription polymerase chain reaction (RT-PCR) was applied for RV detection. Peripheral blood specimens were screened for anti-HSV, RV and CMV antibodies by enzyme-linked immunosorbant assay (ELISA). RESULTS: Rubella virus was detected in nine (18%) lens aspirates, by nRT-PCR which includes six positive by culture. HSV-2 DNA was detected in nine other lens aspirates, while CMV was not detected by PCR. Serological results did not correlate with the presence of viruses in the lens aspirates. This is the first report of detection of HSV-2 DNA in cases of congenital cataract. CONCLUSIONS: Cytomegalovirus may not be playing a significant role in causation of congenital cataract. The role of serology in identifying causative viral infection for congenital cataract needs to be re-evaluated.

Intracanalicular antibiotics may obviate the need for surgical management of chronic suppurative canaliculitis.

Chronic suppurative lacrimal canaliculitis is an important cause of ocular surface discomfort. Treatment with topical antibiotics is often inadequate and surgical treatment by canaliculotomy and canalicular curettage has been the mainstay of treatment in the past. The role of canalicular antibiotic irrigation has been inadequately studied. We report the clinical features, microbiological profile and treatment outcome in a series of 12 patients with suppurative lacrimal canaliculitis. Two patients had Actinomyces infection, five had Nocardia infection and seven patients had polymicrobial infection. Three patients had resolution of canaliculitis on combination broad-spectrum topical antibiotic therapy using ciprofloxacin and fortified cefazolin. In nine patients, topical antibiotic therapy was combined with canalicular irrigation using fortified cefazolin. All patients had excellent resolution of canaliculitis without the need for surgical treatment. Availability of broad-spectrum antibiotics and canalicular irrigation may offer an alternative to surgery in the management of suppurative lacrimal canaliculitis.

Outcome of polymerase chain reaction (PCR) analysis in 100 suspected cases of infectious uveitis.

BACKGROUND: Polymerase chain reaction (PCR) analysis is an important tool in the diagnosis of infectious uveitis. A retrospective, interventional study of PCR analysis of ocular fluid in suspected infectious uveitis cases between January 2014 to July 2016 was done. Nested, real-time and broad range PCR was performed for detection of the genome of Mycobacterium tuberculosis, herpes virus family, Chikungunya virus, Toxoplasma gondii, fungus, eubacterium and propionibacterium acne. RESULTS: Total of 100 cases included, mean age was 39.2 ± 15.4 years. Uveitis was unilateral in 82% and granulomatous in 40%. Mean visual acuity at the initial visit and final visit was 0.73 logMar and 0.63 logMar respectively. PCR analysis confirmed the clinical diagnosis in 70.1% patients. The sensitivity, specificity, positive predictive value and negative predictive value of PCR analysis was 90.2%, 93.9%, 93.9% and 90.2% respectively. The quantitative value of real-time M. tb. Positive PCR ranged from 32c/ml to 2722 c/ml. CONCLUSIONS: PCR assay is an accurate technique with high sensitivity and specificity to diagnose the DNA genome in infectious uveitis.

Development and application of a novel multiplex polymerase chain reaction for semi-quantitation of human Cytomegalovirus in clinical specimens.

Human Cytomegalovirus (HCMV) is an important cause of morbidity and occasional mortality in immunocompromised patients. The aims of the study were to develop and apply a multiplex PCR for semi-quantitation of HCMV in clinical specimens, compare its efficiency with pp65 antigenemia assay and real-time PCR. A multiplex PCR combining the primers targeting three regions of the HCMV genome, viz. the morphological transforming region II (mtr II), UL-83 and glycoprotein O (gO) genes for the detection of the genome of HCMV was standardized with HCMV AD169 strain. This was evaluated against pp65 antigenemia assay by applying it on 70 peripheral blood specimens obtained from 70 post-renal transplant recipients. The multiplex PCR and a real-time PCR were prospectively applied to 31 clinical specimens from 29 immunocompromised patients. The multiplex PCR was specific for HCMV. The level of antigenemia and the copy number of the viral DNA as estimated by real-time PCR in the samples positive for all the three targets was significantly higher than in those that were positive for only one or two of the targets. The multiplex PCR provides a simple and effective means of quantifying HCMV in clinical specimens with efficiency equivalent to the pp65 antigenemia assay and real-time PCR.

Nucleotide polymorphisms associated with Internal Transcribed Spacer (ITS) regions of ocular isolates of Aspergillus flavus.

PURPOSE: To analyse the genetic similarity among ocular isolates of Aspergillus flavus by Polymerase chain reaction based Restriction Fragment Length Polymorphism (PCR-RFLP) and DNA sequencing. MATERIALS AND METHODS: Seven ocular isolates of A. flavus from 5 patients (3 from paient 1, and four isolates from patients no. 2, 3, 4, and 5 respectively) consisting of 2 Aqueous Humor (AH), 2 Vitreous fluid (VF), 1 eviscerated material, 1 corneal button were included in the study. The three specimens from 1 were one each of AH, VF and corneal button. The fungal isolates were amplified using primers targeting ITS region and the amplicons were subjected to PCR-RFLP using Hae-III enzyme and DNA sequencing to analyse the genetic similarity. RESULTS: A. flavus isolates yielded a specific product of 595 bp after amplification. All the seven A. flavus isolates showed similar pattern of digestion with Hae-III . However, DNA sequencing of ITS amplicons revealed 97.7% genetic similarity and 2.3% dissimilarity with nucleotide polymorphisms -- single, double and multiple pertaining to inversion, substitution, insertion and deletion in comparison with that of standard strain of A. flavus ATCC 16883 [Accession Number ]. A. flavus isolated from AH, VF and corneal button from the same patient showed similar nucleotide polymorphisms as against other isolates which exhibited distinct polymorphisms. This pattern of nucleotide polymorphisms in A. flavus isolates is novel and first time reported in literature to the best of our knowledge. CONCLUSION: DNA sequencing proves to be a useful molecular tool in screening for nucleotide polymorphisms among fungal isolates.

Toxoplasma gondii detection by nested polymerase chain reaction in lens aspirate and peripheral blood leukocyte in congenital cataract patients: the first report from a tertiary eye hospital in India.

PURPOSE: To detect T. gondii DNA and specific antibodies in lens aspirates (LA) and peripheral blood leucocytes (PBL) of congenital cataract patients. METHODS: ELISA for T. gondii antibodies on sera nPCR for T. gondii DNA (B1 gene) on LA and PBL were performed for 52 patients. RESULTS: T. gondii DNA was detected in 29 (55.8%) of the 52 patients (LA-14, PBL-13, LA and PBL-2, and specific IgM in 2 sera). nPCR in PBL was more sensitive than ELISA (p<0.005). CONCLUSION: nPCR is a sensitive technique to detect T. gondii from LA and PBL in congenital cataract patients.

Comparative efficacy of PCR-based restriction fragment length polymorphism (RFLP) & multiplex PCR for glycoprotein B (gB) genotyping of human cytomegalovirus.

BACKGROUND & OBJECTIVE: Glycoprotein B (gB), involved in cell-to-cell transmission of human cytomegalovirus (HCMV), is a critical factor in tissue tropism and viral pathogenesis. The aim of the present study was to compare the efficiency of PCR-based RFLP and multiplex nested PCR for gB gene of HCMV to determine their genotype in clinical specimens from patients with HCMV. METHODS: The PCR based RFLP and the multiplex nested PCR were applied on standard strain of HCMV AD169, 4 clinical HCMV isolates and 70 clinical specimens positive for HCMV by pp65 antigenaemia assay or nested PCR for mtr II region or both. RESULTS: Three of the four clinical isolates were genotyped as gB1 and the other as gB3 by both the methods. HCMV genome in all the 70 clinical specimens were genotyped by multiplex nested PCR whereas only 65 were genotyped by PCR-based RFLP. Forty one of 65 clinical specimens, gave concordant results by both methods. In the remaining 24, mixed infection with multiple genotypes was identified by multiplex nested PCR whereas single genotypes were identified by PCR-based RFLP. INTERPRETATION & CONCLUSION: Multiplex nested PCR provided a rapid, sensitive and cost-effective assay for gB genotyping of HCMV and allowed detection of multiple gB genotypes of HCMV in clinical samples compared to PCR-based RFLP.