Internalisation of hepatitis C virus core protein by human conjunctival fibroblasts.

Recent studies indicate that hepatitis C virus (HCV) proteins can mediate innate immune response and inflammation in conjunctival fibroblasts which contributes to the pathology of dry eye condition associated with chronic HCV infection. The present study investigates the phagocytic potential of human conjunctival fibroblasts (HCFj) for HCV core protein. HCFj cells were incubated with HCV core antigen for different periods of time, and fluorescent micrographs were taken to observe protein internalisation. HCFj cells were capable of internalising HCV core antigen within 1 h; this gives an insight into another molecular mechanism which may contribute towards HCV‑associated conjunctival inflammation.

Mebiolgel, a thermoreversible polymer as a scaffold for three dimensional culture of Huh7 cell line with improved hepatocyte differentiation marker expression and HCV replication.

Purpose: A novel three dimensional (3D) culture system purely synthesised from co‑polymer which is free from biological contamination for Huh7 cell cultivation and hepatitis C virus (HCV) replication has been attempted. Materials and Methods: Mebiolgel, a thermo‑reversible gelation polymer was used as a 3D scaffold for culturing Huh7, a liver carcinoma cell line used in our study. The 3D culture of the cells were infected with cell culture derived HCV. Result: The scaffold supported the cell growth as 3D spheroids for up to 63 days. Moreover mebiolgel was found to be improving the hepatocyte differentiation of Huh7 cells at the transcript level. Three dimensional culture was susceptible for HCV infection, and this was confirmed by detecting the HCV replication intermediate viral core antigen. Conclusion: Mebiolgel based culture system was proven to be suited for 3D culture of Huh7 cells by improvising liver specific genotypic expression and was susceptible for HCV replication. Since mebiolgel based Huh 7 express better hepatocyte differentiation markers genotypically, this can be implemented as an alternate for primary hepatocytes in studies such as viral isolation from patient serum.

Optimisation and analysis of polymerase chain reaction based DNA sequencing for genotyping polyoma virus in renal transplant patients: A report from South India.

Purpose: To optimise a polymerase chain reaction (PCR) based DNA sequencing technique for genotyping polyoma virus in clinical specimens obtained from renal transplant patients. Materials and Methods: A hundred and thirty (106 peripheral blood and 24 urine) clinical specimens collected from renal transplant patients were included in the study for detecting the presence of DNA of BK virus (BKV), JC virus (JCV) by PCR targeting the viral protein 1 (VP1) gene. PCR based DNA sequencing was performed to determine the genotypes of polyoma virus and subjected to bioinformatics analysis to determine the amino acid sequences and screen for mutations in the VP1 gene. Results: Polyoma virus was detected in 23 (17.69%) specimens of which 19 (82.60%) were positive for BK virus, 3 (13.04%) for JC virus and 1 for both BK and JC virus. PCR based DNA sequencing detected BK virus genotype I in 12 (50%), genotype IV in 8 (33.3%) and JC virus in 4 (16.6%) clinical specimens. BKV genotype I was the predominant genotype (64.2% in peripheral blood and 33.33% in urine) prevalent in south India. Six novel mutations were found – at position 29, 30 to 47 of BKV genotype I; at position 11 and 15 of BKV genotype IV and at position 2 and 30 of JCV. Conclusion: BKV genotype I is the prominent genotype in India and novel mutations detected in the VP1 gene of BKV and JCV are being reported for the fi rst time in literature.

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.

Chikungunya virus iridocyclitis in Fuchs’ heterochromic iridocyclitis.

We are reporting a case of bilateral Fuchs’ heterochromic iridocyclitis with chikungunya virus infection in the left eye. A 20-year-old female was presented with a past history of fever suggestive of chikungunya with bilateral Fuchs’ heterochromic iridocyclitis and complicated cataract. She had a tripod dendritic pattern of keratic precipitates by confocal microscopy in the left eye with a stippled pattern of keratic precipitates in both eyes. The real-time polymerase chain reaction (RT-PCR) assay in the aqueous humor detected 98 copies/ml of chikungunya virus RNA. The patient underwent clear corneal phacoemulsification with in-the-bag intraocular lens implantation in the left eye with a good visual outcome. This is the first report where the presence of chikungunya virus RNA has been associated with a case of bilateral Fuchs’ heterochromic iridocyclitis.

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

Background & objectives: 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 40C 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 & conclusions: 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.

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.

Aciclovir resistance among Indian strains of Herpes simplex virus as determined using a dye uptake assay.

Resistance to aciclovir (ACV) among Herpes simplex virus (HSV) isolates is increasingly being reported in the literature particularly in immunocompromised patients. However, there is only limited data available from India despite widespread use of ACV in our hospital. A cross-sectional study was hence conducted to determine the aciclovir (ACV) susceptibility of HSV 1 and 2 isolates using a dye uptake (DU) assay. This study showed a 3.0% prevalence of ACV resistance among HSV-1 strains (2/66, median IC 50 0.098 microg/mL) while in HSV-2 strains, it was 7.8% (5/64, median IC 50 0.195 microg/mL). The IC 50 for the HSV-1 and HSV-2 strains resistant to ACV was greater than or equal to 6.25 microg/mL.

Nested reverse transcription polymerase chain reaction for the detection of rubella virus in clinical specimens.

BACKGROUND & OBJECTIVE: Rubella virus (RV) is one of the leading causes of childhood blindness in India. In this study we applied an optimized nested reverse transcription polymerase chain reaction (nRT-PCR) to detect RV in clinical specimens. METHODS: nRT-PCR was optimized using total RNA extracted from standard strain of RV using nested sets of primers specific for E1 open reading frame. nRT-PCR was applied onto 30 lens aspirates and corresponding peripheral blood leucocytes of 30 infants with congenital (29)/ developmental (01) cataract. Serology for anti-RV IgG and IgM antibodies was done. RV isolation was attempted using Vero and SIRC cell cultures. RESULTS: Optimized nRT-PCR was specific for RV and sensitive to detect 10 fg of RV RNA. Among 30 patients, nRT-PCR detected presence of RV in lens aspirates of 6 (20%) and 4 corresponding leucocytes. RV was isolated from 3 (10%) lens aspirates (nRT-PCR positive) of the 30 patients. Sera of these 6 patients showed presence of anti-RV IgG and IgM in one, only anti-RV IgG in 3 others and none in the other two. Of the remaining 24 patients, anti-RV IgG was detected in 3 and no anti-RV IgM antibodies in others. INTERPRETATION & CONCLUSION: Findings of our study showed that the nRT-PCR was a more sensitive and rapid technique to detect RV from lens aspirates compared to conventional methods of virus isolation and serology.

Optimization of a polymerase chain reaction (PCR) for increasing its sensitivity to detect Chlamydia pneumoniae specific genome.

Being an intracellular parasite, Chlamydia pneumoniae disseminates to organs outside the respiratory tract and causes chronic diseases in human. Nucleic acid-based method such as polymerase chain reaction (PCR) as diagnostic test has greater sensitivity and specificity than conventional microbiological techniques. The PCR protocol consisting of touchdown technique to detect C. pneumoniae DNA using major outer membrane protein gene (MOMP) was carried out in our laboratory as described in reference paper, but analytical sensitivity reported in it was not reproducible. Hence, the PCR was optimized after modifications in annealing temperature and magnesium ion concentrations. First round PCR profile with annealing at 56 degrees C for 8 cycles followed by 32 cycles with annealing temperature maintained at 54 degrees C and second round profile modified with annealing temperature maintained at 49 degrees C had resulted in 3-fold increase in clinical sensitivity. The present work highlights the importance of optimization of PCR in laboratory settings.

Cultivation of human corneal limbal stem cells in Mebiol gel--A thermo-reversible gelation polymer.

BACKGROUND & OBJECTIVES: Cultivated limbal stem cell transplantation is being used as a current treatment modality for limbal stem cell deficiency. However, use of allogenic biological material as substrate is associated with risks of transmission of certain diseases and allograft rejection. Therefore development of non-toxic biodegradable synthetic polymers is important. We undertook this study to evaluate the use of a synthetic polymer Mebiol gel as a substrate for the growth of limbal phenotype cells and cornea phenotype cells from limbal explants. METHODS: Human cadaveric limbal explants cells were cultivated on Mebiol gel. The proliferative capacity of cultivated cells was analyzed with thymidine incorporation studies. Immunostaining for presumed limbal stem cell association markers and cornea differentiation markers was performed and confirmed with reverse transcription (RT-PCR). RESULTS: The limbal explants underwent proliferation in vitro. The cultivated cells expressed the presumed limbal stem cell association markers (ABCG2 and p63), the transient amplifying cell markers (connexin 43, integrin alpha9) and the cornea differentiation marker (K3). RT PCR confirmed the immunohistochemical data. INTERPRETATION & CONCLUSION: Our findings showed that the synthetic polymer Mebiol gel was able to support limbal explant proliferation. The cultured cells expressed presumed limbal stem cell association markers, transient amplifying cells and cornea phenotype markers. Mebiol Gel can be used as a scaffold for growing limbal explants.

Application of polymerase chain reaction to differentiate herpes simplex virus 1 and 2 serotypes in culture negative intraocular aspirates.

PURPOSE: To standardize and apply a polymerase chain reaction (PCR) on the glycoprotein D gene to differentiate Herpes simplex virus (HSV) 1 & 2 serotypes in culture negative intraocular specimens. METHODS: Twenty-one intraocular fluids collected from 19 patients were subjected to cultures for HSV and uniplex PCR (uPCR) for DNA polymerase gene. To differentiate HSV serotypes, as 1 & 2, a seminested PCR (snPCR) targeting the glycoprotein D gene was standardised and applied onto 21 intraocular fluids. The specificity of the snPCR was verified by application onto ATCC strains of HSV 1 and 2, clinical isolates and DNA sequencing of the amplified products. All specimens were also tested for the presence of cytomegalovirus (CMV) and varicella zoster virus (VZV) by nucleic acid amplification methods. RESULTS: Four of the 21 intraocular fluids were positive for HSV by uPCR. snPCR detected HSV in three additional specimens (total of seven specimens), and identified three as HSV 1 and four as HSV 2. DNA sequencing of PCR products showed 100% homology with the standard strains of HSV 1 and 2 respectively. None of the samples were positive in culture. Among the other patients, CMV DNA was detected in two and VZV DNA in five others. CONCLUSIONS: The standardized snPCR can be applied directly onto the culture negative specimens for rapid differentiation of HSV serotypes.

Relative susceptibility of six continuous cell lines for cultivation of chlamydia trachomatis strains.

Since susceptibility of a cell line is an important factor for cultivation of Chlamydia trachomatis, McCoy, HeLa, BHK-21, HEp-2, Vero and A549 cell lines were tested for this characteristic. These were inoculated with 150 infection-forming units (IFU) of C. trachomatis A, B, Ba and C serovars. Growth was graded according to the number of IFUs per microscopic field (100X). A549-cell line was not susceptible to infection by any of the serovars. The growth of C. trachomatis was good to very good in McCoy and HeLa cell lines. Vero, BHK-21 and HEp-2 cell lines varied considerably in the susceptibility to infection.

Polymerase chain reaction to detect Chlamydia trachomatis and adenovirus in the nasopharyngeal aspirates from paediatric patients with lower respiratory infections.

Polymerase chain reaction (PCR) was evaluated to detect Adenoviruses and Chlamydia trachomatis on nasopharyngeal aspirates (NPA) obtained 4-5 days after the onset of lower respiratory tract illness in children. Forty-five nasopharyngeal aspirates (NPA) from 45 children with lower respiratory tract infections were processed for the detection of C. trachomatis and Adenovirus by Fluorescent antibody test (FAT), culture and PCR for the cryptic plasmid of C. trachomatis and the gene coding for hexon of Adenoviruses. Seven (13.3%) and 4 (6.6%) of the 45 specimens were positive for C. trachomatis and adenovirus by PCR respectively, which included one specimen each positive for these agents. Cultures were negative for both the organisms. PCRs showed a statistically significant (McNemar test--p= 0.004) higher sensitivity. PCR test is necessary to detect C. trachomatis and adenovirus in nasopharyngeal aspirates obtained 4-5 days after the onset of illness.

Use of uniplex polymerase chain reaction & evaluation of multiplex PCR in the rapid diagnosis of viral retinitis.

BACKGROUND & OBJECTIVES: Polymerase chain reaction (PCR) has been known to be a rapid and accurate diagnostic test for causative viruses of viral retinitis, but cost is the limiting factor. In the present study an attempt was made to standardize a multiplex PCR (mPCR) on intraocular specimens from patients with viral retinitis for the detection of one or more viruses [herpes simplex virus (HSV), varicella zoster virus (VZV) or cytomegalovirus (CMV)] in order to reduce the period of time required for uniplex polymerase chain reaction (uPCR). METHODS: Using the uniplex PCR (uPCR) primers, a nested mPCR was developed and standardized for the simultaneous detection of HSV, VZV and CMV. mPCR and uPCRs were applied on 9 stored specimens and 38 prospective specimens obtained from patients with viral retinitis. RESULTS: The specificity and sensitivity of the mPCR were concordant with that of uPCRs. Clinical specificity and sensitivity of mPCR was further confirmed by the detection of the same herpes viral DNA on the 9 stored specimens. Of the 38 specimens collected prospectively, mPCR detected HSV in 3 (7.9%), VZV in 9 (23.7%), CMV in 5 (13.2%) and both VZV and CMV in 2 (5.3%). Co-infections of two viruses were found in 7 (14.89%) of the 47 specimens. INTERPRETATION & CONCLUSION: mPCR is a rapid, specific and sensitive diagnostic tool in viral retinitis. Compared to uPCR, mPCR is less time-consuming and cost effective.

A hospital based study on the prevalence of conjunctivitis due to Chlamydia trachomatis.

BACKGROUND & OBJECTIVES: Very few studies have been done in India to determine the prevalence of Chlamydia trachomatis causing conjunctivitis using polymerase chain reaction (PCR) methods. Hence the prevalence of primary conjunctivitis due to C. trachomatis among individuals attending ophthalmic hospitals in Chennai was determined and compared with our earlier results. METHODS: A total of 328 conjunctival swabs from 255 (both eyes 73 and one eye 182) patients were investigated by fluorescent antibody test (FAT) on direct smears, culture and PCRs for cryptic plasmid and major outer membrane protein (MOMPI) gene of C. trachomatis. An infant with ophthalmia neonatorum was also included. RESULTS: Among these 328 specimens processed, 16 (4.9%) from 12 (4.7%) patients were positive by cryptic plasmid PCR. Among these, 3 from 2 patients were positive by FAT (direct smear), culture and PCR for MOMP 1 gene. Both eyes of the infant with ophthalmia neonatorum were positive by all the methods. The sensitivity of FAT and culture (18.8%) was lower compared to PCR. INTERPRETATION & CONCLUSION: A significant decrease in the prevalence of adult chlamydial conjunctivitis has occurred in the 10-year period among patients reporting to the ophthalmic hospitals in Chennai. PCR using cryptic plasmid primers was found to be the most sensitive method to detect C. trachomatis in patients with conjunctivitis.

Laboratory methods in the detection of herpes simplex virus (HSV) in keratitis--a 9-year study including polymerase chain reaction (PCR) during last 4 years.

Conventional methods of fluorescent antibody test (FAT) and virus isolation (VI) and molecular method of polymerase chain reaction (PCR) were compared for the detection of HSV in keratitis during a 9-year period. Of 186 corneal scraping specimens, 108 were subjected to FATand VI in the pre-PCR period (initial 5 years) while 78 to FAT, VI and PCR in the PCR period (latter 4 years). HSV was detected by FAT in 44/186 (23.7%), VI in 18/186 (9.7%) and PCR in 27/78 (34.6%) specimens. Overall, HSV was diagnosed in 56/186 (30.1%) specimens. PCR has increased the clinical sensitivity by 12.8%, which is statistically significant (McNemar test, P-0.002). VI should be replaced by PCR. FAT though less sensitive should always be employed as a routine to give an early diagnosis, the results of which could be further confirmed, if necessary, by PCR, which is a more sensitive and specific diagnostic tool.

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.

Prevalence of Chlamydia trachomatis & herpes simplex virus in males with urethritis & females with cervicitis attending STD clinic.

BACKGROUND & OBJECTIVES: Cervicitis and urethritis due to Chlamydia trachomatis are common sexually transmitted diseases. However, there is a paucity of information on urethritis and mucopurulent cervicitis due to herpes simplex virus (HSV) from India. We used polymerase chain reaction (PCR) to find out the prevalence of C. trachomatis and HSV associated urethritis in males and mucopurulent cervicitis in females attending a sexually transmitted diseases (STD) clinic. METHODS: Twenty five endocervical swabs from 25 women with mucopurulent cervicitis and 75 urethral swabs from 72 males with urethritis were processed for the detection of C. trachomatis and HSV by antigen detection by fluorescent antibody test (FAT), culture and PCR. RESULTS: Among the 25 women, one (4.0%) was positive for C. trachomatis and 3 (12.0%) were positive for HSV by PCR. FAT and culture were negative. Nine (12.0%) of the 75 urethral swabs were positive for C. trachomatis and 5 (6.6%) were positive for HSV by PCR. Among the 9 positive by PCR for C. trachomatis, 3 (4.0%) were positive by FAT. Cultures for both organisms were negative. INTERPRETATION & CONCLUSION: Endocervicitis and male urethritis due to C. trachomatis and HSV are not uncommon among high-risk individuals. The diagnosis could be established mainly by PCR.