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.

First case series of emerging Rickettsial neonatal sepsis identified by polymerase chain reaction-based deoxyribonucleic acid sequencing.

Purpose: To detect and identify the aetiological agent in the peripheral blood from the cases of neonatal sepsis. Materials and Methods: Four neonates from geographically different regions of South India presented with signs of neonatal sepsis and all the routine clinical and laboratory investigations were performed. Blood culture by Bac T Alert 3D was negative. To establish the aetiology, polymerase chain reaction (PCR) for eubacterial genome and subsequent amplifi cation with Gram positive and Gram negative primers were performed followed by deoxyribonucleic acid (DNA) sequencing. Results: PCR for the detection of eubacterial genome was positive in all the four neonates and further amplifi cation with designed Gram positive and Gram negative primers revealed the presence of Gram negative bacteria. The amplicons were identifi ed as Orientia tsutsugamushi in three neonates and Coxiella burnetti in the other neonate. Multalin analysis was done to further characterise the strain variation among the three strains. Conclusion: PCR-based DNA sequencing is a rapid and reliable diagnostic tool to identify the aetiological agents of neonatal sepsis. This is the fi rst case series of emerging Rickettsial neonatal sepsis in India.

Diagnosis of Aspergillus fumigatus endophthalmitis from formalin fixed paraffin-embedded tissue by polymerase chain reaction-based restriction fragment length polymorphism.

New molecular biological technique of Polymerase Chain Reaction (PCR) based Restriction Fragment Length Polymorphism (RFLP) can identify the species from paraffin-embedded tissue section. We demonstrated Aspergillus fumigatus fungus by PCR-based RFLP technique from paraffin section of an eyeball of an eight-month-old child removed for endogenous endophthalmitis.

Association of mycobacteria with Eales' disease.

BACKGROUND & OBJECTIVES: Eales' disease is an idiopathic disease resulting in retinal neovascularization, recurrent haemorrhages, with or without retinal detachment predominantly affecting healthy young males (97.6%) in the Indian subcontinent. Inspite of several studies, the aetiology of Eales' disease is not clear. The isolation of Mycobacterium fortuitum from the aqueous humour of a patient with classical Eales' disease, led us to hypothesize that rapid growing nontuberculous mycobacteria (RGNTM), particularly M. fortuitum and M. chelonae could be associated with Eales' disease. We therefore undertook this study to detect DNA of these RGNTM and also of M. tuberculosis in vitreous fluids (VFs) from patients with Eales' disease and non-Eales' disease. METHODS: We developed and optimized seminested polymerase chain reactions (SnPCRs) to detect DNAs of M. fortuitum and M. chelonae on archival ERMs (33) and VFs (19) of Eales' and control patients along with conventional mycobacteriological investigations. RESULTS: In the retrospective study, 70 per cent ERM samples were positive for one or more Mycobacterium spp. tested by snPCR. M. fortuitum and M. chelonae were isolated from two VFs, which were also positive by sn PCR in the prospective study. Statistical evaluation of the results of both retrospective and prospective investigations showed a statistically significant association of Mycobacterium spp. with Eales' disease. INTERPRETATION & CONCLUSION: The results of the present study suggested the involvement of Mycobacterium spp. in the aetiopathogenesis of Eales' disease. Further studies on a larger sample will be required to confirm these findings.

Application of semi-nested polymerase chain reaction targeting internal transcribed spacer region for rapid detection of panfungal genome directly from ocular specimens.

BACKGROUND: The incidence of fungal endophthalmitis has dramatically increased in recent years and rapid detection of fungi using nucleic acid-based amplification techniques is helpful in management. AIM: To evaluate semi-nested polymerase chain reaction (PCR) targeting internal transcribed spacer (ITS) region for detection of panfungal genome in ocular specimens. STATISTICAL ANALYSIS USED: Z test for two proportion. MATERIALS AND METHODS: Standardization of PCR targeting ITS primers was carried out by determining analytical sensitivity and specificity. The sensitivity and specificity of PCR was determined by serial tenfold dilutions of C. albicans (ATCC 24433) DNA and DNA extracts of laboratory isolates of Aspergillus fumigatus, Fusarium lichenicola (4), other fungal and closely related bacterial strains and also human DNA. Semi-nested PCR was applied onto a total of 168 ocular specimens with clinically suspected fungal etiology during 2003-2005. RESULTS AND CONCLUSIONS: PCR was specific and sensitive to detect 1fg of fungal DNA with ITS primers. PCR detected fungal genome in 90 (53.57%) in comparison with the conventional technique, positive in 34 (20.23%) by smear examination and in 42 (25%) by culture. The increase in clinical sensitivity by 28.57% using PCR was found to be statistically significant { P < 0.001 using Z test for two proportion}. The accuracy of the test was found to be 70.85%. PCR proved to be a rapid diagnostic technique for detection of panfungal genome directly from clinical specimens.

Anterior chamber exudative mass due to Scedosporium apiospermum in an immunocompetent individual.

Endogenous intraocular infection of fungal etiology is extremely rare in an immunocompetent individual. Usually, an antecedent history of trauma, surgery, intravenous drug abuse or an immunocompromized state can be elicited. Scedosporium apiospermum is a known cause of keratomycosis after traumatic implantation and can cause fatal disseminated infection in immunocompromized patients. However, cases of S. apiospermum intraocular infection in immunocompetent individuals have been very rarely reported in literature. We report here a case of an anterior chamber exudative mass due to S. apiospermum in an immunocompetent individual which was managed successfully with anterior chamber wash and intravitreal injection of voriconazole.

In-vitro susceptibility testing by agar dilution method to determine the minimum inhibitory concentrations of amphotericin B, fluconazole and ketoconazole against ocular fungal isolates.

PURPOSE: To standardize in-vitro antifungal susceptibility testing by agar dilution method to find out the minimum inhibitory concentration (MIC) of amphotericin B, fluconazole and ketoconazole on ocular fungal isolates. METHODS: A total of 180 ocular fungal isolates (130 filamentous fungi and 50 yeasts) were included. The antifungal drugs such as amphotericin B (0.0625-8 microg/mL), fluconazole (0.2-819.6 microg/mL) and ketoconazole (0.025-6.4 microg/mL) were incorporated in doubling dilutions in the yeast nitrogen base medium. The MIC was determined as the lowest concentration of the antifungal drug preventing growth of macroscopically visible colonies on drug containing plates when there was visible growth on the drug-free control plates. RESULTS: All 50 ocular isolates of yeast were susceptible to amphotericin B, while two (4%) and five (10%) strains were resistant to fluconazole and ketoconazole respectively. Of the 130 filamentous fungi tested, six (4.6%) were resistant to amphotericin B, 49 (37.7%) and 10 (7.6%) were resistant to fluconazole and ketoconazole respectively. Percentile 50 (MIC 50) and Percentile 90 (MIC 90) for all the three antifungal agents were calculated. Aspergillus niger, Aspergillus terreus and Candida krusei were found to be resistant to fluconazole and ketoconazole. CONCLUSION: This technique was found to be reliable, cost effective and easy to perform with consistent results.

Development and application of multiplex polymerase chain reaction for the etiological diagnosis of infectious endophthalmitis.

BACKGROUND: Uniplex polymerase chain reaction (PCR) for detection of bacterial and panfungal genome has been applied onto a large number of intraocular fluids facilitating management of infective endophthalmitis. AIM: To develop and apply a novel, rapid multiplex polymerase chain reaction (mPCR) to detect the presence of eubacterial, Propionibacterium acnes and panfungal genomes in intraocular fluids from patients clinically diagnosed to have infective endophthalmitis. SETTINGS AND DESIGN: Prospective study. MATERIALS AND METHODS: Conventional methods of direct microscopy by KOH/calcofluor mount, Gram's staining and culture were done on 30 (19 Aqueous humor-AH and 11 Vitreous fluid-VF) intraocular specimens and mPCR done for simultaneous detection of eubacterial, P. acnes and panfungal genomes. RESULTS: mPCR detected an infectious etiology in 18 (60%) of 30 intraocular specimens. Eubacterial genome was detected in 12 (40%) specimens, P. acnes genome in 4 (13.3%) specimens and panfungal genome in 2 (6.6%) specimens. mPCR results correlated with those of uniplex PCR. mPCR results were available within 5-6 hours after receipt of specimen, as against 8 hours required for each uniplex PCR with three separate thermalcyclers for their completion. Consumption of Taq polymerase was reduced considerably for mPCR. CONCLUSION: mPCR is a cost effective, single tube method for the simultaneous detection of eubacterial, P. acnes and panfungal genomes in intraocular specimens from patients with infective endophthalmitis. It is a more rapid procedure than uniplex PCRs and requires only a single thermalcycler.