Unusual ulcerative keratitis caused by Prototheca wickerhamii in a diabetic patient

The purpose of the study was to report a case of ulcerative keratitis caused by an unusual algae Prototheca wickerhamii in a diabetic patient. This study design was a case report. A 46-year-old male, who was a known diabetic for 3 years, had an injury to the left cornea with the sparks of fire from wielding at work that developed into an ulcerative keratitis over a period of next 3 months as the patient was not on any medication. Corneal scraping culture report and Vitek 2 system investigation result confirmed it to be a P. wickerhamii infection. The patient was started on intensive topical 1% voriconazole and 5% natamycin for 1 month and with no improvement subsequently underwent penetrating keratoplasty. No recurrence of infection postoperatively was noted. This opportunistic algae rarely known to cause human eye infections is so far reported in either patients with severe systemic immunosuppression causing posterior segment eye involvement or as postcorneal surgery infections. We report an ulcerative keratitis by P. wickerhamii in a diabetic patient post corneal trauma with no prior ocular surgery.

Polymicrobial chronic endophthalmitis diagnosed by culture and molecular technique.

Accurate etiological diagnosis is the key to prevention of ocular morbidity in endophthalmitis cases. A 66 year old male was suffering from chronic endophthalmitis post-cataract surgery. Polymerase chain reaction examination on anterior chamber fluid was positive for Propionibacterium acnes but negative for the panfungal genome. He was advised vitrectomy with intravitreal injections. Polymerase chain reaction of vitreous aspirate was positive for P.acnes as well as panfungal genome. The vitreous sample also grew yeast in culture which was identified as Candida pseudotropicalis. Patient was treated on an alternate day regimen of intravitreal Vancomycin and Amphotericin B in the post-operative period. There was improvement in vision at final follow up. Chronic endophthalmitis can have polymicrobial etiology which will require appropriate diagnostic and therapeutic strategies. The role of molecular testing is vital in these cases as growth in culture is often negative.

Diagnostic appraisal of simultaneous application of two nested PCRs targeting MPB64 gene and IS6110 region for rapid detection of M. tuberculosis genome in culture proven clinical specimens.

Background: Early diagnosis of tuberculosis is critical for its effective management and prevention. Several gene amplifi cation methods are used in the detection of tubercle bacilli from clinical specimens. MPB64 gene and IS6110 region have been identifi ed as potential gene targets for the specifi c detection of Mycobacterium tuberculosis from direct clinical specimens. Objective: The present study was conducted to evaluate the diagnostic utility of simultaneous application of two nested polymerase chain reaction (nPCRs) targeting MPB64 and IS6110 region for the detection of M. tuberculosis genome. Materials and Methods: A total of 100 and 354 clinical specimens from the control group and clinically suspected tuberculosis patients, respectively, were included in the study. nPCRs targeting MPB64 and IS6110 region were performed. Results and Conclusion: All of the 100 clinical specimens from the control group were negative for both nPCRs. Out of the 354 clinical specimens, 339 were positive for both culture and nPCRs, 10 and 5 were positive for culture, and nPCR targeting IS6110 and MPB64 regions, respectively. To conclude, nPCRs targeting MPB64 and IS6110 region are reliable and specifi c targets when applied simultaneously on clinical specimens to attain 100% sensitivity for the detection of M. tuberculosis genome.

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.

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.

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.

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.

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.

Detection and characterization of mutations in Rifampicin resistant Mycobacterium tuberculosis clinical isolates by DNA sequencing.

Background: Multiple Drug Resistant Tuberculosis (MDR-TB) is increasing because of widespread application and results in selection of mutants resistant to other components of short course chemotherapy. Resistance to Rifampicin can be considered as a surrogate marker for MDR-TB and the target gene for detection of rifampicin resistance is the rpo gene. Aims: To detect and characterize mutations in the rpo B region of Rifampicin resistant isolates of Mycobacterium tuberculosis by automated DNA sequencing. Methods: Absolute concentration method was used to determine the MIC of Rifampicin for 44 M. tuberculosis isolates (21 respiratory, 3 ocular, 3 cerebrospinal fluid and 17 biopsies). Automated DNA sequencing was performed in the ABI 310 Genetic Analyser. Results: Five isolates (2 sputa and one each from bronchoalveolar lavage, lymph node and endometrial biopsies) were rifampicin resistant with MIC greater than 128 mg/ml. Three of the five isolates showed mutations. Two of the isolates had the common missense mutation at codon 531(Ser®Leu), the other isolate showed three insertions and two of them did not show any mutation in the sequenced rpo B region. Conclusions: DNA sequencing technique is a rapid, conclusive and more advantageous technique than the conventional susceptibility testing for detection of rifampicin resistance in terms of the risk involved and time consumption.

Application of PCR-based restriction fragment length polymorphism for the identification of mycobacterial isolates.

BACKGROUND AND OBJECTIVE: Conventional identification of mycobacteria is achieved by standard biochemical tests that are time consuming, laborious and is not always conclusive. This study was thus undertaken to standardize a simple, rapid and cost-effective polymerase chain reaction based restriction fragment length polymorphism (PCR-RFLP) using primers coding for the 16S - 23S rRNA spacer region to identify the mycobacterial isolates to the species level. METHODS: The PCR with primers targeting the 16S-23S rRNA spacer region was standardized using the standard mycobacterial strains and applied on 51 clinical isolates. The PCR amplified products were subjected to RFLP using the restriction enzymes, Hae III, MspI and BstXI. The results obtained were compared with those of conventional biochemical tests. RESULTS: PCR was sensitive to detect 2.5 pg of H37Rv DNA (370 bp for slow grower mycobacteria) and 1.5 pg of M. fortuitum DNA (450 bp for rapid grower mycobacteria). Based on the PCRRFLP products obtained the 51 mycobacterial isolates were classified into 41 slow growers and 10 rapid growers. Among the 41 slow growers, 40 were identified as M. tuberculosis, one as M. xenopi and 10 rapid growers as M. fortuitum. INTERPRETATION AND CONCLUSION: PCR using primers targeting the 16S-23S rRNA spacer region was a reliable tool for rapid identification of mycobacterial isolates into slow and rapid growers within 4 h of isolation and further speciation by PCR-RFLP within 6-8 h.

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.

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.

Polymerase chain reaction in the diagnosis of Aspergillus endophthalmitis.

BACKGROUND & OBJECTIVES: Aspergillus endophthalmitis is the commonest type of vision threatening fungal endophthalmitis encountered in India. Since conventional methods lack sensitivity, we evaluated polymerase chain reaction (PCR) against the conventional mycological methods in the diagnosis of Aspergillus endophthalmitis. METHODS: Twenty-seven intraocular specimens from 22 patients with suspected fungal endophthalmitis (proven as non-bacterial origin) and 10 patients with non-infective intraocular disorders (controls) were tested. The intraocular specimens from these patients were subjected to the conventional methods, viz., microscopy and culture for growth of fungi, as well as PCR for the detection and differentiation of species of Aspergillus. RESULTS: None of the controls were positive by microscopy, culture or PCR. Among the 27 test samples, 4 were positive by culture for Aspergillus species, these were also positive by PCR. In addition, PCR detected and identified Aspergillus species in 2 culture negative specimens. The average time required for culture and identification of Aspergillus was 10 days, whereas PCR needed only 24 h. INTERPRETATION & CONCLUSION: This study indicates that PCR was not only a more sensitive, but also a rapid diagnostic tool compared to the conventional mycological methods in the diagnosis of Aspergillus endophthalmitis.

Polymerase chain reaction using IS6110 primer to detect Mycobacterium tuberculosis in clinical samples.

Nucleic acid amplification using IS6110 primers to detect Mycobacterium tuberculosis in clinical specimens has been extensively used as laboratory tool for the diagnosis for tuberculosis. Despite it's dramatic scientific value in practice, it is not as sensitive as expected for the detection of M. tuberculosis. The results of the study suggest that PCR using 123 bp fragment of DNA belonging to IS6110 is specific (95.6%) but only has a sensitivity of 30% to detect M. tuberculosis in clinical specimens.

Polymerase chain reaction using IS6110 primer to detect Mycobacterium tuberculosis in clinical samples.

Nucleic acid amplification using IS6110 primers to detect Mycobacterium tuberculosis in clinical specimens has been extensively used as laboratory tool for the diagnosis for tuberculosis. Despite it's dramatic scientific value in practice, it is not as sensitive as expected for the detection of Mycobacterium tuberculosis. The results of the study suggest that PCR using 123 bp fragment of DNA belonging to IS6110 is specific (95.6%) but only has a sensitivity of 30% to detect M. tuberculosis in clinical specimens.

Spectrum of aetiological agents of postoperative endophthalmitis and antibiotic susceptibility of bacterial isolates.

PURPOSE: To determine the spectrum of infectious agents of postoperative endophthalmitis, the relationship with the time of onset of symptoms after surgery and the antibiotic susceptibilities of the aerobic bacterial isolates. METHODS: A retrospective review of microbiological records from January 1995 to December 1998 yielded 173 isolates from intraocular specimen of 170 patients with culture-proven postoperative endophthalmitis. Antibiotic susceptibility of these isolates was determined for various ocular antibiotics using the Kirby-Bauer disk-diffusion test. Based on the time of onset of illness, clinical presentation was classified into acute, delayed and chronic. RESULTS: Among 170 cases, 71 (41.7%) were attributable to gram-negative, 64 (37.6%) to gram-positive bacteria, and 37 (21.8%) to fungi. Gram-negative bacteria included P. aeruginosa (29;17.1%), other Pseudomonas spp (15;8.8%), non-fermenters (18;10.6%) and others (10;5.8%). Among these, 40 of 72 (55.5%) were sensitive to gentamicin, 47 of 72 (65.2%) to cefotaxime, 47 of 69 (68.1%) to amikacin, 52 of 71 (73.2%) to ciprofloxacin, and 25 of 40 (62.5%) to ceftazidime. The gram-positive bacteria included S. epidermidis (22;12.9%), S. aureus (13;7.6%), P. acnes (10;5.9%), Enterococcus spp (4;2.3%), Streptococcus spp (7;4.1%) and others (8;4.8%). Among these, 41 of 53 (77.3%) were sensitive to gentamicin, 47 of 53 (88.6%) to cefotaxime, 46 of 52 (88.4%) to ciprofloxacin, 38 of 41 (92.6%) to cefazolin and 27 of 37 (72.9%) to ceftazidime. All gram-positive bacteria were sensitive to vancomycin. CONCLUSION: In this large series of postoperative endophthalmitis, gram-negative bacilli followed by fungi accounted for the largest number of cases. A high degree of resistance of gram-negative bacilli to gentamicin, cefotaxime, amikacin and ceftazidime was recorded.