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Purpose: To standardize a novel duplex polymerase chain reaction (PCR) targeting 18S rRNA gene and internal transcribed spacer region for the identification of Pythium insidiosum isolates and also to detect P. insidiosum genome directly from corneal specimens of patients with suspected ocular pythiosis., Methods: A total of 42 nonsporulating molds culturally and morphologically resembling suspected unidentified fungal isolates (corneal buttons 33 and corneal scrapings 9) and 14 clinical specimens (corneal buttons 7 and corneal scrapings 7) clinically suspected to be ocular pythiosis were included in the present study. Standardization of uniplex PCRs and duplex PCRs targeting 18S rRNA gene and internal transcribed spacer region and further application of the standardized PCRs on both clinical isolates and clinical specimens suspected to have fungal keratitis. The sensitivity and specificity of the standardized duplex PCR were calculated using Medcal.net software., Results: The standardized uniplex and duplex PCRs were found specific for the detection of only P. insidiosum DNA, and the analytical sensitivities of the primers were 1.36 Zg. Of the 14 clinical specimens analyzed, 13 were positive in both corneal specimens and their respective P. insidiosum isolates. The specificity of the novel duplex PCR was 100% when applied on corneal specimens and clinical isolates, but the sensitivity was 92.8% (13/14) and 100% (42/42), respectively, for the clinical specimens and fungal isolates from suspected ocular pythiosis patients included in the study., Conclusions: The novel duplex PCR developed in this study will aid in rapid identification of P. insidiosum clinical isolates and clinical specimens from suspected ocular pythiosis specimens, which in turn will help the ophthalmologists to initiate appropriate treatment.

There is an urgent need for a rapid and reliable test to detect actively multiplying Mycobacterium tuberculosis directly from clinical specimens for an early initiation of the appropriate antituberculous treatment. This study was aimed at the optimization and application of nested reverse transcriptase-PCR (nRT-PCR) targeting the messenger RNA of the icl2, hspx, and rRNAP1 genes directly from sputum specimens, and their evaluation against the culture by the BACTEC MicroMGIT mycobacterial culture system. 203 Sputum samples from clinically suspected tuberculosis patients and 30 control specimens (clinically proven viral or bacterial infections other than tuberculosis) were included in this study. The mycobacterial culture was performed by the BACTEC MicroMGIT system following the manufacturer's instructions. The primers for nRT-PCRs targeting icl2, hspx, and rRNAP1 genes were indigenously designed using the Primer-BLAST software, and optimized for sensitivity and specificity. The icl2, hspx, and rRNAP1 genes were able to pick up 63.9%, 67.2%, and 58.75%, respectively, of culture-negative sputum specimens collected from clinically suspected tuberculosis patients. However, three (1.4%) were negative for nRT-PCR, but M. tuberculosis culture positive. All the 30 controls were negative for culture by the BACTEC MicroMGIT method and all three nRT-PCR. The novel nRT-PCRs targeting icl2, hspx, and rRNAP1 genes developed in this study are rapid and reliable diagnostic tools to detect viable M. tuberculosis directly from sputum specimens. However, further study by including a larger number of sputum specimens needs to be carried out to ascertain the diagnostic utility of the novel nRT-PCRs optimized in the study., (Copyright © 2015 Asian African Society for Mycobacteriology. Published by Elsevier Ltd. All rights reserved.)

Purpose: Pterygium is a fibrovascular disease that originates in the conjunctiva and commonly spreads to the corneal surface, thereby posing a threat to eyesight. Despite intensive research, the pathophysiology of this disease remains unclear. Recent research suggests that oncogenic viruses, such as human papillomavirus (HPV), cytomegalovirus, and Epstein–Barr virus (EBV), may play a role in pterygia development. Although there are questions concerning the function of oncogenic viruses in pterygium pathogenesis, existing research shows a lack of consensus on the subject, demonstrating the heterogeneity of pterygium pathophysiology. Therefore, we aimed to simultaneously detect the three common viral pathogens that have been reported in pterygium tissue obtained after excision. Methods: Thirty-five tissue specimens of pterygium from patients undergoing pterygium surgery (as cases) were analyzed for evidence of viral infection with multiplex polymerase chain reaction (PCR), and virus-specific real-time quantitative PCR was used for the samples that were detected positive by multiplex PCR. Results: Of the 35 patients, one sample was positive for EBV and two samples were positive for HPV. Further PCR-based DNA sequencing of the HPV PCR-positive product showed identity with HPV-16. Real-time quantitative PCR on samples that showed EBV or HPV positivity did not yield any detectable copy number. Conclusion: Our study results confirmed that PCR positivity could be due to transient flora, but it was not quantitatively significant to conclude as the causative factor of pterygium pathogenesis. However, additional studies with larger sample populations are warranted to fully determine the role of the virus in pterygium. [ABSTRACT FROM AUTHOR]

Purpose: To assess the clinical and microbiological characteristics of nontuberculous mycobacterial (NTM) keratitis and to evaluate their response to medical therapy. Methods: Sixteen patients of NTM keratitis were retrospectively reviewed from May 2014 to May 2019. Laboratory diagnosis were made using Ziehl-Nielsen acidfast staining, routine culture method of isolation of nontuberculous mycobacteria and further identification of species by PCR (polymerase chain reaction)-based DNA sequencing targeting the heat shock protein-65 (hsp-65) gene. Results: Sixteen patients of microbiologically proven NTM keratitis were included. The average age at the time of presentation was 43.56 years (range, 24--73 years). The mean duration of symptoms was 2.23 months. The commonest risk factor was injury with organic material (43.7) followed by ocular surgery (25%). The majority of the nontuberculous mycobacteria were Mycobacterium abscessus (87.6%) followed by M. fortuitum (6.2%) and M. chelonae (6.2%). The in vitro sensitivity showed maximum sensitivity to Amikacin (AMK; 100%) followed by Azithromycin (AZM; 85.7%), and Clarithromycin (CLR; 85.7%). Out of a total of 16 patients, 12 (75%) had total success with medical therapy while 4 (25%) required surgical intervention. Conclusion: This study is focused on rapid and reliable identification of NTM keratitis through PCR-based identification method to enable effective medical management. The antibiotic susceptibility testing of different subspecies of NTM further reduced the need for surgical intervention. The effective role of AMK either alone or in combination with macrolide antibiotics is also highlighted in this study. [ABSTRACT FROM AUTHOR]

To report a case of bilateral microsporidiosis with coexisting fungal infection in one eye. Retrospective interventional case report. A 61-year-old man with uncontrolled diabetes presented with clinical and microbiological features of non-resolving fungal keratitis in the right eye since 3 months and underwent therapeutic penetrating keratoplasty (TPK) for the same. Fungal filaments along with oval bodies suspicious of microconidia were noted on calcofluor stain. A week following TPK, the patient presented with features of viral keratouveitis in the left eye which on microbiology was confirmed as microsporidiosis. Retrospectively, the right eye microbiology slides were reassessed, which confirmed the coexistence of fungus with microsporidiosis by acid-fast stain and polymerase chain reaction. Structural resemblance of microconidia with microsporidial spores can be misleading, thus creating a need for awareness regarding the possible coexistence along with a need to suspect microsporidiosis in nonresponding clinically resembling viral keratitis. [ABSTRACT FROM AUTHOR]