Role of polymerase chain reaction-based viral detection in pterygia.

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.

Molecular detection and identification of fungal pathogens infections occurring in COVID-19 recovered patients.

The major outbreak of Corona virus disease COVID-19 caused by SARS-CoV-2 had brought about 4.55 million deaths and had shaken the health care system all over the world. From the year 2020 the recovered COVID-19 patients had started to develop microbial infection, most predominantly fungal infection in which Mucormycosis gained immediate attention as it has worsen the mortality rate in humans. In the present study of 53 COVID-19 recovered patients presented with microbial infection, the analysis of frequency distribution of fungal infection preponderantly with Rhizopus oryzae, followed by Aspergillus and Candida species.

Draft Genome Sequence of Mycobacterium tuberculosis Strain SNMICRO 2047-20, Isolated from Intraocular Infection.

Here, we communicate the draft genome sequence of an ocular Mycobacterium tuberculosis strain (SNMICRO 2047-20) that was isolated from the vitreous fluid of a patient diagnosed with endophthalmitis. The genome sequence was 4,391,538 bp long with 3,898 protein-encoding genes and clustered to the East African-Indian lineage.

Clinico-microbiological Profile of Nontuberculous Mycobacterial Keratitis.

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 acid-fast 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.

Coexistence of Fungal Keratitis in Bilateral Sequential Microsporidial Keratitis - A Rare Case Presentation.

AIM: To report a case of bilateral microsporidiosis with coexisting fungal infection in one eye. METHOD: Retrospective interventional case report. RESULTS: 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. CONCLUSION: 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.

First report of Mortierella wolfii causing fungal keratitis from a tertiary eye hospital in India.

A young 33 year old male presented with non-resolving corneal infiltrate for 2 month duration in the right eye. KOH/ Calcoflour wet mount revealed sparsely septate fungal hyphae. Post therapeutic penetrating keratoplasty 3 doses of intracameral voriconazole(100µg/0.1ml) was administered suspecting recurrence. Fungal culture revealed non sporulating mould on SDA. PCR based DNA sequencing targeting the ITS region identified the fungal isolate as Mortierella wolfii (M. wolfii) belonging to zygomycetes. To the best of our knowledge, this is the first report of human fungal keratitis caused by M. wolfii.

Novel Duplex Polymerase Chain Reaction for the Rapid Detection of Pythium insidiosum Directly From Corneal Specimens of Patients With Ocular Pythiosis.

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.

Nested reverse transcriptase-polymerase chain reactions targeting the messenger RNA of icl2, hspx, and rRNAP1 genes to detect viable Mycobacterium tuberculosis directly from clinical specimens.

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.

Detection of novel and reported mutations in the rpoB, katG and inhA genes in multidrug-resistant tuberculosis isolates: A hospital-based study.

The objective of this study was to detect mutations associated with isoniazid (INH) and rifampicin (RIF) resistance in Mycobacterium tuberculosis isolates from newly diagnosed and previously treated tuberculosis patients using a PCR-based DNA sequencing technique. Phenotypic drug susceptibility testing was performed using a BACTEC™ MicroMGIT Culture System in 354 M. tuberculosis isolates. Among the 354 isolates, 18 were multidrug-resistant tuberculosis (MDR-TB). PCR-based DNA sequencing was performed targeting the rpoB gene for RIF and the whole of the katG gene and the promoter and coding region of the inhA gene for INH. Results were analysed using MultAlin analysis to identify the presence of polymorphisms or mutations by comparing with already available GenBank sequences. Only 37.5% of RIF-resistant isolates showed the presence of the most commonly reported mutation (Ser531Leu). The most commonly reported mutation (Ser531Leu) was detected in six MDR-TB isolates. The frequency of mutations associated with INH resistance was 31.5% (17/54) and 29.6% (16/54) for katG and inhA, respectively. Comparing the relative distribution of mutations in the two target loci revealed that 12 isolates (22.2%) had a mutation in both katG and inhA. Apart from previously reported mutations in the katG gene, there were three novel deletion and six novel substitution mutations. As reported in previous studies, Ser531Leu was the most common mutation detected in RIF-resistant isolates. The genetic mechanism of INH resistance in M. tuberculosis is highly complex involving several genes, and much remains to be explored to achieve a better understanding of this complex mechanism.

Whole-Genome Sequencing and Mutation Analysis of Two Extensively Drug-Resistant Sputum Isolates of Mycobacterium tuberculosis (VRFCWCF XDRTB 232 and VRFCWCF XDRTB 1028) from Chennai, India.

We announce the draft genome sequence of two extensively drug-resistant Mycobacterium tuberculosis strains, VRFCWCF XDRTB 232 and VRFCWCF XDRTB 1028, isolated from the sputum samples of a patient clinically suspected to have tuberculosis, and we also report novel mutations that confer drug resistance.

Whole-Genome Sequencing of Streptomycin-Resistant Mycobacterium tuberculosis Isolate VRFCWCF MRTB 180 Reveals Novel and Potential Mutations for Resistance.

We announce the draft genome sequence of a streptomycin monoresistant Mycobacterium tuberculosis strain (VRFCWCF MRTB 180) isolated from sputum of a clinically suspected tuberculosis patient.

Whole Genome Sequence of Polyresistant Mycobacterium tuberculosis CWCFVRF PRTB 19 Sputum Isolate from Chennai, India, Closely Clustering with East African Indian 5 Genogroup.

We announce the draft genome sequence of a polyresistant Mycobacterium tuberculosis strain (CWCFVRF PRTB 19) isolated from the sputum of a clinically suspected tuberculosis patient, and it closely clusters to the East African Indian 5 (EAI5) lineage.

Draft Genome Sequence of Multidrug-Resistant Mycobacterium tuberculosis Strain CWCFVRF MDRTB 670, Isolated from the Sputum of a Patient from Chennai, India, with Clinically Suspected Tuberculosis.

We announce the draft genome sequence of a multidrug-resistant Mycobacterium tuberculosis strain (CWCFVRF MDRTB 670) isolated from sputum from a patient with clinically suspected tuberculosis.

Isolation of Mycobacterium massiliense from a corneal biopsy in India.

INTRODUCTION: Rapidly growing mycobacteria (RGM) are ubiquitous and are usually considered as saprophytes, and have been recovered from the environment, particularly in dust, watery soil and water distribution systems. However, Mycobacterium massiliense is a rare causative agent of ocular infection. CASE PRESENTATION: We report a case of M. massiliense in a 44-year-old female with signs and symptoms of a corneal ulcer. We carried out PCR-based DNA sequencing targeting the hsp 65 gene for the identification of M. massiliense. To confirm the identification, we also performed PCR-based RFLP targeting the hsp65 gene and PCR-based DNA sequencing targeting the internal transcribed spacer region, which showed 97 % nucleotide identity with M. massiliense. CONCLUSION: To the best of our knowledge, this is the first study in India to report the detection of M. massiliense from a corneal biopsy.