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

AbstractThere 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

Virulence genome analysis of Pseudomonas aeruginosa VRFPA10 recovered from patient with scleritis

Infectious keratitis is a major cause of blindness, next to cataract and majority of cases are mainly caused by gram negative bacterium Pseudomonas aeruginosa (P. aeruginosa). In this study, we investigated a P. aeruginosa VRFPA10 genome which exhibited susceptibility to commonly used drugs in vitro but the patient had poor prognosis due to its hyper virulent nature. Genomic analysis of VRFPA10 deciphered multiple virulence factors and P.aeruginosa Genomic Islands (PAGIs) VRFPA10 genome which correlated with hyper virulence nature of the organism. The genome sequence has been deposited in DDBJ/EMBL/GenBank under the accession numbers LFMZ01000001-LFMZ01000044

Culture-positive unilateral panophthalmitis in a serology-positive case of dengue hemorrhagic fever

Dengue fever, a mosquito-borne disease commonly found in the tropics, is one of the most prevalent forms of Flavivirus infection in humans. Symptomatically, it is characterized by fever, arthralgia, headache, and rash. Ophthalmic manifestations can involve both the anterior and posterior segment. Panophthalmitis is rare in dengue hemorrhagic fever, and there is no report of culture-positive panophthalmitis in this setting. Here, we report a case of a serology-positive 33-year-old male patient of dengue hemorrhagic fever who developed sudden onset pain, redness, and proptosis in the right eye. The patient subsequently developed panophthalmitis in his right eye, and Bacillus cereus was isolated from eviscerated sample. This case provides unique insights into pathogenesis of panophthalmitis in dengue and highlights the management options

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