An Update on the Laboratory Diagnosis of Neuromyelitis Optica Spectrum Disorders

Neuromyelitis optica spectrum disorder (NMOSD) is an autoimmune disorder of the central nervous system that is specifically associated with demyelination of spinal cord and optic nerves. The discovery of specific autoantibody markers such as aquaporin-4 IgG and myelin oligodendrocyte glycoprotein IgG has led to several methodologies being developed and validated. There have been numerous investigations of the clinical and radiological presentations used in the clinical diagnosis of NMOSD. However, although various laboratory diagnostic techniques have been standardized and validated, a gold-standard test has yet to be finalized due to uncertain sensitivities and specificities of the methodologies. For this review, the literature was surveyed to compile the standardized laboratory techniques utilized for the differential diagnosis of NMOSD. Enzyme-linked immunosorbent assays enable screening of NMOSD, but they are considered less sensitive than cell-based assays (CBAs), which were found to be highly sensitive and specific. However, CBAs are laborious and prone to batch variations in their results, since the expression levels of protein need to be maintained and monitored meticulously.Standardizing point-of-care devices and peptide-based assays would make it possible to improve the turnaround time and accessibility of the test, especially in resource-poor settings.

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.

First Report on Detection of Dengue Virus in the Donor Cornea.

BACKGROUND: Dengue fever is the most prevalent form of flavivirus infection in humans. We have investigated whether corneoscleral tissue of the donor affected by dengue virus (DENV) harbors the virus. PURPOSE: To identify the risk for viral transmission through corneal transplants in areas where DENV circulates. METHODS: Excised corneoscleral tissue from a cadaver with a history of viral hemorrhagic fever was analyzed using reverse transcriptase-polymerase chain reaction for the presence of DENV and chikungunya virus (CHIV). RESULTS: DENV was detected in RNA extracted from the donor corneoscleral rim. Further genotyping of the viral isolate from the virus-infected cell harvest revealed DENV type 3 as the causative agent. CHIV was not detected. CONCLUSIONS: The data presented in this study recommend the implementation of polymerase chain reaction for detection of DENV and CHIV to analyze excised corneoscleral tissue of a donor with viral hemorrhagic fever.

Expression analysis of toll-like receptors of Dengue-infected cornea by real-time polymerase chain reaction.

BACKGROUND: Toll-like receptors (TLRs) play a significant role based on innate immune mechanism during viral infection. TLR signaling mechanism designates to protect the cells from invading viruses. The expression of TLRs during dengue virus (DENV) infection not yet well explained. This study evaluates the TLR gene expression from DENV-infected patient's cornea. METHODS: Reverse transcriptase PCR was performed for the detection and genotyping of viral nucleic acid from corneal grafts and DENV-infected cell suspension. TLR expression studies were done on DENV-infected cornea by real-time RT2 Profiler PCR Array. RESULTS: The reverse transcriptase PCR and genotyping confirmed the presence of DENV-3. TLR expression studies revealed the upregulated expression of TLR4, TLR7, TLR9 and TLR10. CONCLUSION: Molecular testing of DENV reveals that serological positivity induces transmission of the virus through cornea and stimulates the expression of TLR4, TLR7, TLR9 and TLR10, which may lead to up-regulation of innate pro-inflammatory response in the cornea.

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.

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.

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.

Eales' disease - current concepts in diagnosis and management.

Eales' disease, first described by the British ophthalmologist Henry Eales in 1880, is characterized by three overlapping stages of venous inflammation (vasculitis), occlusion, and retinal neovascularization. Diagnosis is mostly clinical and requires exclusion of other systemic or ocular conditions that could present with similar retinal features. In recent years, immunological, molecular biological, and biochemical studies have indicated the role of human leukocyte antigen, retinal autoimmunity, Mycobacterium tuberculosis genome, and free radical-mediated damage in the etiopathogenesis of this disease. However, its etiology appears to be multifactorial. The management depends on the stage of the disease and consists of medical treatment with oral corticosteroids in the active inflammatory stage and laser photocoagulation in the advanced retinal ischemia and neovascularization stages.

Application of polymerase chain reaction-based restriction fragment length polymorphism in typing ocular rapid-growing nontuberculous mycobacterial isolates from three patients with postoperative endophthalmitis.

PURPOSE: We describe postoperative endophthalmitis caused by rapid-growing nontuberculous mycobacteria (RGNTM) in 3 patients after small-incision cataract surgery with intraocular lens (IOL) implantation performed elsewhere and referred to us for management. Subsequent identification and confirmation was carried out with biochemical tests and polymerase chain reaction-based restriction fragment length polymorphism (PCR-RFLP). MATERIALS AND METHODS: The corneal scraping and eviscerated material of the first patient, the corneal button and the IOL of the second patient, and the corneal scraping of the third patient were processed for routine bacteriologic studies including acid-fast bacilli (AFB) by smear (excepting the IOL) and culture. Subsequent identification of the RGNTM was carried out by using biochemical tests and PCR-RFLP by using primers targeting the heat shock protein 65 region of mycobacteria. RESULTS: AFB smear was positive in all 3 patients. The corneal scraping of the first patient, the corneal button and IOL of the second patient, and the corneal scraping of the third patient were culture positive for RGNTM and were identified as Mycobacterium abscessus in the first and second patients and M. fortuitum sorbitol-positive third biovariant in the third patient. CONCLUSIONS: A clinical suspicion of infection by RGNTM in delayed-onset postoperative endophthalmitis should be considered when resistance to standard therapy is encountered.