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

Therese, K Lily; Gayathri, R; Dhanurekha, L; Ramaswamy, Sridhar; Meenakshi, N.; Hn, Madhavan

doi:10.4103/0255-0857.118887

Cited by 9 publications

(3 citation statements)

References 5 publications

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“…Therese et al in Tamil Nadu concluded that simultaneous use of both targets, IS6110 and MPB64, together on clinical specimens (mostly respiratory specimens) could attain 100% sensitivity in culture proven cases. 21 Addition of MPB64 detected only additional 1.4% of the cases in their study. In a study by Sharma et al the positivity rates of Ziehl-Neelsen smear, culture, and multiplex PCR were 30%, 26.3%, and 91.3%, respectively, in confirmed tubercular lymphadenitis patients.…”

Section: Discussionmentioning

confidence: 83%

Utility of multiplex real-time PCR in the diagnosis of extrapulmonary tuberculosis

Raveendran

Wattal

2016

The Brazilian Journal of Infectious Diseases

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Section: Discussionmentioning

confidence: 83%

Utility of multiplex real-time PCR in the diagnosis of extrapulmonary tuberculosis

Raveendran

Wattal

2016

The Brazilian Journal of Infectious Diseases

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“…Many studies have assessed molecular versus culture methods of detection of bacteria in biological and environmental samples (14,21,22); however, few studies compared alternative methods for confirmation of growth in culture. Many of the methods that are in use for mycobacterial cultures are historical, such as phenotypical differences in colony morphology, biochemical characteristics, and acid-fast staining of smears.…”

Section: Discussionmentioning

confidence: 99%

Efficient, Validated Method for Detection of Mycobacterial Growth in Liquid Culture Media by Use of Bead Beating, Magnetic-Particle-Based Nucleic Acid Isolation, and Quantitative PCR

et al. 2015

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Pathogenic mycobacteria are difficult to culture, requiring specialized media and a long incubation time, and have complex and exceedingly robust cell walls. Mycobacterium avium subsp. paratuberculosis (MAP), the causative agent of Johne's disease, a chronic wasting disease of ruminants, is a typical example. Culture of MAP from the feces and intestinal tissues is a commonly used test for confirmation of infection. Liquid medium offers greater sensitivity than solid medium for detection of MAP; however, support for the BD Bactec 460 system commonly used for this purpose has been discontinued. We previously developed a new liquid culture medium, M7H9C, to replace it, with confirmation of growth reliant on PCR. Here, we report an efficient DNA isolation and quantitative PCR methodology for the specific detection and confirmation of MAP growth in liquid culture media containing egg yolk. The analytical sensitivity was at least 10 4 -fold higher than a commonly used method involving ethanol precipitation of DNA and conventional PCR; this may be partly due to the addition of a bead-beating step to manually disrupt the cell wall of the mycobacteria. The limit of detection, determined using pure cultures of two different MAP strains, was 100 to 1,000 MAP organisms/ml. The diagnostic accuracy was confirmed using a panel of cattle fecal (n ‫؍‬ 54) and sheep fecal and tissue (n ‫؍‬ 90) culture samples. This technique is directly relevant for diagnostic laboratories that perform MAP cultures but may also be applicable to the detection of other species, including M. avium and M. tuberculosis. P aratuberculosis, or Johne's disease, affects many economically important livestock species, including cattle, sheep, goats, and deer (1). It is caused by Mycobacterium avium subsp. paratuberculosis (MAP) and is characterized by chronic enteritis, wasting, and death. Control programs attempting to limit the impact and spread of the disease in many countries are based on the detection of infected animals by using fecal culture, which is one of the most specific antemortem tests for the diagnosis of paratuberculosis infection (2). The gold standard indications for disease confirmation are a pathological lesion consistent with paratuberculosis and a positive culture for MAP from the affected tissues (3).For many mycobacterial species, culture requires long incubation times, from weeks to months, and highly specialized media. For MAP, liquid culture media, such as Bactec 12B medium, have been found to be more sensitive than solid culture media, such as Herrold's egg yolk medium (HEYM) supplemented with mycobactin J (4-6). MAP in cultures with positive growth is confirmed by the demonstration of mycobactin dependency or directly by molecular techniques (7). The advantages of liquid culture media systems include shorter culture incubation times (5, 7), and, importantly, some liquid culture media are capable of supporting the growth of all strains of MAP, including the fastidious sheep strain (8). Monitoring of bacterial growth in liquid cul...

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“…The extracted DNA was stored at -20 degrees C till further use. Nested PCR with 2 sets of primers targeting MPB 64 gene and IS6110 region were carried out [ 14 ]. Real-time PCR for the detection of the Mycobacterial load was estimated in the DNA extracts of the test sample using a commercial kit—Geno Sen’s® MTb Complex RG quantitative and the assay was performed on Rotor-Gene (Hilden, Germany) real-time PCR equipment based on Taqman principle.…”

Section: Methodsmentioning

confidence: 99%

Polymerase chain reaction confirmed mycobacterium tuberculosis intermediate uveitis – analysis of 22 eyes of 14 cases from a tertiary care centre in South India: a retrospective study

Sreenivasan

Jain

Kamalini

et al. 2022

J Ophthal Inflamm Infect

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Background To report the role of Polymerase Chain Reaction in confirming the diagnosis of presumed Mycobacterium Tuberculosis (MTB) Intermediate Uveitis. Method Retrospective analysis of 22 eyes of 14 cases of presumed tubercular intermediate uveitis wherein intraocular fluid was tested for MTB DNA by Nested & Real-time PCR, based on clinical suspicion of tubercular aetiology. QuantiFERON TB gold test and High-Resolution CT Chest were done. Patients were treated with anti-tubercular therapy with oral steroids & immunomodulators. In the study, eleven were male (79%) and three female (21%). The median age was 34 years. Nested PCR for both IS 6110 & MPB 64 was positive in 64% of the cases, IS 6110 positive in 23% and MPB 64 positive in 15%. Real-time PCR was positive in 48% of the cases. Vision improved in 33% of cases, maintained in 57%, and worsened in 10% of cases. Conclusion Presumed Tubercular intermediate uveitis can be confirmed by PCR of intraocular fluids. Anti-tubercular therapy with immunosuppression can improve vision and prevent recurrences in such cases.

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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

Cited by 9 publications

References 5 publications

Utility of multiplex real-time PCR in the diagnosis of extrapulmonary tuberculosis

Utility of multiplex real-time PCR in the diagnosis of extrapulmonary tuberculosis

Efficient, Validated Method for Detection of Mycobacterial Growth in Liquid Culture Media by Use of Bead Beating, Magnetic-Particle-Based Nucleic Acid Isolation, and Quantitative PCR

Polymerase chain reaction confirmed mycobacterium tuberculosis intermediate uveitis – analysis of 22 eyes of 14 cases from a tertiary care centre in South India: a retrospective study

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