Rolling Circle Amplification for Direct Detection of rpoB Gene Mutations in Mycobacterium tuberculosis Isolates from Clinical Specimens

GeneXpert MTB/RIF has revolutionized the tuberculosis diagnosis by simultaneous detection of Mycobacterium tuberculosis and resistance to RIF (rifampicin), a surrogate marker for multidrug-resistant TB in less than two hours. The RIF-resistance pattern in Balochistan, Pakistan, is not documented. This study was aimed to detect RIF-resistant TB and mutations in RNA polymerase beta (rpoB) gene of M. tuberculosis within 81-bp RRDR in Quetta, Pakistan using GeneXpert® MTB/RIF assay. In total, 2300 clinical specimens were collected from suspected TB patients at Fatima Jinnah General and Chest Hospital Quetta, Pakistan between January and August 2017. These specimens were analyzed by GeneXpert® MTB/RIF assay. The data was statistically analyzed using SPSS software. Out of 2300 clinical specimens, M. tuberculosis was positive in in 899 (39.1%) cases by GeneXpert® MTB/RIF assay [positive respiratory cases 42.9% (871/2032) and non-respiratory 10.4% (28/268) with statistically significant difference (χ2= 104.5, p<0.001)]. Among 899 MTB positive cases, 46 (5.1%) were RIF-resistance caused by various rpoB gene mutations within 81-bp RRDR. Most of the RIF-resistant isolates were observed to harbor mutationsin Probe E 78.3% (n=36) whereas mutations in Probe A, B, D were observed 2.2% (n=1), 4.3% (n=2), and 6.5% (n=3), respectively. However, none of cases had RIF-resistance associated with Probe C. Out of 46 RRD cases, 21 (45.7 %) were males and 25 (54.3 %) were females. Additionally, Xpert® test showed higher detection rate than fluorescent microscopy (39.1% vs 31.2%, P<0.05) and detected MTB in 186 (11.8%) smear-negative specimens. Among 42 confirmed TB patients had MDR contact and eight patients were co-infected with HIV. In conclusion, 5.1% of the TB patients showed rifampicin resistance. The most frequent rpoB genetic mutations were observed in codons 531/533 (Probe E, 78.3%) whereas the least within the sequence 511 (Probe A, 2.2%).

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Direct detection of circulating microRNAs in serum of cancer patients by coupling protein-facilitated specific enrichment and rolling circle amplification

Chemical Communications ◽

10.1039/c3cc48996e ◽

2014 ◽

Vol 50 (25) ◽

pp. 3292-3295 ◽

Cited By ~ 39

Author(s):

Cheng-Yi Hong ◽

Xian Chen ◽

Juan Li ◽

Jing-Hua Chen ◽

Guonan Chen ◽

...

Keyword(s):

Cancer Patients ◽

Direct Detection ◽

Rolling Circle Amplification ◽

Circulating Mirnas ◽

Simple Method ◽

Circulating Micrornas ◽

Rolling Circle ◽

Coupling Protein

A simple method for direct detection of circulating miRNAs in serum by coupling p19 protein-facilitated specific enrichment and RCA.

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Evaluation of Gen-Probe Amplified Mycobacterium Tuberculosis Direct Test and PCR for direct detection of Mycobacterium tuberculosis in clinical specimens.

Journal of Clinical Microbiology ◽

10.1128/jcm.32.2.393-397.1994 ◽

1994 ◽

Vol 32 (2) ◽

pp. 393-397 ◽

Cited By ~ 68

Author(s):

N Miller ◽

S G Hernandez ◽

T J Cleary

Keyword(s):

Mycobacterium Tuberculosis ◽

Direct Detection ◽

Direct Test ◽

Clinical Specimens

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Ligation-promoted hyperbranched rolling circle amplification enables ultrasensitive detection of microRNA in clinical specimens

Sensors and Actuators B Chemical ◽

10.1016/j.snb.2018.09.058 ◽

2018 ◽

Vol 277 ◽

pp. 634-639 ◽

Cited By ~ 5

Author(s):

Xiaoe Zhang ◽

Yinong Liu ◽

Yiwen Yang ◽

Junjie Huang ◽

Haitao Wang ◽

...

Keyword(s):

Rolling Circle Amplification ◽

Ultrasensitive Detection ◽

Rolling Circle ◽

Clinical Specimens

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Paradigm for diagnosing mycobacterial disease: direct detection and differentiation of Mycobacterium tuberculosis complex and non-tuberculous mycobacteria in clinical specimens using multiplex real-time PCR

Journal of Clinical Pathology ◽

10.1136/jclinpath-2017-204945 ◽

2018 ◽

Vol 71 (9) ◽

pp. 774-780 ◽

Cited By ~ 10

Author(s):

Jeong-Uk Kim ◽

Dae-Shick Ryu ◽

Choong-Hwan Cha ◽

Seon-Hee Park

Keyword(s):

Mycobacterium Tuberculosis ◽

Real Time ◽

Real Time Pcr ◽

Predictive Value ◽

Direct Detection ◽

Positive Culture ◽

Nucleic Acid Amplification ◽

Mycobacterial Disease ◽

Pcr Assay ◽

Clinical Specimens

AimsMycobacterium tuberculosis and non-tuberculous mycobacteria (NTM) are clinically different, and the rapid detection and differentiation of M. tuberculosis complex (MTBC) and NTM is crucial for patient management and infection control. Given the slow growth of most pathogenic mycobacteria, nucleic acid amplification assays are excellent tools for direct identification of mycobacteria in clinical specimens. Recently, a multiplex real-time PCR assay was developed that can directly detect 20 mycobacterial species in clinical specimens. Here, we evaluated the diagnostic performance of the assay for diagnosing mycobacterial disease under routine laboratory conditions.MethodsA total of 3334 specimens collected from 1437 patients suspected of tuberculosis infection were subjected to acid-fast bacilli staining, conventional culture and the multiplex real-time PCR assay. To evaluate the sensitivity and specificity of the assay, the overall diagnosis of tuberculosis was defined by positive culture plus medical history, and the 2007 American Thoracic Society and Infectious Disease Society of America diagnostic criteria for NTM disease were applied.ResultsThe sensitivity, specificity, positive predictive value and negative predictive value were 87.5%, 99.6%, 96.1% and 98.5%, respectively, for the detection of MTBC isolates and 53.3%, 99.9%, 95.2%, and 98.9%, respectively, for detecting NTM isolates.ConclusionsThus, the assay can correctly differentiate between MTBC and NTM isolates in clinical specimens and would be a useful tool for the rapid differentiation of tuberculosis and NTM disease, despite its limited sensitivity for the diagnosis of NTM disease.

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