Tuberculosis and Molecular Diagnosis (original) (raw)
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Advent in technologies for molecular diagnosis of tuberculosis
The incidence of Tuberculosis varies considerably around the world and most Mycobacterial infections in developing nations are still being caused by Mycobacterium tuberculosis members. A quick and correct diagnosis is of great importance because of the high morbidity. Unfortunately, conventional bacteriological methods are time consuming, their sensitivity is low, and so treatment occasionally becomes empirical. PCR method has high specificity in identifying M. tuberculosis in various specimens. Molecular diagnostic tools for Tuberculosis (TB) have evolved quickly with new innovations which can provide unprecedented opportunities for the rapid, sensitive and specific diagnosis of M. tuberculosis in clinical specimens and the status of its drug sensitivity. Microscopy and culture methods can not be replaced but the molecular assays can be applied in parallel with any new molecular tests for the diagnosis of TB. For extra pulmonary specimens, the use of the amplification methods is advocated, since rapid and accurate laboratory diagnosis is critical. Customization of the diagnostic usefulness of a molecular assay, according to the ease, reliability and need for health care sector is of immense value in a modern clinical Mycobacteriology laboratory.
Diagnosis of Tuberculosis: Molecular Versus Conventional Method
Background: Tuberculosis is as old as mankind. One third of the world’s population carries the bacillus. Tuberculosis remains the single greatest contributor to the world’s morbidity and mortality. Zheil Neelsen stained smears for acid-fast bacilli and culture on Lowenstein Jensen media are the methods being used for the diagnosis of Mycobacterium tuberculosis in most developing countries. Zheil Neelsen smear is rapid but less sensitive and culture is more sensitive and specific but results in delay in definitive diagnosis. Polymerase chain reaction test for the diagnosis of tuberculosis is not well evaluated in developing countries. Objectives: To compare the ability of polymerase chain reaction to diagnose Mycobacterium tuberculosis rapidly in pulmonary and extra-pulmonary clinical specimens, and compare it with the yield of smear positivity and culture. Study design, settings and duration: This was a comparative study which was carried out at PMRC TB Research Centre, King Edward ...
Recent Advances in the Diagnosis of Tuberculosis
Journal of Postgraduate Medicine, Education and Research, 2013
Tuberculosis is an important health problem requiring early diagnosis for timely initiation of therapy and control of disease transmission. Though, conventional techniques, such as detection of acid fast bacilli by Ziehl-Neelsen staining, are very economical, yet have a low sensitivity. Isolation of mycobacteria by culture on Lowenstein Jensen media, considered to be the gold standard, is not only time consuming but has a low sensitivity, especially in extrapulmonary tuberculosis. Recent advances in molecular techniques have revolutionized the diagnostic microbiology. Various new modalities in the diagnosis of tuberculosis, like LED microscopy, microscopically observed drug susceptibility testing (MODS), antigen detection tests along with various molecular methods, like loop mediated isothermal amplification (LAMP), multiplex PCR and Xpert MTB/RIF, are discussed in the present review. How to cite this article Sharma K, Appannanavar SB, Goyal K, Sharma A. Recent Advances in the Diagn...
Recent Advances in Diagnosis of Tuberculosis: A Review
Globally tuberculosis remains a challenge from the point of diagnosis, detection of drug resistance, and treatment. Treatment can only be initiated, when infection is detected and it based on the results of AST, recently there has been a marked increase in the development and testing of novel assays designed to detect Mycobacterium tuberculosis. Although most important advances that would develop tuberculosis (TB) analysis have not been realized, we are beginning to see the innovations that have been prompted by the recognition of the economic potential of the market for new diagnostic tests for TB and considerably increased public and private funding and awareness. In my present review, we focus on the newer tests that are accessible for the analysis of suppressed and active tuberculosis and rapid detection of drug resistance, nucleic acid amplification for identification of M. tuberculosis complex, and rapid tests for detecting drug resistance. PCR-based technologies and hybridization assays used for the recognition of the mycobacteria. Though these newer techniques are useful for a rapid result, emphasizing that culture-based diagnosis is still the 'gold standard' for the diagnosis and follows up on tuberculosis.
Diagnostic Methods for Mycobacterium tuberculosis and Challenges in Its Detection in India
Understanding Tuberculosis - Global Experiences and Innovative Approaches to the Diagnosis, 2012
Tuberculosis (TB) is one of the world's oldest and most important disseminating infectious diseases that still accounts for a high morbidity and mortality among adults. Despite high prevalence, case detection rates are low, posing major hurdles for TB control in developed and developing countries. Traditional diagnosis of TB bacilli depends upon smear positivity in sputum samples, culture and chest radiography. All these tests have known limitations. Conventional tests for detection of drug resistance are slow, tedious and difficult to perform in field conditions. For rapid diagnosis, new methods include newer versions of nucleic acid amplification tests, immune-based assays, skin patch test and rapid culture systems. For drug resistance analysis line-probe assays, bacteriophage-based assays, molecular beacons and microscopic observation drug susceptibility assay are available. An ideal test for TB is still not available and fast emergence of drug resistant tubercle strains aided by the everincreasing HIV AIDS-epidemic in third-world countries has stressed the need of rapid diagnostic test(s) to show the presence of mycobateria in the clinical samples. Microscopy and culture are still the major backbone for laboratory diagnosis of tuberculosis; new methods including molecular diagnostic tests have evolved over a period of time. The majority of molecular tests have been focused on: (i) detection of nucleic acids both DNA and RNA, which are specific to Mycobacterium tuberculosis, by amplification techniques such as polymerase chain reaction (PCR) focusing on detection and molecular epidemiology of M. tuberculosis; and (ii) detection of mutations in the genes which are associated with resistance to anti-tuberculosis drugs by sequencing or nucleic acid hybridization. The development and use of rapid diagnostic tools become increasingly important in addressing the emergence and treatment of multi-drug resistant (MDR) and extreme-drug (XDR) resistant M. tuberculosis strains. Tuberculosis remains one of the most challenging bacterial diseases in spite of development of a realm of antibiotics and diagnostic molecular biology techniques. The tubercle bacillus was discovered more than two hundred years ago and substantial advancements have been made in our knowledge about the development of tuberculosis in human. The organism seems to evolve over a period of time in terms of its ability to survive the action of front line www.intechopen.com Understanding Tuberculosis-Global Experiences and Innovative Approaches to the Diagnosis
Current Trends In The Laboratory Diagnosis Of Tuberculosis
Benin Journal of Postgraduate Medicine, 2009
Mycobacterium tuberculosis (M. tuberculosis) causes tuberculosis and is a very important pathogen of humans. Tuberculosis kills more people than ever, with the increasing number of HIV-infected individuals 1. The fact sheet of global tuberculosis is stunning with one third of the world's population currently infected and new infections occur every second, resulting in new tuberculosis infection in 1% of the world population annually 2. It is projected that newly acquired infections between 2002 and 2010 would be 1 billion persons, of these, 150 million will get sick and 36 million will die of tuberculosis 2. In Sub-Sahara African countries, the incidence of tuberculosis has doubled since the early 1980s 3. Mycobacterium tuberculosis is related and associated with other species of mycobacteria, referred to as the Mycobacteria Tuberculosis Complex (MTB complex). MTB complex includes; M. bovis, (including the vaccination strain BCG) M. tuberculosis, M. africanum, M. microti, and M. canettii 4 .These grouping is based on several parameters which include the analysis of antigenic extracts, target epitopes for monoclonal antibodies and antigenic and DNA relatedness 5 .
In developing countries like Pakistan, rapid diagnosis and effective treatment in life threatening disease such as tuberculosis (TB) is very important. In the study 90 sputum samples from different hospitals were processed using auramine staining, Ziehl-Neelsen staining (ZN), culture on Löwenstein-Jensen (LJ) medium and Polymerase Cain Raction (PCR). Out of all 90 samples, 44 (48.88%) and 56 (62.2%) showed positive sputum smear microscopy, tested by Zeil Neilson and auramine staining respectively. Culturing into Lowenstein Jensen Medium revealed 64 (71.1%) and PCR yield highly significant 83 (92.2%) positive result. In conclusion, it was noted that PCR as a molecular technique is a very rapid, specific and sensitive method for diagnosis of TB as compared to other conventional methods.
Molecular diagnostics in tuberculosis: basis and implications for therapy
Molecular diagnosis & therapy, 2009
The processing of clinical specimens in the mycobacterial diagnostic laboratory has undergone remarkable improvements during the last decade. While microscopy and culture are still the major backbone for laboratory diagnosis of tuberculosis on a worldwide basis, new methods including molecular diagnostic tests have evolved over the last two decades. The majority of molecular tests have been focused on (i) detection of nucleic acids, both DNA and RNA, that are specific to Mycobacterium tuberculosis, by amplification techniques such as polymerase chain reaction (PCR); and (ii) detection of mutations in the genes that are associated with resistance to antituberculosis drugs by sequencing or nucleic acid hybridization. Recent developments in direct and rapid detection of mycobacteria, with emphasis on M. tuberculosis species identification by 16S rRNA gene sequence analysis or oligohybridization and strain typing, as well as detection of drug susceptibility patterns, all contribute to t...
Diagnosis of tuberculosis: Available technologies, limitations, and possibilities
Journal of Clinical Laboratory Analysis, 2003
Rapid diagnosis and treatment are important for preventing transmission of Mycobacterium tuberculosis. However, the diagnosis of tuberculosis continues to pose serious problems, mainly because of difficulties in differentiating between patients with active tuberculosis and those with healed lesions, normal mycobacterium boris BCG (Bacillus Calmette Guerin) vaccinated individuals, and unvaccinated Manteux positives. Physicians still rely on conventional methods such as Ziehl-Neelsen (ZN) staining, fluorochrome staining, sputum culture, gastric lavage, and other non-traditional methods. Although the tuberculin test has aided in the diagnosis of tuberculosis for more than 85 years, its interpretation is difficult because sensitization with nontuberculous mycobacteria leads to false-positive tests. There have been numerous unsuccessful attempts to develop clinically useful serodiagnostic kits for tuberculosis. A number of proteinaceous and nonprotein antigens (such as acyltrehaloses and phenolglycolipids) have been explored from time to time for the development of such assays but they have not proved to be clinically useful. It has been difficult to develop an ELISA utilizing a suitable antigen because M. tuberculosis shares a large number of antigenic proteins with other microorganisms that may or may not be pathogenic. With the advent of molecular biology techniques, there have been significant advances in nucleic acid-based amplification and hybridization, which are helping to rectify existing flaws in the diagnosis of tuberculosis. The detection of mycobacterial DNA in clinical samples by polymerase chain reaction (PCR) is a promising approach for the rapid diagnosis of tuberculous infection. However, the PCR results must be corrected for the presence of inhibitors as well as for DNA contamination. In the modern era of genetics, marked by proteomics and genomics, the day is not far off when DNA chip-based hybridization assays will instantly reveal mycobacterial infections. J. Clin. Lab. Anal. 17: 155–163, 2003. © 2003 Wiley-Liss, Inc.
Jundishapur Journal of Microbiology, 2014
Background: Tuberculosis remains a global epidemic, especially in developing countries, including Iran. Rapid diagnosis of active Mycobacterium tuberculosis infection plays a critical role in controlling the spread of tuberculosis. Conventional methods may take up to several weeks or longer to produce results. In addition to multiplicity of steps involved in conventional detection, including isolation, identification and drug susceptibility testing, the slow growth rate of M. tuberculosis is also responsible for this lengthy time. Objectives: The aim of this study was to compare the polymerase chain reaction (PCR) and culture methods for the detection of M. tuberculosis in different clinical specimens. Materials and Methods: This study was performed on different samples (urine, gastric aspirate, bronchoalveolar lavage, pleural fluid, cerebrospinal fluid, ascetic fluid and joint fluid specimens) of tuberculosis suspected patients. M. tuberculosis DNA was extracted directly from different samples using two different protocols. Next, PCR was performed using three sets of specific primers to detect members of Mycobacterium genus, M. tuberculosis complex and non-tuberculosis Mycobacteria. The results were then compared with that of the culture method, which is considered as the gold standard method. Results: The concordance rate between the three sets of primers was calculated and IS6110/buffer PCR method showed good agreement with the LJ culture method (κ = 0.627, P < 0.0001). The sensitivity of IS6110/buffer PCR was 58.33%, with specificity of 77.78%; the positive and negative predictive values were 100% and 78.26%, respectively. Buffer method for DNA extraction was proved to give a higher accuracy to PCR in comparison with the boiling method. Conclusions: PCR method is a valuable, cost-effective and alternative tool for quick diagnosis of active tuberculosis in different clinical specimens.