Laboratory Diagnosis Of Tuberculosis Research Articles

The use of Kudoh method for culture of Mycobacterium tuberculosis and Mycobacterium africanum in The Gambia.

Mycobacterium tuberculosis culturing remains the gold standard for laboratory diagnosis of tuberculosis. Tuberculosis remains a great public health problem in developing countries like The Gambia, as most of the methods currently used for bacterial isolation are either time-consuming or costly. To evaluate the Kudoh swab method in a West African setting in Gambia, with a particular focus on the method's performance when culturing Mycobacterium africanum West Africa 2 (MAF2) isolates. 75 sputum samples were collected in the Greater Banjul Area and decontaminated in parallel with both the standard N-acetyl-L-Cysteine-NaOH (NALC-NaOH) and the Kudoh swab method in the TB diagnostics laboratory in the Medical Research Council Unit The Gambia between 30th December 2017 and 25th February 2018. These samples were subsequently cultured on standard Löwenstein-Jensen and Modified Ogawa media respectively and incubated at 37°C for mycobacterial growth. Spoligotyping was done to determine if the decontamination and culture methods compared could equally detect Mycobacterium tuberculosis, Mycobacterium africanum West Africa 1 and Mycobacterium africanum West Africa 2. Among the 50 smear positives, 35 (70%) were culture-positive with Kudoh and 32 (64%) were culture positive with NALC-NaOH, whilst 7(28%) of the 25 smear negative samples were culture positive with both methods (Table 2). There was no significant difference in recovery between both methods (McNemar's test, p-value = 0.7003), suggesting that the overall positivity rate between the two methods is comparable. There were no differences in time-to-positivity or contamination rate between the methods. However, Kudoh yielded positive cultures that were negative on LJ and vice versa. All findings were irrespective of mycobacterial lineages. The Kudoh method has comparable sensitivity to the NALC-NaOH method for detecting Mycobacterium tuberculosis complex isolates. It is easy to perform and could be an add on option for mycobacterial culture in the field in The Gambia, since it requires less biosafety equipment.

Evolution of Laboratory Diagnosis of Tuberculosis.

Tuberculosis (TB) is an infectious disease of global public health importance caused by the Mycobacterium tuberculosis complex. Despite advances in diagnosis and treatment, this disease has worsened with the emergence of multidrug-resistant strains of tuberculosis. We aim to present and review the history, progress, and future directions in the diagnosis of tuberculosis by evaluating the current methods of laboratory diagnosis of tuberculosis, with a special emphasis on microscopic examination and cultivation on solid and liquid media, as well as an approach to molecular assays. The microscopic method, although widely used, has its limitations, and the use and evaluation of other techniques are essential for a complete and accurate diagnosis. Bacterial cultures, both in solid and liquid media, are essential methods in the diagnosis of TB. Culture on a solid medium provides specificity and accuracy, while culture on a liquid medium brings rapidity and increased sensitivity. Molecular tests such as LPA and Xpert MTB/RIF have been found to offer significant benefits in the rapid and accurate diagnosis of TB, including drug-resistant forms. These tests allow the identification of resistance mutations and provide essential information for choosing the right treatment. We conclude that combined diagnostic methods, using several techniques and approaches, provide the best result in the laboratory diagnosis of TB. Improving the quality and accessibility of tests, as well as the implementation of advanced technologies, is essential to help improve the sensitivity, efficiency, and accuracy of TB diagnosis.

Comparative Evaluation of Cartridge-based Nucleic Acid Amplification Test Smear Microscopy and Conventional Culture Techniques in Laboratory Diagnosis of Tuberculosis

Comparative Evaluation of Cartridge-based Nucleic Acid Amplification Test Smear Microscopy and Conventional Culture Techniques in Laboratory Diagnosis of Tuberculosis

Innovative laboratory methods for improved tuberculosis diagnosis and drug-susceptibility testing

The laboratory plays a vital role in the diagnosis of all clinical forms of tuberculosis (TB), from microbiological confirmation of Mycobacterium tuberculosis to drug-susceptibility testing (DST) and treatment monitoring. For many decades, laboratory diagnosis of tuberculosis was based on conventional methods such as smear microscopy, and culture-based methods. However, Mycobacterium tuberculosis is a slow-growing organism, requiring 2–4 weeks or longer for cultures to yield results. Therefore, the evaluation of novel and rapid diagnostic methods has been a priority for research and development. In the beginning of 1990s, molecular-based diagnostics became widely available providing rapid detection, identification, and DST of M. tuberculosis. In this paper, we review some of the new diagnostic methods introduced in the clinical laboratory for the diagnosis of tuberculosis. With the global goal of ending TB as a public health challenge by 2030, enhancing diagnostic capabilities for latent and active TB, along with improving DST, would improve identification and management of cases, reducing transmission rates and curbing the spread of drug-resistant strains. These innovations promise to transform TB control efforts, bringing us closer to eradicating this persistent global health threat.

MODERN PROBLEMS OF TUBERCULOSIS, LABORATORY DIAGNOSTICS

In modern conditions, an extraordinary epidemic situation with tuberculosis has developed, which requires the improvement of methods of detection and diagnosis of this disease in order to control infection, morbidity and reduce the epidemiological factors of its spread. Patients who are diagnosed with tuberculosis in a timely manner pose a danger to the environment, and the effectiveness of chemotherapy methods decreases. One patient who secretes mycobacterium tuberculosis can infect 10-15 people during the year, and under favorable conditions for the dispersal of the pathogen, epidemic outbreaks occur, affecting 100 or more people. The problem of timely and effective diagnosis of this pathology is an extremely urgent and priority direction in the system of anti-tuberculosis measures, requiring the doctor to have knowledge of modern research methods.
 The aim of the study - generalization of modern methods of laboratory diagnosis of tuberculosis for timely detection of the disease, its stage, effectiveness of drug treatment.
 Laboratory diagnostics ensures the fulfillment of the main task of diagnosis and treatment of tuberculosis - detection of mycobacterium tuberculosis in a patient.

Modern technologies for studying the genome of mycobacteria

Molecular technologies play a leading role in the laboratory diagnosis of tuberculosis and mycobacteriosis. The successes in studying the genome of Mycobacterium have contributed to significant progress in understanding the evolution, variability, and genetic diversity of pathogens, as well as the development of diagnostic technologies, including research into resistance to anti-tuberculosis drugs.
 The aim of this research is to conduct a comparative study of the spectrum of modern technologies for studying the genomes of mycobacteria and their impact on the efficiency of the laboratory diagnosis of tuberculosis.
 Materials and methods: a search for sources of information was carried out in the PubMed, Medline, Web of Science, and Google Scholar databases. Materials related to the technology of molecular diagnosis of tuberculosis and mycobacteriosis and for determining the susceptibility of pathogens to anti-tuberculosis drugs were selected.
 Results: it was determined that the modern methods for studying the genome of mycobacteria include amplification technologies (PCR analysis), hybridization, restriction, spoligotyping, sequencing, and their various combinations. The main methods are standard and modified protocols of PCR (RAPD-PCR, AP-PCR, rep-PCR, Real-time PCR, Inverse PCR, TB-LAMP, HIP, LM-PCR). Genomic Restriction Analysis can be used in studies of MTBC and NTM strains (RFLP, AFLP analysis, MIRU-VNTR genotyping). The most effective method for genome analysis is WGS. Complex methods that utilize a combination of molecular technologies allow for the direct detection of mycobacteria in clinical samples.
 Conclusions: the widespread application of genomic technologies in the study of mycobacteria will contribute to the effective implementation of the global WHO strategy for the prevention, treatment, and control of tuberculosis and mycobacteriosis

Current innovations in medical laboratory diagnosis of Tuberculosis

Tuberculosis (TB) is a major global health problem, ranking as the second highest cause of death from an infectious disease globally, after the human immunodeficiency virus (HIV). Nigeria ranked first in Africa with burden of TB. Tuberculosis is a major public health problem in Nigeria with about 407,000 people infected. The World Health Organization estimates that 120,000 people developed TB in 2019, with 154,000 death of whom, 13% were HIV positive individuals. Among the incident cases, 56% were from the South-East Asia and Western Pacific Regions and one quarter were from Africa. The African continent accounts for the highest rates of cases and deaths relative to population. Traditionally, tuberculosis is mostly being diagnosed by a combination of chest X-rays, the staining of sputum with special dyes followed by microscopy, the growth of Mycobacterium tuberculosis in culture and the Mantoux test. The sputum smear microscopy is easy to do and is very cheap and combined with chest X-rays has been used for a long time by TB control agencies worldwide. However, the sputum smear microscopy (sputum AFB) test has some problems in HIV-positive patients and children also in patients with low bacterial load and in latent TB infections. TB continues to be a complex disease to diagnose and manage due to the chronicity of the disease, the nature of the host-pathogen relationship, and the resulting diversity in its clinical manifestations. Therefore, this review considers recent innovations in TB laboratory diagnosis in conventional (traditional), non- conventional (molecular), serological (immunological) methods and the latest methods of TB diagnosis.

Evaluation of an AI-Based TB AFB Smear Screening System for Laboratory Diagnosis on Routine Practice.

The most robust and economical method for laboratory diagnosis of tuberculosis (TB) is to identify mycobacteria acid-fast bacilli (AFB) under acid-fast staining, despite its disadvantages of low sensitivity and labor intensity. In recent years, artificial intelligence (AI) has been used in TB-smear microscopy to assist medical technologists with routine AFB smear microscopy. In this study, we evaluated the performance of a TB automated system consisting of a microscopic scanner and recognition program powered by artificial intelligence and machine learning. This AI-based system can detect AFB and classify the level from 0 to 4+. A total of 5930 smears were evaluated on the performance of this automatic system in identifying AFB in daily lab practice. At the first stage, 120 images were analyzed per smear, and the accuracy, sensitivity, and specificity were 91.3%, 60.0%, and 95.7%, respectively. In the second stage, 200 images were analyzed per smear, and the accuracy, sensitivity, and specificity were increased to 93.7%, 77.4%, and 96.6%. After removing disqualifying smears caused by poor staining quality and smear preparation, the accuracy, sensitivity, and specificity were improved to 95.2%, 85.7%, and 96.9%, respectively. Furthermore, the automated system recovered 85 positive smears initially identified as negative by manual screening. Our results suggested that the automated TB system could achieve higher sensitivity and laboratory efficiency than manual microscopy under the quality control of smear preparation. Automated TB smear screening systems can serve as a screening tool at the first screen before manual microcopy.

Impact of the COVID-19 pandemic on laboratory diagnosis of tuberculosis in southern Brazil

Background and objectives: to understand the impact of the COVID-19 pandemic on tuberculosis (TB) diagnosis in different settings is essential to guide the establishment of appropriate TB control strategies. This study aimed to assess the influence of COVID-19 pandemic in laboratory diagnosis of TB in patients tested and diagnosed for TB. Methods: a data survey was carried out in the database of laboratories that perform TB diagnosis for the public health system in Rio Grande city (Rio Grande do Sul, Brazil). Results: there was a decrease of 1,368 to 735 (reduction of 46.3%) in the number of patients tested for TB in public diagnostic services in 2019 and 2020, respectively, and a decrease of 197 to 119 (reduction of 39.6%) in the number of new TB cases diagnosed. In contrast, the positivity rate was 14.4% in 2019 and 16.2% in 2020. Moreover, it was observed that the laboratory that performs the diagnostic service for Primary Health Care was the most affected, when compared to Tertiary Health Care. Conclusion: as a consequence of measures to control the spread of SARS-CoV-2, there was a reduction in TB testing and in the detection of new cases, especially in Primary Health Care, where patients with less need for hospitalization are received.

Use of the Xpert MTB/XDR method in the algorithm of laboratory diagnosis of tuberculosis for rapid detection of cases of multir resistant tuberculosis and tuberculosis with extensively drug in Ukraine

Objective — studying the possibility of implementing the Xpert MTB/XDR method in the algorithm for diagnosing tuberculosis (TB) in Ukraine.
 Materials and methods. 51 strains of M. tuberculosis (MBT) were studied using Xpert MTB/XDR cartridges, the LPA method and the BASTEC system: 16 MTB strains of the control panel with known results of the phDST and gDST; 35 strains of MBT isolated from TB patients with Xpert MTB/RIF ULTRA results. Xpert MTB/RIF ULTRA, Xpert MTB/XDR, LPA analysis methods were used to identify mutations associated with drug resistance (DR).
 Results and discussion. MBT H resistance analysis revealed only 2 discordant results with Xpert MTB/XDR and BASTEC.In 2 cases, studies in Xpert MTB/XDR cartridges revealed a low level of resistance to H, which is associated with mutations either in the intergenic region of oxyR-ahpC and/or in the inhA promoter, since simultaneously with the phenotypic resistance to H in these strains, also resistance to Et, indicating the presence of mutations in the inhA promoter. The low level of resistance to H was confirmed by LPA testing and mutations in the inhA gene were found.Resistance results for Q, Km, Cm and Et obtained with Xpert MTB/XDR cartridges and LPA testing were in agreement.
 Conclusions. Analysis of the DR profile of MBT strains of the international panel and MBT from TB patients with known mutations according to the results of LPA and determined phDST to the first and second line drugs and the results of DR of these MBT, determined using the Xpert MTB/XDR method, revealed its high efficiency.The results of studies on the detection of MBT resistant to H, Q, Am and Et using the Xpert MTB/XDR and LPA methods coincided. The rapid detection of MBT DR to these drugs is a key moment for deciding on the choice of treatment regimens for patients with TB.The method for determining the DR profile of MTB strains using the Xpert MTB/XDR test can be implemented in the TB diagnostic algorithm in Ukraine.

The informativeness of various types of pathological material samples in the laboratory diagnosis of tuberculosis in children

The informativeness of various types of pathological material samples in the laboratory diagnosis of tuberculosis in children

Evaluation of the GeneXpert MTB/RIF assay performance in sputum samples with various characteristics from presumed pulmonary tuberculosis patients in Shiselweni region, Eswatini

Background Tuberculosis is a global health concern and has become more complex to diagnose due to mutations in the causative agent, Mycobacterium tuberculosis. In a setting with high TB prevalence, having a rapid and accurate diagnosis may reduce the rate of infections. The study aimed to evaluate the diagnostic performance of GeneXpert MTB/RIF assay in sputum samples from suspected tuberculosis patients. Methods A total of 1 328 sputum samples were collected from patients, across 12 clinics in the Shiselweni region, Eswatini. One thousand one hundred and ten (1110; 84%) samples were simultaneously processed on GeneXpert MTB/RIF assay and MGIT culture. Results Two hundred and ninety-seven (297) samples tested GeneXpert positive and 813 GeneXpert negatives, while 310 samples tested positive and 800 tested negative on the MGIT culture method. The positive predictive value on GeneXpert MTB/RIF assay was 83% while the negative predictive value was 97.80%. At p-value = .796, sputum quality did not affect the positivity of the GeneXpert MTB/RIF results. Sputum volume had a significant impact on the performance of the GeneXpert MTB/RIF with increased sensitivity in 4 ml and 6 ml samples. Conclusions Although detection of tuberculosis using the GeneXpert MTB/RIF assay in sputum samples is not limited to one specific characteristic, sputum volume assessment should be considered as an integral part of routine laboratory diagnosis of tuberculosis especially in high tuberculosis prevalent settings. However, the ability of the GeneXpert MTB/RIF to provide rapid TB diagnosis is not dependent on sputum quality.

Advances in the laboratory diagnosis of tuberculosis

Despite the advances made in the past 30 years in the laboratory diagnosis of tuberculosis (TB), only a small portion of the overall world population has benefitted. The World Health Organization (WHO) has recommended the use of nucleic acid amplification tests (NAAT) to detect TB instead of smear microscopy, since they can detect TB with greater precision, particularly in patients with paucibacillary disease and in individuals living with HIV. A broad range of molecular TB detection tests are currently being developed and evaluated, some for use in reference laboratories and others for peripheral medical care settings and point-of-care. There has been a surge of molecular tests designed, manufactured, and implemented in countries with a high TB load, and some are specifically meant for use in locations that are close to the patient. In terms of drug susceptibility testing, NAAT and next-generation sequencing may provide faster results than traditional phenotype culture. Further, the results of tests that detect or quantify cytokines released in the inflammatory process in latent tuberculosis infection (LTBI), such as the Interferon-Gamma Release Assay (IGRA), or that quantify IL-6 or other cytokines, depend, as in the tuberculin skin tests (TST), on the prevalence of TB in the tested population. We herein review the recent advances in TB detection tests and resistance to anti-TB drugs.

National Proficiency Testing of Molecular Diagnostics for Tuberculosis and Drug Resistance Detection - China, 2014-2019.

What is already known about this topic? Proficiency testing (PT) is a key component of quality assurance and is essential in ensuring accurate laboratory diagnosis of tuberculosis (TB) and drug-resistant TB. The National Tuberculosis Reference Laboratory (NTRL) developed a novel PT panel to test laboratories’ proficiency for molecular TB diagnostic assays throughout the TB laboratory network. A total of 6 PT rounds for molecular diagnostics were conducted by NTRL from 2014 to 2019. What is added by this report? PT conducted using artificial sputum specimens increased from 120 in the first round to 1,835 in the sixth round. Overall, laboratories demonstrated good proficiency for MTB and drug-resistance detection by molecular diagnostics, which is evident from the qualification rates over the six rounds: 95%, 97%, 96%, 93%, 93%, and 97%, respectively. What are the implications for public health practice? The use of artificial sputum specimens for PT panel production to test TB molecular diagnostics in China is feasible. Most of the participating laboratories provided reliable molecular diagnostic results for MTB and drug-resistance detection. The TB laboratory network can be instrumental in implementing PT expansion and improving the quality of TB molecular diagnosis in China.

Perfil clí­nico, epidemiológico e laboratorial da tuberculose entre 2014 a 2019 no estado da Bahia

Objetivo: Analisar as caracterí­sticas clí­nicas-epidemiológicas dos casos de tuberculose com confirmações laboratoriais, evidenciando a distribuição da doença na Bahia entre 2014 a 2019. Método: Para obtenção dos dados utilizou-se o Sistema de Informação de Agravos e Notificações (SINAN) disponí­vel para consulta no banco de dados Departamento de Informática do Sistema Único de Saúde (DATASUS), além da utilização de boletins epidemiológicos, websites e artigos cientí­ficos indexados nas bases de dados. Resultados: Constatou-se um aumento no coeficiente de incidência da tuberculose nos três últimos anos, no estado da Bahia. A caracterização dos portadores de tuberculose nessa região trata-se de uma população com faixa etária de 20 a 39 anos, predominância pela forma pulmonar e destaca que, quanto aos recursos para diagnóstico laboratorial da tuberculose, a baciloscopia do escarro demostrou maior representatividade. Conclusão: Espera-se que os resultados deste estudo fortaleçam a assistência em saúde, aproximando o paciente das medidas de controle da tuberculose.

Optimization of Lyophilized LAMP and RT-PCR Reaction Mixes for Detection of Tuberculosis

Abstract Undoubtedly, one of the most infectious diseases in the world is tuberculosis. Key factor for tuberculosis control is to prevent possible contagion with rapid diagnosis and effective treatment. The culture method, which it takes several weeks to obtain results, is the gold standard method for laboratory diagnosis of tuberculosis. In order to prevent possible contagion of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. Normally, clinical samples are studied in advanced laboratories designed for this purpose. However, especially after the screening in rural areas, the transmission of the samples to the centers has many negative effects on the clinical material. Therefore, the latest trend molecular techniques in microbiological diagnosis are developing into point of care systems that can be applied in the field without laboratory infrastructure. The major challenge for molecular-based point-of-care tests is the need to store polymerase enzymes and some of the ingredients used in the cold chain. The aim of this study is to increase the resistance of the amplification reaction mixtures by lyophilizing the tuberculosis diagnosis. Lyophilization was performed on Loop-mediated isothermal amplification (LAMP) and Real-time PCR mixtures. For the lyophilization of LAMP and RT-PCR mixtures, two different experimental setups were tried from the literature except for the developed content. Chemicals such as stachyose, trehalose, glycerol and PEG 8000 are widely using as cryoprotectants. As a result, the developed content (0.5% PEG 8000, 2.0 % Stachyose) was determined the best cryoprotectant mixture. Accordingly, amplification mixtures can be produced with the developed lyophilization method and point of care kits can be developed.

Optimization of isothermal amplification method for Mycobacteriumtuberculosisdetection and visualization method for fieldwork

Background/aim Tuberculosis is still one of the most contagious diseases around the world. Key factors of tuberculosis control are rapid diagnostic, efficient treatment, and prevention of contamination by surveillance and monitoring. However, culture is the gold standard method for laboratory diagnosis of tuberculosis; the results are several weeks to obtain. In order to prevent contamination of tuberculosis, diagnosis must be made in short time and treatment should be started as soon as possible. The aim of this study is to optimize the loop-mediated isothermal amplification (LAMP) method, which provides a much faster and more sensitive result than the polymerase chain reaction (PCR) method and allows the replication of target nucleic acid sequences under isothermal conditions without the need for laboratory infrastructure.Materials and methods Sputum samples were homogenized with 5% trypsin solution in CaCl2 to obtain DNA. DNA was purified using QIAGEN QIAamp DNA mini kit. LAMP primers were design using Primer explorer V5 program according to IS6110 gene of Mycobacterium tuberculosis. NEB Bst 3.0 DNA polymerase kit was used for LAMP reactions. Besides, LAMP reactions were compared with TaqMan based RT-PCR method using NEB’s Taq polymerase kit. Finally, for visualization of LAMP products, lateral flow dipsticks that produced by Milenia Biotec, colorimetric 2X LAMP master mix that produced by NEB and 2% w/v agarose gel electrophoresis methods were used.ResultsOptimum amplification temperature for LAMP was found to be 71.4 °C. The detection limit of the method was 102 CFU/mL and sensitivity was determined 100% compared to five different Mycobacterium species. Conclusion The current study indicated that the LAMP-LFD and colorimetric LAMP protocol optimized with sputum samples can be reliable used as a rapid, sensitive and specific assay in the diagnosis of tuberculosis in the field.

Diagnosis of laryngeal tuberculosis in a high TB burden area.

The larynx is the second most commonly affected site in the head and neck region in patients with extrapulmonary tuberculosis (TB). Despite this, the prevalence of laryngeal TB is largely unknown, particularly in areas with a high TB burden. The laboratory diagnosis of TB includes microscopy, culture and molecular testing. The aims of this study were to determine the prevalence of laryngeal TB in patients presenting with laryngeal pathology in a region with a high TB burden and to determine the optimal diagnostic methods for the diagnosis of laryngeal TB. This was a prospective descriptive study of 80 adult patients undergoing direct laryngoscopy and biopsy for laryngeal pathology in the Department of Otorhinolaryngology, Universitas Academic Hospital, Bloemfontein, South Africa over a 1year period. Histopathological and microbiological investigations (microscopy, Xpert MTB/RIF, and TB culture) were performed on all laryngeal biopsies. Five (6.25%) out of 80 patients were diagnosed with laryngeal TB. In one patient, the Xpert MTB/RIF assay was positive on the laryngeal tissue and histology showed granulomas. Two patients had granulomas on histology although the microbiological tests on the tissue were negative. Two patients had only positive tissue cultures for Mycobacterium tuberculosis. None of the biopsies had positive Ziehl-Neelsen stains. The results suggest that the diagnosis of laryngeal TB required a combination of histopathology, culture and PCR and that the Xpert MTB/RIF assay is not a sensitive test for the diagnosis of laryngeal TB.

Effect of specimen processing, growth supplement, and different metabolic population on Mycobacterium tuberculosis laboratory diagnosis.

IntroductionSputum specimen decontamination steps are essential due to the presence of other saprophytic and infectious organisms. However, they negatively affect the mycobacterial recovery. In addition, little is known about the Mycobacterium tuberculosis killing efficacy of the PANTA (polymyxin-B, amphotericin-B, nalidixic acid, trimethoprim, azilocillin) antibiotics. Moreover, M. tuberculosis can be present in more than one metabolic population, but the effect of different growth characteristics on the mycobacterial growth indicator tube (MGIT) based time-to-positive (TTP) is not well studied.MethodsWe performed—(1) experiments using the solid agar and MGIT method to determine the effect of the NALC-NaOH decontamination method, (2) concentration-response studies with each individual antibiotic in the PANTA, and (3) the effect of the M. tuberculosis metabolic population on the TTP. TTP was recorded using the Epicenter software and exponential growth equation was used to calculate the doubling time of the bacteria, whereas, CFU/mL was analyzed using the Inhibitory Sigmoid Emax model for each antibiotic.ResultsDecontamination resulted in 4.36+0.13 log10 CFU/mL difference in cultures treated with NALC-NaOH versus no decontamination process and the limit of detection decreased from 1.47 log10 CFU/mL to the 0.42 log10 CFU/mL following NALC-NaOH treatment. PANTA at currently used antibiotic concentrations, did not had negative effect on mycobacterial recovery. Exponential growth model estimated doubling time for the log-phase growth M. tuberculosis as 2.04 days, for the semi-dormant bacilli as 2.80 days, and 6.37 days for the anaerobic cultures.ConclusionSpecimen decontamination method negatively affect the laboratory diagnosis of M. tuberculosis, polymyxin-B and nalidixic acid have anti-tuberculosis efficacy at high concentrations, and the doubling time of different metabolic population should be considered when deciding the time-in-protocol for the MGIT system.

Rapid detection of mycobacterium tuberculosis complex by real-time polymerase chain reaction (PCR) in pulmonary and extra-pulmonary samples in Casablanca, Morocco

Introductionthe laboratory diagnosis of tuberculosis (TB) relies mainly on conventional techniques. However, it either lacks sensitivity or it is time-consuming. This study aims to evaluate the use of real-time polymerase chain reaction (PCR) targeting IS6110 for Mycobacterium tuberculosis (TB) Complex (MTBC) in the routine diagnosis of TB in our laboratory.Methodsclinical samples were collected from the laboratory of bacteriology at Ibn Rochd University Hospital in Casablanca Morocco. Real-time polymerase chain reaction (PCR) results were compared to AFB smear and culture on Löwenstein-Jensen (LJ) solid media. Sensitivity, specificity, positive and negative predictive value (PPV and NPV) with 95% confidence intervals were calculated using GraphPad Prism.Resultson 171 clinical samples, the study showed positivity of microscopy, culture and real-time PCR for M. TB complex as 19%, 31%, and 32% respectively. Sensitivity, specificity, PPV and NPV for real-time PCR in pulmonary samples were 95.2%, 95.4%, 90.91% and 97.65% respectively. For extra-pulmonary samples, they were: 72.7%, 90.32%, 72.7%, and 90.3%.Conclusionour study shows the effectiveness of using real-time PCR IS6110 in pulmonary and extra pulmonary samples. Future multicentric studies could seek to evaluate the place of this technique on routine diagnosis for better management of TB in Morocco.