Introduction

The clinical manifestations of tuberculosis are quite variable; symptoms are non-specific and depend on both host and microbe-related characteristics as well as their interactions that influence the clinical features of the disease. The contribution of the microbiology laboratory to the diagnosis and management of tuberculosis involves the detection and isolation of mycobacteria, the identification of the mycobacterial species or complex isolated, and the determination of susceptibilities of the organisms to antimycobacterial drugs. Positive smear indicates suitable isolation of the patient to limit the transmission of the TB and multi and extremely drug resistant Mycobacterium tuberculosis strains have to be rapidly detected by molecular tools based on molecular genetic analysis.

Microscopy

The detection of acid-fast bacilli (AFB) in stained smears examined microscopically is the first bacteriologic evidence of the presence of mycobacteria in a clinical specimen. It is the easiest and quickest procedure that can be performed, and it provides the physician with a strong suspicion of the diagnosis of TB. Also, because it gives a quantitative estimation of the number of bacilli being excreted, the smear is of vital clinical and epidemiologic importance in assessing the patient’s infectiousness. Other tests (cultures or molecular tests) are necessary in order to confirm the diagnosis of tuberculosis by the presence of M. tuberculosis complex species in specimen. Smears may be prepared directly from clinical specimens or from concentrated preparations. The acid-fast staining procedure depends on the ability of mycobacteria to retain dye when treated with mineral acid or an acid–alcohol solution. Two procedures are commonly used for acid–fast staining: the carbolfuchsin methods, which include the Ziehl–Neelsen and a fluorochrome procedure using auramine-O or auramine–rhodamine dyes. In reading smears, the microscopist should provide the clinician with a rough estimate of the number of AFB detected. Negative smears, however, do not exclude tuberculosis disease. Other tests with higher sensitivity will have to be performed for detection and identification of the species associated with tuberculosis.

Molecular diagnosis

Correct and rapid diagnosis and treatment of tuberculosis are associated with decrease morbidity, mortality, and on going transmission of infection. However, detection of Mycobacterium tuberculosis (Mtb) and its drug resistance with the use of conventional culture and culture-based phenotypic drug-susceptibility testing is slow and bio hazardous and requires substantial laboratory infrastructure and training. Molecular assays for detection of mutations associated with antibiotic resistance were developed for target genes (See Antibiotic resistance sequencing) (1,2).

Limitation The sensitivity of the investigational genotypic assay to detect resistance is lower when culture-based phenotypic drug-susceptibility testing was considered as the reference comparator. There are at least two potential causes of the phenotypic–genotypic discrepancies — alternative molecular mechanisms of resistance, many of which are still unknown, and limitations of the critical-concentration methods used for phenotypic testing.

1. Cirillo DM, Miotto P, Tortoli E. Evolution of Phenotypic and Molecular Drug Susceptibility Testing. In: Gagneux S, éditeur. Strain Variation in the Mycobacterium tuberculosis Complex: Its Role in Biology, Epidemiology and Control [Internet]. Cham: Springer International Publishing; 2017 [cité 15 mars 2018]. p. 221‑46. Disponible sur: http://link.springer.com/10.1007/978-3-319-64371-7_12

2. Miotto P, Tessema B, Tagliani E, Chindelevitch L, Starks AM, Emerson C, et al. A standardised method for interpreting the association between mutations and phenotypic drug resistance in Mycobacterium tuberculosis. Eur Respir J. déc 2017;50(6):1701354.

GeneXpert

Xpert® MTB/RIF (Cepheid) is an integrated, automated, cartridge-based system, used with GeneXpert instrumentation, for the rapid molecular detection of M. tuberculosis and mutations associated with rifampin resistance. Xpert MTB/RIF is widely used in tuberculosis programs and has contributed to the global increase in detection of rifampin-resistant tuberculosis [1].

The World Health Organization (WHO) recommended the use in all settings of a next-generation Xpert® MTB/RIF Ultra as a replacement for the current Xpert MTB/RIF® cartridge, in 2017. To improve assay sensitivity for the detection of M. tuberculosis, the Ultra assay incorporates two different multi-copy amplification targets (IS6110 and IS1081) and a larger DNA reaction chamber than Xpert MTB/RIF (50μl PCR reaction in Ultra versus 25 μl in Xpert MTB/RIF). Ultra also incorporates fully nested nucleic acid amplification, more rapid thermal cycling, and improved fluidics and enzymes. This has resulted in Ultra having a limit of detection (LOD) of 16 bacterial colony forming units (cfu) per ml (compared to 114 cfu per ml for Xpert MTB/RIF). To improve the accuracy of rifampicin resistance detection, the Ultra incorporates melting temperature-based analysis instead of real-time PCR. Specifically, four probes identify rifampicin resistance mutations in the rifampicin resistance determining region of the rpoB gene by shifting the melting temperature away from the wild type reference value. . The recommendation on the Ultra cartridge is based on a recent WHO Expert Group evaluation of data from a study coordinated by FIND, in collaboration with the Tuberculosis Clinical Diagnostics Research Consortium (CDRC). The study compared the Ultra assay with the current Xpert® MTB/RIF assay for diagnostic accuracy and non-inferiority across 10 study sites in eight countries. 1,520 patients with signs and symptoms of TB were enrolled in these countries for a direct comparison of the performance of Ultra against Xpert MTB/RIF on the same specimen.

Cepheid recently described a new cartridge for the rapid molecular detection of second-line drugs resistance associated mutations [2,3]. This assay will be available and can provide results from unprocessed sputum samples in just over 2 hours, with minimal hands-on technical time.

[1] Boehme CC, Nabeta P, Hillemann D, Nicol MP, Shenai S, Krapp F, et al. Rapid Molecular Detection of Tuberculosis and Rifampin Resistance. New England Journal of Medicine 2010;363:1005–15. doi:10.1056/NEJMoa0907847.

[2] Chakravorty S, Roh SS, Glass J, Smith LE, Simmons AM, Lund K, et al. Detection of Isoniazid-, Fluoroquinolone-, Amikacin-, and Kanamycin-Resistant Tuberculosis in an Automated, Multiplexed 10-Color Assay Suitable for Point-of-Care Use. Journal of Clinical Microbiology 2017;55:183–98. doi:10.1128/JCM.01771-16.

[3] Xie YL, Chakravorty S, Armstrong DT, Hall SL, Via LE, Song T, et al. Evaluation of a Rapid Molecular Drug-Susceptibility Test for Tuberculosis. New England Journal of Medicine 2017;377:1043–54. doi:10.1056/NEJMoa1614915.

Line Probe Assays

Conventional methods for mycobacteriological culture and drug susceptibility testing (DST) are slow and cumbersome, requiring sequential procedures for isolation of mycobacteria from clinical specimens, identification of Mycobacterium tuberculosis complex (MTC), and in vitro testing of strain susceptibility to anti-TB drugs. During this time patients may be inappropriately treated, drug resistant strains may continue to spread, and amplification of resistance may occur. Novel technologies for rapid detection of anti-TB drug resistance have therefore become a priority in TB research and development, and molecular line probe assays focused on rapid detection of antibiotic resistance (mostly for rifampicin, isoniazid, Fluoroquinolones, second line injectable drugs susceptibility testing) (1–4).

Line probe assay technology involves the following steps: First, DNA is extracted from M. tuberculosis isolates or directly from clinical specimens. Next, polymerase chain reaction (PCR) amplification of the resistance-determining region of the gene under question is performed using biotinylated primers. Following amplification, labeled PCR products are hybridized with specific oligonucleotide probes immobilized on a strip. Captured labeled hybrids are detected by colorimetric development, enabling detection of the presence of M. tuberculosiscomplex, as well as the presence of wild-type and mutation probes for resistance. If a mutation is present in one of the target regions, the amplicon will not hybridize with the relevant probe. Mutations are therefore detected by lack of binding to wild-type probes, as well as by binding to specific probes for the most commonly occurring mutations. The post-hybridization reaction leads to the development of coloured bands on the strip at the site of probe binding and is observed by eye. These tests can be performed in a single working day.

1. Crudu V, Stratan E, Romancenco E, Allerheiligen V, Hillemann A, Moraru N. First Evaluation of an Improved Assay for Molecular Genetic Detection of Tuberculosis as Well as Rifampin and Isoniazid Resistances. J Clin Microbiol. 1 avr 2012;50(4):1264‑9.

2. Hillemann D, Rusch-Gerdes S, Richter E. Feasibility of the GenoType MTBDRsl Assay for Fluoroquinolone, Amikacin-Capreomycin, and Ethambutol Resistance Testing of Mycobacterium tuberculosis Strains and Clinical Specimens. J Clin Microbiol. 1 juin 2009;47(6):1767‑72.

3. Hillemann D, Haasis C, Andres S, Behn T, Kranzer K. Validation of the FluoroType MTBDR Assay for Detection of Rifampin and Isoniazid Resistance in Mycobacterium tuberculosis Complex Isolates. Land GA, éditeur. J Clin Microbiol. 28 mars 2018;56(6):e00072-18.

4. Xie YL, Chakravorty S, Armstrong DT, Hall SL, Via LE, Song T, et al. Evaluation of a Rapid Molecular Drug-Susceptibility Test for Tuberculosis. N Engl J Med. 14 sept 2017;377(11):1043‑54.

About culture

Culture amplifies the number of Mycobacterium tuberculosis organisms in a sample. Sample are processed (see Processing specimens for culture) and inoculated either into a liquid culture media (MGIT) to detect positive samples rapidly and to make a semi-quantitative assessment of the bacterial load by determining the time taken for culture tubes to signal positive (time to detection, TTD) in the BACTEC MGIT 960 system for example, and/or into a solid medium as Löwenstein-Jensen slopes to get colonies that can be counted. Kudoh modified Ogawa method is a simple and inexpensive method. It is advantageous for microbiologists because of the low risk of contamination once the sample is inoculated using swabs, and no centrifugation process is necessary. Culture is the reference method. It’s the most sensitive one and enables identification of mycobacteria and phenotypic drug susceptibility testing of first and second line drugs.

About Phenotypic DST

Antimicrobial susceptibility testing is critical in prescribing an effective drug regime for a tuberculosis patient. It is also important in the follow-up of patients who are on antimicrobial therapy but are not responding to therapy. Phenotypic drug susceptibility testing of mycobacteria confirms molecular results. The proportion on solid medium method remains the reference DST method. Drugs can be tested in the MGIT 960 system that enables quicker and less expensive results. All first-line drugs (streptomycin, isoniazid, rifampicin, ethambutol (SIRE); and pyrazinamide (PZA) can be tested in the MGIT 960 system. Additionally, the second line drugs, in particular, the fluoroquinolones (gatifloxacin, levofloxacin, moxifloxacin, ofloxacin) and injectable drugs (amikacin, capreomycin, kanamycin) used in local standard of care can be tested with both systems.

WGS

Whole genome sequencing (WGS) has great potential as a method for rapidly diagnosing drug-resistant tuberculosis (DR-TB) in diverse clinical reference laboratory settings worldwide. The WGS approach overcomes many of the significant challenges associated with conventional phenotypic testing. Unlike other DR-TB molecular assays, which target only the “hot-spot” regions of a few genes to detect resistance to a restricted number of drugs, WGS assays can provide detailed sequence information for multiple gene regions or whole genomes of interest from clinical specimens (1-4).

Recent advances, including the rise of high throughput NGS technologies and massively parallel sequencing, have reduced the time and costs, and have made these technologies reasonable options even for low- and middle-income countries. Further work is being done regarding integration into existing laboratory workflows, technical training and skill requirements for utilization of the technology, and the need for expert guidance regarding the management and clinical interpretation of sequencing data (5, 6).

(1) Whole genome sequencing of Mycobacterium tuberculosis for detection of drug resistance: a systematic review. Papaventsis D, Casali N, Kontsevaya I, Drobniewski F, Cirillo DM, Nikolayevskyy V. Clin Microbiol Infect. 2017 Feb;23(2):61-68. https://www.sciencedirect.com/science/article/pii/S1198743X16303950?via%3Dihub

(2) Whole genome sequencing for M/XDR tuberculosis surveillance and for resistance testing. Walker TM, Merker M, Kohl TA, Crook DW, Niemann S, Peto TEA. Clin Microbiol Infect. mars 2017;23(3):161‑6.

(3) Whole genome sequencing of Mycobacterium tuberculosis. Cabibbe AM, Walker TM, Niemann S, Cirillo DM. Eur Respir J. nov 2018;52(5):1801163.

(4) Mycobacterium tuberculosis Next-Generation Whole Genome Sequencing: Opportunities and Challenges. Thato Iketleng, Richard Lessells, Mlungisi Thabiso Dlamini, Tuelo Mogashoa, Lucy Mupfumi, Sikhulile Moyo, Simani Gaseitsiwe, and Tulio de Oliveira. Tuberculosis Research and Treatment. 2018, Article ID 1298542, 8 pages https://doi.org/10.1155/2018/1298542

(5) Prediction of Susceptibility to First-Line Tuberculosis Drugs by DNA Sequencing. CRyPTIC Consortium and the 100,000 Genomes Project. Allix-Béguec C, Arandjelovic I, Bi L, Beckert P, Bonnet M, Bradley P, Cabibbe AM, Cancino-Muñoz I, Caulfield MJ, Chaiprasert A, Cirillo DM, Clifton DA, Comas I, Crook DW, De Filippo MR, de Neeling H, Diel R, Drobniewski FA, Faksri K, Farhat MR, Fleming J, Fowler P, Fowler TA, Gao Q, Gardy J, Gascoyne-Binzi D, Gibertoni-Cruz AL, Gil-Brusola A, Golubchik T, Gonzalo X, Grandjean L, He G, Guthrie JL, Hoosdally S, Hunt M, Iqbal Z, Ismail N, Johnston J, Khanzada FM, Khor CC, Kohl TA, Kong C, Lipworth S, Liu Q, Maphalala G, Martinez E, Mathys V, Merker M, Miotto P, Mistry N, Moore DAJ, Murray M, Niemann S, Omar SV, Ong RT, Peto TEA, Posey JE, Prammananan T, Pym A, Rodrigues C, Rodrigues M, Rodwell T, Rossolini GM, Sánchez Padilla E, Schito M, Shen X, Shendure J, Sintchenko V, Sloutsky A, Smith EG, Snyder M, Soetaert K, Starks AM, Supply P, Suriyapol P, Tahseen S, Tang P, Teo YY, Thuong TNT, Thwaites G, Tortoli E, van Soolingen D, Walker AS, Walker TM, Wilcox M, Wilson DJ, Wyllie D, Yang Y, Zhang H, Zhao Y, Zhu B. N Engl J Med. 2018 Oct 11;379(15):1403-1415. https://www.nejm.org/doi/10.1056/NEJMoa1800474?url_ver=Z39.88-2003&rfr_id=ori%3Arid%3Acrossref.org&rfr_dat=cr_pub%3Dwww.ncbi.nlm.nih.gov

(6) Integrating standardized whole genome sequence analysis with a global Mycobacterium tuberculosis antibiotic resistance knowledgebase Ezewudo M, Borens A, Chiner-Oms Á, Miotto P, Chindelevitch L, Starks AM, Hanna D, Liwski R, Zignol M, Gilpin C, Niemann S, Kohl TA, Warren RM, Crook D, Gagneux S, Hoffner S, Rodrigues C, Comas I, Engelthaler DM, Alland D, Rigouts L, Lange C, Dheda K, Hasan R, McNerney R, Cirillo DM, Schito M, Rodwell TC, Posey J. Sci Rep. 2018 Oct 18;8(1):15382. https://www.nature.com/articles/s41598-018-33731-1.pdf