Positivity Rate of Pyrosequencing to Diagnose Drug-Resistant Tuberculosis Directly from Sputum with Different Bacterial Load

Selma Zein Syafira, Nabilla Ghina Zavitri, Su Yan, Yunia Sribudiani, Alexander Lezhava, Lidya Chaidir

Abstract


BACKGROUND: Molecular techniques, which detect mutations associated with drug resistance tuberculosis (TB), are promising technologies for rapid diagnosis and monitoring of drug-resistant TB. Pyrosequencing is a potential rapid and robust molecular technique to detect drug resistance but its performance in clinical samples is less investigated. This study aimed to determine the positivity rate of pyrosequencing to diagnose drug-resistant TB directly from sputum samples with different grades of sputum smear microscopy results.

METHODS: Thirty-five sputum specimens from drug-resistant TB suspects were submitted for acid-fast bacilli (AFB) microscopy. All specimens were cultured using microscopic observation drug susceptibility (MODS) culture. Pyrosequencing was performed to DNA extracted from sputum of culture-positive patients.

RESULTS: MODS culture was positive in 19/35 subjects (54.29%) samples; 16 smear-positive and three smear-negative. Using pyrosequencing, Mycobacterium tuberculosis was identified in all culture-positive samples, including smear-negative samples. A complete resistance profile for 16 (82.35%) samples could be generated. Pyrosequencing failed to show results for eis or gyrA promoter in three samples. Nine of 19 patients were multidrug resistant-TB (MDR-TB), 1/19 was rifampicin-resistance TB (RR-TB), and 4/19 were pre-extensively drug-resistant TB (pre-XDR-TB). Two novel mutations in rpoB and rrs (associated with rifampicin and aminoglycoside, respectively) were found in this study.

CONCLUSION: The results of this study demonstrates high positivity rates of pyrosequencing to detect drug-resistant TB directly from sputum samples with different grades of smear microscopy, as the surrogate of bacterial load. The assay can be used as a first prediction test of drug resistance prior to confirmation by phenotypic tests.

KEYWORDS: drug-resistant tuberculosis, pyrosequencing, direct sputum


Full Text:

PDF

References


WHO. TB disease burden. In: Global Tuberculosis Report 2018. Geneva: WHO; 2018. p.27-66, article.

Nguyen L. Antibiotic resistance mechanisms in M. tuberculosis: an update. Archives of Toxicology. 2016; 90: 1585-604, CrossRef.

WHO. Diagnosis and treatment: TB, HIV-associated TB and drug-resistant TB. In: Global Tuberculosis Report 2018. Geneva: WHO; 2018. p.67-102, article.

Azadi D, Motallebirad T, Ghaffari K, Shojaei H. Mycobacteriosis and tuberculosis: laboratory diagnosis. Open Microbiol J. 2018; 12: 41-58, CrossRef.

Dookie N, Rambaran S, Padayatchi N, Mahomed S, Naidoo K. Evolution of drug resistance in Mycobacterium tuberculosis: a review on the molecular determinants of resistance and implications for personalized care. J Antimicrob Chemother. 2018; 73: 1138-51, CrossRef.

Koch A, Cox H, Mizrahi V. Drug-resistant tuberculosis: challenges and opportunities for diagnosis and treatment. Curr Opin Pharmacol. 2018; 42: 7-15, CrossRef.

Al-Mutairi N, Ahmad S, Mokaddas E, Eldeen H, Joseph S. Occurrence of disputed rpoB mutations among Mycobacterium tuberculosis isolates phenotypically susceptible to rifampicin in a country with a low incidence of multidrug-resistant tuberculosis. BMC Infect Dis. 2019; 19: 3, CrossRef.

Agrawal M, Bajaj A, Bhatia V, Dutt S. Comparative study of GeneXpert with ZN stain and culture in samples of suspected pulmonary tuberculosis. J Clin Diagn Res. 2016; 10: DC09-12, CrossRef.

Oommen S, Banaji N. Laboratory diagnosis of tuberculosis: Advances in technology and drug susceptibility testing. Indian J Med Microbiol. 2017; 35: 323-31, CrossRef.

Govindaswamy A, Sakthi D, Pai R, Jeyaseelan L, Michael J. Pyrosequencing: a rapid and effective sequencing method to diagnose drug-resistant tuberculosis. J Med Microbiol. 2018; 67: 1212-6, CrossRef.

Torres J, Paul L, Rodwell T, Victor T, Amallraja A, Elghraoui A, et al. Novel katG mutations causing isoniazid resistance in clinical M. tuberculosis isolates. Emerg Microbes Infect. 2015; 4: e42, CrossRef.

Sheen P, Requena D, Gushiken E, Gilman R, Antiparra R, Lucero B, et al. A multiple genome analysis of Mycobacterium tuberculosis reveals specific novel genes and mutations associated with pyrazinamide resistance. BMC Genomics. 2017; 18: 769, CrossRef.

Zhang S, Chen J, Cui P, Shi W, Zhang W, Zhang Y. Identification of novel mutations associated with clofazimine resistance in Mycobacterium tuberculosis. J Antimicrob Chemother. 2015; 70: 2507-10, CrossRef.

Lyu J, Zhang J, Ren X. Detection and identification of bacterial pathogens directly from sputum samples by pyrosequencing. J Med Microbiol. 2019; 68: 368-73, CrossRef.

Lin S, Rodwell T, Victor T, Rider E, Pham L, Catanzaro A, et al. Pyrosequencing for rapid detection of extensively drug-resistant mycobacterium tuberculosis in clinical isolates and clinical specimens. J Clin Microbiol. 2013; 52: 475-82, CrossRef.

Nimmo C, Shaw L, Doyle R, Williams R, Brien K, Burgess C, et al. Whole genome sequencing Mycobacterium tuberculosis directly from sputum identifies more genetic diversity than sequencing from culture. BMC Genomics. 2019; 20: 389, CrossRef.

Kementerian Kesehatan RI. Peraturan Menteri Kesehatan Nomor 67 Tahun 2016 tentang Penanggulangan Tuberkulosis. Jakarta: Kementerian Kesehatan RI; 2016, article.

Moore D. MODS – a user guide. Lima: Universidad Peruana Cayetano Heredia; 2006.

Zhang Z, Sng L, Yong Y, Lin L, Cheng T, Seong N, et al. Delays in diagnosis and treatment of pulmonary tuberculosis in AFB smear-negative patients with pneumonia. Int J Tuberc Lung Dis. 2017; 21: 544-9, CrossRef.

Reviono R, Setianingsih W, Damayanti K, Ekasari R. The dynamic of tuberculosis case finding in the era of the public–private mix strategy for tuberculosis control in Central Java, Indonesia. Glob Health Action. 2017; 10:1353777, CrossRef.

Rasool G, Khan A, Mohy-Ud-Din R, Riaz M. Detection of Mycobacterium tuberculosis in AFB smear-negative sputum specimens through MTB culture and GeneXpert® MTB/RIF assay. Int J Immunopathol Pharmacol. 2019; 33: 2058738419827174, CrossRef.

Zheng R, Zhu C, Guo Q, Qin L, Wang J, Lu J, et al. Pyrosequencing for rapid detection of Tuberculosis resistance in clinical isolates and Sputum samples from re-treatment Pulmonary Tuberculosis patients. BMC Infect Dis. 2014; 14: 200, CrossRef.

Nambiar R, Shah D, Ajbani K, Kazi M, Sadani M, Shetty A, et al. Evaluation of pyrosequencing for extensive drug resistance-defining anti-tuberculosis drugs for use in public healthcare. Tuberculosis. 2018; 110: 86-90, CrossRef.

Seifert M, Catanzaro D, Catanzaro A, Rodwell T. Genetic mutations associated with isoniazid resistance in mycobacterium tuberculosis: a systematic review. PLOS ONE. 2015; 10: e0119628, CrossRef.

Chiang T, Fan S, Jou R. Performance of an Xpert-based diagnostic algorithm for the rapid detection of drug-resistant tuberculosis among high-risk populations in a low-incidence setting. PLOS ONE. 2018; 13: e0200755, CrossRef.

Ocheretina O, Shen L, Escuyer V, Mabou M, Royal-Mardi G, Collins S, et al. Whole genome sequencing investigation of a tuberculosis outbreak in Port-au-Prince, Haiti caused by a strain with a “low-level” rpoB mutation L511P – insights into a mechanism of resistance escalation. PLOS ONE. 2015; 10: e0129207, CrossRef.

Van Deun A, Aung K, Hossain A, de Rijk P, Gumusboga M, Rigouts L, et al. Disputed rpo B mutations can frequently cause important rifampicin resistance among new tuberculosis patients. Int J Tuberc Lung Dis. 2015; 19: 185-90, CrossRef.

Van Deun A, Decroo T, Kya Jai Maug A, Hossain M, Gumusboga M, Mulders W, et al. The perceived impact of isoniazid resistance on outcome of first-line rifampicin-throughout regimens is largely due to missed rifampicin resistance. PLOS ONE. 2020; 15: e0233500, CrossRef.




DOI: https://doi.org/10.18585/inabj.v12i4.1130

Copyright (c) 2020 The Prodia Education and Research Institute

Creative Commons License
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.

 

Indexed by:

                  

               

                   

 

 

The Prodia Education and Research Institute