XpertW MTB/RIF assay for tuberculosis diagnosis - IngentaConnect

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R. Sarin. Department of Microbiology, Lala Ram Sarup Institute of Tuberculosis and Respiratory Diseases, New Delhi, India. SUMMARY. The present study was ...
INT J TUBERC LUNG DIS 18(8):958–960 Q 2014 The Union http://dx.doi.org/10.5588/ijtld.13.0328

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XpertW MTB/RIF assay for tuberculosis diagnosis: evaluation in an Indian setting V. P. Myneedu, D. Behera, A. K. Verma, M. Bhalla, N. Singh, J. Arora, R. Singhal, M. Mathur, P. Lal, R. Sarin Department of Microbiology, Lala Ram Sarup Institute of Tuberculosis and Respiratory Diseases, New Delhi, India SUMMARY

The present study was conducted to evaluate the performance of the Xpertw MTB/RIF assay and compare Xpert results with solid and MGIT 960 liquid culture system. A total of 134 patients who had failed the Category I or II regimen were recruited for evaluation. Xpert correctly identified all Mycobacteri-

um tuberculosis isolates. The sensitivity and specificity of the Xpert assay for the detection of rifampicin resistance was respectively 98.2% and 97.0% when compared with MGIT 960 results. K E Y W O R D S : Mycobacterium tuberculosis; Xpertw MTB/RIF assay; Lowenstein-Jensen media ¨

TUBERCULOSIS (TB) kills 1.7 million persons worldwide every year. Of all the infectious diseases, TB is responsible for the highest number of casualties. Incorrectly diagnosed or undiagnosed TB patients aggravate the spread of multidrug-resistant (MDR-) and extensively drug-resistant (XDR-) TB, which increases morbidity and mortality. False-negative results and misdiagnosis of TB suspects are common in developing nations, as most TB control programmes use Ziehl-Neelsen (ZN) smear microscopy, which has poor sensitivity and requires multiple visits that cause higher default.1,2 The World Health Organization (WHO) approved Xpertw MTB/RIF assay (Cepheid, Sunnyvale, CA, USA) facilitates rapid and accurate identification of Mycobacterium tuberculosis and the detection of rifampicin (RMP) resistance. Xpert is a fully automated test based on a real-time polymerase chain reaction that can be used to simultaneously detect M. tuberculosis and RMPresistant mutations in the rpoB gene.3 The aim of our study was to evaluate the performance of the Xpert assay and compare Xpert results with smear microscopy, Lowenstein-Jensen (LJ) culture and the ¨ automated BACTECe MGITe (Mycobacterial Growth Indicator Tube) 960 liquid culture results (BD, Franklin Lakes, NJ, USA).

Microbiology, Lala Ram Sarup Institute of Tuberculosis and Respiratory Diseases, a national reference laboratory, from April to November 2011. The study protocol was approved by the Ethics Committee of the Institute. A total of 134 patients who had failed to show clinical improvement on the Category 1 and II drug regimens (Category 1 failures n ¼ 60; Category II failures n ¼ 74) were enrolled in the study.

MATERIALS AND METHODS The study was conducted at the Department of

Laboratory methods Each sputum sample collected was thoroughly mixed and then divided into four parts; one part was immediately tested using Xpert. The remaining three parts were used for ZN smear microscopy, LJ culture and MGIT culture. The laboratory teams performing the tests were blinded to the other test results. Xpert testing was performed according to the manufacturer’s instructions. Sample reagent was added to untreated sputum at a ratio of 2:1, manually agitated and kept for 10 min at room temperature, then shaken again and kept for 5 min; 2 ml of the inactivated material was transferred to the test cartridge and inserted into the test platform. Only electronic results were used for comparison. Smear microscopy was performed with the remainder of the specimen, processed using the N-acetyl-Lcysteine-sodium hydroxide method and cultured on LJ and MGIT media. Cultures positive for M. tuberculosis were further subjected to drug suscepti-

Correspondence to: D Behera, Department of Microbiology, Lala Ram Sarup Institute of Tuberculosis and Respiratory Diseases, Aurobindo Marg, New Delhi 110030, India. Tel: (þ91) 11 2685 4929. Fax: (þ91) 11 2651 7834. e-mail: tbmicro@ gmail.com Article submitted 7 May 2013. Final version accepted 6 April 2014.

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Evaluation of the Xpert MTB/RIF assay

Table 1

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Comparison of the XpertW MTB/RIF assay results with culture methods LJ culture

Xpert MTB/RIF assay M. tuberculosis detected M. tuberculosis-negative Total Sensitivity, % Specificity, % Positive predictive value, % Negative predictive value, % Overall agreement, %

MGIT culture

Positive n

Negative n

Positive n

Negative n

Total n

93 0 93 100 51.9 87.7 100 89.2

13 14 27

95 0 95 100 56.0 89.6 100 90.8

11 14 25

106 14 120

¨ LJ ¼ Lowenstein-Jensen; MGIT ¼ Mycobacteria Growth Indicator Tube.

bility testing (DST) against RMP at a critical concentration of 1.0 lg/ml using MGIT 960. The reference strain M. tuberculosis H37Rv was used as control for each DST batch.4,5

RESULTS Among the 134 patients, 80 (59.70%) sputum samples were AFB-positive using ZN smear, 95 (70.9%) using LJ culture, and 97 (72.4%) using MGIT 960; 2 isolates were identified as nontuberculous mycobacteria (NTM), and 4 LJ and 2 MGIT cultures were contaminated. Of the 134 sputum specimens tested using Xpert, 128 valid (95.52%), 4 invalid (3%), and 2 error (1.5%) results were obtained. M. tuberculosis was identified in 106/ 134 (79.1%) specimens using Xpert. Xpert also correctly identified all M. tuberculosis isolates and the two NTM isolates. Of the 54 AFB-negative samples, 26 (48.1%) were identified as M. tuberculosis using Xpert. Of the samples that were culture-negative using LJ/MGIT (n ¼ 27), 11 (40.7%) were Xpert-positive for M. tuberculosis complex (Table 1). DST results were available for 88 isolates: 54 were RMP-resistant using both MGIT and Xpert, whereas 32 were susceptible using both methods. Two isolates yielded disparate results: one isolate was detected as false-resistant and another as false-susceptible to RMP using Xpert. The sensitivity and specificity of the Xpert assay was found to be respectively 98.2% and 97% when compared with liquid DST (Table 2).

78% and 100%.7,8 Previous studies have shown greater accuracy of Xpert than that of smear microscopy.3 In the present study, 26/54 (48.1%) smear-negative patients were Xpert-positive. Smearnegative patients could be detected earlier using Xpert and initiated on treatment, facilitating the interruption of TB transmission. Sputum samples from 11 patients diagnosed as Xpert-positive were negative using liquid culture (false-positives). Many of the detected M. tuberculosis cases could be due to non-viable organisms, as the patients had been through months of treatment, but the TB DNA could be detected by Xpert. Steingart et al. found that Xpert achieved a pooled sensitivity of 94% and a specificity of 98% for the detection of RMP resistance.9 In our study, the sensitivity and specificity for RMP resistance was respectively 98.2% and 97%. We were not able to sequence the discrepant isolates. Previous studies have found that sequencing often resolves discrepancies in favour of Xpert, with 99.1% sensitivity (RMPresistant) and 100% specificity (RMP susceptibility).3 Despite many advantages, our study had several limitations, such as the lack of data on isoniazid resistance and a suboptimal positive predictive value (PPV) for RMP resistance in settings where MDR-TB prevalence is less than ~20%.

Table 2 Comparison of RMP-resistant results using the XpertW MTB/RIF assay with MGIT 960 (n ¼ 88) RMP resistance MGIT 960 system

DISCUSSION According to the WHO, an estimated 450 000 people developed MDR-TB in 2012 and there were an estimated 170 000 deaths due to MDR-TB. Only 84 000 (19%) of the estimated incident MDR-TB cases in the world were detected.6 In our study, 100% sensitivity was observed for Xpert in smear-positive specimens. Other studies have shown sensitivities of the Xpert assay for smearpositive/culture-positive samples varying between

Test Xpert RMP-resistant RMP-susceptible Total

RMP-resistant n

RMP-susceptible n

54 01 55

01 32 33

Sensitivity, %

98.2

Specificity, %

97

Overall agreement, %

97.7

RMP ¼ rifampicin; MGIT ¼ Mycobacteria Growth Indicator Tube.

960

The International Journal of Tuberculosis and Lung Disease

CONCLUSION Our study showed that the Xpert test was accurate and required only minimal biosafety infrastructure. Additional data on Xpert testing at the various district and sub-district levels (designated microscopy centres) will give a more accurate estimate of the PPV of Xpert.

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Acknowledgements The authors thank the Bill and Melinda Gates Foundation, Seattle, WA, USA, and the Foundation for Innovative New Diagnostics (FIND), Geneva, Switzerland, for providing the Xpertw MTB/RIF assay system and for technical support during the study. Conflict of interest: none declared.

References 1 World Health Organization. Global tuberculosis control, 2010. WHO/HTM/TB/2010.7. Geneva, Switzerland: WHO, 2010. 2 Evans C A. GenXpert – a game changer for tuberculosis control? PLOS MED 2011; 8: e 1001064. 3 Boehme C C, Nabeta P, Hillemann D, et al. Rapid molecular

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detection of tuberculosis and rifampin resistance N Engl J Med 2010; 363: 1005–1015. Kent P T, Kubica G P. 1985. Public health mycobacteriology: a guide for the level III laboratory. Atlanta, GA, USA: US Department of Health and Human Services, Centers for Disease Control. Krunner A, Yates M D, Drobniewski F A. Evaluation of MGIT 960-based antimicrobial testing and determination of critical concentration of first and second-line antimicrobial drug with drug resistant clinical strains of Mycobacterium tuberculosis. J Clin Microbial 2006; 44: 811–818. World Health Organization. Global tuberculosis control, 2013. WHO/HTM/TB/2013.11. Geneva, Switzerland: WHO, 2013. Scott L E, McCarthy K, Gous N, et al. Comparison of Xpertw MTB/RIF with other nucleic acid technologies for diagnosing pulmonary tuberculosis in a high HIV prevalence setting: a prospective study. PLOS MED 2011; 8: e 1001061. Theron G, Peter J, van Zyl-Smit R, et al. Evaluation of the Xpertw MTB/RIF assay for the diagnosis of pulmonary tuberculosis in a high HIV prevalence setting. Am J Respir Crit Care Med 2011; 184: 132–140. Steingart K R, Sohn H, Schiller I, Kloda L A, et al. Xpertw MTB/ RIF assay for pulmonary tuberculosis and rifampicin resistance in adults. Cochrane Database Syst Rev 2014; 1: CD009593.

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Evaluation of the Xpert MTB/RIF assay

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RESUME

Cette e´ tude a e´ t´e entreprise afin d’´evaluer la performance du test Xpertw MTB/RIF et de comparer ses r´esultats avec les syst`emes de culture conventionnel ou MGIT 960 en milieu liquide. Dans cette e´ tude, 134 patients ayant eu un diagnostic de tuberculose cliniquement r´esistante au protocole de Cat´egorie I ou de Cat´egorie II ont e´ t´e recrut e´ s afin d’ e´ valuer le test Xpert. Xpert a

correctement identifi e´ tous les isolats de Mycobacterium tuberculosis. La sensibilit e´ et la spe´ cificite´ du test Xpert pour la de´ tection de la r´esistance a` la rifampicine a e´ t´e de 98,2% et 97,0%, respectivement, par comparaison aux r´esultats du MGIT 960.

RESUMEN

El presente estudio se llevo´ a cabo con el proposito ´ de evaluar la eficacia diagnostica ´ de la prueba Xpertw MTB/RIF y comparar sus resultados con el sistema cla´sico y MGIT 960 de cultivo en medio l´ıquido. Participaron en el estudio 134 pacientes con antecedente de tuberculosis y fracaso terap´eutico a un r´egimen de Categor´ıa I o II para la evaluacion. ´ La prueba

Xpert detecto´ de manera correcta todos los aislados cl´ınicos de Mycobacterium tuberculosis. En comparacion ´ con los resultados del sistema MGIT 960, esta prueba exhibio´ una sensibilidad de 98,2% y una especificidad de 97,0% en la deteccion ´ de la resistencia a rifampicina.