TO THE EDITOR:







In 2010, the WHO endorsed the use of the Xpert MTB/RIF assay (Cepheid; Sunnyvale, CA, USA) in countries with a high burden of tuberculosis, considering it to be a technology capable of revolutionizing the diagnosis of the disease.(1) Xpert MTB/RIF assay results are automatically generated and are reported as either negative or positive for Mycobacterium tuberculosis, and in the latter case, as whether the strain is susceptible or resistant to rifampin. Xpert MTB/RIF assay results are also reported as cycle threshold (CT) values, which correspond to the number of PCR cycles required to detect M. tuberculosis. Each additional cycle represents an approximate 50% decrease in the amount of material present in a sample over the previous cycle, thus providing a semiquantitative measure of bacillary burden, and higher CT values correspond to lower bacillary burden.(2)







Given the WHO recommendation to replace sputum smear microscopy with Xpert MTB/RIF as an initial diagnostic test for tuberculosis (although smear microscopy is still used in some countries), because culture results take several weeks, Xpert MTB/RIF CT values may be the only way to assess bacillary burden.(3-6) The objective of the present study was to assess the association between Xpert MTB/RIF CT values and sputum smear microscopy and to assess the diagnostic performance of Xpert MTB/RIF CT values.







This was a cross-sectional study of prospectively collected data, conducted at the tuberculosis outpatient clinic of a health care center in the city of Alvorada, Brazil, where the incidence of tuberculosis was 84.4 cases/100,000 population between 2017 and 2019.(7) The study was approved by the Research Ethics Committee of the Porto Alegre Hospital de Clínicas, located in the city of Porto Alegre (Protocol no. 160063).







Patients aged 18 years or older who had respiratory symptoms suggestive of pulmonary tuberculosis and were able to produce sputum were invited to participate. Those who could not produce sputum were excluded, as were patients with extrapulmonary tuberculosis. The diagnosis of pulmonary tuberculosis was established in accordance with the Third Brazilian Thoracic Association Guidelines on Tuberculosis.(8)







Data were collected on demographic characteristics, smoking, alcohol abuse, symptoms, and comorbidities. Chest X-rays were classified either as being typical of tuberculosis or as being consistent with tuberculosis. (9) Sputum smears were stained by the Ziehl-Neelsen technique for identification of AFB, and culture was performed using the Ogawa-Kudoh method.(8) The Xpert MTB/RIF assay was performed in accordance with the manufacturer's instructions.(2)







Data analysis was performed with IBM SPSS Statistics, version 18.0 (IBM Corporation, Armonk, NY, USA), and MedCalc, version 16.4.3 (MedCalc Software, Mariakerke, Belgium). On the basis of smear microscopy results (positive or negative), we calculated the sensitivity, specificity, positive predictive value, and negative predictive value of Xpert MTB/RIF CT values, with the corresponding 95% CIs. We also constructed ROC curves to determine the optimal cutoff. In order to calculate sample size, we considered the fact that the sensitivity of a given Xpert MTB/RIF CT cutoff was 85% in a previous study.(4) Therefore, using a 95% CI and a power of 80%, the required sample size was estimated to be 100 patients at least.







During the study period, 407 patients underwent Xpert MTB/RIF testing. Of those, 150 had a positive Xpert MTB/RIF result and were included in the study. Table 1 describes the characteristics of the study participants. There was a statistically significant difference in mean CT between sputum smear-positive and sputum smear-negative patients (17.8 ± 4.8 and 22.3 ± 6.7, respectively; p = 0.002). Sensitivity, specificity, positive predictive value, and negative predictive value of an Xpert CT cutoff of 22.7 were 83.6% (95% CI: 75.8-89.7), 60.7% (95% CI: 40.6-78.5), 90.3% (95% CI: 85.3-93.7), and 45.9% (95% CI: 34.0-58.3), respectively. The area under the ROC curve for this cutoff was 0.70 (95% CI: 0.62-0.77; p = 0.002).







 
















Few studies(4-6) have assessed CT cutoffs as a measure of bacillary burden. The CT cutoffs that have been most widely studied are 28(5,6) and 31.8.(4) Hanrahan et al.(6) demonstrated that a CT cutoff of 28 had good predictive value for smear positivity, with a sensitivity of 89.9% and a specificity of 67.0%. In another study,(5) the authors showed that lower CT values were associated with HIV negativity and low BMI and also used a cutoff of 28, reporting a sensitivity of 95% and a specificity of 54.1%. In the present study, a CT value of 22.7 was found to be the optimal cutoff, which is lower than the cutoffs used in most studies. However, the decision regarding the optimal CT cutoff differs according to the context and the objectives of testing. In order to identify as many smear-positive patients as possible, higher CT values should be chosen. In the context of limited resources, however, patients with lower CT values should be prioritized for respiratory isolation.(4)







In one meta-analysis,(4) cutoffs of 27.7 and 31.8 were shown to have a sensitivity of 85% and 95%, respectively, as well as a specificity of 67% and 35%, for smear-positive samples. However, the authors concluded that the moderate diagnostic accuracy of CT values compared with that of sputum smear microscopy, as well as different needs in contexts with varying prevalence of sputum smear positivity, may preclude the use of CT values as a surrogate for sputum smear microscopy in all contexts.







One of the limitations of the present study was the fact that participants were recruited at a single tuberculosis outpatient clinic. However, we believe that the findings are applicable to similar contexts. In addition, we did not assess whether CT values can predict infectiousness and transmission, although smear positivity alone has been shown to be an imperfect measure of infectiousness, with evidence of transmission from smear-negative but culture-positive cases.(10) Despite these limitations, to our knowledge, this is the first study in Brazil to assess the accuracy of CT values as a surrogate for sputum smear microscopy.







In conclusion, Xpert MTB/RIF CT values are associated with sputum smear results and are lower in smear-positive patients. A CT value cutoff of 22.7 showed good predictive value for smear positivity.







AUTHOR CONTRIBUTIONS







GCP: study design, methodology, investigation, formal analysis, and writing of the original draft; GRP, KGD, and LRM: methodology, investigation, writing, revision, and editing; and DRS: study design, methodology, investigation, formal analysis, and writing of the original draft.







FINANCIAL SUPPORT







This study received financial support from the Fundo de Incentivo à Pesquisa do Hospital de Clínicas de Porto Alegre (FIPE-HCPA, Research Incentive Fund of the Porto Alegre Hospital de Clínicas).







REFERENCES







1. World Health Organization. Global tuberculosis report 2019. Geneva: World Health Organization; 2019.







2. Brasil. Ministério da Saúde. Agência Nacional de Vigilância Sanitária (Anvisa) [homepage on the Internet]. Brasília: Anvisa; c2020 [cited 2020 Jun 2]. Boletim Brasileiro de Avaliação de Tecnologias em Saúde (BRATS) no. 16 Available from: http://portal.anvisa.gov.br/resultado-de-busca?p_p_id=101&p_p_lifecycle=0&p_p_state=maximized&p_p_mode=view&p_p_col_id=column-1&p_p_col_count=1&_101_struts_action=%2Fasset_publisher%2Fview_content&_101_assetEntryId=412399&_101_type=document







3. World Health Organization [homepage on the Internet]. Geneva: World Health Organization; c2011 [cited 2020 Jun 2]. Automated Real-time Nucleic Acid Amplification Technology for Rapid and Simultaneous Detection of Tuberculosis and Rifampicin Resistance: Xpert MTB/RIF System--Policy Statement [Adobe Acrobat document, 36p.]. Available from: https://apps.who.int/iris/bitstream/handle/10665/44586/9789241501545_eng.pdf;jsessionid=726C522135C4C5E1DE0E87F9739C5F2D?sequence=1







4. Lange B, Khan P, Kalmambetova G, Al-Darraji HA, Alland D, Antonenka U, et al. Diagnostic accuracy of the Xpert® MTB/RIF cycle threshold level to predict smear positivity: a meta-analysis. Int J Tuberc Lung Dis. 2017;21(5):493-502. https://doi.org/10.5588/ijtld.16.0702







5. Beynon F, Theron G, Respeito D, Mambuque E, Saavedra B, Bulo H, et al. Correlation of Xpert MTB/RIF with measures to assess Mycobacterium tuberculosis bacillary burden in high HIV burden areas of Southern Africa. Sci Rep. 2018;8(1):5201. https://doi.org/10.1038/s41598-018-23066-2







6. Hanrahan CF, Theron G, Bassett J, Dheda K, Scott L, Stevens W, et al. Xpert MTB/RIF as a measure of sputum bacillary burden. Variation by HIV status and immunosuppression. Am J Respir Crit Care Med. 2014;189(11):1426-1434. https://doi.org/10.1164/rccm.201312-2140OC







7. Brasil. Ministério da Saúde. Secretaria de Vigilância em Saúde [homepage on the Internet]. Brasília: Ministério da Saúde, c2020 [updated 2020 Mar; cited 2020 Jun 2]. Boletim Epidemiológico: Tuberculose 2020 [Adobe Acrobat document, 40p.]. Available from: https://antigo.saude.gov.br/images/pdf/2020/marco/24/Boletim-tuberculose-2020-marcas--1-.pdf







8. Conde MB, Melo FA, Marques AM, Cardoso NC, Pinheiro VG, Dalcin Pde T, et al. III Brazilian Thoracic Association Guidelines on tuberculosis. J Bras Pneumol. 2009;35(10):1018-1048. https://doi.org/10.1590/S1806-37132009001000011







9. Diagnostic Standards and Classification of Tuberculosis in Adults and Children. This official statement of the American Thoracic Society and the Centers for Disease Control and Prevention was adopted by the ATS Board of Directors, July 1999. This statement was endorsed by the Council of the Infectious Disease Society of America, September 1999. Am J Respir Crit Care Med. 2000 Apr;161(4 Pt 1):1376-95. https://doi.org/10.1164/ajrccm.161.4.16141







Transmission of Mycobacterium tuberculosis from patients smear-negative for acid-fast bacilli [published correction appears in Lancet 1999 May 15;353(9165):1714]. Lancet. 1999;353(9151):444-449. https://doi.org/10.1016/S0140-6736(98)03406-0