• 1.

    Evans CA, 2011. GeneXpert—a game-changer for tuberculosis control? PLoS Med 8: e1001064.

  • 2.

    McCarthy K, Fielding K, Churchyard GJ, Grant AD, 2018. Empiric tuberculosis treatment in South African primary health care facilities—for whom, where, when and why: implications for the development of tuberculosis diagnostic tests. PLoS One 13: e0191608.

    • Search Google Scholar
    • Export Citation
  • 3.

    Wingfield T, Boccia D, Tovar M, Gavino A, Zevallos K, Montoya R, Lönnroth K, Evans CA, 2014. Defining catastrophic costs and comparing their importance for adverse tuberculosis outcome with multi-drug resistance: a prospective cohort study, Peru. PLoS Med 11: e1001675.

    • Search Google Scholar
    • Export Citation
  • 4.

    Martinson NA, Karstaedt A, Venter WF, Omar T, King P, Mbengo T, Marais E, McIntyre J, Chaisson R, Hale M, 2007. Causes of death in hospitalized adults with a premortem diagnosis of tuberculosis: an autopsy study. AIDS 21: 20432050.

    • Search Google Scholar
    • Export Citation
  • 5.

    Moore DAJ 2006. Microscopic-observation drug-susceptibility assay for the diagnosis of TB. N Engl J Med 355: 15391550.

  • 6.

    DiNardo AR 2018. Diagnostic and treatment monitoring potential of a stool-based quantitative PCR assay for pulmonary tuberculosis. Am J Trop Med Hyg 99: 310316, doi:10.4269/ajtmh.18-0004.

    • Search Google Scholar
    • Export Citation
  • 7.

    Oberhelman RA 2015. A controlled study of tuberculosis diagnosis in HIV-infected and uninfected children in Peru. PLoS One 10: e0120915.

  • 8.

    Burman WJ, Reves RR, 2000. Review of false-positive cultures for Mycobacterium tuberculosis and recommendations for avoiding unnecessary treatment. Clin Infect Dis 31: 13901395.

    • Search Google Scholar
    • Export Citation
  • 9.

    de Boer AS, Blommerde B, de Haas PEW, Sebek MMGG, Lambregts-van Weezenbeek KSB, Dessens M, van Soolingen D, 2002. False-positive Mycobacterium tuberculosis cultures in 44 laboratories in The Netherlands (1993 to 2000): incidence, risk factors, and consequences. J Clin Microbiol 40: 40044009.

    • Search Google Scholar
    • Export Citation
  • 10.

    Steingart KR, Schiller I, Horne DJ, Pai M, Boehme CC, Dendukuri N, 2014. Xpert® MTB/RIF assay for pulmonary tuberculosis and rifampicin resistance in adults. Cochrane Database Syst Rev 1: CD009593.

    • Search Google Scholar
    • Export Citation
  • 11.

    Mayta H 2003. Evaluation of a PCR-based universal heteroduplex generator assay as a tool for rapid detection of multidrug-resistant Mycobacterium tuberculosis in Peru. J Clin Microbiol 41: 57745777.

    • Search Google Scholar
    • Export Citation
  • 12.

    Datta S, Shah L, Gilman RH, Evans CA, 2017. Comparison of sputum collection methods for tuberculosis diagnosis: a systematic review and pairwise and network meta-analysis. Lancet Glob Health 5: e760e771.

    • Search Google Scholar
    • Export Citation
  • 13.

    Chow F, Espiritu N, Gilman RH, Gutierrez R, Lopez S, Escombe AR, Evans CAW, Moore DAJ, 2006. La cuerda dulce—a tolerability and acceptability study of a novel approach to specimen collection for diagnosis of paediatric pulmonary tuberculosis. BMC Infect Dis 6: 67.

    • Search Google Scholar
    • Export Citation
  • 14.

    Paris L 2017. Urine lipoarabinomannan glycan in HIV-negative patients with pulmonary tuberculosis correlates with disease severity. Sci Transl Med 9: eaal2807.

    • Search Google Scholar
    • Export Citation
  • 15.

    Wolf H 2008. Diagnosis of pediatric pulmonary tuberculosis by stool PCR. Am J Trop Med Hyg 79: 893898.

  • 16.

    Cordova J 2010. Evaluation of molecular tools for detection and drug susceptibility testing of Mycobacterium tuberculosis in stool specimens from patients with pulmonary tuberculosis. J Clin Microbiol 48: 18201826.

    • Search Google Scholar
    • Export Citation
  • 17.

    Oberhelman RA 2010. Diagnostic approaches for paediatric tuberculosis by use of different specimen types, culture methods, and PCR: a prospective case-control study. Lancet Infect Dis 10: 612620.

    • Search Google Scholar
    • Export Citation
  • 18.

    Nicol MP, Spiers K, Workman L, Isaacs W, Munro J, Black F, Zemanay W, Zar HJ, 2013. Xpert MTB/RIF testing of stool samples for the diagnosis of pulmonary tuberculosis in children. Clin Infect Dis 57: e18e21.

    • Search Google Scholar
    • Export Citation
  • 19.

    LaCourse SM 2018. Stool Xpert MTB/RIF and urine lipoarabinomannan for the diagnosis of tuberculosis in hospitalized HIV-infected children. AIDS 32: 6978.

    • Search Google Scholar
    • Export Citation
  • 20.

    Ramos E 2010. Optimizing tuberculosis testing for basic laboratories. Am J Trop Med Hyg 83: 896901.

  • 21.

    Friedrich SO Pan African Consortium for the Evaluation of Anti-tuberculosis Antibiotics (PanACEA), 2013. Assessment of the sensitivity and specificity of Xpert MTB/RIF assay as an early sputum biomarker of response to tuberculosis treatment. Lancet. Respir Med 1: 462470.

    • Search Google Scholar
    • Export Citation
  • 22.

    Datta S, Sherman JM, Bravard M, Valencia T, Gilman RH, Evans CA, 2015. Clinical evaluation of tuberculosis viability microscopy for assessing treatment response. Clin Infect Dis 60: 11861195.

    • Search Google Scholar
    • Export Citation
  • 23.

    Hellyer TJ, DesJardin LE, Teixeira L, Perkins MD, Cave MD, Eisenach KD, 1999. Detection of viable Mycobacterium tuberculosis by reverse transcriptase-strand displacement amplification of mRNA. J Clin Microbiol 37: 518523.

    • Search Google Scholar
    • Export Citation

 

 

 

 

The Potential for Testing Stool to Reduce Tuberculosis Missed Diagnoses and Misdiagnoses

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  • 1 Section of Infectious Diseases and Immunity, Imperial College London, and Wellcome Trust Imperial College Centre for Global Health Research, London, United Kingdom;
  • 2 Innovation for Health and Development (IFHAD), Laboratory of Research and Development, Universidad Peruana Cayetano Heredia, Lima, Peru;
  • 3 Innovación Por la Salud Y Desarrollo (IPSYD), Asociación Benéfica PRISMA, Lima, Peru

Tuberculosis kills more people than any other infection, mainly in resource-constrained settings where diagnosing tuberculosis and detecting drug resistance are particularly challenging.1 All tuberculosis tests frequently have false-negative results, causing some patients to miss out on the tuberculosis treatment they need, patients with suspected tuberculosis to require multiple tests before diagnosis, delayed diagnosis, and a significant minority of patients to start treatment of tuberculosis empirically, without bacteriological confirmation of diagnosis or assessment of drug susceptibility.2 Inevitably, some of the patients receiving empirical tuberculosis therapy do so inappropriately, unnecessarily experiencing stigma, costs, and toxicity, and sometimes causing them to miss out on potentially life-saving therapy for their actual disease process.3,4

Difficulties diagnosing tuberculosis and determining drug susceptibility partly result from the inadequacy of appropriate technology tests. Chest radiography has moderate sensitivity and poor specificity. The only laboratory test that most patients have access to globally is smear microscopy, which for more than a century has had such low sensitivity that approximately half of people with tuberculosis have false-negative results.5 Culture is the most sensitive test and provides the most clinically reliable assessment of drug sensitivity, but it is technically demanding, potentially hazardous to laboratory personnel, and generally takes weeks to complete.1 Polymerase chain reaction (PCR) has great potential because it takes only a few hours, has intermediate sensitivity between smear and culture for sputum analysis, and predicts drug resistance fairly reliably.1 However, the need for carefully selected controls in the evaluation of all laboratory diagnostic tests has been largely neglected, which is important because culture and in-house PCR are both prone to false-positive and false-negative results.611

Poor tuberculosis test performance is confounded by the quality of diagnostic specimens.12 Most tuberculosis affects the lungs, for which sputum is the main diagnostic specimen, and strategies to increase the quality of sputum collection have recently been clarified.12 However, it is difficult or impossible to collect sputum from some adults and most young children, for whom the relative merits of testing saliva, induced sputum, urine, nasogastric aspirates, or swallowed string are poorly defined.13,14 Most sputum is swallowed, and both tuberculosis bacteria and their DNA survive passage through the intestinal tract, so stool samples may be tested for the evidence of pulmonary tuberculosis and drug susceptibility.1517 However, the feasibility of using stool to diagnose pulmonary tuberculosis is limited by the observations that swallowed sputum in stool is likely to be considerably diluted within the intestinal tract, culture-based detection may be impaired by gastric acid killing mycobacteria, stool flora can contaminate cultures, and stool may contain PCR inhibitors.17

In this issue of The American Journal of Tropical Medicine and Hygiene, Andrew DiNardo et al.6 report an important advance in this field. They adapted a DNA extraction and concentration technique developed for soil samples with an in-house PCR technique to study detection of mycobacteria in stool. This considerably improved the limit of the detection of Mycobacterium tuberculosis DNA in stool to be comparable with that of the most widely used techniques for sputum. Indeed, in patients with sputum culture–positive tuberculosis, stool PCR was considerably more sensitive than sputum microscopy—a remarkable finding. These results are comparable with those from pilot studies in pediatric populations in which PCR was used to compare small-volume stool samples with induced sputum samples.18 Concordant with these findings, in HIV-coinfected children, PCR testing was shown to have similar diagnostic yield for stool, expectorated sputum, and gastric aspirates.19 If validated, the novel sample processing approach described by DiNardo et al. may be transformative for diagnosing tuberculosis by stool PCR and perhaps also by PCR of other specimens. This is important because the highest risk patients with tuberculosis such as children and people living with human immunodeficiency virus usually have paucibacillary sputum, which urgently requires new techniques for concentrating mycobacterial DNA. Currently, centrifugation is the main technique used for concentrating mycobacteria in clinical specimens, but this is inefficient and biohazardous, and alternatives such as filtration or flotation have so far been problematic.20 Although this study offers a significant advance, some aspects of the methodology will require refinement before progressing from proof-of-concept to implementation. It is also noteworthy that most of the few participants who were unable to produce sputum or had presumed false-negative sputum tuberculosis test results were also negative on stool testing. Indeed, even if a new test had 100% sensitivity and specificity for tuberculosis, this would be hard to demonstrate in people with suspected sputum-scarce or paucibacillary tuberculosis because in these groups definitively proving the presence or absence of tuberculosis is often impossible.

The tuberculosis diagnostic landscape is advancing rapidly but tools to monitor treatment response are relatively neglected. Monitoring treatment response in patients with pulmonary tuberculosis provides information about patient clinical status, has the potential to predict and enable prevention of treatment failure, and also guides infection control. The tool most commonly used to monitor treatment response is sputum-smear microscopy, which relies on the patient producing good quality sputum, and is generally only useful for patients who were smear-positive at the start of treatment. DiNardo et al. report that patients who remained stool PCR positive after 2 months of therapy were 2.8 times more likely to have drug resistance or treatment failure, although sensitivity was only 44%.6 Poor treatment response has multiple causes, but the most common is inadequate treatment of unrecognized drug-resistant tuberculosis. Therefore, rapid drug susceptibility testing should be provided to all patients at the start of treatment. Even when this policy is optimally implemented, tuberculosis treatment sometimes fails. Changes in symptoms, body weight, and microbiological indicators during treatment are poorly characterized, so when inadequate treatment is commenced, it often continues for months before incipient treatment failure is ascertained. Neither conventional tuberculosis microscopy nor PCR discriminate between live and dead tuberculosis bacilli, so these assays may have intrinsically limited reliability for predicting treatment failure, whether testing sputum or stool.6,21 Consequently, culture is the gold standard for identifying whether treatment is killing tuberculosis bacteria in vivo. However, culture is slow, so results provide out-of-date information. Therefore, finding tools for the early prediction of treatment failure is a priority. Viability microscopy and RNA-PCR have shown promise to monitor treatment response within 2 weeks of treatment initiation with same-day results, and the former can be used in microscopy centers with infrastructure that is already widely available.22,23 The novel technique for nucleic acid concentration that has been adapted from soil processing as evaluated by DiNardo et al may have important potential for more accurately assessing tuberculosis treatment response if it can be adapted to concentrate RNA from stool samples.23

In addition to its potential for predicting treatment failure, the improved sensitivity of stool PCR reported by DiNardo et al. has the potential to increase the laboratory confirmation of tuberculosis. This should decrease the number of cases with empirical treatment, increase the proportion of patients with tuberculosis who receive prompt and appropriate therapy, prevent people with other diagnoses receiving unnecessary tuberculosis therapy instead of the specific treatment that they actually need, and reduce the risk of inadvertent mistreatment of drug-resistant tuberculosis causing selection of drug resistance, treatment failure, death, and spread of tuberculosis to others.

Acknowledgments:

We thank the following for suggestions concerning this editorial: Dougles Huff, Matthew Saunders, Renee Newby, Justine Zhang, and Trent Herdman.

REFERENCES

  • 1.

    Evans CA, 2011. GeneXpert—a game-changer for tuberculosis control? PLoS Med 8: e1001064.

  • 2.

    McCarthy K, Fielding K, Churchyard GJ, Grant AD, 2018. Empiric tuberculosis treatment in South African primary health care facilities—for whom, where, when and why: implications for the development of tuberculosis diagnostic tests. PLoS One 13: e0191608.

    • Search Google Scholar
    • Export Citation
  • 3.

    Wingfield T, Boccia D, Tovar M, Gavino A, Zevallos K, Montoya R, Lönnroth K, Evans CA, 2014. Defining catastrophic costs and comparing their importance for adverse tuberculosis outcome with multi-drug resistance: a prospective cohort study, Peru. PLoS Med 11: e1001675.

    • Search Google Scholar
    • Export Citation
  • 4.

    Martinson NA, Karstaedt A, Venter WF, Omar T, King P, Mbengo T, Marais E, McIntyre J, Chaisson R, Hale M, 2007. Causes of death in hospitalized adults with a premortem diagnosis of tuberculosis: an autopsy study. AIDS 21: 20432050.

    • Search Google Scholar
    • Export Citation
  • 5.

    Moore DAJ 2006. Microscopic-observation drug-susceptibility assay for the diagnosis of TB. N Engl J Med 355: 15391550.

  • 6.

    DiNardo AR 2018. Diagnostic and treatment monitoring potential of a stool-based quantitative PCR assay for pulmonary tuberculosis. Am J Trop Med Hyg 99: 310316, doi:10.4269/ajtmh.18-0004.

    • Search Google Scholar
    • Export Citation
  • 7.

    Oberhelman RA 2015. A controlled study of tuberculosis diagnosis in HIV-infected and uninfected children in Peru. PLoS One 10: e0120915.

  • 8.

    Burman WJ, Reves RR, 2000. Review of false-positive cultures for Mycobacterium tuberculosis and recommendations for avoiding unnecessary treatment. Clin Infect Dis 31: 13901395.

    • Search Google Scholar
    • Export Citation
  • 9.

    de Boer AS, Blommerde B, de Haas PEW, Sebek MMGG, Lambregts-van Weezenbeek KSB, Dessens M, van Soolingen D, 2002. False-positive Mycobacterium tuberculosis cultures in 44 laboratories in The Netherlands (1993 to 2000): incidence, risk factors, and consequences. J Clin Microbiol 40: 40044009.

    • Search Google Scholar
    • Export Citation
  • 10.

    Steingart KR, Schiller I, Horne DJ, Pai M, Boehme CC, Dendukuri N, 2014. Xpert® MTB/RIF assay for pulmonary tuberculosis and rifampicin resistance in adults. Cochrane Database Syst Rev 1: CD009593.

    • Search Google Scholar
    • Export Citation
  • 11.

    Mayta H 2003. Evaluation of a PCR-based universal heteroduplex generator assay as a tool for rapid detection of multidrug-resistant Mycobacterium tuberculosis in Peru. J Clin Microbiol 41: 57745777.

    • Search Google Scholar
    • Export Citation
  • 12.

    Datta S, Shah L, Gilman RH, Evans CA, 2017. Comparison of sputum collection methods for tuberculosis diagnosis: a systematic review and pairwise and network meta-analysis. Lancet Glob Health 5: e760e771.

    • Search Google Scholar
    • Export Citation
  • 13.

    Chow F, Espiritu N, Gilman RH, Gutierrez R, Lopez S, Escombe AR, Evans CAW, Moore DAJ, 2006. La cuerda dulce—a tolerability and acceptability study of a novel approach to specimen collection for diagnosis of paediatric pulmonary tuberculosis. BMC Infect Dis 6: 67.

    • Search Google Scholar
    • Export Citation
  • 14.

    Paris L 2017. Urine lipoarabinomannan glycan in HIV-negative patients with pulmonary tuberculosis correlates with disease severity. Sci Transl Med 9: eaal2807.

    • Search Google Scholar
    • Export Citation
  • 15.

    Wolf H 2008. Diagnosis of pediatric pulmonary tuberculosis by stool PCR. Am J Trop Med Hyg 79: 893898.

  • 16.

    Cordova J 2010. Evaluation of molecular tools for detection and drug susceptibility testing of Mycobacterium tuberculosis in stool specimens from patients with pulmonary tuberculosis. J Clin Microbiol 48: 18201826.

    • Search Google Scholar
    • Export Citation
  • 17.

    Oberhelman RA 2010. Diagnostic approaches for paediatric tuberculosis by use of different specimen types, culture methods, and PCR: a prospective case-control study. Lancet Infect Dis 10: 612620.

    • Search Google Scholar
    • Export Citation
  • 18.

    Nicol MP, Spiers K, Workman L, Isaacs W, Munro J, Black F, Zemanay W, Zar HJ, 2013. Xpert MTB/RIF testing of stool samples for the diagnosis of pulmonary tuberculosis in children. Clin Infect Dis 57: e18e21.

    • Search Google Scholar
    • Export Citation
  • 19.

    LaCourse SM 2018. Stool Xpert MTB/RIF and urine lipoarabinomannan for the diagnosis of tuberculosis in hospitalized HIV-infected children. AIDS 32: 6978.

    • Search Google Scholar
    • Export Citation
  • 20.

    Ramos E 2010. Optimizing tuberculosis testing for basic laboratories. Am J Trop Med Hyg 83: 896901.

  • 21.

    Friedrich SO Pan African Consortium for the Evaluation of Anti-tuberculosis Antibiotics (PanACEA), 2013. Assessment of the sensitivity and specificity of Xpert MTB/RIF assay as an early sputum biomarker of response to tuberculosis treatment. Lancet. Respir Med 1: 462470.

    • Search Google Scholar
    • Export Citation
  • 22.

    Datta S, Sherman JM, Bravard M, Valencia T, Gilman RH, Evans CA, 2015. Clinical evaluation of tuberculosis viability microscopy for assessing treatment response. Clin Infect Dis 60: 11861195.

    • Search Google Scholar
    • Export Citation
  • 23.

    Hellyer TJ, DesJardin LE, Teixeira L, Perkins MD, Cave MD, Eisenach KD, 1999. Detection of viable Mycobacterium tuberculosis by reverse transcriptase-strand displacement amplification of mRNA. J Clin Microbiol 37: 518523.

    • Search Google Scholar
    • Export Citation

Author Notes

Address correspondence to Carlton A. Evans, Section of Infectious Diseases and Immunity, Imperial College London, Hammersmith Hospital Campus, London W12 0NN, United Kingdom. E-mail: carlton.evans@ifhad.org

Financial support: The Wellcome Trust (awards 057434/Z/99/Z, 070005/Z/02/Z, 078340/Z/05/Z, 105788/Z/14/Z, and 201251/Z/16/Z); DFID-CSCF; the Joint Global Health Trials consortium (MRC, DFID, and Wellcome Trust award MR/K007467/1); the STOP TB partnership’s TB REACH initiative funded by the Government of Canada and the Bill & Melinda Gates Foundation (awards W5_PER_CDT1_PRISMA and OPP1118545); and IFHAD (Innovation For Health And Development).

Authors’ addresses: Sumona Datta and Luis E. Gonzales-Huerta, Section of Infectious Diseases and Immunity, Imperial College London, and Wellcome Trust Imperial College Centre for Global Health Research, London, United Kingdom, Innovation for Health and Development (IFHAD), Laboratory of Research and Development, Universidad Peruana Cayetano Heredia, Lima, Peru, and Innovación Por la Salud Y Desarrollo (IPSYD), Asociación Benéfica PRISMA, Lima, Peru, E-mails: sumona.datta@ifhad.org and luis.gonzales@ifhad.org. Carlton A. Evans, Section of Infectious Diseases and Immunity, Imperial College London, United Kingdom, E-mail: carlton.evans@ifhad.org.

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