Visualizing viable Mycobacterium tuberculosis in sputum to monitor isolation measures

Accepted Manuscript Visualizing viable Mycobacterium tuberculosis in sputum to monitor isolation measures Dr Benjamin Wyplosz, MD, PhD, Faiza Mougari, MD, May Al Rawi, Céline Baillon, PharmD, Dhiba Marigot Outtandy, MD, Damien Le Du, MD, Mathilde Jachym, MD, Vincent Hervé, MD, Laurent Raskine, MD, Emmanuelle Cambau, MD, PhD PII:

S0163-4453(16)30313-9

DOI:

10.1016/j.jinf.2016.11.014

Reference:

YJINF 3850

To appear in:

Journal of Infection

Received Date: 30 October 2016 Revised Date:

27 November 2016

Accepted Date: 30 November 2016

Please cite this article as: Wyplosz B, Mougari F, Al Rawi M, Baillon C, Outtandy DM, Le Du D, Jachym M, Hervé V, Raskine L, Cambau E, Visualizing viable Mycobacterium tuberculosis in sputum to monitor isolation measures, Journal of Infection (2017), doi: 10.1016/j.jinf.2016.11.014. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

Visualizing viable Mycobacterium tuberculosis in sputum to monitor

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isolation measures

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Authors: Benjamin Wyplosz1 (MD, PhD), Faiza Mougari2, 3,4 (MD), May Al Rawi2,4, Céline

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Baillon5 (PharmD), Dhiba Marigot Outtandy6 (MD), Damien Le Du6 (MD), Mathilde

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Jachym6 (MD), Vincent Hervé5 (MD), Laurent Raskine2,4 (MD), Emmanuelle Cambau

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(MD, PhD)

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AP-HP, CHU Bicêtre, Service de maladies infectieuses et tropicales, Le Kremlin-Bicêtre, France

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AP-HP, Hôpital Lariboisière, Laboratory of Bacteriology, Paris, France

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Université Paris Diderot, INSERM IAME UMR1137, Sorbonne Paris Cité, Paris, France.

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National Reference Center for mycobacteria and antimycobacterial resistance, Paris, France.

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Centre Médico-chirugical de Bligny, Laboratoire de bactériologie, Briis-sous-Forges, France

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Centre Médico-chirugical de Bligny, Sanatorium, Briis-sous-Forges, France

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Corresponding author:

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Dr Benjamin Wyplosz, MD, PhD

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Service de maladies infectieuses et tropicales

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CHU Bicêtre, Le Kremlin-Bicêtre, France

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Telephone: + 33 1 45 21 74 25 and +33 6 60 84 02 18, Fax: + 33 1 45 21 74 23

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Running title: Viability test for M. tuberculosis in sputum

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Total word count for the body of the manuscript: 1026

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Highlights 3-5 bullet points (84 words) •

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mainly on the infectiousness of respiratory specimens •

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Discontinuing isolation measures for tuberculosis patients under treatment relies

Assessing the viability of Mycobacterium tuberculosis in sputum takes between 10 days (when bacilli are viable) and 42 days (when bacilli are dead) in liquid media.

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Adapting a commercialized kit, we monitored accurately (specificity 100 %), rapidly (<1h) and at a low cost (<1€) the viability of bacilli in the sputa of 18 tuberculosis

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patients.

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The viability test could help worldwide in the decision to maintain isolation measures.

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Keywords: tuberculosis, isolation, hospital infection control, viability stain, treatment

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response

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ACCEPTED MANUSCRIPT In this Journal, Awoniyi and colleagues evaluated cytokine responses against novel Mtb

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antigens as diagnostic markers for TB disease. However a practical issue for physicians

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looking after patients with TB is being able to distinguish smear positive/culture negative

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patients during the early phases of treatment (1). Isolation measures are mandatory for

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patients hospitalized with pulmonary tuberculosis (TB), in order to prevent transmission (2).

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Although the average time to culture conversion is one month for patients with drug-

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susceptible TB (3), 5%-30% cases require more than two months (4) and up to 6 months can

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be required for MDR-TB (5). Recommendations are to maintain isolation until patients are no

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more infectious, which is ascertained when cultures of respiratory specimens become negative

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(2). Microscopic examination of acid-fast bacilli (AFB) is mainly used as a surrogate marker

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(6, 7) but because AFB staining do not differentiate alive from dead bacilli, the interpretation

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of AFB-positive smears is misleading. After 1-2 months of treatment, AFB can be considered

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dead while they are still alive and isolation measures are thus erroneously discontinued.

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Conversely, AFB can be considered viable while they have already been killed by the

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treatment and patients kept inadequately in isolation (8). To overcome the misleading results,

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attempts have been made to develop viability biomarkers for Mtb. Studies using NAAT

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targeting bacterial RNA/DNA gave convincing results but their cost is not affordable in most

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part of the world (9). Those using fluorescein diacetate are easiest but studies have shown

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either conflicting results, or have been evaluated only for a short period of time after initiation

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of therapy (10).

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Our objective was to develop a viability test to predict the culture results of Mtb by

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visualizing live bacilli in sputum of smear-positive tuberculosis patients under treatment. We

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evaluated a fluorescent staining able to differentiate dead bacilli from alive, which was

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previously used for detection of industrial environmental pathogens. We firstly adapted the kit

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to mycobacteriology and assessed, in in vitro experiments, the concordance of the test with

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the culture results. Then, we studied in an observational prospective study its accurateness to

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predict culture results in patients undergoing antituberculous therapy. Briefly, the Live/Dead® BacLight™ Bacterial viability test (Invitrogen, Biocentric,

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France) permits to visualize the bacteria using SYTO-9 and propidium iodide (PI) fluorescent

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dyes, which both bind to DNA but penetrate specifically cytoplasmic membrane: SYTO-9

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penetrates all bacilli, either viable or not, whereas the PI penetrates only in cells with

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damaged membrane. Consequently, the viable bacteria are impermeable to PI and only

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fluoresced due to SYTO-9 appearing green under the fluorescent microscope, whereas dead

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bacteria are marked by both fluorescent dyes and appear red.

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A bacterial suspension (McFarland standard 1.0, approximately 108CFU/ml) of M.

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tuberculosis H37Rv was submitted to heat inactivation for five hours at 96°C, one sample

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taken every hour. A same suspension was submitted to the activity of isoniazid at 1 mg/L

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(Sigma-Aldrich, Quentin Falavier, France), one sample was taken after 7 and 14 days of

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contact. Then, 10µl of each fluorescent dye (SYTO-9:6 mM, PI:30mM) were mixed with

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20µl of the bacterial suspension sample and incubated for 15 minutes at room temperature

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hidden from light. The results were visualized under a fluorescent microscope (Prototype

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Optical LED fluorescent microscope, RAL Diagnostics, Martillac, France) at x40 and x100

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magnification using B-2A filters and G-2A filters (excitation spectrum 450-490 and 510-560

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nm, respectively) with 7µl of the mix spotted on pre-circled slides. An example of visual

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results is shown in Figure1.

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We measured the proportion of green (alive) bacilli/total green + red (dead) bacilli

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after heat inactivation. This proportion decreased from 90% to 40% after 1h but three hours

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were necessary to reach 0%. After isoniazid contact, the median (mean) proportion of live

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bacilli decreased from 80% (80% +0.12%) to 33 % (40% +0.12%) after 7 days and to 13%

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(13% +0.15%) after 14 days.

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ACCEPTED MANUSCRIPT We included 18 patients (mean age: 37 ±10.5 years) admitted for smear-positive

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pulmonary tuberculosis and hospitalized at Bligny Hospital (Briis-sous-Forge, France).

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Fifteen (83%) patients were infected with a pan-susceptible M. tuberculosis isolate and started

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a standard regimen; one patient was infected with an isolate showing high-level resistance to

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isoniazid and two patients with MDR strains received regimens adapted to antibiotic

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susceptibility testing results. The patients were followed until the discharge. Respiratory

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specimens were routinely collected and submitted to the standard procedures (11) (Auramine

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smear microscopy and culture in liquid and solid media after decontamination) as well as the

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microscopic direct viability test. A 10-µl sample was used for viability staining according to

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the protocol used in the in vitro experiments.

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After starting treatment, seven patients (39%) and three patients (17%) were still

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smear-positive after four and eight weeks of treatment, respectively. The number of patients

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who converted to culture-negative according to time is shown in the Figure 2A. Within the

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observational period of 8 weeks, 15 patients were discharged from the hospital when they did

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not cough anymore (n=11, 61%), or when three smear-negative sputa were obtained (n=4,

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22%). Fifteen patients (83%) converted to culture-negative but three cases, with susceptible

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Mtb, remained smear- and culture- positive (Figure 2B). A decline in viability was

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demonstrated as an increase in time-to-positive culture from a median of 10 days to 30 days

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after 6 weeks (Figure 2C). The microscopic test showed a significant decrease in Mtb

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viability (Figure 2D, number of green bacilli) according to time, which correlated to the

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increase in the median time to positivity of cultures (Figure 2C). The viability test accurately

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detected all respiratory samples that subsequently grew positive-culture (specificity 100%).

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This is the first study showing utility of the microscopic viability test for monitoring

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the infectiousness of smear-positive pulmonary tuberculosis patients under treatment. We

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showed that the viability test correctly predicted all culture-positive samples in the first two

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ACCEPTED MANUSCRIPT months after treatment. Since the test is quick (less than 1 hour for the test versus a median of

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23 days for culture) and easy to perform, it would be useful to help physicians to maintain

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isolation in clinical settings while avoiding unnecessary cultures. Since the test is also cheap

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(<1€ for reagents) this issue could be particularly valuable in countries with limited resources

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and where cultures are unavailable.

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120 Acknowledgments:

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The authors thank their colleagues at Bligny Hospital in the bacteriology laboratory and in the

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sanatorium clinical ward. The authors thank also the technicians and nurses of Laboratory at

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Lariboisière and Bligny hospitals.

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All patients included were treated according to Good Clinical Practices and under Hospital

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regulations for patient information and consent (standard of care).

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All authors declare having no financial and personal relationships with other people or

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organizations that could inappropriately influence their work.

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Funding source:

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The National Reference Center for mycobacteria receives an annual grant from Institut de

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Veille Sanitaire.

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Meeting presentation: our data have never been presented in a meeting

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Figure 1. Viability microscopic direct test using fluorescent microscopy (kit LIVE/DEAD®

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BacLight™) for a AFB-smear positive sputum specimen.

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Bacteria were incubated with SYTO-9 fluorescent dye, which penetrates all bacteria viable or

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not, and with propidium iodide (PI) fluorescent dye which only penetrates in cells with

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damaged membrane. Consequently, the viable bacteria, that are impermeable to PI, are only

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marked by SYTO-9 and appear green under the fluorescent microscope, whereas dead

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bacteria are marked by both fluorescent dyes and appear red.

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Figure 2. Clinical results of the microscopic viability test assessed for sputa collected in

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18 smear-positive patients according to the weeks under antituberculous therapy.

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A) Number of patients treated for pulmonary tuberculosis who converted to culturenegative specimens according to time

B) AFB-positivity according to the WHO semi-logarithmic scale (<1, 1-9, 10-99 and

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≥100 AFB/microscope field by Auramine staining) for all specimens collected C) Time to culture to be positive (liquid or solid culture, the time of positivity being the

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first to be positive) of all specimens collected. The median time decreased

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significantly (Kruskall-Wallis test, p<0.0001).

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D) Viability of M. tuberculosis bacilli on direct microscopic examination of sputum

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smears estimated by the number of green (stained by SYTO9) bacilli per spot, in all

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specimens collected

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References

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1. Awoniyi DO, Teuchert A, Sutherland JS, Mayanja-Kizza H, Howe R, Mihret A, et al. Evaluation of cytokine responses against novel Mtb antigens as diagnostic markers for TB disease. Journal of Infection.73(3):219-30. 2. Jensen PA, Lambert LA, Iademarco MF, Ridzon R. Guidelines for preventing the transmission of Mycobacterium tuberculosis in health-care settings, 2005. MMWR Recomm Rep. 2005 Dec 30;54(RR-17):1-141. PubMed PMID: 16382216. 3. Al-Moamary MS, Black W, Bessuille E, Elwood RK, Vedal S. The significance of the persistent presence of acid-fast bacilli in sputum smears in pulmonary tuberculosis. Chest. 1999 Sep;116(3):726-31. PubMed PMID: 10492279. 4. Lee HY, Chae KO, Lee CH, Choi SM, Lee J, Park YS, et al. Culture conversion rate at 2 months of treatment according to diagnostic methods among patients with culture-positive pulmonary tuberculosis. PLoS One. 2014;9(8):e103768. PubMed PMID: 25105410. Pubmed Central PMCID: 4126681. Epub 2014/08/12. eng. 5. Guglielmetti L, Le Dû D, Jachym M, Henry B, Martin D, Caumes E, et al. Compassionate use of bedaquiline for the treatment of MDR- and XDR-tuberculosis: an interim analysis of a French cohort. Clinical Infectious Diseases. 2014 October 15, 2014. 6. Migliori GB, Zellweger JP, Abubakar I, Ibraim E, Caminero JA, De Vries G, et al. European Union Standards for Tuberculosis Care. European Respiratory Journal. 2012 201204-01 00:00:00;39(4):807-19. 7. National Institute for Health and Clinical Excellence. Tuberculosis. NICE guidelines [NG33]2016 May 2016:[1-178 pp.]. Available from: https://www.nice.org.uk/guidance/ng33. 8. Iseman MD. An unholy trinity--three negative sputum smears and release from tuberculosis isolation. Clin Infect Dis. 1997 Sep;25(3):671-2. PubMed PMID: 9314459. 9. Wallis RS, Doherty TM, Onyebujoh P, Vahedi M, Laang H, Olesen O, et al. Biomarkers for tuberculosis disease activity, cure, and relapse. Lancet Infect Dis. 2009 Mar;9(3):162-72. PubMed PMID: 19246020. Epub 2009/02/28. eng. 10. Datta S, Sherman JM, Bravard MA, Valencia T, Gilman RH, Evans CA. Clinical evaluation of tuberculosis viability microscopy for assessing treatment response. Clin Infect Dis. 2015 Apr 15;60(8):1186-95. PubMed PMID: 25537870. Pubmed Central PMCID: 4370166. Epub 2014/12/30. eng. 11. European Centre for Disease Prevention and Control. Mastering the basics of TB control: development of a handbook on TB diagnostic methods. Stockholm, Sweden: ECDC, 2011.

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ACCEPTED MANUSCRIPT Figure 1 Viability detection using the kit LIVE/DEAD® BacLight™ and fluorescent microscopy for monitoring the decrease in infectiousness under treatment by distinguishing

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viable bacilli (appearing green) from dead ones (appearing red).

Figure 2. Clinical results of the microscopic viability test assessed for sputa collected in 18 smear-positive patients according to the weeks under antituberculous therapy. B. Bacilli richness

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D. Number of green bacilli per spot Green bacilli per spot (N)

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