Fluorescein diacetate vital staining for detecting viability of acid-fast bacilli in patients on antituberculosis treatment

International Journal of Mycobacteriology

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Fluorescein diacetate vital staining for detecting viability of acid-fast bacilli in patients on antituberculosis treatment Swapna Kanade a, Gita Nataraj a,*, Milind Ubale a,b, Preeti Mehta a a b

Department of Microbiology, Seth GS Medical College and KEM Hospital, Mumbai, India Department of Microbiology, Rajiv Gandhi Medical College, Thane, India

A R T I C L E I N F O

A B S T R A C T

Article history:

Objective: To determine the performance of FDA vital staining method in detecting viability

Received 30 May 2016

of acid fast bacilli in comparison with culture and also its correlation with conventional

Accepted 8 June 2016

drug susceptibility testing results for isoniazid and rifampicin. Design: The study was

Available online xxxx

conducted from Jan 2014 to June 2015. Sputum samples of patients taking either Cat I or Cat II anti-TB treatment were tested by Ziehl Neelsen and FDA staining, culture and

Keywords:

susceptibility to rifampicin and isoniazid at the end of intensive phase. Results: Of the

Fluorescein diacetate

100 ZN positive specimens, 74 were FDA positive of which 70 were reported positive by both

Tuberculosis

the readers. All specimens having higher grades (2+ and 3+) by ZN were positive by FDA

Vital staining

staining. 87.88% of 1+ and 45% of scanty smears were positive by FDA staining. MTB was isolated in 75 specimens of which 72 were positive by FDA. Two culture negative specimens were FDA positive. FDA staining had sensitivity and specificity of 96% and 92% respectively. 70.66% of the total culture isolates were MDRTB strains. 2.66% strains demonstrated monoresistance to each of INH and rifampicin. Conclusion: FDA microscopy can be used as a simple and rapid TB treatment monitoring tool in resource limited settings to identify those patients requiring immediate culture and DST test. Ó 2016 Asian-African Society for Mycobacteriology. Production and hosting by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/ licenses/by-nc-nd/4.0/).

Introduction Tuberculosis (TB) remains a global health problem in the 21st century, and multidrug-resistant TB (MDR-TB) has amplified the problem. Globally, 480,000 new cases of MDR-TB occur annually, and of these, an estimated 9.0% also exhibit extensively drug-resistant TB [1]. Early diagno-

sis and effective anti-TB treatment (ATT) is necessary to control this epidemic. TB treatment is administered for a long duration, usually a minimum of 6 months. Follow-up at regular intervals is required during the course of treatment to monitor response, as well as for early detection of drug resistance. Presence of viable organisms during the course of treatment, especially after the intensive

* Corresponding author at: Department of Microbiology, 5th Floor, Multistory Building, Parel, Mumbai 400012, Maharashtra, India. E-mail address: [email protected] (G. Nataraj). Peer review under responsibility of Asian African Society for Mycobacteriology. http://dx.doi.org/10.1016/j.ijmyco.2016.06.003 2212-5531/Ó 2016 Asian-African Society for Mycobacteriology. Production and hosting by Elsevier Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Please cite this article in press as: S Kanade et al. Fluorescein diacetate vital staining for detecting viability of acid-fast bacilli in patients on antituberculosis treatment. Int. J. Mycobacteriol. (2016), http://dx.doi.org/10.1016/j.ijmyco.2016.06.003

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phase, indicates resistance to the drugs administered to the patient [2]. Culture is the reference standard for detecting viability of Mycobacterium tuberculosis (MTB), the causative organism of TB, but it takes weeks to obtain results. Automated liquidculture systems, such as MGIT 960, reduce the turnaround time to 2–3 weeks. Most commercially available molecular techniques detect DNA, which is present and amplified from both viable and dead bacilli and, therefore, cannot report organism viability. Furthermore, culture and molecular techniques require expensive infrastructure, equipment, reagents, and lab expertise. Peripheral health centers (PHCs) in resource-limited settings usually lack these facilities and rely only on microscopy as a tool for TB diagnosis and prognosis. Once the patient has started ATT, the first follow-up sputum examination using either Ziehl-Neelsen (ZN) or fluorescent-staining methods is performed at the end of the intensive phase, i.e., 2 months for Cat I patients and 3 months for Cat II patients [3]. In a previous study, sputum conversion by microscopy after a 2month intensive phase was 75.0%, with a range from 61.7% to 90.9% in patients with initially strong and weak positive smears, respectively [4]. Both staining techniques stain live and dead bacilli. A positive follow-up smear-microscopy result can occur, even if a patient is responding to treatment. Limited diagnostic facilities at PHCs force the treatment provider to continue ATT for a further period of 1 month. This unnecessary prolongation of treatment may result in extra burden on the Revised National Tuberculosis Control Program (RNTCP), unnecessary exposure to highly toxic drugs for patients, and a missed diagnosis of drug-resistant TB. Fluorescein diacetate (FDA) is a vital stain, with the technique based on the principle of intracellular FDA hydrolysis. Only living cells actively convert the nonfluorescent FDA into the green fluorescent compound fluorescein following enzymatic activity, which is considered a sign of viability [5,6]. The presence of fluorescent bacilli in follow-up specimens is, therefore, due to viable bacilli and may be indicative of a drug-resistant strain. This study was undertaken to determine the performance of FDA vital staining in detecting viability of acid-fast bacilli (AFB) in comparison with culture as the gold standard. Additionally, the results of FDA staining were correlated with those of conventional drug-susceptibility testing (DST) results for isoniazid and rifampicin.

Materials and methods This study was conducted in a tertiary care hospital in Mumbai, India, after obtaining ethics committee permission. Patients taking either Cat I or Cat II medication under directly observed treatment submitted two sputum specimens for ZN staining at the end of the intensive phase [7,8]. Patients submitting an insufficient quantity of specimen (<2 mL) for performance of all three tests were excluded from analysis.

Microscopy Patients whose follow-up sputum specimens demonstrated the presence of AFB in any of the two specimens submitted

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for examination and who were willing to provide written informed consent were included in the study. To verify the FDA vital staining technique, 20 smear-positive sputum specimens from newly diagnosed treatment-naı¨ve pulmonary TB patients and smears prepared from 10 fresh MTB isolates, including one from the standard strain H37Rv on Lo¨wenstein-Jensen (LJ) medium, were stained using the FDA-staining method. All 30 smears were FDA positive. All 20 sputum specimens were then treated with 5% phenol overnight to kill mycobacteria, followed by FDA staining. These 20 smears were FDA negative. Two smears were prepared from each of the AFB-positive specimens and stained with FDA staining. FDA staining was performed as described by Tsukiyama et al. [9]. All reagents were procured from Sigma–Aldrich (St. Louis, MO, USA). FDA stock solution was used to prepare fresh FDA-working solution. The air-dried, nonfixed smears were stained by FDAworking solution for 30 min, washed with distilled water, and decolorized with 0.5% acid alcohol for 3 min. After washing, the smear was counterstained with 0.5% KMnO4. To render the smear noninfectious before examination, the slide was covered with 5% phenol for 10 min [10]. Smears were examined by fluorescent microscopy at 1000 magnification using an Olympus CX 21 microscope equipped with a Fluo Led Blue (480 nm) light-emitting diode (LED) cassette (Fraen Corporation, Srl, Italy). The presence of bright yellow or greenish rods against the black background indicated positive results. A smear was defined as FDA positive if at least one fluorescent bacillus per 100 high-power fields was observed [11]. Staining and reporting of smears was performed by two independent, trained observers and two technical experts. The results were recorded, but blinded from each other. The technical experts were also blinded to culture results, which were available after a few weeks.

Culture and DST Sputum specimens were digested and decontaminated using the NALC-NaOH method and inoculated on LJ medium for MTB isolation as per World Health Organization (WHO) guidelines [12]. All cultures were read daily for the first week for contamination and rapidly growing mycobacterial species and then weekly thereafter, until growth was detected or after 8 weeks, whichever was later. The recovered isolates were confirmed as MTB using the SD BIOLINE TB Ag MPT 64 rapid assay [13]. The susceptibility of these isolates to isoniazid and rifampicin was tested using the 1% proportion method on LJ medium as described by Canetti et al. [14]. All procedures were performed in certified Class II biological safety cabinets, and biosafety Level 2 practices were followed.

Results One hundred consecutive ZN-smear-positive patients whose specimens gave confirmed results for all three techniques (i.e., FDA staining, culture, and DST) were included for analysis. Of the 100 ZN-positive specimens, 74 were FDA positive, and 70 of these specimens were reported as FDA positive by

Please cite this article in press as: S Kanade et al. Fluorescein diacetate vital staining for detecting viability of acid-fast bacilli in patients on antituberculosis treatment. Int. J. Mycobacteriol. (2016), http://dx.doi.org/10.1016/j.ijmyco.2016.06.003

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Table 1 – Comparison of FDA staining and culture results with grading of ZN staining. ZN grading

ZN-P

CP

CN

FDA-P

FDA-N

Scanty 1+ 2+ 3+ Total

40 33 15 12 100

18 30 15 12 75

22 3 0 0 25

18 29 15 12 74

22 4 0 0 26

CN = culture negative; CP = culture positive; FDA = fluorescein diacetate; N = negative; P = positive; ZN = Ziehl-Neelsen.

Table 2 – Comparison of FDA staining with culture results in both Cat I and Cat II patients. FDA

Pos Neg Total

Patients on Cat II

Patients on Cat I

CP

CN

Total

CP

CN

Total

49 1 50

1 3 4

50 4 54

23 2 25

1 20 21

24 22 46

CN = culture negative; CP = culture positive; FDA = fluorescein diacetate; Neg = negative; Pos = positive.

both readers. When FDA positivity was compared with the grading of ZN-stained smears, all specimens (100%) having higher grades (2+ and 3+) were positive by FDA staining, with 87.88% (29/33) of 1+ and 45% (18/40) of scanty smears also positive by FDA staining (Table 1). MTB was isolated in 75 specimens, of which 72 were positive by FDA. Additionally, two culture-negative specimens were FDA positive. Considering culture as the gold standard, FDA staining had a sensitivity of 96% and a specificity of 92%. Culture positivity in patients taking Cat I or Cat II antiTB regimens was 54.35% and 92.60%, respectively (Table 2). MDR-TB strains were present in 70.66% of the total culture isolates. Of these, 69.81% and 30.19% were on Cat II or Cat I regimen, respectively, and 2.66% of strains demonstrated monoresistance to isoniazid and rifampicin.

Discussion Long turnaround times for cultures and false-positive rates associated with DNA-based molecular-amplification assays for detection of viable bacteria in specimens necessitates the need for other rapid, yet accurate tests. This study on FDA staining, which is a simple and rapid technique, demonstrated that the test exhibited good sensitivity and specificity when compared with culture, for the detection of viable mycobacteria. FDA is a salt of the green fluorophore fluorescein. The principle of FDA staining is based on the ability of FDA to pass through the hydrophobic cell membrane of viable mycobacteria, followed by rapid hydrolysis of FDA by cell esterases [15,16], which are assumed to be present only in live cells. Fluorescein accumulates in the cytoplasm due to the intact cell wall and reports positive FDA results. When viewed under an LED fluorescent microscope, live bacilli demonstrate yellowish-green fluorescence, while dead bacilli lack functional acetyl esterase activity and do not fluoresce. Here, FDA vital staining demonstrated 96% sensitivity, 92% specificity, 94.74% positive-predictive value, and

79.31% negative-predictive value. Other studies have reported comparable sensitivity and specificity [6,11,17,18]. ZN staining and FDA staining demonstrated 25% and 2% false-positive rates, respectively, for viable bacteria as compared with results obtained from culture. Microscopy is now available in all TB centers, and LED microscopy is being phased in under RNTCP in India. It is recommended that in follow-up patients, AFB-positive smears should be stained by FDA immediately to identify microscopy defined treatment failure and, where possible, be confirmed by culture. When FDA was compared with culture results, five discordant results were observed, with all having grading of either scanty or 1+ according to the ZN technique. Two were FDA positive, but culture negative, and three were FDA negative, but culture positive. The two FDA positive/culture negative results could either be false positives or true positives. False-positive FDA results may be due to spontaneous hydrolysis of FDA during storage. This was addressed by preparing working solution weekly using acetone and avoiding contact with water, as well as by storing them in aliquots at 20 °C. In these two cases, culture negativity might be the result of over decontamination of specimens. Furthermore, the viability of bacilli as defined by FDA positivity (fluorochrome activation by enzymatic activity) may not always correspond to bacillus viability as defined by culture (ability to multiply). This is reportedly observed more commonly in early treatment phases [19]. Three specimens that were positive by culture were false negative according to FDA staining. This may be due to poor esterase activity seen in some bacilli. Due to the long turnaround time of culture results, repeat FDA staining in discordant cases was not performed. This study involved follow-up patients on anti-TB treatment. The correlation between the results of ZN staining and FDA staining and the good performance of FDA staining in comparison with those obtained by culture gave rise to two suggestions. Follow-up patients with ZN-smear grades of 2+ or 3+ should be immediately referred for culture and

Please cite this article in press as: S Kanade et al. Fluorescein diacetate vital staining for detecting viability of acid-fast bacilli in patients on antituberculosis treatment. Int. J. Mycobacteriol. (2016), http://dx.doi.org/10.1016/j.ijmyco.2016.06.003

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DST based on the results of ZN-stained smears alone, without the additional FDA staining. This is because all specimens with a ZN-smear grade of 2+ or 3+ were FDA positive. The presence of viable bacilli at follow-up examination is probably a strong indicator of drug-resistant TB. The results of DST indicated a high proportion of MDR-TB (70.66%) strains. All the specimens carrying drug-resistant strains demonstrated viable bacilli by FDA staining. The association between high smear grades and DR-TB has also been reported by others [20,21]. For high-burden settings and where better diagnostic modalities to rapidly screen for DR-TB, such as Xpert MTB/ RIF assays are not available or where culture facilities are limited, it is suggested that follow-up ZN smears demonstrating scanty or 1+ grades should be screened by FDA, and those that are FDA positive referred for culture. Twenty-four percent of patients were FDA, as well as culture positive, but were sensitive to both drugs. This may be due to delayed smear conversion, which is observed more commonly in patients taking Cat I treatment [22]. Of the 74 FDA-positive specimens, concordance in results of both the readers were observed in 70, with all four discordant results exhibiting scanty FDA grading. An additional finding of this study was the high culture positivity (92.6%) in patients taking Cat II treatment. Of these, 74% had MDR-TB and 4% demonstrated monoresistance to rifampicin. As per RNTCP guidelines, Cat II regimens are initiated when patients have either taken >1 month of Cat I treatment in the past or failed to respond to Cat I after 5 months of treatment [23]. It is possible that these patients were harboring a drug-resistant strain to begin with, which could not be detected due to the nonavailability of recommended DST methods, such as MGIT 960, line-probe assays, or Xpert MTB/RIF assays, at peripheral centers. The addition of streptomycin to an already failed regimen would probably not eliminate these strains; therefore, there is an urgent need for baseline DST at diagnosis in MDR-TB suspects. FDA staining may also play a role in infection control. As per WHO guidelines, two consecutive negative smears and cultures taken 30 days apart are considered as criteria to decide noninfectiousness [2]; however, culture has a turnaround time running into weeks or months. Recently, the Centers for Disease Control recommended the use of two consecutive negative test results by Xpert MTB/RIF assay to consider a patient noninfectious [23]; however, the overall cost for implementing this in India may be prohibitive. In resource-constrained settings and given that FDA staining exhibited good sensitivity and specificity as compared with culture results, this method can be used for determining discontinuation of infection-control measures. The FDA vital staining technique has some limitations. The stock solution of the staining reagent needs to be frozen at 20 °C, and the working solution needs to be prepared daily. Fluorescence fades rapidly; therefore, smears should be examined as early as possible. Additionally, this technique does not identify the underlying cause of resistance. In this study, only positive ZN-stained smears were included. The cost of the reagents required for FDA staining is similar to that of AFB or fluorescent staining. A fluorescent microscope is likely available at many centers, as LED technology is recommended by the WHO [24]. The procedures for prepara-

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tion of the stock, as well as those for the working solution for FDA staining, and interpretation of the smear results require minimal training. Therefore, FDA staining can be used in peripheral laboratories having only microscopy facilities. In conclusion, FDA microscopy can be used as a simple and rapid TB-treatment-monitoring tool in resource-limited settings to identify those patients requiring immediate culture and DST. However, studies involving larger numbers of samples will be needed to establish the value of FDA microscopy in the diagnosis of MDR-TB under RNTCP.

Conflicts of interest The authors declare no conflicts of interest.

Acknowledgments The authors would like to acknowledge funds obtained from Research Society of Seth G. S. Medical College and King Edward Memorial Hospital.

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Please cite this article in press as: S Kanade et al. Fluorescein diacetate vital staining for detecting viability of acid-fast bacilli in patients on antituberculosis treatment. Int. J. Mycobacteriol. (2016), http://dx.doi.org/10.1016/j.ijmyco.2016.06.003