Bilayered Medium for Rapid Isolation of Mycobacterium tuberculosis

Bilayered Medium for Rapid Isolation of Mycobacterium tuberculosis Abstract Background A bilayered medium (BLM) seemed to indicate a rapid recovery time from Mycobacterium tuberculosis (M. tuberculosis) for the patients in question, namely, 48 hours. Method Sputum specimens collected from 112 patients with clinically suspected pulmonary tuberculosis were subjected to Ziehl-Neelsen staining and inoculation on Löwenstein-Jensen medium (LJM) and BLM. Results BLM grew all the 36 (100%) smear positive samples, while LJM grew only 20 (55.5%). On the first 2 days of incubation, BLM grew M. tuberculosis colonies in 14 specimens and in 36 specimens after 3 to 6 days. From the seventh day until the end of the incubation period, BLM grew M. tuberculosis in only 9 specimens. The mean (SD) time for detection on BLM was 6 (5) days, whereas on LJM, it was 22 (12) days. Conclusions BLM is more sensitive than LJM in positive and negative smears. It was also much faster than other methods in detecting the presence of M. tuberculosis. M. tuberculosis, culture, rapid, bilayered medium In recent decades, there has been a resurgence of tuberculosis (TB) with the emergence of multidrug-resistant Mycobacterium tuberculosis (M.tuberculosis) strains. As a result, there are now significant ongoing efforts to research and develop more-efficient tools to isolate and identify M. tuberculosis.1 Löwenstein-Jensen medium (LJM) is still considered the criterion standard for the diagnosis of TB in low-income countries despite its disadvantages, namely, that it is time consuming to perform and has low sensitivity, especially in smear-negative pulmonary TB and extrapulmonary TB. Automated broth culture systems for mycobacterial detection using the mycobacteria growth indicator tube (MGIT) and BacT/ALERT systems (bioMérieux SA) have been major improvements. These systems decreased the time for detection of positive cultures. The authors of studies of those systems reported high isolation rates reaching 100%.2,3 However, automated systems are expensive and require staff training and infrastructure; some even produce radioactive waste. Also, international guidelines recommend that all specimens that are cultured on automated liquid systems should be inoculated on solid medium due to higher contamination probability of liquid media. These obstacles render it difficult to use automated systems in routine diagnosis in most developing countries.4,5 Consequently, many trials have been performed to develop a stronger medium. The aim of such trials has always been to develop a more rapid, sensitive, and economical method to detect M. tuberculosis. A novel bilayered medium (BLM), developed in India in 2008, allegedly had a rapid recovery time, namely, 48 hours. This medium consists of a lower layer of LJM and an upper layer of Middlebrook (MB) 7H10 with an added indicator to detect growth by color change. To date, we know of no available cited studies of this medium except another study by the same authors.6 In this study, we aimed to evaluate the BLM, comparing it with the conventional LJM regarding time of recovery and isolation rates.7 Materials and Methods This study was conducted during a 3-month period from August 2014 through November 2014 at El-Maamorah Chest Hospital in Alexandria, Egypt. The hospital is the only referral hospital in Alexandria for diagnosis and treatment of patients with TB. Specimens were taken from 114 patients with clinically and radiologically suspected pulmonary tuberculosis (PT) who had not yet received any antituberculous treatment. The cohort included 79 males and 35 females, their age ranging from 17 years through 70 years. Written informed consent was obtained from each individual participant; each patient also filled out a full questionnaire. Specimen Collection We obtained 1 early-morning sputum specimen, spontaneously produced, from each patient. Patients were instructed to rinse their mouths by gargling with then takes a deep breath, holding it momentarily, and then lowering the head and bringing up sputum by a deep and vigorous cough. Specimens were collected in obtained in leakproof, sterile, labeled containers, each with a 50-mL–wide mouth. The specimens were checked regarding whether they were mucoid or mucopurulent material from the bronchial tree, with minimum amounts of oral or nasal material, and whether they were free of food particles and other extraneous matter, before they were stored in the refrigerator at 4°C.8 Specimen Processing Each sputum specimen was divided into 2 portions. We processed those specimens as follows:9,10 Acid-Fast Staining Ziehl-Neelsen (ZN) Stain and Culture Sputum smears were prepared, stained and examined according to preestablished guidelines.9,10 The second portion of the sputum specimen was initially digested and decontaminated with NALC–2%NaOH. It was then cultured onto LJM medium with and without paranitrobenzoic acid (PNB) and BLM. LJM was prepared according to standard procedures.9,10 BLM consisted of a lower layer of 5-mL LJM without malachite green (MG) and an upper layer of 2-mL MB 7H10 without malachite green.7 Inoculation Culture media were inoculated using Pasteur pipette with approximately 0.5 mL of sputum after the decontamination process. Bottles of the 3 media (LJM, LJM with PNB, and BLM) were incubated at 37°C in a slant position. Screw caps were loosened for at least 1 week to permit circulation of carbon dioxide for the initiation of growth. To prevent dehydration, caps were tightened and loosened briefly once a week. Culture Examination and Identification LJM. All cultures in LJM bottles were observed for evidences of bacterial growth after 48 to 72 hours to detect any contaminants. We then examined those cultures on the seventh day, to determine which cultures exhibited rapid growth, and then once weekly and as long as 8 weeks later to determine which cultures showed slow growth before discarding the bottle as having negative results. Typical colonies of M. tuberculosis were rough, tough, crumbly, dry, nonpigmented (buff colored), and slow-growing (growth appearing after the seventh day of inoculation).11 BLM. BLM bottles were examined daily and discarded as having negative results after 30 days. There was an earlier appearance of color change away from red, followed by the appearance of translucent or dark-red colonies, or vice versa. The formation of red color in the medium occurred due to reduction of tetrazolium, the growth indicator. Some cultures showed color change only; others showed colored colonies, which were small, translucent, or red, partially above the surface of the media and partially seen submerged in the top layer or totally embedded in the top layer. With further incubation, confluent growth occurred. For confirmation, ZN films were made from all cultures. For those cultures that tested positive and exhibited color change only, the ZN films were taken from the water condensation in the bottle.7 Biochemical Tests For confirmation of M. tuberculosis colonies, 3 biochemical tests were carried out (catalase, nitrate reduction, and niacin). M. tuberculosis had negative results via catalase testing and positive results on nitrate reduction testing and niacin paper strip testing.9 Statistical Analysis We collected data and entered them into the computer using the Statistical Package for the Social Sciences (SPSS) program for statistical analysis, version 21 (SPSS Inc). Data were entered as numerical or categorical, as appropriate. Pearson χ2 testing was used to test the association of categorical variables. Box-and-whiskers plots and error-bar graphs were used. We used kappa testing to determine agreement. Area under the receiver operator characteristic (ROC) curve was used for sensitivity analysis using MedCalc Statistical Software, version 14.8.1 (MedCalc Software bvba). In the present study, an alpha level was set to 5% with a significance level of 95%, and a beta error was accepted up to 20% with a power of study of 80%. Results The present study evaluated BLM in the diagnosis of PT against routine culture results, as determined by LJM. Of the sputum specimens collected from 114 patients with clinically suspected pulmonary TB, on culture, 1 sputum sample grew Mycobacteria; mycobacterium other than TB (TB[MOTT]) on LJM and BLM, and LJM and BLM each showed 1 case of contamination (data not shown). When comparing examined media to ZN smear results, BLM showed high superiority. All specimens that tested culture positive (45 cases) grew on BLM. BLM grew all 36 smear-positive specimens (100%) and 9 smear-negative ones (11.8%). LJM grew only 20 smear-positive specimens (55.5%) and 3 smear-negative ones (3.9%). LJM failed to grow 16 of 36 smear-positive specimens (44.4%) and 6 smear-negative specimens (7.9%) that had tested positive via BLM (Table 1). The percentage of agreement between the 2 culture media was 80.5%. Table 1. Distribution of 112 Sputum Specimens on BLM and LJM According to Their ZN Smear Results Media Smear Result Total (n = 112) Positive (n = 36) Negative (n = 76) BLM No. (%) Growth 36 (100) 9 (11.8) 45 (40.2) No Growth 0 67 (88.2) 67 (59.8) LJM Growth 20 (55.5) 3 (3.9) 23 (20.5) No Growth 16 (44.4) 73 (96.1) 89 (79.5) Media Smear Result Total (n = 112) Positive (n = 36) Negative (n = 76) BLM No. (%) Growth 36 (100) 9 (11.8) 45 (40.2) No Growth 0 67 (88.2) 67 (59.8) LJM Growth 20 (55.5) 3 (3.9) 23 (20.5) No Growth 16 (44.4) 73 (96.1) 89 (79.5) BLM, bilayered medium; LJM, Löwenstein-Jensen medium; ZN, Ziehl-Neelsen. n= refers to the number of specimens positive and negative with ZN smear. View Large Table 1. Distribution of 112 Sputum Specimens on BLM and LJM According to Their ZN Smear Results Media Smear Result Total (n = 112) Positive (n = 36) Negative (n = 76) BLM No. (%) Growth 36 (100) 9 (11.8) 45 (40.2) No Growth 0 67 (88.2) 67 (59.8) LJM Growth 20 (55.5) 3 (3.9) 23 (20.5) No Growth 16 (44.4) 73 (96.1) 89 (79.5) Media Smear Result Total (n = 112) Positive (n = 36) Negative (n = 76) BLM No. (%) Growth 36 (100) 9 (11.8) 45 (40.2) No Growth 0 67 (88.2) 67 (59.8) LJM Growth 20 (55.5) 3 (3.9) 23 (20.5) No Growth 16 (44.4) 73 (96.1) 89 (79.5) BLM, bilayered medium; LJM, Löwenstein-Jensen medium; ZN, Ziehl-Neelsen. n= refers to the number of specimens positive and negative with ZN smear. View Large The sensitivity, negative predictive value (NPV), and overall accuracy of BLM were significantly higher than those of LJM. The sensitivity, NPV, and accuracy of BLM were 100%. The sensitivity of LJM was 55.55%, its NPV was 82.0%, and its overall accuracy was 83.0%. There was perfect raw agreement between ZN and BLM (κ = 0.845). Its specific agreement was 93.0% for negative agreement and 96.8% for positive agreement. There was perfect raw agreement between ZN and LJM (κ = 0.641). Its negative agreement was 88.5% and its positive agreement was 67.8% (Table 2). Table 2. Agreement Between Culture Results of BLM and LJM BLM Result LJM Result Negative Positive Total No. (%) Negative 67 (75.3) 0 67 (59.8) Positive 22 (24.7) 23 (100) 45 (40.2) Total 89 (100) 23 (100) 112 (100) BLM Result LJM Result Negative Positive Total No. (%) Negative 67 (75.3) 0 67 (59.8) Positive 22 (24.7) 23 (100) 45 (40.2) Total 89 (100) 23 (100) 112 (100) BLM, bilayered medium; LJM, Löwenstein-Jensen medium. View Large Table 2. Agreement Between Culture Results of BLM and LJM BLM Result LJM Result Negative Positive Total No. (%) Negative 67 (75.3) 0 67 (59.8) Positive 22 (24.7) 23 (100) 45 (40.2) Total 89 (100) 23 (100) 112 (100) BLM Result LJM Result Negative Positive Total No. (%) Negative 67 (75.3) 0 67 (59.8) Positive 22 (24.7) 23 (100) 45 (40.2) Total 89 (100) 23 (100) 112 (100) BLM, bilayered medium; LJM, Löwenstein-Jensen medium. View Large BLM showed better performance than LJM in successfully discriminating between smear-positive specimens (n = 36) and smear-negative specimens (n = 76). Area under the curve (AUC) for BLM was 0.947, whereas that of LJM was 0.792; the 95% confidence interval (CI) was .89 to .98 for BLM and .70 to .86 for LJM (Table 3). This CI value was statistically significant (z = 3.519 [Figure 1], P ˂.001 [Table 4]). Table 3. Sensitivity Analysis of Performance of BLM and LJM Against Direct Smear: Comparison of ROC Curve Classification Variable Direct Smear AUC SE 95% CI LJM 0.792 0.0422 .70–.86 BLM 0.947 0.0177 .89–.98 Classification Variable Direct Smear AUC SE 95% CI LJM 0.792 0.0422 .70–.86 BLM 0.947 0.0177 .89–.98 ROC, receiver operating characteristic; LJM, Löwenstein-Jensen medium; BLM, bilayered medium; AUC, area under the curve; CI, confidence interval. View Large Table 3. Sensitivity Analysis of Performance of BLM and LJM Against Direct Smear: Comparison of ROC Curve Classification Variable Direct Smear AUC SE 95% CI LJM 0.792 0.0422 .70–.86 BLM 0.947 0.0177 .89–.98 Classification Variable Direct Smear AUC SE 95% CI LJM 0.792 0.0422 .70–.86 BLM 0.947 0.0177 .89–.98 ROC, receiver operating characteristic; LJM, Löwenstein-Jensen medium; BLM, bilayered medium; AUC, area under the curve; CI, confidence interval. View Large Table 4. Sensitivity Analysis of Performance of BLM and LJM Against Direct Smear: Pairwise Comparison of ROC Curves LJM and BLM Variables Value Difference between areas 0.155 SE 0.0440 95% CI .07–.24 z statistic 3.519 Significance level P < .001 LJM and BLM Variables Value Difference between areas 0.155 SE 0.0440 95% CI .07–.24 z statistic 3.519 Significance level P < .001 BLM, bilayered medium; LJM, Löwenstein-Jensen medium; ROC, receiver operating characteristic; CI, confidence interval View Large Table 4. Sensitivity Analysis of Performance of BLM and LJM Against Direct Smear: Pairwise Comparison of ROC Curves LJM and BLM Variables Value Difference between areas 0.155 SE 0.0440 95% CI .07–.24 z statistic 3.519 Significance level P < .001 LJM and BLM Variables Value Difference between areas 0.155 SE 0.0440 95% CI .07–.24 z statistic 3.519 Significance level P < .001 BLM, bilayered medium; LJM, Löwenstein-Jensen medium; ROC, receiver operating characteristic; CI, confidence interval View Large Figure 1 View largeDownload slide Receiver operating characteristic (ROC) curves of Löwenstein-Jensen medium (LJM) and bilayered medium (BLM). Figure 1 View largeDownload slide Receiver operating characteristic (ROC) curves of Löwenstein-Jensen medium (LJM) and bilayered medium (BLM). Regarding recovery time, BLM could grow M. tuberculosis as quickly as 2 days after incubation. Within 1 to 2 days of incubation, BLM started to grow M. tuberculosis colonies in 12 smear-positive specimens and 2 smear-negative specimens. Within 6 days, it grew 80% of all smear-positive specimens. LJM did not isolate any colonies for these specimens during this time frame. A total of 32 smear-positive and 4 smear-negative BLM cultures tested positive after 3 to 6 days of incubation. In the period from day 7 to day 30, the end of the incubation period, BLM grew M. tuberculosis in only 4 smear-positive specimens and in only 5 smear-negative specimens. LJM gave the maximum yield of M. tuberculosis colonies (15 for smear-positive specimens and 3 for smear-negative specimens) in the period from day 15 until the end of incubation (Figure 2). Figure 2 View largeDownload slide Time taken for growth on bilayered medium (BLM) and Löwenstein-Jensen medium (LJM) in relation to their smear results. A, Smear positive. B, Smear negative. Figure 2 View largeDownload slide Time taken for growth on bilayered medium (BLM) and Löwenstein-Jensen medium (LJM) in relation to their smear results. A, Smear positive. B, Smear negative. The shorter recovery time with BLM, compared with LJM, was discovered to be statistically significant (Pearson χ2 = 39.941, Monte Carlo Sig P < .001). The difference in the mean (SD) time for detection of M. tuberculosis was considerably shorter on BLM (5.54 [4.95] days), compared with LJM (21.86 [11.56] days). The minimum recovery time for detection of M. tuberculosis with LJM was 8 days; the maximum was 40 days. In contrast, the minimum recovery time for detection of M. tuberculosis on BLM was 2 days; the maximum was 19 days. Most colonies on BML were embedded inside the agar bottles (21 colonies [46.7%]); a smaller number (10 colonies [22.2%]) grew surface colonies. Fourteen cultures (31.1%) were only detected by a color change of the agar. Within the first 2 days of incubation, cultures that only showed color change were most numerous (11 cultures). After 3 to 6 days of incubation, 20 cultures showed positivity with apparat colonies, whereas only 2 cultures showed only a color change. After that, only colonies indicated positivity of culture results (Figure 3). Figure 3 View largeDownload slide Colonial morphologic characteristics for 45 cultures that tested positive on bilayered medium (BLM). Figure 3 View largeDownload slide Colonial morphologic characteristics for 45 cultures that tested positive on bilayered medium (BLM). On BLM medium, all ZN smears yielded positive results via culture, regardless of smear grading. LJM could detect 12.5% from the few ZN smears with positive results; this percentage increased with increasing smear positivity to be 38.5% for (+), 81.81% for (++), and 100% for (+++) (Table 5). Table 5. Relationship Between ZN Smear Grading Results of 36 Smear-Positive Specimens and Their Culture Yield on LJM and BLM Positive Culture Result ZN Grading No. (%) Scanty (n = 7) + (n = 13) ++ (n = 11) + + + (n = 5) LJM (n = 20) 1 (12.5) 5 (38.5) 9 (81.8) 5 (100) BLM (n = 36) 7 (100) 13 (100) 11 (100) 5 (100) Positive Culture Result ZN Grading No. (%) Scanty (n = 7) + (n = 13) ++ (n = 11) + + + (n = 5) LJM (n = 20) 1 (12.5) 5 (38.5) 9 (81.8) 5 (100) BLM (n = 36) 7 (100) 13 (100) 11 (100) 5 (100) LJM, Löwenstein-Jensen medium; BLM, bilayered medium; ZN, Ziehl-Neelsen. View Large Table 5. Relationship Between ZN Smear Grading Results of 36 Smear-Positive Specimens and Their Culture Yield on LJM and BLM Positive Culture Result ZN Grading No. (%) Scanty (n = 7) + (n = 13) ++ (n = 11) + + + (n = 5) LJM (n = 20) 1 (12.5) 5 (38.5) 9 (81.8) 5 (100) BLM (n = 36) 7 (100) 13 (100) 11 (100) 5 (100) Positive Culture Result ZN Grading No. (%) Scanty (n = 7) + (n = 13) ++ (n = 11) + + + (n = 5) LJM (n = 20) 1 (12.5) 5 (38.5) 9 (81.8) 5 (100) BLM (n = 36) 7 (100) 13 (100) 11 (100) 5 (100) LJM, Löwenstein-Jensen medium; BLM, bilayered medium; ZN, Ziehl-Neelsen. View Large Discussion In the present study, we attempted to assess the feasibility of using BLM as a primary isolation medium for mycobacteria. We compared BLM with the LJM (the criterion standard for M. tuberculosis detection) against the ZN smear as a reference standard. It should be mentioned that ZN might yield false-negative results. It requires a high number of bacilli in a specimen (5000 to 10,000/mL) for detection of bacteria. Results are also affected by a smaller size of specimens and unequal distribution of bacilli in each specimen.12 Consequently, a higher level of positive results in ZN smears is predictive of positive culture results. Among different studies, the isolation rate was reported to range from 76.80% to 85.18% for smear-positive specimens and from 10.20% to 66.66% among smear-negative specimens.13-15 The effect of smear positivity was also apparent in the results of the present study: the isolation rate was discovered to be 56.76% for smear-positive specimens and 3.90% for smear-negative ones. Other factors contributing to failure of growth from smear-positive specimens (false-negative results) may be due to patients who received antitubercular treatment, exposure of sputum specimens to sunlight or heat, contamination before inoculation, or excessive decontamination procedures. These factors were not applicable in this study.12 LJM had a relatively lower isolation rate: it detected 21.0% of specimens. This figure is close to those reported by Chihota et al,14 Ghatole et al,13 and Naveen et al2 (22.80%, 23.17%, and 34.74% respectively). The total isolation rate, on BLM, was almost double that on LJM (40.3%). BLM yielded only positive results in 22 specimens that tested negative on LJM, whereas none of the specimens yielded colonies on LJM only. So, if we were relying only on LJM, we would have missed 22 cases. Other studies that examined combined mycobacterial culture medium also reported favorable isolation rates. Ghatole et al13 examined a combined medium of MB7H11 slant and MB7H9 broth and reported isolation rate to be 97.05% for smear-positive specimens and 91.66% for smear-negative specimens. Cui et al15 evaluated another combined culture media (LJM slant + MB 7H9 broth) and reported isolation rates of M. tuberculosis to be 87.6% and 20.04% among smear-positive and smear-negative specimens, respectively. Both authorship groups recorded lower LJM sensitivity and NPV. BLM yielded a much more rapid recovery time for M. tuberculosis, compared with LJM. These other combined media, however, yielded a longer time for recovery, almost similar to that yielded by LJM. Ghatole et al13 recovered M. tuberculosis within a mean (SD) of 21 (4.44) days in smear-positive cases and 36 (3.44) days in smear-negative cases. Cui et al15 reported the median time for M. tuberculosis detection to be 14 days and 21 days among smear-positive and smear-negative specimens, respectively. Thus, the BLM seems to have a shorter time for M. tuberculosis detection than the other nonautomated combined mycobacterial culture media.≈ In addition to the previously mentioned advantages of the short recovery time and the high isolation rate of BLM noted in the present study, another advantage could be added. The presence of the color growth indicator (triphenyltetrazolium chloride [TTC]) helped in decreasing the recovery time and rendered reading isolated colonies more objective, via the red-color change occurring in the upper layer of BLM that can detected by the naked eye. The color indicator in the present study differs from indicators used in other studies. For example, nitrate reductase assay must be added to the culture during incubation to indicate growth via color change. This finding does not reflect the mycobaterial growth in real time.16,17 Other growth indicators such as resazurin solution, used in resazurin tube assay, require more mycobacteria to produce any colorimetric change; also the transition from one color to another can be difficult to determine.18 The combined medium evaluated by Cui et al15 used the same color indicator as in the present study. It had the same advantages but could not provide quantitative data as accurately, due to the presence of liquid media. However, we note several disadvantages of BLM. First, BLM preparation is cumbersome and requires more time and procedures to prepare than LJM. Second, BLM needs to be freshly prepared, for fear of degradation of antibiotics and antifungals, in contrast with LJM, which can be preserved in the refrigerator as long as 1 month. Third, when attempting to examine ZN films and perform biochemical confirmatory tests on colonies using BLM, we faced a problem with embedded colonies in the upper layer of the agar. We needed to cut through this layer to extract colonies, which was necessary and difficult to carry. Also, in cultures with positive results that have only a red-color change, smears and biochemical tests were performed from water condensation in the culture bottle with the growing microorganism, a process which we sometimes had to repeat. In culture that only tested positive with a color change, results appeared more quickly. These results occurred probably because it is quicker and easier to observe a color change than to observe an actual colony. When adopting ZN staining results as a reference standard, BLM is more sensitive than LJM in smears with positive and negative results. BLM also detected the presence of M. tuberculosis much more rapidly than LJM. In addition, BLM is less expensive than other methods of diagnosis that yield colonies in a comparable period of time. Despite some difficulties we faced in its preparation and diagnosis, we recommend that further studies be performed to simplify the confirmation of isolates. Abbreviations TB tuberculosis LJM Löwenstein-Jensen medium MGIT mycobacteria growth indicator tube BLM bilayered medium MB Middlebrook PT pulmonary tuberculosis ZN Ziehl-Neelsen PNB para-nitrobenzoic acid MG malachite green SPSS Statistical Package for the Social Sciences ROC receiver operator characteristic TB(MOTT) tuberculosis (mycobacterium other than tuberculosis) NPV negative predictive value AUC area under the curve CI confidence interval TTC triphenyltetrazolium chloride. References 1. World Health Organization (WHO) . Global tuberculosis report . Geneva , 2016 . WHO website. http://apps.who.int/iris/bitstream/10665/250441/1/9789241565394-eng.pdf. Accessed 7 Feb, 2018. 2. Naveen G , Peerapur BV . Comparison of the Lowenstein-Jensen medium, the middlebrook 7H10 medium and MB/BacT for the isolation of Mycobacterium tuberculosis (MTB) from clinical specimens . J Clin Diagn Res . 2012 ; 6 ( 10 ): 1704 – 1709 . 3. Dunn JJ , Starke JR , Revell PA . Laboratory diagnosis of Mycobacterium tuberculosis infection and disease in children . J Clin Microbiol . 2016 ; 54 ( 6 ): 1434 – 1441 . 4. Shenai S . WHO recommended tools to improve diagnosis of active and drug resistant tuberculosis . Acta Med Int . 2015 ; 2 ( 2 ): 118 – 129 . 5. World Health Organization . Use of Liquid TB Culture and Drug Susceptibility Testing (DST) in Low and Medium Income Settings: Summary Report of the Expert Group Meeting on the Use of Liquid Culture Media . Geneva : World Health Organization ; 2007 . 6. Chatterjee M , Bhattacharya S , Karak K , Dastidar SG . Effects of different methods of decontamination for successful cultivation of Mycobacterium tuberculosis . Indian J Med Res . 2013 ; 138 ( 4 ): 541 – 548 . 7. Bhattacharya S , Roy R , Chowdhury NR , Dasgupta A , Dastidar SG . Comparison of a novel bilayered medium with the conventional media for cultivation of Mycobacterium tuberculosis . Indian J Med Res . 2009 ; 130 ( 5 ): 561 – 566 . 8. Global Laboratory Initiative . Mycobacteriology laboratory manual . Global Laboratory Initiative ; 2014 . GLI website. http://www.who.int/tb/laboratory/mycobacteriology-laboratory-manual.pdf. Accessed on January 18, 2018 . 9. World Health Organization . Laboratory Service in Tuberculosis Control Part III: Culture , 1998 : Available from: http://apps.who.int/iris/bitstream/10665/65942/1/WHO_TB_98.258_(part1).pdf. Accessed 7 Feb, 2018. 10. Kent PT , Kubica GF. Public health mycobacteriology: a guide for the level III laboratory . In: Kubica GP , editor. Atlanta, GA : U.S. Dept. of Health and Human Services, Public Health Service, Centers for Disease Control ; 1985 . 11. Forbes B , Sahm D , Weissfeld A. Bailey and Scott’s Diagnostic Microbiology . 12th edn. St. Louis, MO : Mosby ; 2007 . 12. Toman K. Toman’s Tuberculosis: Case Detection, Treatment, and Monitoring: Questions and Answers . Geneva, Switzerland : World Health Organization ; 2004 . 13. Ghatole M , Sable C , Kamale P , Kandle S , Jahagirdar V , Yemul V . Evaluation of biphasic culture system for mycobacterial isolation from the sputum of patients with pulmonary tuberculosis . Indian J Med Microbiol . 2005 ; 23 ( 2 ): 111 – 113 . 14. Chihota VN , Grant AD , Fielding K , et al. Liquid vs. solid culture for tuberculosis: performance and cost in a resource-constrained setting . Int J Tuberc Lung Dis . 2010 ; 14 ( 8 ): 1024 – 1031 . 15. Cui Z , Wang J , Zhu C , et al. Evaluation of a novel biphasic culture medium for recovery of mycobacteria: a multi-center study . PLoS One . 2012 ; 7 ( 4 ): e36331 . 16. Kumar M , Khan IA , Verma V , Kalyan N , Qazi GN . Rapid, inexpensive MIC determination of Mycobacterium tuberculosis isolates by using microplate nitrate reductase assay . Diagn Microbiol Infect Dis . 2005 ; 53 ( 2 ): 121 – 124 . 17. Affolabi D , Odoun M , Sanoussi N , et al. Rapid and inexpensive detection of multidrug-resistant Mycobacterium tuberculosis with the nitrate reductase assay using liquid medium and direct application to sputum samples . J Clin Microbiol . 2008 ; 46 ( 10 ): 3243 – 3245 . 18. Jadaun GP , Agarwal C , Sharma H , et al. Determination of ethambutol MICs for Mycobacterium tuberculosis and Mycobacterium avium isolates by resazurin microtitre assay . J Antimicrob Chemother . 2007 ; 60 ( 1 ): 152 – 155 . © American Society for Clinical Pathology 2018. All rights reserved. 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Bilayered Medium for Rapid Isolation of Mycobacterium tuberculosis

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Abstract

Abstract Background A bilayered medium (BLM) seemed to indicate a rapid recovery time from Mycobacterium tuberculosis (M. tuberculosis) for the patients in question, namely, 48 hours. Method Sputum specimens collected from 112 patients with clinically suspected pulmonary tuberculosis were subjected to Ziehl-Neelsen staining and inoculation on Löwenstein-Jensen medium (LJM) and BLM. Results BLM grew all the 36 (100%) smear positive samples, while LJM grew only 20 (55.5%). On the first 2 days of incubation, BLM grew M. tuberculosis colonies in 14 specimens and in 36 specimens after 3 to 6 days. From the seventh day until the end of the incubation period, BLM grew M. tuberculosis in only 9 specimens. The mean (SD) time for detection on BLM was 6 (5) days, whereas on LJM, it was 22 (12) days. Conclusions BLM is more sensitive than LJM in positive and negative smears. It was also much faster than other methods in detecting the presence of M. tuberculosis. M. tuberculosis, culture, rapid, bilayered medium In recent decades, there has been a resurgence of tuberculosis (TB) with the emergence of multidrug-resistant Mycobacterium tuberculosis (M.tuberculosis) strains. As a result, there are now significant ongoing efforts to research and develop more-efficient tools to isolate and identify M. tuberculosis.1 Löwenstein-Jensen medium (LJM) is still considered the criterion standard for the diagnosis of TB in low-income countries despite its disadvantages, namely, that it is time consuming to perform and has low sensitivity, especially in smear-negative pulmonary TB and extrapulmonary TB. Automated broth culture systems for mycobacterial detection using the mycobacteria growth indicator tube (MGIT) and BacT/ALERT systems (bioMérieux SA) have been major improvements. These systems decreased the time for detection of positive cultures. The authors of studies of those systems reported high isolation rates reaching 100%.2,3 However, automated systems are expensive and require staff training and infrastructure; some even produce radioactive waste. Also, international guidelines recommend that all specimens that are cultured on automated liquid systems should be inoculated on solid medium due to higher contamination probability of liquid media. These obstacles render it difficult to use automated systems in routine diagnosis in most developing countries.4,5 Consequently, many trials have been performed to develop a stronger medium. The aim of such trials has always been to develop a more rapid, sensitive, and economical method to detect M. tuberculosis. A novel bilayered medium (BLM), developed in India in 2008, allegedly had a rapid recovery time, namely, 48 hours. This medium consists of a lower layer of LJM and an upper layer of Middlebrook (MB) 7H10 with an added indicator to detect growth by color change. To date, we know of no available cited studies of this medium except another study by the same authors.6 In this study, we aimed to evaluate the BLM, comparing it with the conventional LJM regarding time of recovery and isolation rates.7 Materials and Methods This study was conducted during a 3-month period from August 2014 through November 2014 at El-Maamorah Chest Hospital in Alexandria, Egypt. The hospital is the only referral hospital in Alexandria for diagnosis and treatment of patients with TB. Specimens were taken from 114 patients with clinically and radiologically suspected pulmonary tuberculosis (PT) who had not yet received any antituberculous treatment. The cohort included 79 males and 35 females, their age ranging from 17 years through 70 years. Written informed consent was obtained from each individual participant; each patient also filled out a full questionnaire. Specimen Collection We obtained 1 early-morning sputum specimen, spontaneously produced, from each patient. Patients were instructed to rinse their mouths by gargling with then takes a deep breath, holding it momentarily, and then lowering the head and bringing up sputum by a deep and vigorous cough. Specimens were collected in obtained in leakproof, sterile, labeled containers, each with a 50-mL–wide mouth. The specimens were checked regarding whether they were mucoid or mucopurulent material from the bronchial tree, with minimum amounts of oral or nasal material, and whether they were free of food particles and other extraneous matter, before they were stored in the refrigerator at 4°C.8 Specimen Processing Each sputum specimen was divided into 2 portions. We processed those specimens as follows:9,10 Acid-Fast Staining Ziehl-Neelsen (ZN) Stain and Culture Sputum smears were prepared, stained and examined according to preestablished guidelines.9,10 The second portion of the sputum specimen was initially digested and decontaminated with NALC–2%NaOH. It was then cultured onto LJM medium with and without paranitrobenzoic acid (PNB) and BLM. LJM was prepared according to standard procedures.9,10 BLM consisted of a lower layer of 5-mL LJM without malachite green (MG) and an upper layer of 2-mL MB 7H10 without malachite green.7 Inoculation Culture media were inoculated using Pasteur pipette with approximately 0.5 mL of sputum after the decontamination process. Bottles of the 3 media (LJM, LJM with PNB, and BLM) were incubated at 37°C in a slant position. Screw caps were loosened for at least 1 week to permit circulation of carbon dioxide for the initiation of growth. To prevent dehydration, caps were tightened and loosened briefly once a week. Culture Examination and Identification LJM. All cultures in LJM bottles were observed for evidences of bacterial growth after 48 to 72 hours to detect any contaminants. We then examined those cultures on the seventh day, to determine which cultures exhibited rapid growth, and then once weekly and as long as 8 weeks later to determine which cultures showed slow growth before discarding the bottle as having negative results. Typical colonies of M. tuberculosis were rough, tough, crumbly, dry, nonpigmented (buff colored), and slow-growing (growth appearing after the seventh day of inoculation).11 BLM. BLM bottles were examined daily and discarded as having negative results after 30 days. There was an earlier appearance of color change away from red, followed by the appearance of translucent or dark-red colonies, or vice versa. The formation of red color in the medium occurred due to reduction of tetrazolium, the growth indicator. Some cultures showed color change only; others showed colored colonies, which were small, translucent, or red, partially above the surface of the media and partially seen submerged in the top layer or totally embedded in the top layer. With further incubation, confluent growth occurred. For confirmation, ZN films were made from all cultures. For those cultures that tested positive and exhibited color change only, the ZN films were taken from the water condensation in the bottle.7 Biochemical Tests For confirmation of M. tuberculosis colonies, 3 biochemical tests were carried out (catalase, nitrate reduction, and niacin). M. tuberculosis had negative results via catalase testing and positive results on nitrate reduction testing and niacin paper strip testing.9 Statistical Analysis We collected data and entered them into the computer using the Statistical Package for the Social Sciences (SPSS) program for statistical analysis, version 21 (SPSS Inc). Data were entered as numerical or categorical, as appropriate. Pearson χ2 testing was used to test the association of categorical variables. Box-and-whiskers plots and error-bar graphs were used. We used kappa testing to determine agreement. Area under the receiver operator characteristic (ROC) curve was used for sensitivity analysis using MedCalc Statistical Software, version 14.8.1 (MedCalc Software bvba). In the present study, an alpha level was set to 5% with a significance level of 95%, and a beta error was accepted up to 20% with a power of study of 80%. Results The present study evaluated BLM in the diagnosis of PT against routine culture results, as determined by LJM. Of the sputum specimens collected from 114 patients with clinically suspected pulmonary TB, on culture, 1 sputum sample grew Mycobacteria; mycobacterium other than TB (TB[MOTT]) on LJM and BLM, and LJM and BLM each showed 1 case of contamination (data not shown). When comparing examined media to ZN smear results, BLM showed high superiority. All specimens that tested culture positive (45 cases) grew on BLM. BLM grew all 36 smear-positive specimens (100%) and 9 smear-negative ones (11.8%). LJM grew only 20 smear-positive specimens (55.5%) and 3 smear-negative ones (3.9%). LJM failed to grow 16 of 36 smear-positive specimens (44.4%) and 6 smear-negative specimens (7.9%) that had tested positive via BLM (Table 1). The percentage of agreement between the 2 culture media was 80.5%. Table 1. Distribution of 112 Sputum Specimens on BLM and LJM According to Their ZN Smear Results Media Smear Result Total (n = 112) Positive (n = 36) Negative (n = 76) BLM No. (%) Growth 36 (100) 9 (11.8) 45 (40.2) No Growth 0 67 (88.2) 67 (59.8) LJM Growth 20 (55.5) 3 (3.9) 23 (20.5) No Growth 16 (44.4) 73 (96.1) 89 (79.5) Media Smear Result Total (n = 112) Positive (n = 36) Negative (n = 76) BLM No. (%) Growth 36 (100) 9 (11.8) 45 (40.2) No Growth 0 67 (88.2) 67 (59.8) LJM Growth 20 (55.5) 3 (3.9) 23 (20.5) No Growth 16 (44.4) 73 (96.1) 89 (79.5) BLM, bilayered medium; LJM, Löwenstein-Jensen medium; ZN, Ziehl-Neelsen. n= refers to the number of specimens positive and negative with ZN smear. View Large Table 1. Distribution of 112 Sputum Specimens on BLM and LJM According to Their ZN Smear Results Media Smear Result Total (n = 112) Positive (n = 36) Negative (n = 76) BLM No. (%) Growth 36 (100) 9 (11.8) 45 (40.2) No Growth 0 67 (88.2) 67 (59.8) LJM Growth 20 (55.5) 3 (3.9) 23 (20.5) No Growth 16 (44.4) 73 (96.1) 89 (79.5) Media Smear Result Total (n = 112) Positive (n = 36) Negative (n = 76) BLM No. (%) Growth 36 (100) 9 (11.8) 45 (40.2) No Growth 0 67 (88.2) 67 (59.8) LJM Growth 20 (55.5) 3 (3.9) 23 (20.5) No Growth 16 (44.4) 73 (96.1) 89 (79.5) BLM, bilayered medium; LJM, Löwenstein-Jensen medium; ZN, Ziehl-Neelsen. n= refers to the number of specimens positive and negative with ZN smear. View Large The sensitivity, negative predictive value (NPV), and overall accuracy of BLM were significantly higher than those of LJM. The sensitivity, NPV, and accuracy of BLM were 100%. The sensitivity of LJM was 55.55%, its NPV was 82.0%, and its overall accuracy was 83.0%. There was perfect raw agreement between ZN and BLM (κ = 0.845). Its specific agreement was 93.0% for negative agreement and 96.8% for positive agreement. There was perfect raw agreement between ZN and LJM (κ = 0.641). Its negative agreement was 88.5% and its positive agreement was 67.8% (Table 2). Table 2. Agreement Between Culture Results of BLM and LJM BLM Result LJM Result Negative Positive Total No. (%) Negative 67 (75.3) 0 67 (59.8) Positive 22 (24.7) 23 (100) 45 (40.2) Total 89 (100) 23 (100) 112 (100) BLM Result LJM Result Negative Positive Total No. (%) Negative 67 (75.3) 0 67 (59.8) Positive 22 (24.7) 23 (100) 45 (40.2) Total 89 (100) 23 (100) 112 (100) BLM, bilayered medium; LJM, Löwenstein-Jensen medium. View Large Table 2. Agreement Between Culture Results of BLM and LJM BLM Result LJM Result Negative Positive Total No. (%) Negative 67 (75.3) 0 67 (59.8) Positive 22 (24.7) 23 (100) 45 (40.2) Total 89 (100) 23 (100) 112 (100) BLM Result LJM Result Negative Positive Total No. (%) Negative 67 (75.3) 0 67 (59.8) Positive 22 (24.7) 23 (100) 45 (40.2) Total 89 (100) 23 (100) 112 (100) BLM, bilayered medium; LJM, Löwenstein-Jensen medium. View Large BLM showed better performance than LJM in successfully discriminating between smear-positive specimens (n = 36) and smear-negative specimens (n = 76). Area under the curve (AUC) for BLM was 0.947, whereas that of LJM was 0.792; the 95% confidence interval (CI) was .89 to .98 for BLM and .70 to .86 for LJM (Table 3). This CI value was statistically significant (z = 3.519 [Figure 1], P ˂.001 [Table 4]). Table 3. Sensitivity Analysis of Performance of BLM and LJM Against Direct Smear: Comparison of ROC Curve Classification Variable Direct Smear AUC SE 95% CI LJM 0.792 0.0422 .70–.86 BLM 0.947 0.0177 .89–.98 Classification Variable Direct Smear AUC SE 95% CI LJM 0.792 0.0422 .70–.86 BLM 0.947 0.0177 .89–.98 ROC, receiver operating characteristic; LJM, Löwenstein-Jensen medium; BLM, bilayered medium; AUC, area under the curve; CI, confidence interval. View Large Table 3. Sensitivity Analysis of Performance of BLM and LJM Against Direct Smear: Comparison of ROC Curve Classification Variable Direct Smear AUC SE 95% CI LJM 0.792 0.0422 .70–.86 BLM 0.947 0.0177 .89–.98 Classification Variable Direct Smear AUC SE 95% CI LJM 0.792 0.0422 .70–.86 BLM 0.947 0.0177 .89–.98 ROC, receiver operating characteristic; LJM, Löwenstein-Jensen medium; BLM, bilayered medium; AUC, area under the curve; CI, confidence interval. View Large Table 4. Sensitivity Analysis of Performance of BLM and LJM Against Direct Smear: Pairwise Comparison of ROC Curves LJM and BLM Variables Value Difference between areas 0.155 SE 0.0440 95% CI .07–.24 z statistic 3.519 Significance level P < .001 LJM and BLM Variables Value Difference between areas 0.155 SE 0.0440 95% CI .07–.24 z statistic 3.519 Significance level P < .001 BLM, bilayered medium; LJM, Löwenstein-Jensen medium; ROC, receiver operating characteristic; CI, confidence interval View Large Table 4. Sensitivity Analysis of Performance of BLM and LJM Against Direct Smear: Pairwise Comparison of ROC Curves LJM and BLM Variables Value Difference between areas 0.155 SE 0.0440 95% CI .07–.24 z statistic 3.519 Significance level P < .001 LJM and BLM Variables Value Difference between areas 0.155 SE 0.0440 95% CI .07–.24 z statistic 3.519 Significance level P < .001 BLM, bilayered medium; LJM, Löwenstein-Jensen medium; ROC, receiver operating characteristic; CI, confidence interval View Large Figure 1 View largeDownload slide Receiver operating characteristic (ROC) curves of Löwenstein-Jensen medium (LJM) and bilayered medium (BLM). Figure 1 View largeDownload slide Receiver operating characteristic (ROC) curves of Löwenstein-Jensen medium (LJM) and bilayered medium (BLM). Regarding recovery time, BLM could grow M. tuberculosis as quickly as 2 days after incubation. Within 1 to 2 days of incubation, BLM started to grow M. tuberculosis colonies in 12 smear-positive specimens and 2 smear-negative specimens. Within 6 days, it grew 80% of all smear-positive specimens. LJM did not isolate any colonies for these specimens during this time frame. A total of 32 smear-positive and 4 smear-negative BLM cultures tested positive after 3 to 6 days of incubation. In the period from day 7 to day 30, the end of the incubation period, BLM grew M. tuberculosis in only 4 smear-positive specimens and in only 5 smear-negative specimens. LJM gave the maximum yield of M. tuberculosis colonies (15 for smear-positive specimens and 3 for smear-negative specimens) in the period from day 15 until the end of incubation (Figure 2). Figure 2 View largeDownload slide Time taken for growth on bilayered medium (BLM) and Löwenstein-Jensen medium (LJM) in relation to their smear results. A, Smear positive. B, Smear negative. Figure 2 View largeDownload slide Time taken for growth on bilayered medium (BLM) and Löwenstein-Jensen medium (LJM) in relation to their smear results. A, Smear positive. B, Smear negative. The shorter recovery time with BLM, compared with LJM, was discovered to be statistically significant (Pearson χ2 = 39.941, Monte Carlo Sig P < .001). The difference in the mean (SD) time for detection of M. tuberculosis was considerably shorter on BLM (5.54 [4.95] days), compared with LJM (21.86 [11.56] days). The minimum recovery time for detection of M. tuberculosis with LJM was 8 days; the maximum was 40 days. In contrast, the minimum recovery time for detection of M. tuberculosis on BLM was 2 days; the maximum was 19 days. Most colonies on BML were embedded inside the agar bottles (21 colonies [46.7%]); a smaller number (10 colonies [22.2%]) grew surface colonies. Fourteen cultures (31.1%) were only detected by a color change of the agar. Within the first 2 days of incubation, cultures that only showed color change were most numerous (11 cultures). After 3 to 6 days of incubation, 20 cultures showed positivity with apparat colonies, whereas only 2 cultures showed only a color change. After that, only colonies indicated positivity of culture results (Figure 3). Figure 3 View largeDownload slide Colonial morphologic characteristics for 45 cultures that tested positive on bilayered medium (BLM). Figure 3 View largeDownload slide Colonial morphologic characteristics for 45 cultures that tested positive on bilayered medium (BLM). On BLM medium, all ZN smears yielded positive results via culture, regardless of smear grading. LJM could detect 12.5% from the few ZN smears with positive results; this percentage increased with increasing smear positivity to be 38.5% for (+), 81.81% for (++), and 100% for (+++) (Table 5). Table 5. Relationship Between ZN Smear Grading Results of 36 Smear-Positive Specimens and Their Culture Yield on LJM and BLM Positive Culture Result ZN Grading No. (%) Scanty (n = 7) + (n = 13) ++ (n = 11) + + + (n = 5) LJM (n = 20) 1 (12.5) 5 (38.5) 9 (81.8) 5 (100) BLM (n = 36) 7 (100) 13 (100) 11 (100) 5 (100) Positive Culture Result ZN Grading No. (%) Scanty (n = 7) + (n = 13) ++ (n = 11) + + + (n = 5) LJM (n = 20) 1 (12.5) 5 (38.5) 9 (81.8) 5 (100) BLM (n = 36) 7 (100) 13 (100) 11 (100) 5 (100) LJM, Löwenstein-Jensen medium; BLM, bilayered medium; ZN, Ziehl-Neelsen. View Large Table 5. Relationship Between ZN Smear Grading Results of 36 Smear-Positive Specimens and Their Culture Yield on LJM and BLM Positive Culture Result ZN Grading No. (%) Scanty (n = 7) + (n = 13) ++ (n = 11) + + + (n = 5) LJM (n = 20) 1 (12.5) 5 (38.5) 9 (81.8) 5 (100) BLM (n = 36) 7 (100) 13 (100) 11 (100) 5 (100) Positive Culture Result ZN Grading No. (%) Scanty (n = 7) + (n = 13) ++ (n = 11) + + + (n = 5) LJM (n = 20) 1 (12.5) 5 (38.5) 9 (81.8) 5 (100) BLM (n = 36) 7 (100) 13 (100) 11 (100) 5 (100) LJM, Löwenstein-Jensen medium; BLM, bilayered medium; ZN, Ziehl-Neelsen. View Large Discussion In the present study, we attempted to assess the feasibility of using BLM as a primary isolation medium for mycobacteria. We compared BLM with the LJM (the criterion standard for M. tuberculosis detection) against the ZN smear as a reference standard. It should be mentioned that ZN might yield false-negative results. It requires a high number of bacilli in a specimen (5000 to 10,000/mL) for detection of bacteria. Results are also affected by a smaller size of specimens and unequal distribution of bacilli in each specimen.12 Consequently, a higher level of positive results in ZN smears is predictive of positive culture results. Among different studies, the isolation rate was reported to range from 76.80% to 85.18% for smear-positive specimens and from 10.20% to 66.66% among smear-negative specimens.13-15 The effect of smear positivity was also apparent in the results of the present study: the isolation rate was discovered to be 56.76% for smear-positive specimens and 3.90% for smear-negative ones. Other factors contributing to failure of growth from smear-positive specimens (false-negative results) may be due to patients who received antitubercular treatment, exposure of sputum specimens to sunlight or heat, contamination before inoculation, or excessive decontamination procedures. These factors were not applicable in this study.12 LJM had a relatively lower isolation rate: it detected 21.0% of specimens. This figure is close to those reported by Chihota et al,14 Ghatole et al,13 and Naveen et al2 (22.80%, 23.17%, and 34.74% respectively). The total isolation rate, on BLM, was almost double that on LJM (40.3%). BLM yielded only positive results in 22 specimens that tested negative on LJM, whereas none of the specimens yielded colonies on LJM only. So, if we were relying only on LJM, we would have missed 22 cases. Other studies that examined combined mycobacterial culture medium also reported favorable isolation rates. Ghatole et al13 examined a combined medium of MB7H11 slant and MB7H9 broth and reported isolation rate to be 97.05% for smear-positive specimens and 91.66% for smear-negative specimens. Cui et al15 evaluated another combined culture media (LJM slant + MB 7H9 broth) and reported isolation rates of M. tuberculosis to be 87.6% and 20.04% among smear-positive and smear-negative specimens, respectively. Both authorship groups recorded lower LJM sensitivity and NPV. BLM yielded a much more rapid recovery time for M. tuberculosis, compared with LJM. These other combined media, however, yielded a longer time for recovery, almost similar to that yielded by LJM. Ghatole et al13 recovered M. tuberculosis within a mean (SD) of 21 (4.44) days in smear-positive cases and 36 (3.44) days in smear-negative cases. Cui et al15 reported the median time for M. tuberculosis detection to be 14 days and 21 days among smear-positive and smear-negative specimens, respectively. Thus, the BLM seems to have a shorter time for M. tuberculosis detection than the other nonautomated combined mycobacterial culture media.≈ In addition to the previously mentioned advantages of the short recovery time and the high isolation rate of BLM noted in the present study, another advantage could be added. The presence of the color growth indicator (triphenyltetrazolium chloride [TTC]) helped in decreasing the recovery time and rendered reading isolated colonies more objective, via the red-color change occurring in the upper layer of BLM that can detected by the naked eye. The color indicator in the present study differs from indicators used in other studies. For example, nitrate reductase assay must be added to the culture during incubation to indicate growth via color change. This finding does not reflect the mycobaterial growth in real time.16,17 Other growth indicators such as resazurin solution, used in resazurin tube assay, require more mycobacteria to produce any colorimetric change; also the transition from one color to another can be difficult to determine.18 The combined medium evaluated by Cui et al15 used the same color indicator as in the present study. It had the same advantages but could not provide quantitative data as accurately, due to the presence of liquid media. However, we note several disadvantages of BLM. First, BLM preparation is cumbersome and requires more time and procedures to prepare than LJM. Second, BLM needs to be freshly prepared, for fear of degradation of antibiotics and antifungals, in contrast with LJM, which can be preserved in the refrigerator as long as 1 month. Third, when attempting to examine ZN films and perform biochemical confirmatory tests on colonies using BLM, we faced a problem with embedded colonies in the upper layer of the agar. We needed to cut through this layer to extract colonies, which was necessary and difficult to carry. Also, in cultures with positive results that have only a red-color change, smears and biochemical tests were performed from water condensation in the culture bottle with the growing microorganism, a process which we sometimes had to repeat. In culture that only tested positive with a color change, results appeared more quickly. These results occurred probably because it is quicker and easier to observe a color change than to observe an actual colony. When adopting ZN staining results as a reference standard, BLM is more sensitive than LJM in smears with positive and negative results. BLM also detected the presence of M. tuberculosis much more rapidly than LJM. In addition, BLM is less expensive than other methods of diagnosis that yield colonies in a comparable period of time. Despite some difficulties we faced in its preparation and diagnosis, we recommend that further studies be performed to simplify the confirmation of isolates. Abbreviations TB tuberculosis LJM Löwenstein-Jensen medium MGIT mycobacteria growth indicator tube BLM bilayered medium MB Middlebrook PT pulmonary tuberculosis ZN Ziehl-Neelsen PNB para-nitrobenzoic acid MG malachite green SPSS Statistical Package for the Social Sciences ROC receiver operator characteristic TB(MOTT) tuberculosis (mycobacterium other than tuberculosis) NPV negative predictive value AUC area under the curve CI confidence interval TTC triphenyltetrazolium chloride. References 1. World Health Organization (WHO) . Global tuberculosis report . Geneva , 2016 . WHO website. http://apps.who.int/iris/bitstream/10665/250441/1/9789241565394-eng.pdf. Accessed 7 Feb, 2018. 2. Naveen G , Peerapur BV . Comparison of the Lowenstein-Jensen medium, the middlebrook 7H10 medium and MB/BacT for the isolation of Mycobacterium tuberculosis (MTB) from clinical specimens . J Clin Diagn Res . 2012 ; 6 ( 10 ): 1704 – 1709 . 3. Dunn JJ , Starke JR , Revell PA . Laboratory diagnosis of Mycobacterium tuberculosis infection and disease in children . J Clin Microbiol . 2016 ; 54 ( 6 ): 1434 – 1441 . 4. Shenai S . WHO recommended tools to improve diagnosis of active and drug resistant tuberculosis . Acta Med Int . 2015 ; 2 ( 2 ): 118 – 129 . 5. World Health Organization . Use of Liquid TB Culture and Drug Susceptibility Testing (DST) in Low and Medium Income Settings: Summary Report of the Expert Group Meeting on the Use of Liquid Culture Media . Geneva : World Health Organization ; 2007 . 6. Chatterjee M , Bhattacharya S , Karak K , Dastidar SG . Effects of different methods of decontamination for successful cultivation of Mycobacterium tuberculosis . Indian J Med Res . 2013 ; 138 ( 4 ): 541 – 548 . 7. Bhattacharya S , Roy R , Chowdhury NR , Dasgupta A , Dastidar SG . Comparison of a novel bilayered medium with the conventional media for cultivation of Mycobacterium tuberculosis . Indian J Med Res . 2009 ; 130 ( 5 ): 561 – 566 . 8. Global Laboratory Initiative . Mycobacteriology laboratory manual . Global Laboratory Initiative ; 2014 . GLI website. http://www.who.int/tb/laboratory/mycobacteriology-laboratory-manual.pdf. Accessed on January 18, 2018 . 9. World Health Organization . Laboratory Service in Tuberculosis Control Part III: Culture , 1998 : Available from: http://apps.who.int/iris/bitstream/10665/65942/1/WHO_TB_98.258_(part1).pdf. Accessed 7 Feb, 2018. 10. Kent PT , Kubica GF. Public health mycobacteriology: a guide for the level III laboratory . In: Kubica GP , editor. Atlanta, GA : U.S. Dept. of Health and Human Services, Public Health Service, Centers for Disease Control ; 1985 . 11. Forbes B , Sahm D , Weissfeld A. Bailey and Scott’s Diagnostic Microbiology . 12th edn. St. Louis, MO : Mosby ; 2007 . 12. Toman K. Toman’s Tuberculosis: Case Detection, Treatment, and Monitoring: Questions and Answers . Geneva, Switzerland : World Health Organization ; 2004 . 13. Ghatole M , Sable C , Kamale P , Kandle S , Jahagirdar V , Yemul V . Evaluation of biphasic culture system for mycobacterial isolation from the sputum of patients with pulmonary tuberculosis . Indian J Med Microbiol . 2005 ; 23 ( 2 ): 111 – 113 . 14. Chihota VN , Grant AD , Fielding K , et al. Liquid vs. solid culture for tuberculosis: performance and cost in a resource-constrained setting . Int J Tuberc Lung Dis . 2010 ; 14 ( 8 ): 1024 – 1031 . 15. Cui Z , Wang J , Zhu C , et al. Evaluation of a novel biphasic culture medium for recovery of mycobacteria: a multi-center study . PLoS One . 2012 ; 7 ( 4 ): e36331 . 16. Kumar M , Khan IA , Verma V , Kalyan N , Qazi GN . Rapid, inexpensive MIC determination of Mycobacterium tuberculosis isolates by using microplate nitrate reductase assay . Diagn Microbiol Infect Dis . 2005 ; 53 ( 2 ): 121 – 124 . 17. Affolabi D , Odoun M , Sanoussi N , et al. Rapid and inexpensive detection of multidrug-resistant Mycobacterium tuberculosis with the nitrate reductase assay using liquid medium and direct application to sputum samples . J Clin Microbiol . 2008 ; 46 ( 10 ): 3243 – 3245 . 18. Jadaun GP , Agarwal C , Sharma H , et al. Determination of ethambutol MICs for Mycobacterium tuberculosis and Mycobacterium avium isolates by resazurin microtitre assay . J Antimicrob Chemother . 2007 ; 60 ( 1 ): 152 – 155 . © American Society for Clinical Pathology 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

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Laboratory MedicineOxford University Press

Published: Mar 8, 2018

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