|Year : 2019 | Volume
| Issue : 1 | Page : 44-51
Light-emitting diode-fluorescent microscopy: Determining its sensitivity and specificity in diagnosis of pulmonary tuberculosis in a high-burden tuberculosis region and resource-limited country like India
Shariq Ahmed1, Indu Shukla1, Nazish Fatima1, Sumit K Varshney1, Mohammad Shameem2, Uzma Tayyaba1
1 Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
2 Department of TB and Respiratory Diseases, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh, Uttar Pradesh, India
|Date of Submission||12-Apr-2018|
|Date of Decision||27-May-2018|
|Date of Acceptance||24-Jun-2018|
|Date of Web Publication||14-Feb-2019|
Department of Microbiology, Jawaharlal Nehru Medical College, Aligarh Muslim University, Aligarh - 202 001, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: Tuberculosis (TB) remains a major global health problem. India accounts for the one-fourth of the global TB burden. For resource-limited countries like India having high burden of TB, smear microscopy is the most commonly used method for microbiological diagnosis. Although culture being highly sensitive and specific method, it is time-consuming. Under Revised National TB Control Program (RNTCP), Ziehl–Neelsen (ZN) stain-based microscopy and light-emitting diode-based fluorescent microscopy (LED-FM) are being recommended. Aim: The present study was done to determine the sensitivity and specificity of ZN stain-based microscopy and FM (LED-FM) keeping solid culture as gold standard. Materials and Methods: A total of 1503 sputum samples were collected from suspected cases of pulmonary TB (new or previously treated). They were processed and subjected to ZN (conventional light microscopy), Auramine-O (AO) staining (LED-FM) and solid culture on Lowenstein–Jensen medium for detection of acid-fast bacilli. Positive smears were graded according to RNTCP guidelines. Results: Sensitivity, specificity, positive predictive value, and negative predictive value of ZN staining technique were 84.93%, 95.74%, 96.50%, and 82.16%, respectively, and that of FM was 95.25%, 91.33%, 93.81%, and 93.31%, respectively. Conclusion: LED-FM being more sensitive, especially in detecting the paucibacillary cases and less time-consuming, has advantage over ZN method. Nation-wide implementation will lead to improved diagnosis, thus better control of TB.
Keywords: Auramine-O, light-emitting diode-based fluorescent microscopy, sensitivity, specificity, Ziehl–Neelsen
|How to cite this article:|
Ahmed S, Shukla I, Fatima N, Varshney SK, Shameem M, Tayyaba U. Light-emitting diode-fluorescent microscopy: Determining its sensitivity and specificity in diagnosis of pulmonary tuberculosis in a high-burden tuberculosis region and resource-limited country like India. CHRISMED J Health Res 2019;6:44-51
|How to cite this URL:|
Ahmed S, Shukla I, Fatima N, Varshney SK, Shameem M, Tayyaba U. Light-emitting diode-fluorescent microscopy: Determining its sensitivity and specificity in diagnosis of pulmonary tuberculosis in a high-burden tuberculosis region and resource-limited country like India. CHRISMED J Health Res [serial online] 2019 [cited 2019 May 22];6:44-51. Available from: http://www.cjhr.org/text.asp?2019/6/1/44/252289
| Introduction|| |
Tuberculosis (TB) remains a major global health problem. With a population of around 1.24 billion, India is the largest country in the Southeast Asia region. It ranks first among the high TB burden countries contributed 26% of estimated global incident TB cases in 2012. India accounts for the one-fourth of the global TB burden, i.e., 2.2 million out of 9.6 million new cases annually. In India, more than 40% of population is infected (prevalence of infection) with Mycobacterium tuberculosis.
Smear microscopy being the most commonly used method for microbiological diagnosis of TB for the last several decades has enormous value in TB diagnosis but with limited sensitivity, more so in children.,,, Under Revised National TB Control Program (RNTCP), two methods of microscopy are currently being used Ziehl–Neelsen (ZN) stain-based microscopy using conventional microscope and light-emitting diode-based fluorescent microscopy (LED-FM). Culture though highly sensitive and specific method for TB diagnosis requires 2–8 weeks to yield results and hence does not help in early diagnosis. However, culture can distinguish viable and nonviable bacilli of expectoration, but its use is very limited in resource-limited settings because this method requires safety equipment, containment laboratories, and trained personnel. Hence, demonstration of acid fast-bacilli (AFB) in smears from clinical specimens provides preliminary diagnostic information within an hour but is less sensitive as it does not discriminate between Mycobacterium species and lacks specificity.,,
The major limitation of ZN microscopy is the lack of sensitivity, which varies from 20% to 78%; the lower figures are from ordinary laboratories, whereas the higher figures are from research laboratories, showing that adoption of the right method with sincerity is what is necessary for increasing the sensitivity of the method., FM introduce in some laboratories detects 10% more TB cases than optical microscopy and requires only 25% of the time taken to read a ZN-stained smear. Examination of sputum smears stained with ZN requires on an average 5–10 min, consuming considerable working hours from the laboratories with limited resources. The newer alternative technique to ZN smear microscopy, FM, is known to increase the sensitivity (10% higher) when compared with ZN microscopy methods while speeding up the whole process to consume much lesser time.,
Considering the above-mentioned points signifying the importance of microscopy in early diagnosis of TB in resource-limited country like India, the present study was conducted to find the sensitivity and specificity of ZN and fluorescent staining methods keeping solid Lowenstein–Jensen (LJ) culture as gold standard.
| Materials and Methods|| |
The study was approved by the Institutional Ethics and Research Advisory Committee. Informed consent was not required in this study since it was part of routine investigations done in the department.
The present study was conducted in our culture and drug susceptibility testing laboratory (RNTCP certified), department of microbiology, on the sputum samples received of the multidrug-resistant TB (MDR-TB)-suspected patients from the outpatient and inpatient departments of the hospital and from various TB units in and around Aligarh region from January 2015 to August 2016. They were divided into new case – A TB patient who has never had treatment for TB or has taken anti tubercular drugs (ATD) for <1 month and previously treated (follow-up) patients who have received 1 month or more ATD in the past (according to the programmatic management of drug-resistant TB guidelines).
Collection of specimens
The patients were advised to collect 4–5 ml of early morning sputum in a sterilized 50-ml falcon tube. They were instructed to rinse their mouth with pure water and clean their teeth before collection to avoid contamination with food and other particles. The two consecutive days' sputum samples were collected as per the RNTCP criteria.
- One spot specimen when the patient first attends the hospital
- One next early morning specimen.
Microscopic examination of specimen
New clean slide was used for each specimens and smear was made from the thick and purulent portion of the sputum specimen. Smear was air-dried and heat fixed inside the biosafety cabinet. 1% carbol-fuchsin solution was poured on the slide and it was heated intermittently until the steam arose. The smear was washed with water and it was decolorized with 3% acid alcohol till smear was faint pink in color, and then after washing with water, it was counterstained by 1% filtered malachite green solution for 30 s, washed and dried, and examined under oil immersion lens. Grading of the smear was done according to [Table 1].
|Table 1: Grading of acid-fast bacilli smear as per Revised National Tuberculosis Control Program criteria|
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In fluorescent staining method, smears from specimens were air-dried and heat fixed by flaming. The slides were then flooded with freshly filtered auramine phenol (0.3 g auramine in 100 ml 3% phenol) for 7–10 min without heating. The smears were washed in running water and decolorized with 3% acid in alcohol for 3–5 min. The slide was washed well in running water and counterstained with 0.1% potassium permanganate for 3 s and washed and air-dried. Smears were then examined under ×40 with LED ultraviolet (UV) microscope, and grading was done according to [Table 2].
|Table 2: Recommended number of Acid-fast bacillus and fields for grading of Ziehl-Neelsen and fluorescent-stained slides|
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Culture on solid Lowenstein–Jensen media
The specimens were then processed by a standard NALC–NaOH digestion–decontamination method; briefly, an equal volume of 2% NaOH and 1.45% sodium citrate containing 0.5% NALC was added to each tube. The contents within the tubes were then mixed by vortexing and then incubated at room temperature for 15 min. The tubes were then shaken by hand at regular intervals. Phosphate-buffered saline (pH 6.8) was added up to 45 ml and then centrifuged at ×3000 g for 15 min. The supernatant was carefully poured off, the resulting sediments were then resuspended in 1.5 ml of phosphate-buffered saline, and the suspensions were used to inoculate into LJ culture media.
Data were analyzed using MedCalc developed by MedCalc Software (Acacialaan 22, 8400 Ostend, Belgium). Data were presented as frequency (percentage) and mean (standard deviation). Sensitivity, specificity, negative predictive value, and positive predictive value with 95% confidence intervals were calculated. P < 0.05 was taken as statistically significant.
| Results|| |
A total of 1503 sputum samples of MDR-TB-suspected patients were collected; out of which, majority of patients, i.e., 1088 (72.39%) were male and only 415 (27.61%) were female. Microscopic examination of sputum sample was done by ZN technique. Out of a total 1503 samples, 781 (51.96%) were found to be positive for AFB [Table 3] and 722 (48.04%) were negative for AFB. Microscopic examination of sputum sample was also done by Auramine-O (AO) technique; 904 (60.15%) samples were found to be positive for AFB [Table 4] and 599 (39.85%) were negative for AFB.
|Table 3: AFB sputum smear examination of multidrug-resistant tuberculosis suspected patients (Ziehl-Neelsen staining) (n=1503)|
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|Table 4: Acid-fast bacilli sputum smear examination of multidrug-resistant tuberculosis-suspected patients (fluorescent staining) (n=1503)|
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A higher number of samples, i.e., 904 (60.15%) were positive by AO staining (LED-FM) than microscopic smear examination by ZN staining 781 (51.96%) [Table 5]. Paucibacillary (PB) cases (scanty and 1+) were 395 (50.57%) on ZN microscopy and 513 (56.74%) on FM. Multibacillary cases were 386 (49.42%) on ZN microscopy and 391 (43.25%) on FM. FM is better in detecting PB cases [Table 6].
|Table 5: Comparison of Ziehl-Neelsen and Auramine staining reports (n=1503)|
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Among the new cases, 59.06% smears were positive by ZN staining method and 66.35% were positive by fluorescent staining method. Similarly, higher positivity was seen among the previously treated cases by fluorescent (59.15%) than by ZN (52.12%).
The gain in positivity among the new cases who were PB (scanty, 1+) was 15.49% by FM over ZN microscopy, whereas it was 6.60% among the previously treated cases.
Multibacillary (2+, 3+) cases showed no such gain by FM among both new (0.01%) and previously treated patients (0.38%) [Table 7].
|Table 7: Distribution of positive slides by grading and technique used among new and previously treated follow-up cases|
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Culture on LJ medium 843 (56.09%) showed growth of mycobacteria on LJ medium, while 611 (40.65%) did not show any growth on LJ medium, and 49 samples were either contaminated or reported as dry [Table 8].
|Table 8: Mycobacterial culture examination of multidrug-resistant tuberculosis suspected patients on Lowenstein-Jensen medium (n=1503)|
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While comparing ZN staining, fluorescent staining on smears and culture examination on LJ medium 49 samples that contaminated/dry were excluded from the study. FM showed that 856 (58.87%) samples were positive for AFB, 843 (57.98%) were culture positive on LJ, and 742 (51.03%) samples were positive on ZN-stained smears for AFB. More increase in positivity of smear was seen among the new cases (14.92%) by FM as compared to ZN than in previously treated patients. Similarly, more cultures were positive in new cases than previously treated cases as compared to ZN [Table 9].
|Table 9: Comparison of Ziehl-Neelsen smear examination, fluorescent microscopy, and mycobacterial culture on Lowenstein-Jensen medium of multidrug-resistant tuberculosis suspected patients (n=1454)|
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On comparing the smear examination result (ZN staining and FM) with mycobacterial culture. It was seen that 716 (49.24%) were both culture ZN smear and culture positive and 585 (40.23%) were both ZN smear and culture negative. One hundred and twenty-seven (8.73%) samples were ZN smear negative and culture positive. On the contrary, 26 (1.79%) samples were ZN smear positive and culture negative. Similarly, 803 (55.22%) samples were both FM and culture positive and 558 (38.38%) samples were both FM and culture negative. Forty (2.75%) samples were FM positive and culture negative. On the contrary, 53 (3.65%) samples were FM positive and culture negative [Table 10].
|Table 10: Profile of Ziehl-Neelsen, smear, fluorescent microscopy, and mycobacterial culture examination of multidrug-resistant tuberculosis-suspected cases (n=1454)|
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Sensitivity, specificity, positive predictive value, negative predictive value of ZN staining technique came out to be 84.93%, 95.74%, 96.50% and 82.16% when compared with mycobacterial culture which was taken as gold standard. Similarly, sensitivity, specificity, positive predictive value, and negative predictive value of AO staining technique were 95.25%, 91.33%, 93.81%, and 93.31%, respectively [Table 11] and [Table 12].
|Table 11: Sensitivity and specificity of Ziehl-Neelsen microscopy method|
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Among the 53 fluorescent-positive smear that came out to be culture negative, 43 (81.13%) were PB case (scanty, 1+), whereas 10 (18.87%) were multibacillary cases.
Similarly, among the 26 smears positive by ZN microscopy that were culture negative, 16 (61.54%) were PB cases, whereas 10 (38.46%) were multibacillary cases.
| Discussion|| |
The most important tool in the diagnosis of TB is direct microscopic examination of appropriately stained sputum specimens for AFB. The technique is simple and inexpensive and detects those cases of TB, which are infectious. Sputum microscopy is also useful to assess the response to treatment and to establish cure or failure at the end of treatment. Although microscopy has a low sensitivity rate, it is still the most commonly used AFB detection method especially in resource-limited developing countries.
AFB-microscopy needs 5000–10,000 AFB per milliliter of sputum for direct microscopy to be positive.,,, Sputum smear microscopy detects most infectious cases and specificity is very high (97%–99%) in settings where the burden of TB is high.,, In our present study, out of 1503 MDR-TB-suspected patients, 781 (51.96%) came out smear positive for AFB and 722 (48.04%) were sputum negative when they were subjected to ZN staining technique, whereas out of 1503, 904 (60.15%) were sputum smear positive and 599 (39.85%) were sputum smear negative when subjected fluorescent staining technique. Odubanjo and Dada-Adegbola 2011 showed 38.7% of smear positive on ZN staining. In the study conducted by Goyal and Kumar 2013, ZN smear positivity rate and AO smear positivity rate were 7.47% (29/388) and 14.69% (57/388), respectively. Golia et al. 2013 showed ZN smear positivity rate 10.57% and AO smear positivity rate 16.56%. Similar results have also been reported by studies done by Kumar et al. 2012, whereas higher smear positivity rates were shown by Prasanthi and Kumari 2005 (50% by ZN and 69% by AO) and Ulukanligil et al. 2000 (67.6% by ZN and 85.2% by AO). In present study, the higher percentage of smear positivity by AO staining LED-FM than by ZN staining is seen and it is in concordance with the other studies as shown above.
We found AO staining LED-FM to be 7.48% more effective than ZN staining. Similarly, Golia et al. 2013 had shown AO staining to be 6.15% more effective than ZN staining.
In our study, ZN microscopy was able to detect 395 PB cases (scanty and 1+) whereas FM was able to detect 513 PB cases. Similar findings were seen in their study by Golia et al. 2013 where AO staining could detect 26 PB cases, whereas ZN staining detected only 3 of them.
Among the new and previously treated follow-up cases, 7.27% gain in smear positivity by LED-FM was seen in former and the gain was 7.03% in latter.
A substantial increase in positivity of smear in positivity PB (scanty, 1+) cases both among new cases (15.49%) and previously treated cases (6.60%) was seen by LED-FM as compared to ZN microscopy. Multibacillary (2+, 3+) cases showed no such gain by FM among both new (0.01%) and previously treated patients (0.38%). Hence, we can conclude that LED-FM detects PB cases better than ZN microscopy. Failure to detect and hence to treat PB cases can be effectively prevented by the use of AO staining.
The effective life span of LED-FM is thousands of hours and is relatively inexpensive, and therefore, it holds multiple advantages and mercury vapor lamp and standard light microscopy. Since mercury vapor and halogen lamp are often used after their effective life span with diminished power to cause fl uorescence LED having larger life span may result in better quality FM.,,,
Since in LED-FM, the smear is examined in ×40 as compared to ×100 in conventional light microscopy, the field of examination was wider and hence it took less time (25%) in screening of the smears by the former. Fluorescence bacilli had a better contrast in LED-FM than conventional ZN staining which leads to easy and better detection. Both these factors can be attributed to better detection of PB cases as seen in our study. Therefore, implementation of FM in high-burden and resource-limited countries like India would be quite fruitful.,
It is reported that smear detects AFB only at a concentration of around 10,000 bacilli per milliliter of specimen, but only as few as 100 bacilli per milliliter may be required for positive culture. In our study, out of 1503 samples, 843 (56.09%) were positive on culture, 611 (40.65%) were negative, and 49 (3.26%) were contaminated/dry. Yadav et al. 2013 reported out of 269 cultures, 251 (93%) were M. tuberculosis, 8 (3%) were culture negative, 3 (1%) demonstrated were nontuberculous mycobacteria, and the remaining 7 (3%) were contaminated. Sharma et al. showed that of the 62 fluorescent-positive samples, 54 (87.10%) were positive and 8 (12.90%) were negative on culture. Raizada et al. 2014 depicted of all 640 samples by culture yielded at least one positive growth for M. tuberculosis complex in 256 (80%) patients. Samples from 51 (16%) patients had no growth, and in 13 (4%) patients, the cultures were contaminated.
In our study, positivity rate of ZN, AO staining, and mycobacterial culture positive were 742 (51.03%), 856 (58.87%), and 843 (57.98%), respectively. Laifangbam et al. 2013 showed that positivity rates for ZN, AO, and culture were 39 (36.1%), 80 (74.1%), and 78 (72.2%), respectively, in HIV-TB-co-infected cases. FM showed more positivity than culture in both the studies.
In our study, 716 (49.24%) samples were positive on both culture and ZN microscopy, whereas 803 (55.22%) samples were positive on both FM and culture. Five hundred and eight-five (40.23%) samples were both ZN microscopy and culture negative. Similarly, 558 (38.38%) samples were both FM and culture negative. 127 (8.73%) samples were ZN smear negative and culture positive and 26 (1.79%) samples were ZN smear and culture negative. Similarly, 40 (2.75%) samples were FM negative and 53 (3.65%) samples were fluorescent positive and culture negative. Odubanjo and Dada-Adegbola. 2011 reported that 78 (60.5%) samples were positive on AFB microscopy alone, 13 (10.0%) on culture alone, and 38 (29.5%) on both culture and AFB microscopy. Fifty-one cases were positive on culture. Thakur et al. 2015 also reported 68.70% samples as ZN smear positive and 31.29% as ZN smear negative, and among these ZN smear-negative specimens, 18.67% were found to be culture positive in their study.
As seen in the present study that positive culture yield is less than the positivity on FM in previously treated cases (57.86% by LED-FM, 56.74% by culture), the reason could be that during treatment, only the dead/killed bacteria remain in the respiratory specimen (sputum) which were detected by microscopy but were unable to grow in LJ media. This phenomenon is presumably due to the expectoration and microscopic visualization of dead bacilli. Smear microscopy cannot distinguish viable from dead bacilli. A significant proportion of patients on treatment may, however, continue to cough up dead bacilli from necrotic lung cavities, thus remaining “smear positive” although responding to therapy.
Fluorescent AFB can be seen at lower magnification than ZN-stained AFB. FM smears can be examined in a fraction (about 25%) of the time needed for ZN smears as well. Recent development of simple FM systems based on light-emitting diodes (LED-FM) which have long life span, do not produce UV light, and have minimal power requirements could facilitate the implementation of FM in high-burden and resource-limited countries.,
In the present study, sensitivity and specificity of FM and ZN staining was 95.25%, 91.33% and 84.93%, 95.74%, respectively. Odubanjo and Dada-Adegbola 2011 showed specificity of 71.6% and sensitivity of 74.5%. Previous studies from other developing countries showed similar range of specificity and sensitivity values for AFB microscopy. Shea et al. 2009 showed a specificity of 100% and a sensitivity of 93.1% in Uganda while Farnia et al. 2002 showed a specificity of 96% and a sensitivity of 78% in Iran. Sensitivity and specificity for ZN were 48.7% and 96.6%, respectively, whereas those for AO were 100% and 93.3%, respectively, in HIV-TB-co-infected cases. Fluorescent LED microscopy showed 84% sensitivity and 98% specificity against culture as the reference standard. Ulukanligil et al. 2000 showed sensitivity and specificity of FM 83% and 99% and of ZN 61% and 100%.
In our study, among the 53 fluorescent-positive smears, 43 (81.13%) were PB case (scanty, 1+) that came out to be culture negative, whereas 10 (18.87%) were multibacillary cases. Similarly, among the 26 smears positive by ZN microscopy, 16 (61.54%) were PB cases that came out to be culture negative and10 (38.46%) were multibacillary cases. Such observation implies that by LED-FM, chances of false-positive smear may be more as compared to ZN microscopy. But in our study majority of the cases that were culture negative and microscopy positive belonged to previously treated category which could be the reason of their being culture negative as discussed above.
As shown in our study and similar other studies, ZN being more specific and LED-FM more sensitive, it is preferable to err on the side of increased sensitivity and to treat a small number of patients who do not have TB. There are real consequences to a false-positive result: patients incorrectly diagnosed with TB have the cost and inconvenience of taking a 6-month course of treatment, and troublesome side effects are common, but life-threatening treatment-related events (hepatitis and Stevens–Johnson syndrome) are infrequent (5% incidence).
Cuevas et al. 2011 expressed some concerns over using solid and liquid culture as an adequate comparator. They pointed out that culture system has higher specificity than smear microscopy might be debatable. The WHO manual for laboratory services in TB control states that the probability of obtaining a positive culture is related to the number of AFB in the specimen, with only about 50% of cultures of specimens with 1–2 AFB per 100 fields being identified as positive, increasing to 80% and 96.7% for specimens with “scanty” (1–9 AFB per 100 fields) and “+” AFB grades, respectively. This is unfortunately an inherent weakness of solid culture (and, to a lesser extent, liquid culture) as a diagnostic reference standard.
Therefore, they stated that judgments on the performance of new diagnostic tests must be done with considerations of these limitations. The “false-positive” LED-FM results are thus most likely due to technician error, but we cannot discount the possibility that these were cases with TB or nontuberculous mycobacteria with a false-negative solid medium result that would have been detected using liquid medium. There might also be cases of TB that would have been missed by both culture technologies (because of delayed processing/destruction of marginally viable bacilli during decontamination or bacterial overgrowth). Our study also showed similar results which relate to the above-mentioned problems stated by Cuevas et al. 2011.
Finally, LED-FM should be reviewed in light of WHO endorsed new rapid test like automated nucleic acid amplification test, GeneXpert-TB. The possibility of Xpert being available in microscopy centers and thus replacing smear microscopy in the upcoming recent time is highly unlikely in developing country like India, especially when the resources are scarce, and burden of TB and MDR-TB is quite high. Cost-effectiveness considerations favor the use of smear microscopy as the initial diagnostic tool. Despite the reduced specificity of LED-FM, this approach still has operational advantages that make it an attractive tool for TB laboratory diagnosis.
| Conclusion|| |
From the present study, we can conclude that in resource-limited countries like India, microscopy has strong implications in the diagnosis of TB. LED-FM being more sensitive, especially in detecting the PB cases and less time consuming, has advantage over ZN method. Screening of smear in FM is done under low power of magnification (×40), and fluorescence has been found to be less time consuming compared to ZN method (×100) in the diagnosis of TB. The fluorescing bacilli are easily identifiable and cause less eye strain. Higher specificity of ZN makes it better staining option in doubtful cases. Although the culture of mycobacteria is a most sensitive method than smear microscopy, it is time-consuming and requires proper laboratory set-ups, which is not possible in remote rural areas with poor resource settings. Hence, we can conclude that the FM method is quite economical in terms of both time and expense and it is recommended for laboratories handling large number of sputum specimens.
The authors would like to thank the technical members of Intermediate Reference Laboratory, Department of microbiology, Jawaharlal Nehru medical college, Aligarh Muslim University, Aligarh, Uttar Pradesh for their technical support during research work.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5], [Table 6], [Table 7], [Table 8], [Table 9], [Table 10], [Table 11], [Table 12]