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 Table of Contents  
ORIGINAL ARTICLE
Year : 2019  |  Volume : 8  |  Issue : 4  |  Page : 277-281

Same-day sputum smear microscopy for the diagnosis of pulmonary tuberculosis – Light-emitting diode fluorescent staining with direct sputum versus Ziehl–Neelsen staining with concentration sputum


Department of Microbiology, GSL Medical College, Rajahmundry, Andhra Pradesh, India

Date of Submission12-Mar-2019
Date of Acceptance26-Sep-2019
Date of Web Publication15-Oct-2019

Correspondence Address:
Dr. T Jaya Chandra
Department of Microbiology and Central Research Lab, GSL Medical College, Rajahmundry, Andhra Pradesh
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/ijhas.IJHAS_12_19

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  Abstract 


OBJECTIVE: The objective of the study was to evaluate the utility of fluorescent staining (FS) technique with direct sputum for the diagnosis of pulmonary tuberculosis (TB).
MATERIALS AND METHODS: Three sputum specimens (spot, second spot, and morning) were collected from the study participants. One smear was prepared from the direct sample and one using the N-acetyl-L-cysteine-sodium hydroxide concentration method. The direct smears were stained using FS, and Ziehl–Neelsen (ZN) staining was used to stain smears prepared after concentration method. Smear results were pooled and compared in two categories: standard spot-morning and same-day smears. The Chi-square test was used to evaluate the statistical difference in smear-positive cases.
RESULTS: Among 2922 participants included in the study, smear positivity was, respectively, 10.5% and 10.2% for FS and ZN smears using spot-morning approach. Using the same-day approach, the smear positivity was, respectively, 10.4% and 9.9% for FS and ZN smears; statistically, the difference was not statistically significant (P > 0.05) between the staining techniques in both the approaches.
CONCLUSION: More smear positivity is the advantage of using FS technique. Given this advantage, National TB Control Programs of high-TB-burden countries should consider installing light-emitting diode FS technique in peripheral settings also.

Keywords: Concentrated sputum, direct sputum, fluorescent staining, sample, Ziehl–Neelsen staining


How to cite this article:
Chandra T J. Same-day sputum smear microscopy for the diagnosis of pulmonary tuberculosis – Light-emitting diode fluorescent staining with direct sputum versus Ziehl–Neelsen staining with concentration sputum. Int J Health Allied Sci 2019;8:277-81

How to cite this URL:
Chandra T J. Same-day sputum smear microscopy for the diagnosis of pulmonary tuberculosis – Light-emitting diode fluorescent staining with direct sputum versus Ziehl–Neelsen staining with concentration sputum. Int J Health Allied Sci [serial online] 2019 [cited 2019 Nov 22];8:277-81. Available from: http://www.ijhas.in/text.asp?2019/8/4/277/269242




  Introduction Top


Tuberculosis (TB) is an important global health issue, and Mycobacterium tuberculosis complex is the causative agent. People with a low standard of living and poverty are the contributory factors for the spread of TB. Several diagnostic tests such as culture (Lowenstein–Jensen [LJ] medium and BACTEC), molecular methods (polymerase chain reaction, line probe assay LPA), and serodiagnostic techniques (enzyme-linked immunosorbent assay, quantiFERON-TB Gold, and interferon-γ assay) are available for the diagnosis of TB. High sensitivity and specificity are the major advantages of these methods. In addition to prolonged time period, culture on LJ medium requires sophisticated infrastructure, skilled workforce, and accreditation from Revised National TB Control Program (RNTCP). The other diagnostic techniques may be rapid, but high cost is the major drawback, and the serodiagnostic tests were banned by the World Health Organization (WHO) and RNTCP.[1] Due to the advantages such as rapidity and ease of technique, sputum microscopy is considered to be the key diagnostic tool for the diagnosis of pulmonary TB.[2],[3]

Different sputum microscopy techniques such as Ziehl–Neelsen (ZN) staining,[2] modified ZN staining,[4] and fluorescent staining (FS)[3] were reported for the diagnosis of pulmonary TB. It was well documented that the diagnostic yield using light-emitting diode (LED) FS was more compared to ZN staining.[5] However, in most of the peripheral health settings, ZN staining was the only alternative for the diagnosis of pulmonary TB.[4] Many researchers have suggested that the performance of sputum microscopy can be improved significantly if the sputum is liquefied with chemical reagents, followed by concentration either by centrifugation or by sedimentation before acid-fast staining.[6],[7]

The present study was undertaken to evaluate the utility of direct sputum using LED FS technique with spot-morning and same-day smears for the diagnosis of pulmonary TB.


  Methods Top


The study was conducted in the Department of Microbiology, GSL Medical College, Rajahmundry, Andhra Pradesh, India, from January 2013 to December 2016. The study protocol was approved by the Institutional Research and Ethics Committee of GSL Medical College. Informed written consent in the presence of a witness was provided by all the volunteers who participated in the study.

Individuals with signs and symptoms suggestive of TB were included in the study. The participants were informed to submit good quality sputum sample. The visual difference between sputum and saliva and how to produce good quality sputum sample were practically demonstrated as the patient education is imperative for obtaining the correct sputum sample. All the individuals were requested to provide three sputum samples: a spot sample at the first visit to the hospital, a second spot sample collected 1 h after the spot sample, and a morning sample collected after waking early in the morning. After providing two spot samples, the patients were given prelabeled sample containers to collect the morning sample at home.

Immediately after collection of the sputum, direct smear was prepared. After smear preparation, the samples were processed using N-acetyl-L-cysteine (NALC)-sodium hydroxide (NaoH) method,[3] and smear was prepared with sediment. A total of two smears were prepared with each sample, i.e., six smears for each patient. [Figure 1] shows the algorithm for the procedure used. The direct smears were stained using FS technique, and ZN staining was used to stain sputum smears prepared after concentration method. Smear results were pooled and compared in two categories: standard spot-morning and same-day approaches.
Figure 1: Algorithm of the study. ZN = Ziehl–Neelsen; FS = Fluorescent staining

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Ziehl–Neelsen staining

In the ZN staining method, smears were flooded with filtered 1% carbol fuchsin and heated until steaming, then left to steam for 5 min.[8] After rinsing the slides with a gentle stream of water, 25% sulfuric acid was used to decolorize the smears for 2–4 min; if necessary, the decolorization step was repeated for another 1–3 min. The slides were rinsed as before, and counterstained with 0.1% methylene blue for 30 s. The slides were then washed, air-dried, and examined under oil immersion. The smears were graded according to the guidelines in the RNTCP technical manual.[8]

Fluorescent staining

For fluorescent staining, the slides were placed on the staining rack without touching each other, flooded with freshly filtered auramine phenol, and left for 7–10 min.[9] They were then washed with running water (care was taken to control the flow of water so as to prevent washing away the smear). The slides were decolorized by covering completely with acid alcohol for 2 min twice, then washed again with running water to remove the acid alcohol, after which they were counterstained with 0.1% potassium permanganate for 30 s and washed as before with water. The slides were then air-dried and observed under a ×40 objective, and the smears were graded according to RNTCP technical manual guidelines.[9]

N-acetyl-L-cysteine sodium hydroxide method

With the NALC-NaOH method, the sputum specimen was mixed with equal parts of NALC-NaOH solution for 15 s on vortex mixture. A sufficient quantity of phosphate-buffered saline (PBS) was then added to fill the tube within 1 cm of the top. The cap was closed tightly, and the tube was inverted to mix the solution, which was then centrifuged at 3600 × g for 15 min. The supernatant was decanted into a container with 5% phenol solution, and the sediment was suspended in 1–2 ml of PBS and used for smear preparation.

Statistical analysis

Data were analyzed using SPSS, version 16 (SPSS, Inc., Chicago, IL, USA) with patient as the unit of analysis. Chi-square test was used to find the statistical difference in the smear-positive cases between the staining techniques. P <0.05 was considered to be statistically significant.


  Results Top


Among the 2922 study participants, with concentration sputum using ZN staining, the smear positivity was 10.2% and 9.9%, and with direct sputum using FS technique, the smear positivity was 10.5% and 10.4%, respectively, in spot-morning and same-day smears [Table 1]; statistically, the difference was not significant (P > 0.05) between the staining techniques in both the approaches.
Table 1: Smear results for direct sputum and concentration sputum (n=2922)

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  Discussion Top


Sputum microscopy is a simple, rapid, inexpensive technique, which is highly specific (97%–99%) in areas with a very high prevalence of TB and identifies most of the infected patients.[10],[11],[12],[13] Hence, sputum microscopy is the only alternative for the diagnosis of pulmonary TB, especially in the high-TB-burden countries.[2]

It was reported that the utility of same-day approach was almost similar with the standard spot-morning approach for the diagnosis of pulmonary TB.[2],[3],[4] In this study, with direct sputum using FS technique, there was a 3.7% and 4.8% improvement in smear positivity, respectively, for spot-morning and same-day approaches (P > 0.05). As per Chandra et al.[3] report, using ZN staining with concentration sputum, the smear positivity was 10.4% (332) and 10.3% (328), and using FS with direct sputum, the smear positivity was 10.8% (343) and 10.6% (338), respectively, for spot-morning and same-day approaches; i.e., a 3.3% and 3% improvement in smear positivity, respectively, for the approaches. Reza et al.[5] reported that the smear-positive cases were increased from 14% to 17.8% after introducing LED FS; the increment in the smear positivity rate was 30%. In Reza et al.[5] study, the investigators compared the results of direct sputum smears, whereas in this study, the comparison was between the concentration sputum using ZN staining and direct sputum using LED FS, with the aim to assess the efficacy of direct LED FS technique for the diagnosis of pulmonary TB. Hence, the improvement in smear positivity was less compared to Reza et al.[5] report but on a par with Chandra et al.[3] study. However, this number is significant in TB eradication.

Various physical and chemical sputum-processing techniques were reported. Of these, technique using NALC with 2% NaOH was considered to be the best.[14] Hence, NALC–NaOH technique was used in this study for the processing of the sputum samples.

In field conditions, laboratory technicians are less motivated to perform repeat sputum microscopy, especially if the first-sputum smear results are negative for acid-fast bacilli.[15] Van Deun et al.[16] quoted that copying the patients previous smear results for the same patients subsequent smear examination is the practice followed in overburden laboratories. This leads to the manipulation of smear results and causes a significant hurdle to implement concentration technique, especially in the field condition. In addition to these, Mittal et al.[17] reported that processing of sputum samples by concentration method is not possible in India due to the problem of installing biosafety cabinets which are mandatory for sputum processing. This is another hurdle found to implement concentration technique, especially in the field conditions.

In addition to low cost,[18] rapidity in smear reading,[19] higher the chance to detect acid-fast bacilli in paucibacillary samples,[20] and so on, are the advantages with LED FS, and this study reported more smear positivity with direct sputum using LED FS compared to concentration sputum using ZN technique (P > 0.05). As per Bhalla et al.[21] report, sensitivity and specificity were reported to be 81.6, 83.5 and 83.1, 82.4, respectively, for ZN and LED FS techniques. Other investigators also reported low specificity which is the limitation of LED FS.[19],[21] Culture on LJ medium may be the gold standard when sensitivity and specificity were only considered, but the turnaround time is significantly low. However, as per the literature, there was 25%–66% time saving using LED FS compared to ZN technique.[22],[23] Hence, rapidity in smear reading should be considered in high-TB-burden countries, and this is significant favoring factor of LED FS technique. Hence, culture issue was not considered as limitation of the current research, though it was not included.

The WHO recommended LED FS for the detection of pulmonary TB in high-TB-burden countries.[24] The Southeast Asia office of International Union Against Tuberculosis and Lung Diseases in partnership with RNTCP and National Task Force implemented a project called LIGHT (LED fluorescence microscopy in gaining TB cases in high-workload TB hospitals), involved in replacing existing RNTCP ZN staining with LED FS procedure in all medical colleges that have designated microscopy centers for the diagnosis of pulmonary TB and facility for the treatment of TB. In addition, it was reported in the literature that concentration techniques for the diagnosis of pulmonary TB can be initiated by RNTCP in medical colleges due to the availability of workforce and infrastructure.[3]


  Conclusion Top


However, lack of trained workforce is the significant hurdle to implement concentration method in medical colleges. It is simple to train the laboratory technicians and easy to install LED FS facility in designated microscopy centers. With this, the National TB Control Programs of high-TB-burden countries should consider installing LED FS in peripheral settings also.

Financial support and sponsorship

Nil.

Conflicts of interest

There are no conflicts of interest.



 
  References Top

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Pai M, Das J. Management of tuberculosis in India: Time for a deeper dive into quality. Natl Med J India 2013;26:65-8.  Back to cited text no. 1
    
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Chandra TJ, Raj RS, Sharma YV. Same day sputum smear microscopy approach with modified ZN staining for the diagnosis of pulmonary tuberculosis in a microscopy centre at Rajahmundry. Indian J Med Microbiol 2014;32:153-6.  Back to cited text no. 4
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5.
Reza LW, Satyanarayna S, Enarson DA, Kumar AM, Sagili K, Kumar S, et al. LED-fluorescence microscopy for diagnosis of pulmonary tuberculosis under programmatic conditions in India. PLoS One 2013;8:e75566.  Back to cited text no. 5
    
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RNTCP. Central TB Division, Directorate General of Health Services Ministry of Health and Family Welfare. Manual for Sputum Smear Fluorescence Microscopy. New Delhi, India: Ministry of Health and Family Welfare. Available from: http://www.tbcindia.nic.in/showfile.php?lid=2988. [Last accessed on 2015 Dec 26].  Back to cited text no. 9
    
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Long R, Scalcini M, Manfreda J, Jean-Baptiste M, Hershfield E. The impact of HIV on the usefulness of sputum smears for the diagnosis of tuberculosis. Am J Public Health 1991;81:1326-8.  Back to cited text no. 10
    
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Uddin MK, Chowdhury MR, Ahmed S, Rahman MT, Khatun R, van Leth F, et al. Comparison of direct versus concentrated smear microscopy in detection of pulmonary tuberculosis. BMC Res Notes 2013;6:291.  Back to cited text no. 14
    
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