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 Table of Contents  
ORIGINAL ARTICLE
Year : 2021  |  Volume : 7  |  Issue : 2  |  Page : 82-86

Evaluation of hypertonic saline–sodium hydroxide method for isolation of Mycobacterium tuberculosis on Lowenstein–Jensen medium


Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry, India

Date of Submission12-Feb-2021
Date of Acceptance28-Jun-2021
Date of Web Publication30-Dec-2021

Correspondence Address:
Noyal Mariya Joseph
Department of Microbiology, Jawaharlal Institute of Postgraduate Medical Education and Research, Puducherry
India
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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/jcrsm.jcrsm_6_21

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  Abstract 


Background: Decontamination of sputum specimen is a crucial procedure for successful isolation of Mycobacterium tuberculosis (MTB) using Lowenstein–Jensen (LJ) media. The aim of this study was to evaluate hypertonic saline–sodium hydroxide (HSSH) method for decontamination of sputum specimens for the isolation of MTB on LJ medium.
Methods: The sputum specimens were divided into three aliquots and were decontaminated by HSSH method, N-acetyl L-cysteine-sodium hydroxide (NALC-NaOH) method, and modified Petroff's method. We have compared the smear positivity, isolation rate, and contamination rate of HSSH method with that of NALC-NaOH method and modified Petroff's method for decontamination of sputum specimens for LJ culture.
Results: Sputum samples from 270 patients were processed by the three decontamination methods, of which 24 (8.9%) were smear and culture positive by one or more decontamination methods. Of the 24 smear-positive cases, 23 (95.8%) were positive by NALC-NaOH method, 21 (87.5%) were positive by HSSH method, and 17 (70.8%) were positive by modified Petroff's method. The contamination rate of HSSH method, NALC-NaOH method, and modified Petroff's method was 2.2% (6/270), 8.9% (24/270), and 6.7% (18/270), respectively. The MTB isolation rate of HSSH method, NALC-NaOH method, and modified Petroff's method was 7.0% (19/270), 4.1% (11/270), and 3.3% (9/270), respectively.
Conclusion: Among the three decontamination methods, a significantly higher isolation rate and a lower contamination rate were observed with HSSH method. Therefore, we recommend HSSH method for decontamination of sputum samples for the isolation of MTB using LJ medium.

Keywords: Hypertonic saline–sodium hydroxide method, Lowenstein–Jensen culture, Mycobacterium tuberculosis, N-acetyl L-cysteine-sodium hydroxide method


How to cite this article:
Athira C, Joseph NM. Evaluation of hypertonic saline–sodium hydroxide method for isolation of Mycobacterium tuberculosis on Lowenstein–Jensen medium. J Curr Res Sci Med 2021;7:82-6

How to cite this URL:
Athira C, Joseph NM. Evaluation of hypertonic saline–sodium hydroxide method for isolation of Mycobacterium tuberculosis on Lowenstein–Jensen medium. J Curr Res Sci Med [serial online] 2021 [cited 2022 Jan 20];7:82-6. Available from: https://www.jcrsmed.org/text.asp?2021/7/2/82/334462




  Introduction Top


Tuberculosis (TB) is an infectious disease caused by Mycobacterium tuberculosis (MTB).[1] It can affect both the pulmonary (lungs) and extrapulmonary sites.[2] It is considered as a global health problem.[3] It causes millions of death per year. As per the Global TB reports, in 2016, there were about 1.3 lakh multidrug-resistant cases reported. Among these, 79,000 cases presented with pulmonary manifestations. Of 1.1 lakh HIV-associated TB cases, 37,000 deaths were reported, and India has the second position in the incidence of HIV-associated TB cases worldwide.[4] As per the WHO reports in 2016, 10.4 million people suffered TB in 2016, and 56% of them were from five countries (India, Pakistan, Indonesia, the Philippines, and China). 90% were adults and 65% were males among the reported cases. There were 1.3 million deaths among HIV-negative people who suffered TB. In addition, there were 374,000 deaths reported among TB patients who were HIV positive.[5]

Timely diagnosis of TB can be the most important aspect for treatment and control of the spread of disease. Several diagnostic procedures including stained smear examination and culture methods are available for the detection of MTB from the sputum specimens in case of pulmonary TB.[6] Currently, sputum smear examination for the detection of acid–fast bacilli (AFB) is used as the case finding tool under the Revised National Tuberculosis Control Programme (RNTCP). Sensitivity of direct smear stained by Ziehl–Neelsen (ZN) stain is only 40%–60%, which can result in false-negative reports in patients with paucibacillary TB.[7] Due to this limitation of smear examination, culture isolation of MTB is considered as gold standard for diagnosis of TB.[8] The usefulness of culture can be compromised by the presence of contaminant bacteria in clinical samples, which can over grow in culture and can interfere with isolation and identification of MTB.[9] Therefore, it is necessary to establish efficient decontamination methods, to avoid any delay in isolation of MTB and initiation of treatment.

An ideal decontamination method should be reliable, specific, sensitive, inexpensive, and easy to carry out. There are many conventional methods available for the decontamination of sputum.[10] RNTCP recommends routine use of modified Petroff's method using 4% sodium hydroxide (NaOH) for Lowenstein–Jensen (LJ) culture; however, as the concentration of NaOH is higher, there is increased chance of damage to the mycobacteria during the decontamination process.[9] N-acetyl L-cysteine (NALC) with 2% NaOH (NALC-NaOH) is another decontamination method, which is routinely advocated for culture of mycobacteria using Mycobacteria Growth Indicator Tube 960 system.[9] The NALC used in this method facilitates proper digestion and homogenization of the sputum specimen, while the low concentration of NaOH prevents unwanted damage to mycobacteria. Recently, a decontamination method using hypertonic saline and sodium hydroxide (HSSH) has been reported to be associated with better recovery of MTB from sputum specimens.[8] This method is relatively easy to perform and less expensive compared to NALC-NaOH method.

The aim of this study was to evaluate HSSH method for decontamination of sputum specimens for the isolation of MTB on LJ medium. We have compared the smear positivity, isolation rate, and contamination rate of HSSH method with that of NALC-NaOH method and modified Petroff's method for decontamination of sputum specimens for the isolation of MTB on LJ medium.


  Materials and Methods Top


The present study was conducted in the Department of Microbiology. The study was approved by the Institutional Ethics Committee (No. JIP/IEC/2016/1074) with waiver of consent as the study involved only samples routinely sent to the laboratory for mycobacteria culture. All consecutive sputum samples from TB suspects sent to the Mycobacteriology Laboratory during the study period, i.e. January 2017–November 2017, were included in the study. Samples with inadequate volume (<3 ml), poor quality (predominantly saliva), and from patients already undergoing antitubercular therapy were excluded.

Specimens were processed immediately or within 24 h (refrigerated at 2°C–8°C) in LJ culture. The sputum specimens were divided into three aliquots and were decontaminated by HSSH method, NALC-NaOH method, and modified Petroff's method.[4] In brief, the HSSH method was performed by taking 1 ml of sputum sample and adding equal amount of 7% NaCl and 4% NaOH into 15 ml centrifuge tube. It was vortexed well for 15–20 s and the tubes were incubated at 37°C for 30 min, followed by neutralization with Phosphate Buffer Saline (PBS) (pH 6.8), and the volume was made up to 15 ml. The contents were vortexed again for 5 s and centrifuged at 3400 g for 15 min. The supernatant fluid was discarded, and the sediment was suspended with 200 μl PBS solution and vortexed for 5 s.

The sediment obtained by all the three methods was used for smear examination and culture using LJ medium. All LJ culture bottles with growth were subjected to ZN staining and examined for the presence of AFB. The culture isolates were identified as MTB complex or nontuberculous mycobacteria by immunochromatographic test for MPT64 antigen. Contamination was checked by blood agar subculture. Sputum specimens from which MTB was isolated by any of the three decontamination methods were considered as positive. The sensitivity of HSSH method was compared with that of NALC-NaOH method and modified Petroff's method.


  Results Top


Sputum samples from a total of 270 patients were processed by the three decontamination methods, viz., NALC-NaOH method, HSSH method, and modified Petroff's method. Of the 270 sputum samples, 24 (8.9%) were positive for AFB and 24 (8.9%) were culture positive for MTB by one or more decontamination methods.

Patients' demographics

The mean age of the patients was 40.68 ± 21.24 (range 1–82 years). The mean age of the culture-confirmed cases of TB was 41.83 ± 16.72 and those who were culture negative for MTB was 40.57 ± 21.66 (P = 0.734). Our study included samples from 84 (31.1%) female and 186 (68.9%) male patients. MTB was isolated from 11.9% (10/84) of female patients, while it was isolated from 7.5% (14/186) of male patients (P = 0.242).

Smear positivity

Of the 270 sputum samples, overall, 24 (8.8%) were smear positive for AFB by different decontamination methods. Of the 24 smear-positive cases, 23 (95.8%) were positive by NALC-NaOH method, 21 (87.5%) were positive by HSSH method, and 17 (70.8%) were positive by modified Petroff's method [Table 1].
Table 1: Comparison of the smear positivity of hypertonic saline–sodium hydroxide method with N-acetyl L-cysteine-sodium hydroxide method and modified Petroff's method

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Contamination rates

The contamination rate of HSSH method, NALC-NaOH method, and modified Petroff's method was 2.2% (6/270), 8.9% (24/270), and 6.7% (18/270), respectively. The contamination rate of the HSSH method was significantly lesser than that of modified Petroff's method and NALC-NaOH method [Table 2].
Table 2: Comparison of the contamination rate of hypertonic saline–sodium hydroxide method with N-acetyl L-cysteine-sodium hydroxide method and modified Petroff's method

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Mean time to contamination of cultures of HSSH method, NALC-NaOH method, and modified Petroff's method was 1.67 ± 0.52, 3.08 ± 1.47, and 2.72 ± 1.45, respectively.

Mycobacterium tuberculosis isolation rate

Of the 270 sputum samples, MTB was isolated from 24 samples by one or more decontamination methods. Out of the 24 MTB isolates, 3 were isolated by all three methods, 9 were isolated by any two methods, 8 were isolated only by HSSH method, and 2 each were isolated only by NALC and modified Petroff's method.

The MTB isolation rate of HSSH method, NALC-NaOH method, and modified Petroff's method was 7.0% (19/270), 4.1% (11/270), and 3.3% (9/270), respectively. The HSSH method missed 5 MTB isolates (3 smear-positive cases), while the NALC-NaOH method and modified Petroff's method missed 13 (10 smear-positive cases) and 15 MTB (14 smear-positive cases) isolates, respectively. The comparison of the MTB culture positivity of HSSH method with NALC-NaOH method and modified Petroff's method is shown in [Table 3].
Table 3: Comparison of the Mycobacterium tuberculosis culture positivity of hypertonic saline–sodium hydroxide method with N-acetyl L-cysteine-sodium hydroxide method and modified Petroff's method

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The sensitivities of HSSH, NALC-NaOH, and modified Petroff's methods were 79.2%, 45.8%, and 37.5%, respectively. This suggests the superiority of the HSSH method over the other two decontamination methods.

The mean time to detection of MTB by HSSH, NALC-NaOH, and modified Petroff's methods was 3.84 ± 1.01, 3.73 ± 1.01, and 5.22 ± 0.67, respectively.


  Discussion Top


The present study included 270 sputum samples from TB suspects. All samples were subjected to three decontamination methods, viz., HSSH method, NALC-NaOH method, and modified Petroff's method, which were analyzed by smear microscopy and LJ culture.

Gender-based analysis of culture positivity showed that MTB isolation was more among female patients (11.9%) than male patients (7.5%). In a study conducted in Puducherry by Hochberg et al., they observed that alcoholic males are more prone to TB than that of females. However, at the same time, they have noted that females (61.5%) of age group between 15 and 49 years were more affected than males (57.4%) by TB due to malnutrition.[11] A study by Kaulagekar and Radkar suggested that about 3.1 million females were severely affected by TB due to malnutrition, repeated delivery, HIV, delay in seeking treatment, fear, etc.[12]

According to the WHO data, about 3.5 million females within age limit of 20–59 fell ill with TB. Among HIV-related TB deaths, 40% were female patients, and there was a higher risk in pregnancy period. TB pregnant women who co-infected with HIV were at the higher risk of infant mortality. Genital TB is one of the important causes of infertility. Socioeconomic factors such as cultural and financial barriers, poverty, malnutrition, increasing tobacco use, and diabetes may increase TB burden among females. Especially economically backward and reproductively active females are under higher risk.[13]

Because of the low cost, simplicity, and minimum requirements, microscopy for detection of AFB is largely used for definitive diagnosis of MTB from samples. However, microscopy has low sensitivity requiring 5000–10,000 bacilli/ml to give positive reports.[14] It is very difficult to isolate mycobacteria from the sputum samples contaminated with normal flora. It is necessary to subject such specimens to different decontamination methods to remove contaminant normal flora.

In this current study, we compared smear positivity of HSSH method with modified Petroff's and NALC-NaOH methods. There was no statistically significant difference in the smear positivity of these three methods. According to a study from India by Someshwaran et al., both HSSH method and modified Petroff's method showed similar smear positivity among 143 samples tested.[7] According to the study conducted by Ganoza et al., the sensitivity of AFB smears had increased from 28.6% using the direct method to 71.4% by HSSH method and 66.7% by NALC-NaOH method.[8]

In concordance with other studies, LJ contamination rate was less in HSSH method when compared with NALC-NaOH and modified Petroff's methods. A study by Chaudhary and Mishra compared HSSH method and modified Petroff's method and observed that the contamination rate was less in HSSH method (6%) when compared with modified Petroff's method (8%).[15] A study by Morcillo et al. also showed there was less contamination rate by HSSH method (1.2%) over NALC-NaOH method (2.5%).[16]

Culture in LJ media is considered as the gold standard method for isolation of MTB. To obtain a positive culture, sample should contain a least 10 bacilli/ml, since MTB is a slow-growing bacterium which requires 4–6 weeks to show positivity in cultures.[16] In the present study, we compared LJ culture results of the three decontamination methods, viz., NALC-NaOH method, HSSH method, and modified Petroff's method. Among 270 samples which were processed by all the three decontamination methods, the MTB isolation rate of HSSH method was higher than the other two decontamination methods. This could be due to the lower concentration of NaOH used in HSSH method (1.3%).[15] Higher NaOH concentration may kill about 60%–70% of MTB. Out of 270 samples, 19 (7.0%) were positive by HSSH method, whereas 11 (4.1) were positive by NALC-NaOH method and 9 (3.3%) were positive by modified Petroff's method. However, in a study by Morcillo et al., similar culture-positive results were observed for both HSSH method and NALC-NaOH method (2.3%).[16] A study by Chaudhary and Mishra which compared HSSH method and modified Petroff's method showed culture positivity of 84% and 70%, respectively, but this difference was not statistically significant because they included less samples in their study.[15]

Ganoza et al. have reported that the culture sensitivity of HSSH method and NALC-NaOH method were 95.2% and 76.2%, respectively.[8] In our study also, HSSH method (79.2%) showed higher sensitivity over NALC-NaOH method (45.8%) and modified Petroff's method (37.5%%). However, Morcillo et al. observed that both HSSH method and NALC-NaOH methods had similar culture sensitivity of about 88%.[16]

In our study, we compared mean time to detection of MTB between HSSH method, NALC-NaOH method, and modified Petroff's method. The mean time to detection of MTB by HSSH method and NALC-NaOH method was similar, but the mean time to detection of modified Petroff's method was about 1 week more than that of the other two decontamination methods. In agreement with our findings, Chaudhary and Mishra also have observed that 28.5% cultures turned out to be positive at 4th week by HSSH method, whereas in modified Petroff's method, only 5.7% cultures were positive.[15]


  Conclusion Top


Among the three decontamination methods, a significantly higher isolation rate of MTB was observed with HSSH method. In addition, the culture contamination rate was also very less by using HSSH method for decontaminating sputum samples. Lower time to detection by HSSH method eliminates the longer turnaround time by using modified Petroff's method. HSSH method is practically more feasible in terms of resource and task force as well. It is also less expensive compared to the other two methods. Therefore, considering the discussed variabilities and outcomes, we recommend HSSH method for decontamination of sputum samples for the better and faster isolation of MTB by using LJ medium.

Financial support and sponsorship

The authors thank the Jawaharlal Institute of Postgraduate Medical Education and Research for supporting this project with the intramural research grant (No. JIP/Res/Intra-MSc/02/2016-17/21).

Conflicts of interest

There are no conflicts of interest.



 
  References Top

1.
World Health Organization. Global Tuberculosis Report 2016. Geneva: World Health Organization; 2016.  Back to cited text no. 1
    
2.
Lee JY. Diagnosis and treatment of extrapulmonary tuberculosis. Tuberc Respir Dis (Seoul) 2015;78:47-55.  Back to cited text no. 2
    
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Dodd PJ, Looker C, Plumb ID, Bond V, Schaap A, Shanaube K, et al. Age- and sex-specific social contact patterns and incidence of Mycobacterium tuberculosis infection. Am J Epidemiol 2016;183:156-66.  Back to cited text no. 3
    
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Central TB Division. TB India 2017. New Delhi: Central TB Division; 2017.  Back to cited text no. 4
    
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WHO. Global Tuberculosis Report 2017. Geneva: World Health Organization; 2017.  Back to cited text no. 5
    
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Gautam G, Singh TS, Sharma TD, Ghimire M, Regmi SM. Decontamination of sputum sample for the isolation of Mycobacterium tuberculosis. J Gandaki Med Coll Nepal 2010;3:32-5.  Back to cited text no. 6
    
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Someshwaran R, Deshpande SA, Gnanaprakash K. Evaluation of sputum decontamination methods to facilitate the Mycobacterium tuberculosis detection in a tertiary care hospital. Int J Curr Microbiol App Sci 2016;5:889-94.  Back to cited text no. 7
    
8.
Ganoza CA, Ricaldi JN, Chauca J, Rojas G, Munayco C, Agapito J, et al. Novel hypertonic saline-sodium hydroxide (HS-SH) method for decontamination and concentration of sputum samples for Mycobacterium tuberculosis microscopy and culture. J Med Microbiol 2008;57:1094-8.  Back to cited text no. 8
    
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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;138:541-8.  Back to cited text no. 9
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Appalaraju B, Rajamani S. Evaluation of novel hypertonic saline sodium hydroxide (HS-SH) method for decontamination of sputum samples for detection of Mycobacterium tuberculosis in a tertiary care hospital. Univ J Pre Para Clin Sci 2016;2:1-6.  Back to cited text no. 10
    
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Hochberg NS, Sarkar S, Horsburgh CR Jr., Knudsen S, Pleskunas J, Sahu S, et al. Comorbidities in pulmonary tuberculosis cases in Puducherry and Tamil Nadu, India: Opportunities for intervention. PLoS One 2017;12:e0183195.  Back to cited text no. 11
    
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Kaulagekar A, Radkar A. Social status makes a difference: Tuberculosis scenario during National Family Health Survey-2. Indian J Tuberc 2007;54:17-23.  Back to cited text no. 12
    
13.
World Health Organization. Tuberculosis in Women. Geneva: World Health Organization; 2015.  Back to cited text no. 13
    
14.
Tripathi K, Tripathi PC, Nema S, Shrivastava AK, Dwiwedi K, Dhanvijay AK. Modified Petroff's Method: An excellent simplified decontamination technique in comparison with Petroff's Method. Int J Recent Trends Sci Technol 2014;10:461-4.  Back to cited text no. 14
    
15.
Chaudhary SK, Mishra B. Comparison of hypertonic saline-sodium hydroxide method with modified Petroff's method for the decontamination and concentration of sputum samples. Int J Infect Microbiol 2013;2:78-81.  Back to cited text no. 15
    
16.
Morcillo N, Imperiale B, Palomino JC. New simple decontamination method improves microscopic detection and culture of mycobacteria in clinical practice. Infect Drug Resist 2008;1:21-6.  Back to cited text no. 16
    



 
 
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