Year : 2009 | Volume
: 52 | Issue : 1 | Page : 59--61
Transport and storage of sputum specimen by using cetylpyridinium chloride for isolation of mycobacteria
Nita Pal, Babita Sharma, Bharti Malhotra, Suman Rishi
Department of Microbiology, SMS Medical College, Jaipur, Rajasthan, India
82, Green Nagar, Durgapura, Jaipur
Of the 191 sputum specimens that were collected from pulmonary tuberculosis patients, 78.65% (140/178) specimens were culture positive when processed within 48 h by the NaOH method. The culture positivity in the same specimen that were stored with cetylpyridinium chloride (CPC) and processed after 7-8 days was 70.22% (125/178), whereas those stored without CPC and processed by the NaOH method was 46.62% (83/178). The difference in number of positive cultures obtained before storage and after storage (without CPC) was statistically significant (P = 0.001). Culture positivity by the CPC method was comparable with that of NaOH method before storage and the difference was not statistically significant (P = 0.35).
|How to cite this article:|
Pal N, Sharma B, Malhotra B, Rishi S. Transport and storage of sputum specimen by using cetylpyridinium chloride for isolation of mycobacteria.Indian J Pathol Microbiol 2009;52:59-61
|How to cite this URL:|
Pal N, Sharma B, Malhotra B, Rishi S. Transport and storage of sputum specimen by using cetylpyridinium chloride for isolation of mycobacteria. Indian J Pathol Microbiol [serial online] 2009 [cited 2021 Dec 2 ];52:59-61
Available from: https://www.ijpmonline.org/text.asp?2009/52/1/59/44966
Tuberculosis remains one of the major public health problems worldwide and for the successful treatment of pulmonary tuberculosis patients, especially those with previous history of antituberculosis therapy, drug susceptibility testing is essential. Drug susceptibility testing first requires the isolation of M. tuberculosis for which the specimen should be transported at 2-4°C and processed within 24-48 h of collection.  Few laboratories are performing these tests; therefore, the sputum specimens have to be sent to central mycobacteriology laboratories for culture and drug susceptibility tests and there is usually a delay in the transporting and processing of the specimens for about a week. This leads to an increased contamination rate and loss of positive cultures. 
Therefore, a simple inexpensive storage method for transport of sputum specimens that preserves the viability of tubercle bacilli in sputum specimen up to 8 days will be useful. Cetylpyridinium chloride (CPC) is a quaternary ammonium compound, when added to sputum specimen at final concentration of 0.5%, does not kill tubercle bacilli for 14 days. 
The present study was undertaken to assess the usefulness of cetylpyridinium chloride (CPC) for storage of sputum specimen at room temperature up to 7-8 days under our climatic conditions (summer temperature range: 30-48°C).
Materials and Methods
One hundred and ninety-one sputum specimens were selected from the specimens that were received in sufficient quantity in the Department of Microbiology, SMS Medical College, Jaipur (Rajasthan) from May to August 2006.
Each sputum specimen was homogenized using sterile glass beads and divided in three aliquots. One of these aliquots was processed by routine 4%NaOH method  on the same day or the next day and inoculated onto two Lowenstein-Jensen (LJ) slopes.
To the second aliquot, an equal amount of CPC-NaCl reagent was added and mixed thoroughly. The second and third aliquots (without any preservative) were kept at ambient temperature for 7-8 days and then processed for culture by the respective methods.
CPC method: The sputum specimen treated with CPC-NaCl reagent was transferred in 50-ml screw-capped centrifuge tubes; sterile distilled water was filled till brim of the tube, capped and centrifuged at 3000 x g for 20 min. A loopful of the sediment was inoculated onto the two LJ slopes. 
The third sputum aliquot (without preservative) was subjected to the routine NaOH method  and inoculated onto the two LJ slopes.
The LJ slopes were incubated for 8 weeks and examined weekly for growth.
Note: Only LJ medium was used for culture because on 7H10 or agar based media the residual CPC in the inoculum remains active and partially inhibits the growth of tubercle bacilli. 
Out of the 191 sputum specimen, 13 were excluded from the study as they were found to be culture negative by all the three culture methods. Prior to storage, 78.65% (140/178) of sputum specimens were positive on culture, 15.16% (27/178) were negative and 6.17% (11/178) were contaminated [Table 1]. Culture positivity after storage for 7-8 days with CPC was 70.22% (125/178) and for specimen without any preservative, it was 46.62% (83/178).
The number of culture positives obtained by the CPC and NaOH methods after storage are presented in [Table 2]. The number of culture positives obtained by the CPC methods after storage (125/178) was comparable to that obtained by the NaOH method before storage (140/178) and the difference was not statistically significant (Chi -square test: P = 0.35). The number of culture positives before and after storage (without CPC) was 140 and 83 and the difference is highly significant ( P = 0.001).
In the present study, the CPC method was compared with the NaOH method that is routinely followed in most of the referral laboratories in India. Using the NaOH method, Paramasivan et al .  had shown that the culture positivity decreased from 88% before storage to 68% after storage for 7 days and in the study, of Selvakumar et al .,  the culture positivity decreased from 43.18% before storage to 31.81% after storage. Similarly, in the present study, the culture positivity decreased from 78.65% before storage to 46.62% after storage for 7-8 days.
By CPC, 70.22% (125/178) sputum specimens were positive compared to78.65% (140/178) by the NaOH method before storage. Reduction in culture positivity was statistically insignificant ( P = 0.35). In the study conducted by Selvakumar et al ., a similar reduction in culture positivity from 43.18% (95/220) to 38.63% (85/220) was reported. While in another study by Selvakumar et al .,  the yield of positive culture by CPC method 24.80% (31/125) was slightly higher than in NaOH method before storage 21.60% (27/125). Higher rate of positive cultures was observed when sputum specimen were treated with CPC method than by NaOH method by Pardini et al .  and Bobadilla-del-Valle et al . 
The culture positives obtained by the CPC method after storage in the present study were comparable to the results obtained by the NaOH method before storage ( P = 0.35). This is in conformity with two studies of Selvakumar et al . ,
The proportion of contaminated cultures by the NaOH method before storage was similar to the CPC method ( P = 0.22), while after storage without CPC contamination, the proportion was statistically significant ( P = 0.05). Similar contamination rate was reported in studies of Selvakumar et al . ,
For transport of sputum specimens requiring storage up to 7-8 days, the CPC-NaCl method has advantages over the standard NaOH method in reducing the contamination and yielding more positive cultures. Other advantages of the CPC method are that the reagent is stable at room temperature, easy to prepare, inexpensive and self-sterilizing. Moreover, the routine NaOH method requires careful processing of the specimen in different steps that are time bound, whereas in the CPC method, the specimens need only a single step centrifugation before inoculation. The CPC-NaCl reagent is also known to eliminate pathogenic fungi from the sputum specimens. 
The results suggest that when sputum specimens need to be processed after storage at room temperature up to 7-8 days, the yield of positive cultures would be higher with the CPC method than without the CPC method.
However, a large-scale study must be conducted under field conditions to investigate the effect of storage of sputum specimens with CPC reagent during transport before they are processed for culture.
|1||Bobadilla-del-Valle M, Ponce-de-León A, Kato-Maeda M, Hernαndez-Cruz A, Calava-Mercado JJ, Chαvez-Mazari B, et al . Comparison of sodium carbonate, cetylpyridinium chloride and sodium borate for preservation of sputa for culture of Mycobacterium tuberculosis . J Clin Microbiol 2003;41:4487-8.|
|2||Paramasivan CN, Narayana SL, Prabhaker, Rajgopal MS, Somasundaram PR, Tripathy SP. Effect of storage of sputum specimen at room temperature on smears and culture results. Tubercle 1983;64:119-24.|
|3||Tazir M, David HL, Boulahbal F. Evaluation of the chloride and bromide salts of cetylpridinium for the transportation of sputum in tuberculosis bacteriology. Tubercle 1979;60:31-6.|
|4||Kent PT, Kubica GP. Public Health Mycobacteriology. A Guide for the level III laboratory. US Health Service Centers for Disease Control. Atlanta GA: 1985.|
|5||Smithwick RW, Stratigos CB, David HL. Use of cetylpyridinium chloride and sodium chloride for decontamination of sputum specimens that are transported to the laboratory for the isolation of Mycobacterium tuberculosis . J Clin Microbiol 1975;1:411-3.|
|6||Selvakumar N, Vanajakumar, Narayana AS, Suryanarayana D, Umapathy KC, Paramasivan CN, et al . Use of cetylpridinium chlorides for storage of sputum specimens and isolation of M. tuberculosis . Indian J Tuberc 1993;40:95-7.|
|7||Selvakumar N, Vanajakumar, Gopi PG, Venkataramu KV, Datta M, Paramasivan CN, et al . Isolation of tubercle bacilli from sputum samples of patients in field studies by the cetylpyridinium chloride and sodium chloride methods. Indian J Med Res 1995;102:149-51.|
|8||Pardini M, Varaine F, Iona E, Arzumanian E, Checchi F, Oggioni MR, et al . Cetyl-pyridinium chloride is useful for isolation of Mycobacterium tuberculosis from sputa subjected to long-term storage. J Clin Microbiol 2005;43:442-4.|
|9||Phillips BJ, Kaplan W. Effect of on pathogenic fungi and Nocardia asteroids in sputum. J Clin Microbiol 1976;3:272-6.|