|Year : 2012 | Volume
| Issue : 2 | Page : 97-100
Comparison of modified Petroff's and N-acetyl-L-cysteine-sodium hydroxide methods for sputum decontamination in tertiary care hospital in India
Mukesh Sharma1, Rabindra Nath Misra1, Nageswari Rajesh Gandham1, Savita Vivek Jadhav1, Kalpana Angadi1, Vivian Wilson2
1 Department of Microbiology, Padm. Dr. D.Y. Patil Medical College & Research Centre, Pimpri, Pune, India
2 Department of Pulmonary Medicine, Padm. Dr. D.Y. Patil Medical College & Research Centre, Pimpri, Pune, India
|Date of Web Publication||10-Nov-2012|
Department of Microbiology, Padm Dr. D.Y. Patil Medical College & Research Centre, Pimpri, Pune
Source of Support: None, Conflict of Interest: None
Background : Tuberculosis is the second leading cause of death worldwide, killing nearly two million people each year. Sputum decontamination with N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) is expected to improve detection of Mycobacterium tuberculosis (M. tb) by culture better than that with modified Petroff's; which is widely used in laboratories. In this study, sputum samples collected from suspected cases of pulmonary tuberculosis (TB) were cultured directly on Lowenstein-Jensen (LJ) medium and after decontamination by both the methods and the results of smear and culture positivity were evaluated to assess whether the NALC-NaOH treatment method improves smear and culture. Materials and Methods : For each decontamination method, 30 samples were obtained from suspected cases of Pulmonary TB, from Pad. Dr. D.Y. Patil Medical College and Hospital. Two sputum samples from each patient were collected on day 1 and 2. These samples then underwent decontamination process by performing the 4% NaOH, NALC-2% NaOH treatment methods and direct inoculation. After each process a smear was made and culture was done on LJ medium. Results: The modified Petroff's and NALC-NaOH treatment methods did not significantly affect the AFB smear positivity of the sputum samples (66% and 72.3%, respectively). (However, the culture positivity for M. tb on LJ medium was significantly different by the three processes. With NALC-NaOH and modified Petroff's it was 63% and 46%, respectively, while with direct culture it was 23%. Conclusion: NALC-NaOH treatment is better than modified Petroff's treatment for the detection of M. tb by culture. However, AFB microscopy does not seem to be significantly different by either process.
Keywords: Decontamination, Mycobacterium tuberculosis, tuberculosis, Ziehl-Neelsen Staining
|How to cite this article:|
Sharma M, Misra RN, Gandham NR, Jadhav SV, Angadi K, Wilson V. Comparison of modified Petroff's and N-acetyl-L-cysteine-sodium hydroxide methods for sputum decontamination in tertiary care hospital in India. Med J DY Patil Univ 2012;5:97-100
|How to cite this URL:|
Sharma M, Misra RN, Gandham NR, Jadhav SV, Angadi K, Wilson V. Comparison of modified Petroff's and N-acetyl-L-cysteine-sodium hydroxide methods for sputum decontamination in tertiary care hospital in India. Med J DY Patil Univ [serial online] 2012 [cited 2017 Apr 26];5:97-100. Available from: http://www.mjdrdypu.org/text.asp?2012/5/2/97/103323
| Introduction|| |
Tuberculosis (TB) is a common and often deadly infectious disease caused by mycobacteria in humans, mainly by Mycobacterium tuberculosis.  TB is a major air-borne, infectious bacterial disease. It remains a major worldwide health problem with global mortality ranging from 1.6 to 2.2 million lives per year. Direct smear microscopy for acid-fast bacilli (AFB) is rapid, inexpensive, highly specific, and capable of identifying the most infectious cases of TB. , The only disadvantage of this method is low sensitivity (varying from 50 to 80%) relative to culture. ,,,,
The Gold standard for diagnosing pulmonary TB remains culture. Decontamination of clinical specimens such as sputum is an important and critical step in the isolation of mycobacteria, and the mildest decontamination procedure which provides sufficient control of contaminants is likely to yield best results.  Decontamination and concentration of sputum specimens by modified Petroff's method is one of the most commonly used methods for M. tuberculosis culture.  N-acetyl-L-cysteine-sodium hydroxide (NALC-NaOH) solution is recommended as a gentle but effective digesting and decontaminating agent. Addition of a large volume of phosphate buffer (PB) makes strong shifts in pH, "washes" the specimen, dilutes toxic substances and decreases the specific gravity of the specimen so that centrifugation is more effective. Therefore, it is generally accepted that the NALC-NaOH method of Kent and Kubica et al.  should be given preference over the modified Petroff's methods. The technical and procedural factors can influence the sensitivity of each method. The main objective of this study was to evaluate the performance of NALC-NaOH and modified Petroff's methods in our laboratory.
| Materials and Methods|| |
This comparative study was conducted in the Department of Microbiology, Dr. D.Y. Patil Medical College and Hospital, Pimpri, Pune, India, from April 2011 to June 2011. Thirty suspected case of pulmonary tuberculosis was selected for this study and sputum specimens were taken as per Revised National Tuberculosis Control Programme (RNTCP) guidelines.
These samples had previously undergone acid-fast microscopy (direct smear) by RNTCP. However, it was repeated to confirm the results. This was done according to the Ziehl-Neelsen technique recommended by World Health Organization (1998). Acid-fast microscopy was also carried out after decontamination and centrifugation of the samples (concentrated smear) as established by Salem et al. (1990). 
Specimen Digestion and Decontamination
Specimens were vortex mixed and equally divided into two parts. One part of the specimen was processed by modified Petroff's method and other part was processed by NALC-NaOH method. In addition, for this study we inoculated all the samples directly on Lowenstein-Jensen (LJ) medium without doing decontamination method.
NALC-2% sodium hydroxide-sodium citrate solution was prepared as described by Kent and Kubica.  To the 50- ml tube containing 3-5 ml of seeded sputum sample, an equal volume of NALC-NaOH citrate reagent was added and the tube was vortexed briefly. Following 15 min of incubation at room temperature, the volume was brought to 50 ml with 0.067 M phosphate buffer (pH 6.8) and the contents were mixed by inversion. Bacteria were pelleted by centrifugation at 3,000 x g for 15 min. The supernatant was discarded, and the pelleted material was resuspended in 1 ml of PB. From the pellet 0.5 ml was inoculated on LJ slopes and the smear was made. The culture slants were incubated at 37°C.
Modified Petroff's Method
All the sputum samples were processed by the modified Petroff's method for culture of M. tuberculosis.  In brief, 3-5 ml of sputum was homogenized for 15 min in a shaker using an equal volume of 4% NaOH. After centrifugation at 3,000 rpm for 15 min, the deposit was neutralized with 20 ml of sterile distilled water. The samples were again centrifuged. From the sediment, LJ medium was inoculated and smear was made. The culture slants were incubated at 37°C.
All slopes were observed for occurrence of growth daily for first week and then at weekly intervals for 8 weeks. The isolates were identified by following tests: rate of growth, optimum temperature of growth, colony morphology, pigmentation, niacin test, and nitrate reduction tests, which confirmed that all isolates are M. tuberculosis.
Absence of growth at the end of 8 th weeks was regarded as negative culture. Contamination, if any, was recorded separately. The number of culture failures for a certain decontamination method, included the number of specimens with negative culture as well as number of contaminated cultures.
| Results|| |
Out of 30 specimens, 20 (66.70%) were smear positive and 10 (32.30%) were smear negative by direct microscopy. After decontamination by both the methods again smear was made, to rule out any change in smear microscopy, which showed positivity by NALC-NaOH 21 (70%) and modified Petroff's 20 (66.70%) [Figure 1] and [Figure 2].
Maximum number of positive culture 19 (63.7%) were obtained by NALC-NaOH technique. In modified Petroff's method it was 46.7% (14 strains) while in direct method 10% (3 strains) [Table 1]. The total number of culture failures (which includes both contamination and negative cultures) were 11 (36.7%) in NALC-NaOH as against 16 (53.3%) in modified Petroff's and 27 (90%) in direct methods as shown in [Table 2]. The contamination rate was lowest in NALC-NaOH 4 (13.2%), whereas it was higher in modified Petroff's 7 (23.1%) and 23 (79.20%) direct method.
|Table 2: Showing comparison of the three procedures (in regards rate of contamination, negative cultures and culture failures)|
Click here to view
| Discussion|| |
Sputum culture is an important tool for TB control programs; it is more sensitive than smear microscopy in diagnosing TB, and facilitates drug susceptibility testing. Because sputum samples pass through the oropharynx during collection, culture contamination limits the diagnostic yield of sputum culture for TB. In addition to oral bacteria and yeast, the sputum decontamination process, used to process sputum specimens for the isolation of mycobacteria also kills mycobacteria, and the percentage of organisms killed varies according to the method used and the mycobacteial species present in the specimens. , In this study, we used the NALC-NaOH sputum digestion method of Kent and Kubica et al. as it is widely used and recommended by standard laboratory manuals, and because the final 2% NaOH concentration employed results in the destruction of fewer mycobacteria than 4% NaOH used by modified Petroff's method.
In the analysis of 30 samples, the decontaminated by the NALC-NaOH method provided isolation of M. tuberculosis in a higher percentage (63.7%) than obtained by Kang et al. and Chakravorty et al. (39.7%) study. But result from Pathak, Deshmukh and Menon (1973) who reported superiority of swab method Nassau (Nussau, 1954) 92.3% over NALC-NaOH 79.4% method. 
As per our observation in this study we noticed that in NALC-NaOH growth were higher 19 (63.7%) during V and VI wk as compare to modified Petroff's method 11 (36.3%) the probable reason are, 4% NaOH is used for modified Petroff's method as compared NALC-NaOH method which uses 2% NaOH while concentrating the sputum may kill or seriously injure few Mycobacteria. Hence recovery by NALC-NaOH was faster and better than modified Petroff's method. In our study we found smear positivity higher than the culture positivity. The reason might be that microscopy sometimes gives false positive results and in our condition, it cannot distinguish between dead and live bacteria. In such cases, the patients might be treated with antitubercular drugs and in the microscopy of these samples, the AFB might be dead. For these reason, the dead isolates did not grow in the L-J culture media. This also reveals that AFB microscopy does not always give accurate results for the diagnosis of TB. The contamination rate by NALC-NaOH method was 13.2% in our study which is lower than that reported by other workers. Engback et al. (1961) reported 30.4% while Pathak et al. (1973) reported 27% as contamination rate with NALC-NaOH method.  Additionally one sample was found positive by NALC-NaOH method, which was found negative using Petroff's and Direct method in smear microscopy and culture. In our study the NALC-NaOH method for AFB smear and culture improves the sensitivity when compared with the Direct and modified Petroff's method.
In conclusion, the results of this study suggest that treatment with NALC-NaOH method has lower contamination rate and higher yield by culture as compared to the other two methods, thus making it more suitable for routine use.
| References|| |
|1.||Kumar V, Abbas AK, Fauto N, Mitchell RN. Robbins Basic Pathology. 8 th ed. Saunders: Elsevier; 2007;p.516-22. |
|2.||Somoskövi A, Hotaling JE, Fitzgerald M, O'Donnell D, Parsons LM, Salfinger M. Lessons from a proficiency testing event for acid-fast microscopy. Chest 2001;120:250-7. |
|3.||Bruchfeld J, Aderaye G, Palme IB, Bjorvatn B, Källenius G, Lindquist L. Sputum concentration improves diagnosis of tuberculosis in a setting with a high prevalence of HIV. Trans R Soc Trop Med Hyg 2000;94:677-80. |
|4.||Murray SJ, Barrett A, Magee JG, Freeman R. Optimization of acid fast smears for the direct detection of mycobacteria in clinical samples. J Clin Pathol 2003;56:613-5. |
|5.||Peterson EM, Nakasone A, Platon-DeLeon JM, Jang Y, de La Maza LM, Desmond E. Comparison of direct and concentrated acid-fast smears to identify specimens culture positive for Mycobacterium spp. J Clin Microbiol 1999;37:3564-8. |
|6.||Woods GL, Pentony E, Boxley MJ, Gatson AM. Concentration of sputum by cytocentrifugation for preparation of smears for detection of acid-fast bacilli does not increase sensitivity of the fluorochrome stain. J Clin Microbiol 1995;33:1915-6. |
|7.||Saceanu CA, Pfeiffer NC, McLean T. Evaluation of sputum smears concentrated by cytocentrifugation for detection of acid-fast bacilli. J Clin Microbiol 1993;31:2371-4. |
|8.||Rothlauf MV, Brown GL, Blair EB. Isolation of mycobacteria from undecontaminated specimens with selective 7H10 medium. J Clin Microbiol 1981;13:76-9. |
|9.||National Tuberculosis Institute, Monograph Series I. Manual for establishing and functioning of a tuberculosis culture laboratory; Indian J Tuberc (New Delhi) 1983. p. 19-23. |
|10.||Kent PT, Kubica GP. Public health mycobacteriology. A guide for the level III laboratory. Atlanta, GA: Centers for Disease Control and Prevention; 1985. |
|11.||Salem JI, Marója MF, Carvalho FF, Lima MO, Litaiff LR, Cardoso MS, et al. Valor relativo do exame direto, após concentração e por cultivo de escarro no diagnóstico bacteriológico da tuberculose pulmonar no Amazonas. J Pneumol 1990;16:133-6. |
|12.||Tuberculosis Chemotherapy Center, Madras. A concurrent comparison of isoniazid plus PAS with three regimens of isoniazid alone in the domiciliary treatment of pulmonary tuberculosis in South India. Bull World Health Organ 1960;23:535-85. |
|13.||Buijtels PC, Petit PL. Comparison of NaOH-N-acetyl cysteine and sulfuric acid decontamination methods for recovery of mycobacteria from clinical specimens. J Microbiol Methods 2005;62:83-8. |
|14.||Grandjean L, Martin L, Gilman RH, Valencia T, Herrera B, Quino W, et al. Tuberculosis diagnosis and multidrug resistance testing by direct sputum culture in selective broth without decontamination or centrifugation J Clin Microbiol 2008;46:2339-44. |
|15.||Nassau E, Parsons ER, Johnson GD. Bacteriolgical examination in tuberculosis. Tubercle (London): Tubercle; 1954. p. 35 (Colonial suppl. No. 3). |
|16.||Pathak SK, Deshmukh PA, Menon CR. A comparison of different culture techniques. Ind J Tubercle 1973;20:85-7. |
[Figure 1], [Figure 2]
[Table 1], [Table 2]
|This article has been cited by|
||Sociodemographic, microbial and phylogentic studies of Mycobacterium tuberculosis cases diagnosed in El-Minia governorate, Egypt
| ||Y Ahmad Fatma,M Abd El Baky Rehab,F M Gad Gamal,A Soliman Youssef |
| ||African Journal of Microbiology Research. 2016; 10(7): 191 |
|[Pubmed] | [DOI]|
||Prevalence of pulmonary tuberculosis in Wardha district of Maharashtra, Central India
| ||Pratibha Narang,Deepak K. Mendiratta,Naresh K. Tyagi,Ullhas N. Jajoo,Atul T. Tayade,Pratapsingh H. Parihar,Rahul Narang,Pranav S. Mishra,Sharda M. Anjinappa,Vineet K. Chadha |
| ||Journal of Epidemiology and Global Health. 2015; 5(4): S11 |
|[Pubmed] | [DOI]|