|Year : 2014 | Volume
| Issue : 4 | Page : 404-407
Is universal sample processing methodology better than conventional techniques for detection of tuberculosis?
V Mittal1, F Haider2, S Singhal2, S Jamal2
1 Department of Microbiology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow, Uttar Pradesh, India
2 Department of Microbiology, Era's Lucknow Medical College and Hospital, Lucknow, Uttar Pradesh, India
|Date of Submission||20-Aug-2013|
|Date of Acceptance||20-Jan-2014|
|Date of Web Publication||4-Oct-2014|
Department of Microbiology, Dr. Ram Manohar Lohia Institute of Medical Sciences, Lucknow, Uttar Pradesh
Source of Support: None, Conflict of Interest: None
Background: The identification of infectious cases is a crucial first step for tuberculosis control programmes worldwide. It relies exclusively on the detection of acid-fast bacilli in sputum by smear microscopy. Therefore, there is an urgent and definite need to improve the sensitivity of smear microscopy. Objective: The USP method was compared with the two most commonly used conventional methods of smear microscopy namely; direct smear microscopy and the microscopy by modified Petroff's method. Materials and Methods: Two samples from each patient were taken from 197 patients of presumptive tuberculosis. One smear was made for direct Ziehl-Neelsen staining and two smears were made after processing by two concentration methods i.e., modified Petroff's and USP solution. LJ media were inoculated for culture after processing by both concentration methods. Results: Among 197 cases 93 were culture positive by either method. Out of 93 culture-positive sample, 78.5% were direct smear positive, 89% were 4%NaOH smear positive and 96% were USP smear-positive samples but difference in diagnostic accuracy of USP (96%) and modified Petroff method (93%) is not statistically significant (P > 0.01). Conclusion: The present study evaluated the smear microscopy by USP method with two conventional methods, direct microscopy and microscopy by modified Petroff's method. The study concludes that although USP method is more sensitive than conventional methods, it is not feasible to include it in diagnosis of early tuberculosis within RNTCP.
Keywords: Modified Petroff method, tuberculosis, USP, Ziehl-Neelsen staining
|How to cite this article:|
Mittal V, Haider F, Singhal S, Jamal S. Is universal sample processing methodology better than conventional techniques for detection of tuberculosis?. Indian J Med Microbiol 2014;32:404-7
|How to cite this URL:|
Mittal V, Haider F, Singhal S, Jamal S. Is universal sample processing methodology better than conventional techniques for detection of tuberculosis?. Indian J Med Microbiol [serial online] 2014 [cited 2020 Oct 31];32:404-7. Available from: https://www.ijmm.org/text.asp?2014/32/4/404/142249
| ~ Introduction|| |
Tuberculosis (TB) is a serious public health problem causing immense morbidity, mortality and distress to individuals, families and communities. The identification of infectious cases is a crucial first step for tuberculosis (TB) control programmes worldwide. It relies exclusively on the detection of acid-fast bacilli (AFB) in sputum by smear microscopy. This is the key diagnostic tool used for case detection in Revised National Tuberculosis Control Programme (RNTCP) in India. 
Most laboratories use smears of unconcentrated sputum (direct smears) with Ziehl-Neelsen staining which is a less sensitive method as 5-10,000 bacilli per ml are required to get reproducible results.  It is estimated that less than 20% of approximately eight million predicted annual cases of TB worldwide are identified as smear positive. The targets of a 90% case detection rate and treatment success are not likely to be achieved with the existing methods of smear microscopy. 
Therefore, there is an urgent and definite need to improve the sensitivity of smear microscopy. There is a novel specimen processing technology, called universal sample processing (USP),  for TB diagnosis in both pulmonary and extrapulmonary specimens. This enables highly sensitive smear microscopy (with a sensitivity of detection in the order of 300-400 AFB/ml of specimen) and culturing of the tubercle bacilli. The present study was undertaken to evaluate the performance of the USP method in a clinical setting with different specimens and compared with the two most commonly used conventional methods of smear microscopy namely, direct smear microscopy and the smear microscopy by concentration method (modified Petroff's method) for detection of TB bacilli.
| ~ Materials and Methods|| |
In this prospective cross-sectional study total 210 sputum samples of presumptive new cases of tuberculosis were included from patients attending OPD of different departments of a tertiary care center of Lucknow from August 2011 to December 2011. The study was conducted in accordance with the ethical rules of Institute. Informed written consent to participate in the study was obtained from all patients.
According to RNTCP India, two samples, one early morning and other spot sample from each patient, were collected in a universal container. All the received sputum specimens were divided into three parts. One part was taken for direct smear preparation on a new slide and Ziehl-Neelsen staining  was performed. Second and third portions were processed for decontamination using the modified Petroff method and USP method. Modified Petroff method consists 4% NaOH as a decontaminating reagent. USP solution consists of 4 M guanidinium hydrochloride (GuHCl) (Amresco), 50 mM Tris-Cl, (Sigma), 25 mM EDTA (SRL), 0.5% Sarkosyl (Sigma) and 0.2 M mercaptoethanol (Sisco SRL).
Smears were made from sediments and Ziehl-Neelsen staining was performed. Then Lowenstein Jensen media (LJ media) were inoculated with 5-mm loop full of the centrifuged sediments processed by the two concentration methods separately. Bottle caps were tightened to minimise evaporation and drying of media. All cultures were incubated at 37⁰C for 12 weeks.
Out of 210 samples, 13 cultures were contaminated by both methods probably due to improper exposure time with decontaminating agent and not using the cold centrifuge for centrifugation. Hence, these cases were excluded from the study. The USP method was evaluated for its sensitivity/efficiency using these 197 sputum specimens by the use of smear microscopy and culture.
Statistical analysis was done using culture as gold standard test for diagnosis of TB. A positive LJ slant by any one method of processing (modified Petroff or USP method) for the same specimen was considered a true positive. LJ slants that were negative by both methods for the same specimen were considered true negative samples. We calculated the sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV) and diagnostic accuracy on the basis of culture results using the statistical Software SPSS.
| ~ Results|| |
Detection of TB by modified Petroff method
Of the 122 negative samples by the direct smear method (smear-negative samples), the 4%NaOH method detected 11 additional positive samples. In our study, 4% NaOH method had 89.25% sensitivity as compared to the direct method which had 78.49% sensitivity; the 11% enhancement in sensitivity was highly significant (P < 0.001). Diagnostic accuracy of four percent NaOH method was also more (~93%) than direct method (~89%) [Table 1]. There was also the concentrating effect in smear status. The smear grade status was enhanced by the 4% NaOH smear method; slides graded as scanty, 1+, or 2 + by direct smear microscopy generally were graded as 1+, 2+, or 3+ by 4% NaOH smear microscopy.
|Table 1: Analysis of smear microscopy for detection of tuberculosis by different methods |
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Detection of TB by USP method
Of the 122 negative samples by the direct smear method (smear-negative samples), the USP method detected 18 additional positive samples out of them 1 was 2+. 3 were 1+ and rest 14 smears had scanty bacilli. In our study USP method had 95.7% sensitivity as compared to direct method which had 78.49% sensitivity; the 17% enhancement in sensitivity was highly significant (P < 0.001) [Table 1].
The USP smear method was simultaneously compared with the modified Petroff smear microscopy. Seven specimens which were smear negative by the 4% NaOH method were detected positive by the USP method, All of these specimens which were missed by the modified Petroff method of smear microscopy had small numbers of bacilli, as they predominantly belonged to the scanty or 1+ category. Thus, the USP method showed an enhancement in sensitivity over that of the modified Petroff method. But 7% enhancement in sensitivity of USP method as compared to modified Petroff was not found to be statistically significant (P > 0.01). Specificity by all methods was almost equal.
In our study, diagnostic accuracy of USP method was best (~96%) as compared to other methods but not much difference with modified Petroff method (~93%) was seen.
There was also concentrating effect in smear status. The smear grade status was enhanced by the USP smear method; slides graded as scanty, 1+, or 2 + by direct smear microscopy generally were graded as 1+, 2+, or 3+ by USP smear microscopy. Among the 197 specimens, 75 specimens were positive by both the direct and USP methods of smear microscopy. Of these, only 33 belonged to the 3+ category by the direct smear method, in contrast to 41 by the USP method. Thus, 10% of the specimens that were positive by the direct smear method were upgraded to the 3+ category by the USP method. The same trend was observed between the USP method and modified Petroff smear microscopy; among 86 specimens positive by both the modified Petroff and USP methods of smear microscopy, eight were upgraded to next category by the USP method although two were downgraded also. Furthermore, none of the specimens which were positive by all of the methods of smear microscopy belonged to the scanty category according to USP smear microscopy.
Evaluation between cultures by USP and modified Petroff methods
Total numbers of specimens, culture positive by either method, were 93. Out of which 88 were positive by USP method or modified Petroff method. Out of 88 samples, 77 samples were positive by both culture methods. Out of 93 culture-positive sample, 78.5% were direct smear positive, 89% were 4% NaOH smear positive and 96% were USP smear-positive samples so increase yield over direct smear by USP was 17.21% (P = 0.003) and by 4%NaOH was 10.76% (P = 0.044) but yield over 4%NaOH by USP was 6.45% (P = 0.093). In our study, contamination rate in the USP method was higher (7.61%) than modified Petroff methods (2.03%).
| ~ Discussion|| |
Chakravorty and Tyagi  described a new methodology of microscopy by USP method that can be reliably applied to all types of clinical specimens for diagnosing tuberculosis in laboratories with diverse infrastructure capabilities. In other study, USP method exhibited a highly significant enhancement in sensitivity compared to direct method and NALC-NaOH method of smear microscopy.  In our study, USP methods was definitely exhibited a highly significant enhancement in sensitivity compared to direct method. It detected 18 additional positive samples proving that the direct smear method sometimes fails to detect specimens with high bacterial loads due to technical errors during smear preparation or faulty reading of slides. But in our study USP methods have not significant enhancement in sensitivity as compared to modified Petroff method. Diagnostic accuracy of USP method and modified Petroff method had not much difference also. This was also reported by Cattamanchi et al. 
In culture methods also, both the USP and modified Petroff method showed equal percentages
of positivity (42.47%). In our study, contamination rate by modified Petroff methods was 2.03% which is acceptable. However, the USP method yielded more contaminated cultures (7.61%). It was due to mixing of five chemicals to make solution. Although Chakravorty et al. have reported very low contamination rate (0.88%) by the USP method in their study but we had opposite results. One more disadvantage with USP method was its cost because NaOH is a very economical and readily available reagent but USP solution has to be made by mixing of many chemicals which are quite expensive.
For laboratories conducting TB testing, the most important hazard (or risk) is the generation of infectious aerosols since infection with Mycobacterium tuberculosis occurs primarily through the inhalation of infectious aerosols. According to WHO expert group, biological safety cabinet (BSCs) are not mandatory for performing direct sputum-smear microscopy. The Expert Group found that with good microbiological technique, direct sputum-smear microscopy entails a low risk of generating infectious aerosols, and such procedures may therefore be performed on an open bench. But processing of samples by concentrated methods requires biological safety cabinet  In our country, DOTS centre under RNTCP has done sputum smear microscopy by direct method. It is not possible to install BSC at every DOTS centre for processing of samples by concentration methods.
| ~ Conclusions|| |
The present study evaluated the smear microscopy by USP method with two conventional methods, direct microscopy and microscopy by modified Petroff's method. The study concludes that although USP method is more sensitive than conventional methods, it is not feasible to include it in diagnosis of early tuberculosis within RNTCP.
| ~ References|| |
|1.||TB India 2013, Revised National Tuberculosis Control Programme annual status report, publisher Central TB division, New Delhi, 2013. Available: www.tbcindia.nic.in/pdfs/tbindia2013.pdf. [Last accessed 2013 October 4]. |
|2.||Katoch VM. Smear microscopy to diagnose tuberculosis. Indian J Med Res 2006;123:735-8. |
|3.||Chakravorty S, Tyagi JS. Novel multipurpose methodology for detection of mycobacteria in pulmonary and extrapulmonary specimens by smear microscopy, culture, and PCR. J Clin Microbiol 2005;43:2697-702. |
|4.||Chakravorty S, Dudeja M, Hanif M, Tyagi JS. Utility of universal sample processing methodology, combining smear microscopy, culture, and PCR, for diagnosis of pulmonary tuberculosis. J Clin Microbiol 2005;43:2703-8. |
|5.||Cattamanchi A, Davis JL, Worodria W, Yoo S, Matovu J, Kiidha J, et al. Poor performance of universal sample processing method for diagnosis of pulmonary tuberculosis by smear microscopy and culture in Uganda. J Clin Microbiol 2008;46:3325-9. |
|6.||WHO, Tuberculosis laboratory biosafety manual, GPS publishing Italy 2012. Available: www.who.int/tb/publications/ 2012/tb_biosafety/en/index.html. [Last accessed 2013 October 9]. |