Screening tuberculosis suspects: How many sputum specimens are adequate?


Background: The aim of the study was to assess whether the number of sputum samples for screening of tuberculosis suspects could be minimized. Materials and Methods: It was a hospital-based cross-sectional study conducted from December 2006 to May 2008. Chest symptomatic 2810 patients were screened for tuberculosis (TB) by subjecting them to sputum microscopy using 2 sputum samples as well as 3 samples following standard procedure for sputum collection, staining, and AFB identification. Results: First sputum smear alone could detect 91.8% cases while first 2 samples together could detect 96% cases. Maximum no. of cases were diagnosed by only 2 specimen sputum smears, and added diagnostic value of third specimen was small i.e., 4%. Conclusions: Hence, 2 sputum samples as diagnostic criteria for screening of TB suspects is one way of improving the efficient use of scarce resources.

Keywords: Cough symptomatic, cough, morning specimen, OPD patients, screening of tuberculosis, spot specimen, sputum samples, tuberculosis

How to cite this article:
Hamid S, Hussain SA, Imtiyaz A. Screening tuberculosis suspects: How many sputum specimens are adequate?. Ann Trop Med Public Health 2012;5:317-20


How to cite this URL:
Hamid S, Hussain SA, Imtiyaz A. Screening tuberculosis suspects: How many sputum specimens are adequate?. Ann Trop Med Public Health [serial online] 2012 [cited 2020 Nov 24];5:317-20. Available from:



The purpose of case detection in tuberculosis control is to identify and treat the source of infection, i.e. sputum smear-positive cases, in order to reduce the prevalence and spread of infection. The main strategy recommended for case detection in developing countries, where 95% of tuberculosis cases occur, is sputum smear microscopy among out-patients with prolonged cough who voluntarily attend health facilities. [1],[2],[3]

Most standard laboratory tests [4] and guide lines [5],[6] for mycobacteriology laboratories recommend that at least 3 sputum specimens, preferably collected on successive days, be submitted to the laboratory for AFB smear and culture for patients suspected to have tuberculosis. Unfortunately, there has been a paucity of published data analyzing the validity of this recommendation. [7]

Recently, the number of sputum specimens suggested has become a matter of debate to assess the benefit and to reduce hospital expenses in the diagnosis of pulmonary tuberculosis (Nelson et al, 1998, Finch and Beaty, 1997, Cascina et al, 2000, craft et al 2000, Harvell et al 2000). [8] Studies have shown that examination of 2 consecutive specimens (on spot and overnight sputum) is sufficient to detect a large number of infectious cases in the community. [9] Hence, in this study, an attempt has been made to see the yield of screening TB suspects using 2 sputum samples versus 3 samples.


The objectives of the study were:

  1. To find out how many sputum samples are adequate for diagnosis of tuberculosis
  2. To find the best possible combination of sputum smears for diagnosis of tuberculosis.
Materials and Methods

The present study was carried out in district tuberculosis center Srinagar and was a hospital-based cross-sectional study. The study was conducted for a period of one and half year starting from December 2006. The approach that was used to identify acid-fast bacilli in the sputum samples of the chest symptomatic patients in hospital was Ziehl Nielson direct staining method. During the study, sputum samples of all the chest symptomatic patients (2810) were collected and sent for ZN staining and sputum microscopy. Patients on treatment attending the hospital for follow-up were excluded. The sputum specimen was collected on spot from each symptomatic patient. The patient was then instructed to return on the following day for the examination of an overnight sputum specimen and to provide a second spot specimen. The smears were then processed and read by trained laboratory technicians. The sputum sample results, thus obtained, were collected and evaluated to see which cough symptomatic came positive by first 2 smears, which by first and third or by second and third or by all the 3 sputum smears. The statistical significance analysis was performed using chi-square test. Two-tailed P-value ≤ 0.05 were considered significant.


The total number of new adult patients who attended the OPD during the study period were 70000, and the total suspects subjected to sputum microscopy were 2810, which accounted for 4% of the total OPD attendees [Table 1].

Table 1: General description

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Among 2810 cough symptomatic, 367 were sputum positive, giving the sputum positivity rate as 13.1%. Among the 367 sputum smear-positive cases, all the 3 sputum smears were positive in 86.6% (318) cases [Table 2]. Only and any 2 sputum-positive were seen in total in 13.4% (49) cases, whereas first 2 smears were positive in 1.4% (5) cases. First and third smear only positive were in 3.8% (14) cases.

Table 2: Pattern of sputum smear positivity with duration of cough

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Second and third smears positive only in total was seen in 8.2% (30) cases. Overall, 2 smears-positive in total was seen in 13.1 cases. Also, first smear alone positive was seen in total in 91.8% cases. Second smear alone was positive in overall 96.2% cases. Third smear only positive was seen in total in 98.6% cases. Findings were statistically significant. From all cough symptomatic, taking first sample alone, case yield is 91.8%, seconds smear takes the yield to 96.2%, showing a percentage increase as 4.4% [Table 3].

Table 3: Overall incremental increase in number of sputum +ve cases using different combinations of sputum +ve samples in all cough symptomatic

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Taking 2 samples together and using different combinations, the highest incremental increase is seen with first and third sample (6.8%).

By adding third sample to first and second, incremental increase in case yield is 3.8%.


Prompt and accurate diagnoses together with effective treatment are the essential elements of TB care and control. Because of its low complexity and low cost, sputum smear microscopy is well suited to resource-poor settings fortunately; in high burden settings, the finding of AFB by microscopy in sputum smears is highly specific. [10],[11],[12] Poor sensitivity, however, remains the problem. Also, there is a debate about how many sputum samples taken are adequate to get good yield. Consequently, the optimization of smear microscopy techniques and the development of new diagnostic tools are both areas of active investigation. [13],[14]

2 Sputum versus 3 sputum samples

Our study shows that maximum number i.e. 96% of cases are diagnosed by only 2 sputum smears with the 3rd smear adding only a small number of cases i.e., only 4 percent of cases are further diagnosed. Several other studies have shown that added diagnostic value of third specimen is small. [15]

Thus, under routine conditions, 2 sputum smears can be recommended in place of 3 smears for screening chest symptomatic. Mase et al[15] in a systematic review of studies that quantified the diagnostic yield of each of the 3 sputum specimens, noticed that reducing the sputum smear samples from 3 to 2 could benefit TB control program and potentially increase case-detection in resource-poor settings for several reasons like decreasing the burden and improving quality of service, reduction in the number of smears requiring rechecking in external quality assessment schemes, time saving, and reduction in costs.

Diagnostic incremental yield

The present study showed that 1 st sputum smear sample alone could detect overall 91.8% cases, which is consistent with the studies by Cascina et al,[16] and Mathew et al[17] It was further observed that First two sputum smears: Could detect on an average 96% cases. Similar results were found in a study by Wu et al,[18] Addition of 3 rd sputum smear sample to first 2 smear samples increased the diagnostic yield by only 3.8% in our study. Wu et al,[18] showed increase in diagnostic yield of 3.2% by adding 3 rd smear to first 2, and Yilmaz et al,[19] observed the additional diagnostic yield by 3 rd smear to be 4.2%.

Thus, it is evident that not much added advantage in the case yield is obtained while adding 3 rd sputum sample, and the difference in the percentage increase of 3 rd sample in chest symptomatic of varying cough duration was almost similar. Hence, we also conclude that 2 sputum specimens can be safely recommended for confirmation of AFB positivity from chest symptomatic.

Comparison of different combinations of two smears: (I st spot and morning, I st and 2 nd spot, morning and 2 nd spot).

Gopi et al, observed that examination of 2 specimens- first spot and early morning spots [2 visits] or early morning and 2 nd spot specimens [single visit] yield the highest number of cases. [20] However, in our study, we found the highest incremental increase by using 2 spots together, while as with the other 2 combinations (I st spot and morning, morning and 2 nd spot), the incremental increase was less. The remarkably high diagnostic value of the 2 spot smears combination is surprising. The addition of morning specimen to either first spot or 2 nd spot gives surprisingly low incremental yield. The scenario many reflect poor instructions for specimens collected at home or very good instructions for the 2 spot specimens and might be also due to improved cough technique by the time of the 3 rd specimen. Thus, our findings although corroborate with a recent systematic review, which suggested that reducing the recommended number of specimens from 3 to 2 could also potentially increase case detection by improving the quality of examination of first and 2 nd specimens. [15] However, on basis of our findings, we suggest use of 2 spot specimens rather than I and II but needs further investigations.


The present study has shown that just 2 sputum samples are adequate enough to result in a comparable overall yield as is seen while using 3 sputum samples for screening in a cough symptomatic. This can be one way of improving efficient use of scarce resources i.e., by reducing the number of sputum examinations ordered per patient. Surprisingly, our study showed that using of 2 spot specimens can yield better than any other possible permutation of samples used that needs further evaluation.



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Source of Support: None, Conflict of Interest: None


DOI: 10.4103/1755-6783.102035


[Table 1], [Table 2], [Table 3]

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