|Year : 2021 | Volume
| Issue : 3 | Page : 145-150
Study of quality of sputum being submitted for smear examination
Maneesha Regati1, E Srikanth2, Karanam Gowrinath3
1 Department of Geriatric Medicine, Amrita Institute of Medical Sciences, Kochi, Kerala, India
2 Department of Pulmonary Medicine, Narayana Medical College, Nellore, Andhra Pradesh, India
3 Department of Pulmonary Medicine, Apollo Specialty Hospitals, Nellore, Andhra Pradesh, India
|Date of Submission||01-Apr-2021|
|Date of Acceptance||26-May-2021|
|Date of Web Publication||13-Sep-2021|
Consultant Pulmonologist, Department of Pulmonary Medicine, Apollo Specialty Hospitals, # 16/111/1133, Muttukur Road, Pinakini Nagar, Nellore 524 004, Andhra Pradesh
Source of Support: None, Conflict of Interest: None
Background: One of the key factors influencing the results of sputum smear microscopy is the quality of sputum being submitted.
Methods: This is a prospective study of quality of sputum specimens being submitted for smear examination under Revised National Tuberculosis Control Programme and its impact on diagnostic yield of sputum smear microscopy. Based on sputum Gram's staining results, the submitted specimen was classified as 'sputum' and 'saliva'.
Results: During the period 21 April to 20 June 2012, 238 sputum specimens submitted for evaluation were studied. Of these, 196 (82%) specimens were submitted for diagnosis and 42 (18%) specimens were obtained during follow-up evaluation. Overall, 42/238 (17.6%) specimens were in fact 'saliva' and not sputum. Among specimens submitted for diagnosis (n = 196), 28/167 (16.8%) specimens reported as 'sputum acid-fast bacilli (AFB) negative' were in fact saliva. Among the specimens submitted on follow-up examination (n = 42), 14 of the 38 (36.8%) specimens reported as 'sputum AFB negative' were actually saliva. A significantly higher proportion of sputum smears submitted on follow-up examination were actually 'saliva' as compared to specimens submitted for diagnosis (14/42 [33.3%] vs. 28/196 [14.3%], χ2 = 6.738; P = 0.014). None of the saliva samples were reported to be 'sputum positive'.
Conclusions: The fact that a significant proportion of specimens that are reported smear negative are in fact saliva and not sputum highlights the importance of sputum quality in smear microscopy results. Follow-up specimens are more likely to be saliva possibly because directly observed therapy (DOTS) treatment would have resulted in clinical improvements and patients may not be producing significant sputum anymore.
Keywords: Acid-fast bacilli, diagnosis, saliva, sputum, tuberculosis
|How to cite this article:|
Regati M, Srikanth E, Gowrinath K. Study of quality of sputum being submitted for smear examination. J Clin Sci Res 2021;10:145-50
| Introduction|| |
The essential component of tuberculosis (TB) control is the detection of the most infectious cases of TB i.e., patients who are sputum smear-positive pulmonary cases. Identifying the sources of infection in the community by detecting individuals who are discharging large numbers of tubercle bacilli facilitates institution of specific anti-TB treatment. Sputum is the product of secretions from mucous glands and goblet cells in the bronchial wall. It is composed of 95% water and 5% solids. Sputum smear microscopy is one of the key components of Revised National TB Control Programme (RNTCP) for case finding. The RNTCP aims at achieving 70% case detection rate by utilising sputum smear microscopy as the tool. Under the RNTCP, a designated microscopy centre (DMC) has been established to cater the need for approximately 100,000 population (50,000 for tribal, difficult and mountainous areas). Each DMC is staffed by a RNTCP trained laboratory technician. Furthermore, DMCs have been established in every medical college in the country both in the government and private sectors to provide quality assured diagnostic services under the programme. To ensure quality a comprehensive sputum smear microscopy, quality assurance protocol that includes on site evaluation, panel testing and random blinded rechecking of routine slides has been developed and is being practised. This system has ensured that sputum smear examination carried out anywhere in the country at an RNTCP DMC is quality assured and reliable.
Critical review of published literature reveals that only about 50% of patients with pulmonary TB produce sputum adequate enough for smear examination. One of the key factors influencing the results of sputum smear microscopy is the quality of sputum being submitted. While a good quality sputum examined will reduce chances of false-negative results, the inadequate sputum sample will be falsely negative and repeat samples will have to be submitted resulting in delay in diagnosis.
While sputum smear microscopy is a reliable, reproducible and verifiable tool for the diagnosis of pulmonary TB, sparse published data are available assessing the performance of DMCs in real time under field conditions. The present study was, therefore, conceived to study the quality of sputum being submitted for smear examination under RNTCP in a private and government medical college DMCs under the RNTCP.
| Material and Methods|| |
The study was conducted in the DMCs at Department of Pulmonary Medicine, Narayana Medical College (NMC), Nellore, a private Medical College and Department of Medicine, Sri Venkateswara Institute of Medical Sciences (SVIMS), Tirupati, a Government Medical College in Andhra Pradesh during the period April 2012–June 2012. The Institutional Ethics Committee approval from NMC, Nellore, and SVIMS, Tirupati, was taken before start of the study. Informed consent was obtained from all patients participating in the study.
Consecutive patients above 18 years age of both genders referred for sputum smear examination to the DMC under RNTCP located at NMC, Nellore, and SVIMS, Tirupati, were included in the study. Patients unwilling to participate in the study were excluded from the study.
As per the RNTCP algorithm [Figure 1] in all the participants, two sputum specimens were collected over 2 consecutive days. Sputum was obtained after giving standard instructions and under supervision by the investigator/laboratory technician/nurse. The sputum was collected in an open place meant for this purpose. The patients were asked to rinse their mouth clear of food particles. They were instructed to inhale deeply (2–3 times) and produce sputum by deep coughing which is spit into the pre-labelled container without spilling. Patients not able to expectorate with deep breathing were demonstrated the technique of how they should place their palms on the waist, squat or sit and continue deep breathing again. Tapping or thumping of the back was also utilised to encourage expectoration.
Of the two sputum specimens, one was collected on the spot and the other was an early morning specimen collected at home by the patient. Ziehl–Neelsen method staining was used for staining the sputum smears thus obtained. Pulmonary TB was diagnosed if at least one of the two sputum smears tested positive for acid-fast bacilli (AFB).
As per the RNTCP Laboratory Manual, gross visual examination of the submitted sputum specimen was carried out to categorise it as 'sputum' or 'saliva'. The visual appearance of the sputum sample was recorded on the laboratory form in the space provided. A 'good sputum sample' was thick (semi-solid), coughed outdeeply from the lungs, purulent (yellowish mucus) and sufficient in amount. A poor quality sputum sample was one that contained only saliva (watery) or nasal mucus and was small in quantity (<2 mL). If the specimen was suggestive of being saliva, the patient was asked to produce another sputum specimen.
As per the RNTCP standard operating procedure, the sputum was spread over the central area of the slide using a continuous rotatory movement of the applicator. The size of sputum was kept at about 20 mm × 10 mm as per recommendations. The dried sputum was fixed by holding with a forceps and passing it over the flame for 5 times for about 4 s.
Two smears were prepared from each of the sputum obtained from all the participants – one for staining by the Ziehl–Neelsen method to detect AFB and the other for sputum Gram's staining to assess quality. Sputum specimens were processed and examined on site within 2–3 h of collection, to avoid cellular changes in sputum. These details were recorded in a case record form.
Each Gram stained sputum specimen was examined for 3–5 min under low power field (LPF) (×100) magnification for the number of squamous epithelial cells per LPF and for the number of neutrophils per LPF. The AFB screening was blinded to the results of visual and microscopic quality assessment. On sputum Gram's staining, specimens with <10 squamous epithelial cells per LPF and more than 25 neutrophils per LPF with the presence of bronchial epithelial cells and alveolar macrophages were categorised as 'sputum'. Specimens not fulfilling these criteria were categorised as 'saliva'. Sputum smear grading was recorded as per the RNTCP recording format as 'scanty', '1+','2+'and '3+' [Table 1].
Data were recorded on a pre-designed pro forma and managed using Microsoft Excel 2007 (Microsoft Corp, Redmond, WA). All the entries were double checked for any possible error. Continuous variables are summarised as mean ± standard deviation; categorical variables are summarised as percentages. The proportion of sputum samples that are not sputum but 'saliva' will be expressed as percentage. This number would reflect the false-negative sputum smear result. The yield of sputum smear results as per the sputum quality in specimens submitted for 'diagnosis'; sputum quality in specimens submitted for diagnosis and follow-up examination and by gender was studied by Chi-square test with Yates correction. Statistical software IBM SPSS, Version 20 (IBM SPSS Statistics, Somers NY, USA) was used for statistical analysis.
| Results|| |
During the period 21 April–20 June 2012, a total of 238 sputum specimens submitted for evaluation at the NMCH, Nellore (n = 100; 42%) and SVIMS, Tirupati (n = 138; 58%) were studied. Their median (interquartile range) age was 46.5 (34–57.5) years. There were 174 (73.1%) males. Of these, 196 (82%) specimens were submitted for diagnosis and 42 (18%) specimens were obtained during follow-up evaluation. Overall, 33 of the 238 sputum specimens (14%) tested positive for AFB.
Based on sputum Gram's staining results, the submitted specimen was classified as 'sputum' and 'saliva'. Overall, 42/238 (17.6%) specimens were in fact 'saliva' and not sputum. This included 28/196 (14.3%) specimens submitted for diagnosis; and 14/42 (33.3%) specimens submitted on follow-up.
Sputum quality in 196 specimens submitted for 'diagnosis' and on follow-up examination is shown in [Table 2]a and [Table 2]b. Among specimens submitted for diagnosis (n = 196), a statistically significant proportion of specimens reported to have a 'negative AFB smear' (28/167; 16.8%) were in fact saliva (P = 0.017). However, similar findings were not observed among specimens submitted on follow-up examination.
There were no false-positive results; i.e., none of the saliva samples were reported to be sputum positive. A significantly higher proportion of sputum smears submitted on follow-up (14/42; 33.3%) examination were actually saliva as compared to specimens submitted for diagnosis (28/196; 14.3%) (P = 0.014). Among males 32/174 (18.4%) of the sputum specimens were saliva compared to 10/64 (15.6%) in females, there was no gender difference in specimens in the quality of sputum (P = 0.562).
| Discussion|| |
In many high TB burden countries including India, pulmonary TB is diagnosed by TB microscopy of stained sputum smears. To be consistently detected by sputum smear microscopy, the AFB must be present in concentrations more than 100,000/mL. Sputum smear microscopy is considered to be a reliable, reproducible low cost method of detecting Mycobacterium tuberculosis. This is particularly true when adequate sputum specimens are carefully examined by well-trained microscopists. Poor quality of sputum submitted for testing has been recognised as an important contributing factor for smear negativity. The main consequences of false-negative sputum smear results include delay in starting of specific anti-TB treatment resulting in morbidity, spread of TB in the community and death. Furthermore, patients with a false-negative sputum smear would also be subject to unnecessary, costly investigations.
Several factors influence the sputum quality. In a study conducted at the federal TB Centre in Rawalpindi, Pakistan, lower smear positivity in women as compared to men was found to be mainly a function of poor-quality specimen submission. By provision of brief instructions, smear positivity in women was increased substantially. In another study from laboratories in Moldova, Mongolia, Uganda and Zimbabwe, women and persons at extremes of age were found to have lower sputum smear positivity and this was related to the quality of sputum specimens submitted. Thus, it is important to ensure that a good quality sputum specimen is submitted for the diagnosis of TB. In a randomised controlled trial (n = 174) conducted in an urban TB clinic in Indonesia in patients with suspected pulmonary TB, the contribution of patient education to the diagnostic yield of sputum microscopy was evaluated. Sputum samples from the intervention group were of better quality, and TB was diagnosed in 50.6% of patients who received additional counselling compared with 35.5% of patients in the control group following routine diagnostic procedure.
In another study from Pakistan, 3055 patients with suspected TB were randomly assigned over 3 months to receive sputum-submission guidance before specimen submission or to submit specimens without specific guidance. Significantly more individuals in the intervention group tested smear positive than did those in the control group (12.1% vs. 8.8%, P = 0.003).
The median age of the pulmonary TB suspects included in the present study was 46.5 years. This finding is in concordance with the observation that TB affects persons in their economically most productive years. Men outnumbered women in the present study. Whether this reflects a true gender difference or is merely due to the fact that more men utilise hospital services in India than women needs to be evaluated further.
Careful visual assessment by trained staff has been found to be useful to categorise sputum specimens as adequate in other studies. Under the RNTCP, specimens submitted to the DMC are visually screened to categorise them as sputum or saliva, and only those specimens classified as sputum are processed further to test for the presence of AFB. The same protocol was followed at both the DMCs in the present study. Yet, a significant proportion of specimens submitted for diagnosis (28/196 [14.3%]) was found to be 'saliva' and not sputum on Gram's staining in the present study. Even though the patients had apparently been educated regarding providing an optimum sputum specimen and careful visual inspection of the specimen submitted was carried out before considering it for AFB staining, 14.3% collected as adequate sputum turned out to be saliva on Gram's staining. A statistically significant proportion of specimens reported to have a 'negative AFB smear' (28/167; 16.8%) were in fact saliva (P = 0.017) [Table 1]. However, there were no false-positive results and none of the specimens found to be saliva were reported to be AFB positive. These observations suggest that inadequate sputum is an important contributor to sputum smear-negative results.
In the present study, a significantly higher proportion of sputum smears submitted on follow-up examination were actually saliva as compared to specimens submitted for diagnosis (14/42 [33.3%] vs. 28/196 [14.3%] [P = 0.014]) [Table 2]. This is likely to be due to the fact that DOTS treatment would have resulted in an improvement in the patients' clinical condition, consequent to which, the patients may not be producing adequate sputum.
Unlike the observations in another study, where female gender was observed to affect the quality of sputum submitted for smear microscopy, no gender difference was observed in the quality of sputum in the present study. Several factors such as the educational status and motivation status of the patients, ability of the laboratory staff to effectively communicate and educate the patients regarding the provision of an appropriate sputum specimen could have been the reasons for this observation.
The fact that a significant proportion of specimens that are reported smear negative are in fact saliva and not sputum highlights the importance of sputum quality in smear microscopy results. Follow-up specimens are more likely to be saliva possibly because DOTS treatment would have resulted in clinical improvements and patients may not be producing significant sputum any more. The results from the present study highlight the importance of focussed counselling to the patient as to how to provide a good quality sputum sample. This will facilitate increased diagnostic yield and reduce false-negative reporting.
Focussed counselling by clinicians reinforced by dedicated efforts by laboratory technicians regarding proper methods of sputum collection can be help in obtaining appropriate sputum specimens for testing. Ensuring optimum workload for laboratory staff so that adequate time is spent on assessing the quality of sputum specimens submitted before AFB staining and enhancing the motivation of laboratory technicians also have potential for enhancing the yield of sputum smear examination in the diagnosis of TB.
The study was conducted under Indian Council of Medical Research (ICMR), STS2012 grant (Reference ID: 2012-02396) awarded to Maneesha R. A part of this work was presented in the 41st Annual Conference of Andhra Pradesh Chapter of the Association of Physicians of India (APAPICON 2013), at Mahaboob Nagar on 10–11, August 2013 (Maneesha R, Srikanth E, Gowrinath K, Mohan A. Study of quality of sputum being submitted for smear examination under RNTCP. J Clin Sci Res 2013; 2 (Suppl 2):S17).
The study was carried out by Maneesha Regati under the Indian Council of Medical Research (ICMR), Short-term Research Studentship STS2012 grant: Reference ID: 2012-02396.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Table 1], [Table 2]