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A comparative study of sputum induction in suspected pulmonary tuberculosis.


Tuberculosis (TB) remains a major global health problem and causes ill-health among millions of people each year and ranks as the second leading cause of death from infectious disease worldwide. Though India is the second most populous country in the world, it has more new TB cases annually than any other country. In 2011, out of the estimated global annual incidence of 9 million TB cases, 2.3 million were estimated to have occurred in India. As per the World Health Organization (WHO) global TB report 2010, India is 17th among 22 high burden countries in terms of TB incidence rate. By the end of 2015, The Revised National Tuberculosis Control Programme (RNTCP) aims to achieve detection and treatment of at least 90% of estimated TB cases in the community, including HIV associated TB. Delayed or inappropriate diagnosis leads to increased load of disease in the community (Yazdani et al., 2002; Long et al., 1999). Quality sputum microscopy is pre-eminent in the diagnosis of pulmonary TB under RNTCP.

The WHO recommends the detection of Acid Fast Bacilli (AFB) in respiratory specimens as the initial approach to the diagnosis of TB. However, this method has low sensitivity and a little value in patients who cannot produce sputum spontaneously.

A fairly good number of cases, strongly suspected of pulmonary TB are not confirmed bacteriologically. One reason for this may be related to the difficulties of obtaining adequate quantity of sputum specimen from TB suspects either due to dry cough or inadequate or scanty sputum (<2 ml) (Anderson et al., 1995). Thus sputum remains negative in about 25-50% of all cases of active pulmonary TB (Menzies, 2003). The aim of sputum induction is to collect adequate sample of secretions from the lower airways in subjects who do not produce sputum spontaneously. In view of this it is of utmost importance to find a best possible method to obtain secretions from the lower airways in subjects who do not produce sputum spontaneously for early diagnosis. The value of sputum induction and analysis is not restricted to the recognition of sputum eosinophilia but also may be used to direct novel anti-neutrophilic therapies. Thus, it is time for sputum induction to move from the research laboratory to the clinic (Gupta and Seema, 2005).

Materials and Methods

Study sample--Three groups, each consisting of 40 TB suspected patients.

Study period--One year.

Ethical committee clearance was taken before starting the study.

Study protocol

Sputum induction was compared between nebulized levosalbutamol, 0.9% normal saline, and 3% hypertonic saline in chest symptomatics with dry cough or scanty sputum suspected of pulmonary TB. All the patients were randomly allotted into the three groups after meeting the inclusion criteria. After taking a detailed history and doing a clinical examination the patients were given levosalbutamol, normal saline, and hypertonic saline depending on group allotted to him/her. Each patient was given nebulization and spot samples followed by two samples on two consecutive days were collected. The nebulization was given by Pulmo-Aide compressor based nebulizer. The protocol for sputum induction was followed as used by Gupta and Seema (2005). The induced sputa were submitted for Ziehl-Neelsen (Z-N) staining as per RNTCP guidelines.

Study design

The present study was conducted with the aim to compare sputum induction in chest symptomatics with dry cough or scanty sputum (<2 ml saliva) with nebulized (a) 0.9% normal saline, (b) 3% hypertonic saline, and (c) levosalbutamol (1.26 mg).

Inclusion criteria

Age 6-65 years. Persistent cough for 3 weeks or more, with or without one or more of the following symptoms are suggestives of pulmonary TB--weight loss, tiredness, fever particularly with rise of temperature in the evening, night sweats, chest pain, loss of appetite, coughing up of blood, and presence of dry cough or scanty sputum (<2 ml saliva).

Exclusion criteria

Chest symptomatics who expectorate mucoid sputum (>2 ml). Patients who have taken anti-tuberculosis treatment, previously. Presence of other causes of hemoptysis, chest pain, and dyspnea like cardiac, renal, or chronic infective conditions of lung other than pulmonary TB.

A detailed history was taken and thorough clinical examination was done as per the proforma (Annexure I). The questionnaire used in this study was framed to collect information on respiratory symptoms and identifying patients who meet the definition of "Suspected Pulmonary TB" with dry cough and scanty sputum. The questionnaire was adopted from Francis J. Curry National Tuberculosis Center (Annexure II).

Nebulization as a method of sputum induction was chosen as it is safe and can be used in children, postprandial, and as an Outpatient Department (OPD) procedure which is inexpensive and less time consuming.

Each patient was given nebulization for two consecutive days and three sputa samples collected were:

a) First nebulized spot sample.

b) Next day early morning home collection sample.

c) Second nebulized spot sample.

Sputum may sometimes not be produced for 30 min to 24 h later, depending on the hydration level of the patient. Therefore patient was sent home with sample cups and direction for first sputum of next day morning.

Sputum induction was performed on all patients selected by the above mentioned criteria after a verbal informed consent. A brief description of the procedure was given to patients.

It was performed in a well-ventilated room with windows open and before giving nebulization the patients were asked to gargle with saline to clear debris from the mouth and oropharynx. All the subjects were asked to inhale mist with deep breaths delivered by the nebulizer and cough vigorously, if spontaneous coughing does not occur. Inhalation was continued until an adequate amount of sputum sample (minimum 5-10 ml) was obtained or for a maximum period of 30 min. The nostrils of the subjects were closed with clip to prevent nasal inhalation. The inhalation procedure was interrupted every 5-10 min, so that patient could expectorate sputum. The patient was observed all the time during the procedure for signs of respiratory distress. The sputum sample was collected into a clean sterile calibrated container. The container was labeled on its side. The nebulizer equipment was decontaminated after each session by thorough washing and soaking the reservoir, tubing and mask in glutaraldehyde overnight. The induced sputum samples were submitted to the laboratory. All the sputum specimens were examined for AFB by Z-N staining in the laboratory as per RNTCP guidelines.

Z-N method of staining sputum smears

Briefly the method consists of a good smear made from the induced sputum evenly distributed (3 x 2) using a broom sticks. Smear is air dried and fixed by heating briefly for 3-5 times for 3-4 sec each. It is stained with 1% carbol fuchsin and heated until steam starts rising, allowed to stand for 5 min and then rinsed with water. At this point the sputum smear on the slide looks red in color. The smear is decolorized with 25% sulphuric acid, rinsed with water and counter stained with 0.1% methylene blue, and then gently rinsed with water before drying for examination under microscope for detecting AFB and grading (Table 1).


In the levosalbutamol group, sputum induction was successful in 90% of cases (Table 2). 60% of the cases were positive with first spot nebulization, the carried-over effect of the first spot nebulization was 50%, and the total cumulative effect was seen in 47.5% of cases (Table 3).

In the 0.9% normal saline group, sputum induction was successful in 50% of cases (Table 2). 25% of cases were positive with first spot nebulization, the carried-over effect of the first spot nebulization was 25%, and the total cumulative effect was only 20% (Table 4).

In the 3% hypertonic saline group, sputum induction was successful in 97.5% of cases (Table 2). 75% of cases were positive with first spot nebulization, the carried-over effect of the first nebulization was 67.5%, and the total cumulative effect was 60% (Table 5).

In the levosalbutamol group, 88.2% males and 91.3% females induced sputum. In the 0.9% normal saline group, 59.1% males and 38.9% females induced sputum and in the 3% hypertonic saline group, 100% males and 94.1% females induced sputum.

The levosalbutamol group showed 40% (1 +), 17.5% (2+), and 20% (3+). The 0.9% normal saline group showed 22.5% (1+), 12.5% (2+), and 7.5% (3+). The 3% hypertonic saline group showed 57.5% (1+), 12.5% (2+), and 20% (3+) (Table 6).

The results of levosalbutamol group showed 77.5% positivity, the 0.9% normal saline group showed 42.5% positivity, and the 3% hypertonic saline group showed 90% positivity (Table 7).


In recent years, sputum induction and its subsequent processing has been refined as a non-invasive research tool providing important information about inflammatory events in the lower airways, and it has been used for studying various diseases (Li et al., 1999). The development of standardized methods for sputum induction has improved the quality and reproducibility of sputum samples (Brightling, 2006).

The WHO recommends the detection of AFB in respiratory specimens as the initial approach to the diagnosis of TB (WHO, 1993). However, this method has low sensitivity and has little value in patients who cannot produce sputum spontaneously. In HIV-seronegative patients with suspected TB who do not produce sputum or have negative AFB smears, diagnosis of TB becomes difficult and in most cases they are treated empirically which may result in resistance, unnecessary cost, and toxicity especially in resource restricted countries. Moreover, HIV-seropositive patients who do not produce sputum undergo expensive and more invasive procedures (Conde et al., 2000). Thus, it is time for sputum induction to move from research laboratory to the clinic. There are many methods to improve the bacteriological diagnosis of smear negative pulmonary TB i.e., bronchoscopy, laryngeal swabs, and gastric and trans tracheal aspirates. These diagnostic tools have their own limitations (McWilliams et al., 2002).

Directly observed treatment, shortcourse (DOTS) strategy prioritizes the identification and cure of infectious cases i.e., those patients with sputum smear 'positive', pulmonary TB and considers sputum smear microscopy as the primary tool for diagnosing TB as it is more specific and has less inter-reader variability than the X-ray. It also categorizes the TB cases on the basis of sputum smear microscopy and history.

Sputum induction by various inhalants in patients with suspected pulmonary TB included agents like 10% normal saline, sulfur dioxide, pancreatic dornase, acetyl cysteine, and sterile water (Yue and Cohen, 1967). The present study was carried out with the aim of comparing different inhalants to induce good quality and quantity of sputum. Levosalbutamol ((32-agonist), 0.9% normal saline, and 3% hypertonic saline were used in a randomized way in chest symptomatics with dry cough or scanty sputum (<2 ml) suspected of having pulmonary TB. Sputum induction was successful in 90% of cases (Table 2). Yazdani et al. (2002) found that oral salbutamol produced adequate sputum and 88% among the dry cough/scanty sputum had positive smears. In the present study, 77.5% had positive smears. These findings show relevance to the above quoted studies.

[beta]2-agonists enhance mucociliary transport in healthy subjects (Sadoul et al., 1981). Sputum induction by [beta]2-agonist is a useful method to improve quality microscopy (Yazdani et al., 2002). The concentration of the saline for sputum induction has ranged from 0.9% to 7% (Bacci et al., 1996). The present study used 0.9% normal saline and 3% hypertonic saline in the chest symptomatics. 3% hypertonic saline was used as it is safe in young children, postprandial, and an OPD procedure with no to minimal adverse effects like bronchoconstriction (Gupta and Seema, 2005). In the present study, 3% hypertonic saline induced sputum in 97.5% of cases as compared to 50% of cases with the normal saline group (Table 2). These findings are also in agreement with the study done by Gupta and Seema (2005).

Analysis of the three inhalants with respect to sex distribution revealed that maximum sputum induction in males (100%) and females (94.1%) was shown by the hypertonic saline group. In the levosalbutamol group 91.3% of females induced sputum. This result correlates with the study done by Long et al. (1999) where they found more females to be smear positive after oral administration of salbutamol.

Analyzing the overall results with smear grading the qualitative yield in the levosalbutamol group (20%, 3 +ve) was comparable to the hypertonic saline group. Thus sputum induction by 32-agonists may be a useful method to improve quality microscopy especially in chest symptomatics with co-morbid conditions like asthma where induction with hypertonic saline may not be tolerable. Also the easy availability of levosalbutamol in sterile respules makes it a better choice for sputum induction in rural/urban set up. The optimum dose for sputum induction needs to be standardized by further studies.


It is concluded from this study that sputum induction with 3% hypertonic saline is a safe, simple, and cost effective method of obtaining adequate amount of quality sputum in patients with dry cough or scanty sputum and this can be used as an adjunct to improve sputum smear positivity and hence optimize treatment in suspected pulmonary TB. The results indicate that 3% hypertonic saline induces more sputum, yielding more positivity (90%) than levosalbutamol. The effect of second intervention (82.5% positivity) and cumulative effect (60% positivity) was higher in 3% hypertonic saline group.

With regard to gradation of sputum there is no difference between levosalbutamol and hypertonic saline.
Annexure I
Case sheet proforma


(A) Presenting complaints
  Chest pain

(B) History
  --Grade/type chills/rigor
  --Evening rise of temperature
Loss of appetite/weight
Night sweats
Hoarseness of voice
Pedal edema
Puffiness of face
Abdominal distension
Right hypochondrial pain
Bleeding disorders

(C) Past history
    If present/when?
    Duration of treatment?
    Complete/incomplete treatment?
    Cause of incomplete treatment?
  Hypertension/diabetes mellitus
  Contact with case of TB

(D) Family history

(E) Personal history

(F) Occupational history

(G) Treatment history

(H) Vital signs
  Blood pressure

(I) General examination

  Comfortable/dyspnoeic at rest
  Pedal/sacral edema
  Lymphadenopathy--scalene node/cervical node
  Neck-Thyroid swelling/tracheal tug

(J) Examination of the respiratory system
  (1) Upper respiratory tract
  (2) Respiratory system proper
    (a) Inspection
    (b) Palpation
    (c) Percussion
    (d) Ausculatation

(K) Examination of other systems

(L) Sputum induction
  (a) 1st spot nebulization
  (b) Early morning home collection
  (c) 2nd spot nebulization

Annexure II

1. Do you have a cough that has
  lasted longer than 3 weeks?                    Yes/No
2. Have you lost your appetite?                  Yes/No
3. Have you lost weight without dieting?         Yes/No
4. Have you had fever,
  chills, or night sweats?                       Yes/No
5. Have you coughed up mucous or blood?          Yes/No
6. Have you been feeling very tired?             Yes/No
7. Have you ever had a positive TB skin test?    Yes/No
8. Have you ever hand an abnormal chest X-ray?   Yes/No
9. Have you recently had the mucous
  you coughed up tested for TB?                  Yes/No
10. Have you ever been told you had TB?          Yes/No
11. Have you ever taken medicine for TB?         Yes/No
12. Have you ever lived with or had
close contact with Someone who had TB?           Yes/No

Ethical Approval

Institutional ethics committee clearance was taken before starting this research project.

Conflict of Interests

The authors declare that they have no conflict of interest.

Authors' Contributions

All authors contributed equally to this study.


We wish to thank the Management of Mahavir Hospital and Research Centre for the facilities provided.


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Bacci E, Cianchetti S, Paggiaro PL, Carnevali S, Bancalari L, Dente FL, et al., 1996. Comparison between hypertonic and isotonic saline-induced sputum in the evaluation of airway inflammation in subjects with moderate asthma. Clinical and Experimental Allergy, 26(12): 1395-1400.

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Conde MB, Soares SL, Mello FC, Rezende VM, Almeida LL, Reingold AL, et al., 2000. Comparison of sputum induction with f ber-opitc bronchoscopy in the diagnosis of tuberculosis: Experience at an acquired immune deficiency syndrome reference center in Rio de Janeiro, Brazil. American Journal of Respiratory and Critical Care Medicine, 162(6): 2238-2240.

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Pitchenik AE, Ganjei P Torres A, Evans DA, Rubin E, Baier H, 1986. Sputum examination for the diagnosis of Pneumocystis carinii pneumonia in the acquired immunodeficiency syndrome. The American Review of Repsiratory Disease, 133(2): 226-229.

Sadoul P Puchelle E, Zahm JM, Jacquot J, Aug F, Polu JM, 1981. Effect of terbutaline on mucociliary transport and sputum properties in chronic bronchitis. Chest, 80(Suppl. 6): 885-889.

Scheicher ME, Terra Filho J, Vianna EO, 2003. Sputum induction: Review of literature and proposal for a protocol. Sao Paulo Medical Journal, 121(5): 213-219.

World Health Organization, 1993. Treatment of Tuberculosis: Guidelines for National Programmes. Third Edition, Geneva.

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Mohammed SS Ansari *, Mohammed Hidayath, Waseem Kawoosa, Arif Ghouse Department of Pulmonary and Critical Care Medicine, Mahavir Hospital and Research Centre, 10-1-1, Mahavir Marg, AC Guards, Hyderabad 500 004, Andhra Pradesh, India.

* Correspondence:

Accepted: 27th Jul 2013; Published: 5th Aug 2013
Table 1: Grading of AFB smear.

Examination              Result      Grading      No. of fields
                                                  to be examined

More than 10 AFB per    Positive       3+              20
  oil immersion field
1-10 AFB per oil        Positive       2+              50
  immersion field
10-99 AFB per 100       Positive       1+              100
  oil immersion field
1-9 AFB per 100          Scanty    Record exact        200
  oil immersion field              number seen
No AFB per 100          Negative        --             100
  oil immersion field

Table 2: Sputum induction.

Nebulisation            Induced       Not        Total

Levosalbutamol          36 (90%)    4 (10%)    40 (100%)
0.9% normal saline      20 (50%)    20 (50%)   40 (100%)
3% hypertonic saline   39 (97.5%)   1 (2.5%)   40 (100%)

Table 3: Cumulative effects of levosalbutamol.

1st        Home      2nd     Positive     Negative      Total
spot    collection   spot

+ve        +ve       +ve    19 (47.5%)   21 (52.5%)   40 (100%)
+ve        -ve       +ve     3 (7.5%)    37 (92.5%)   40 (100%)
-ve        +ve       -ve      0 (0%)     40 (100%)    40 (100%)
-ve        +ve       +ve     6 (15%)      34 (85%)    40 (100%)
-ve        -ve       +ve     1 (2.5%)    39 (97.5%)   40 (100%)

Table 4: Cumulative effect of normal saline.

1st        Home       2nd    Positive     Negative      Total
spot    collection    spot

+ve         +ve       +ve     8 (20%)     32 (80%)    40 (100%)
+ve         -ve       +ve     0 (0%)     40 (100%)    40 (100%)
-ve         +ve       -ve     0 (0%)     40 (100%)    40 (100%)
-ve         +ve       +ve    7 (17.5%)   33 (82.5%)   40 (100%)
-ve         -ve       +ve     0 (0%)     40 (100%)    40 (100%)

Table 5: Cumulative effect of hypertonic saline.

1st       Home      2nd    Positive    Negative      Total
spot   collection   spot

+ve       +ve       +ve    24 (60%)    16 (40%)    40 (100%)
+ve       -ve       +ve    3 (7.5%)   37 (92.5%)   40 (100%)
-ve       +ve       -ve     0 (0%)    40 (100%)    40 (100%)
-ve       +ve       +ve    6 (15%)     34 (85%)    40 (100%)
-ve       -ve       +ve     0 (0%)    40 (100%)    40 (100%)

Table 6: Comparision of overall results with smear grading.

Group               1 +          2+          3+

Levosalbutamol    16 (40%)    7 (17.5%)   8 (20%)
0.9% normal      9 (22.5%)    5 (12.5%)   3 (7.5%)
3% hypertonic    23 (57.5%)   5 (12.5%)   8 (20%)

Group             Negative      Total

Levosalbutamol   9 (22.5%)    40 (100%)
0.9% normal      23 (57.5%)   40 (100%)
3% hypertonic     4 (11.1)    40 (100%)

Table 7: Overall results of positivity.

Group                   Positive     Negative      Total

Levosalbutamol         31 (77.5%)   9 (22.5%)    40 (100%)
0.9% normal saline     17 (42.5%)   23 (57.5%)   40 (100%)
3% hypertonic saline    36 (90%)     4 (10%)     40 (100%)
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Article Details
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Title Annotation:Research Article
Author:Ansari, Mohammed S.S.; Hidayath, Mohammed; Kawoosa, Waseem; Ghouse, Arif
Publication:Biology and Medicine
Article Type:Report
Geographic Code:1USA
Date:Jan 1, 2013
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