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Role of mycobacterium culture in the diagnosis of smear negative pulmonary tuberculosis suspects attending tertiary care setting.

Byline: Aamir Nazir, Iffat Shabbir and Tayyaba Rahat

Abstract Background: Sputum smear testing for acid fast bacilli is the gold standard for the diagnosis of tuberculosis. The role of culture in cases that are negative on sputum smear testing is being emphasized for early diagnosis of disease and prompt initiation of treatment.

Objectives: To correlate the role of culture in diagnosing smear-negative pulmonary tuberculosis suspects.

Study type, settings and duration: Descriptive study carried out in chest clinic of Sir Ganga Ram Hospital, Lahore in collaboration with Institute of Public Health Lahore from January 2012 to June 2012.

Materials and Methods: Adults over 15 year of either gender having symptoms and X-ray findings consistent with pulmonary tuberculosis were selected. The sputa of sputum smear negative cases were inoculated on Lowenstein-Jensen medium culture medium to isolate the organism and correlate the culture positivity and negativity in smear negative cases.

Results: A total of 88 sputa were negative for acid fast bacilli on smear which were subjected to culture on Lowenstein-Jensen medium. Of these 31(35%) grew acid fast bacilli and were thus confirmed positive by culture while rest 57 remained negative on culture. There was no significant difference in the symptoms and extent of lesions on X-ray between those who were culture positive or negative.

Conclusion: Sputum culture should be done for the diagnosis or exclusion of smear negative clinically suspected and X-ray positive cases of pulmonary tuberculosis.

Policy message: All clinically suspected and X-ray positive but sputum smear negative cases should undergo sputum culture for Mycobacterium tuberculosis on Lowenstein-Jensen medium. The facilities to culture the micro organism should be made available at district level by the tuberculosis control program.

Key words: Tuberculosis (TB), Mycobacterium tuberculosis (MTB), pulmonary tuberculosis (PTB), acid fast bacilli (AFB), Ziehl Neelsen staining (ZN), Lowenstein-Jensen medium (LJ).


Carly and correct diagnosis of tuberculosis is essential for an effective tuberculosis control program1. Tuberculosis is diagnosed on finding acid-fast bacilli (AFB) on direct microscopic examination of sputum smear but this is not a very sensitive technique2 and almost 50% cases with tuberculosis can present with negative results3,4.

DOTS strategy relies on sputum smear microscopy in pulmonary tuberculosis cases. In many cases despite clinical suspicion of pulmonary tuberculosis, the sputum smear is negative for AFB and clinicians are not sure whether to treat or not treat such cases and thus the diagnosis of pulmonary tuberculosis is missed. Clinicians use symptoms of fever, productive cough and weight loss to predict the risk of tuberculosis in the patients who have a smear-negative disease and due to the smaller mycobacterial burden in smear- negative-disease, these patients may have different clinical and radiographic findings5-9.

For confirming the diagnosis in sputum negative cases clinicians rely on chest X-ray or newer expensive diagnostic tests having poor sensitivity and specificity. In DOTS strategy, chest X-ray is discouraged for the diagnosis of tuberculosis and is used to diagnose smear-negative pulmonary tuberculosis suspects whose sputum is negative for AFB10. As the sensitivity of Z-N staining is low, therefore, using chest X-ray, a large proportion of cases would be over diagnosed11. Even when restricting chest X-ray for smear negative suspects the proportion of over diagnosis still remains high12.

Culture for Mycobacterium tuberculosis is the gold standard for the diagnosis of tuberculosis13 and this can be used in diagnosing smear negative and other difficult cases. This study was done to correlate the role of culture in diagnosing smear-negative pulmonary tuberculosis suspects.

Materials and Methods

This descriptive observational study was conducted in the outpatient's department of model Chest Clinic, Sir Ganga Ram Hospital Lahore and Punjab Tuberculosis Reference Laboratory, Institute of Public Health, Lahore from January 2012 to June 2012.

A total of 140 new pulmonary tuberculosis suspects aged 15 years and above, belonging to both gender and having symptoms and X-ray findings consistent with pulmonary tuberculosis were selected. Cases with previous history of tuberculosis and those currently receiving anti-tuberculosis treatment were excluded from the study. Informed written consent was taken from the patients. Demographic data, current symptoms, and relevant co-morbid conditions were collected using a questionnaire.

Both hospitals had a bio-safety level-2 cabinet where sputa were examined using ZN staining technique by a trained microscopist and were cross checked at the reference lab. The same sputum was processed for culture by digestion, decontamination and concentration following modified Petroff`'s method and inoculated on LJ culture media for six weeks to isolate the organisms. Readings were taken after 7 days for eight weeks. The isolates were identified by growth rate and colony morphology. Sputum specimen were refrigerated at 4-6oC in cases where processing was delayed. Standard strain of H 37 RV was used for the quality control.

All smear positive cases were excluded from the study while, remaining were given a course of broad spectrum antibiotic and were asked to repeat chest X-ray after two weeks. If the patients improved clinically and radiologically they were excluded from the study. Rest of the patients were given anti-tuberculosis treatment according to DOTS strategy.


Out of 140 patients, 49 were smear positive and 3 responded to broad spectrum antibiotics, therefore, these patients were excluded from the study. Sputum smear was negative in 88 patients and these comprised the study population. Out of 88 smear negative patients, 31(35.22%) showed growth of Mycobacterium on culture while, 57(64.77%) patients remained culture negative. Out of 31 culture positive patients, there were 14(45.16%) males and 17(54.83%) females. X-ray chest of 88 smear negative patients showed that 48.3% had minimal lesions and 32.2% had advanced lesions but the type of lesions did not show any significant difference between those who were culture positive and culture negative (Table-1).

Similarly the symptoms between these two groups also did not show any statistical difference (Table-2). As per protocol of DOTs strategy, 64.77% patients who were smear negative and their clinical and radiological pictures did not improve after antibiotic therapy, received a full course of anti tuberculosis treatment despite being negative culture, indicating over diagnosis and over treatment.

Table1: Comparison of X-ray lesions in smear-negative cases with culture results. (n=88)

Extent of X-ray###Culture

Lesion###+ve (n=31)###-ve (n=57)

Minimal###15 (48.38%)###24(42.10 %)###X2 =

Moderately advanced###6 (19.35 %)###13(22.80 %)###0.337

Far advanced###10 (32.24%)###20(35.08%)###p value=


Table 2: Comparison of respiratory symptoms of smear - negative cases with culture results. (n=88)

###Culture Results

Patients with





Cough###30(96.77 %)###50(87.7%)###X2 = 1.992

###p value= 0.158

Sputum###26(83.87 %)###47(82.45%)

production###X2 = 3.549

###p value= 0.060

Breathlessness###24(77.41 %)###44(77.2%)

###X2 = 1.832

###p value= 0.176

Haemoptysis###12(38.70 %)###16(28.11%)

###X2 = 1.048

###p value= 0.306

Chest pain###19(61.29 %)###35(61.4%)

###X2 = 0.000

###p value= 0.992

Fever###28(90.32 %)###47(82.5%)

###X2 = 0.377

###p value= 0.539

Night sweats###17(54.83 %)###33(57.89%)

###X2 = 0.076

###p value= 0.782

Loss of appetite###22(78.96 %)###34(59.6%)

###X2 = 1.112

###p value= 0.292

Weight loss###28(90.32 %)###51(89.5%)

###X2 = 0.016

###p value= 0.900

Malaise###30(96.77 %)###55(96.5%)###

###X2 = 8.471

###p value= 0.005


The present study showed that 35% cases that were negative on sputum microscopy were positive on culture indicating the usefulness of culture in microscopy negative cases. The study also highlights that almost 65% patients who remained culture negative also received a full course of anti tuberculosis treatment, indicating over diagnosis and over treatment. The phenomenon of culture positivity in smear negative cases has been reported by others too where it ranges between 24-62%14-16 in different geographical locations.

X-ray lesions in smear negative culture positive patients vary from minimal to advanced cavitations17-20. In the present study 48.3% cases had minimal lesion on X-rays and similar figures of minimal lesions were reported by Kim et al (60%)21 and Gonzalez et al (54%)19. Present study showed that 32.24% of smear negative culture positive patients had far advanced lesions which, are much higher than 5-10% reported in one study21 but is similar to another study from Lahore22.

Sensitivity and specificity of X-ray to pick TB cases depends on the bacterial count, site of infection, delay in diagnosis, and gender of patient, quality of the film and experience/ skill of the technician. At sites where TB culture facility is not available in DOTS, clinicians will have to rely on X-ray for the diagnosis and treatment of cases. With a 35% picking of TB on culture there is a strong need for the establishment of culture facilities and its use in smear negative pulmonary tuberculosis to reduce the burden of unnecessary treatment.


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Aamir Nazir1, Iffat Shabbir2, Tayyaba Rahat2 Model Chest Clinic1, PMRC TB Research Centre2, Sir Ganga Ram Hospital, Lahore. Corresponding Author: Iffat Shabbir PMRC TB Research Centre Sir Ganga Ram Hospital , Lahore. Email:
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Publication:Pakistan Journal of Medical Research
Article Type:Report
Geographic Code:9PAKI
Date:Mar 31, 2014
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