|Year : 2013 | Volume
| Issue : 3 | Page : 203-207
Detection of Mycobacterium tuberculosis by three methods and their correlation to chest X-ray findings and CD4 T-lymphocyte counts in human immunodeficiency virus-pulmonary tuberculosis coinfection
Supriya Laifangbam1, Lokhendro Singh Huidrom1, Sulochana Devi Khumanthem2
1 Department of Microbiology, Jawaharlal Nehru Institute of Medical Sciences, Imphal, Manipur, India
2 Department of Microbiology, Regional Institute of Medical Sciences, Imphal, Manipur, India
|Date of Web Publication||19-Feb-2014|
Brain and Spinal Cord Centre, Regional Institute of Medical Sciences Road, Imphal - 795 004, Manipur
Source of Support: None, Conflict of Interest: None
Aims: 1) To compare three microbiological methods of detection of Mycobacterium tuberculosis 2) To find their correlation with (a) chest x-ray (CXR) findings and (b) absolute CD4 counts. Materials and Methods: 147 human immunodeficiency virus (HIV)-pulmonary tuberculosis (PTB) suspects were enrolled. Sputum from 108 of them were subjected to light microscopy of Ziehl-Neelsen (ZN) stained smear, fluorescence microscopy of Auramine-O (AO)-stained smear, and culture on Lowenstein-Jensen media for detection of M. tuberculosis. The results were analyzed statistically and compared with CXRs and CD4 counts. Results: Of 147 eligible patients, 108 were studied and 78 were diagnosed as HIV-PTB coinfection. Positivity rates for ZN, AO, and culture were 39 (36.1%), 80 (74.1%), and 78 (72.2%), respectively. Sensitivity and specificity for ZN were 48.7% and 96.6%, respectively, whereas those for AO were 100% and 93.3%, respectively. Additional yield of AO over ZN was 42 (38.8%). AO was able to detect all the culture positives. The difference in case-yields between ZN and AO has been found to be highly significant at P value 0.05 and χ2 test = 3.84. The sensitiveness of these methods of detection was higher for patients showing active disease on CXR and those with CD4 count > 200 cells/μL. Conclusion: Fluorescence microscopy is recommended in HIV-PTB coinfection to enhance case detection and management.
Keywords: CD4 count, Fluorescence microscopy, HIV, PTB, Pulmonary tuberculosis
|How to cite this article:|
Laifangbam S, Huidrom LS, Khumanthem SD. Detection of Mycobacterium tuberculosis by three methods and their correlation to chest X-ray findings and CD4 T-lymphocyte counts in human immunodeficiency virus-pulmonary tuberculosis coinfection. J Med Soc 2013;27:203-7
|How to cite this URL:|
Laifangbam S, Huidrom LS, Khumanthem SD. Detection of Mycobacterium tuberculosis by three methods and their correlation to chest X-ray findings and CD4 T-lymphocyte counts in human immunodeficiency virus-pulmonary tuberculosis coinfection. J Med Soc [serial online] 2013 [cited 2020 Oct 20];27:203-7. Available from: https://www.jmedsoc.org/text.asp?2013/27/3/203/127394
| Introduction|| |
Manipur is one of the six high prevalence states of India regarding human immunodeficiency virus (HIV) infection. Pulmonary tuberculosis (PTB) is highly common among HIV positive persons in India.  Difficulty in diagnosis of smear-negative PTB is a major contributor to the death of HIV-PTB coinfected patients. World Health Organization (WHO) states that sputum positivity for tuberculosis (TB) decreases with atypical chest x-ray (CXR) findings as CD4 T-lymphocyte count decreases with progression of HIV infection. In this study, an attempt has been made to compare three different microbiological methods of detection of Mycobacterium tuberculosis and how they correlate with CXR and CD4 counts among HIV-PTB coinfected patients. We aim to find how to enhance TB detection so that treatment and prognosis of HIV-PTB coinfection are improved.
| Materials and Methods|| |
This hospital-based, comparative, cross-sectional study was conducted in the Department of Microbiology, Regional Institute of Medical Sciences (RIMS), Imphal, Manipur, India, from January 2005 to June 2006, on 147 patients confirmed with HIV infection as per National AIDS Control Organization (NACO) guidelines and suspected of PTB. HIV positive patients attending the Respiratory Diseases Department, RIMS, and having cough, fever, night sweats, weight loss, difficulty in breathing, and/or radiological changes in CXR were included in the study. Those who have not done a CD4 count test during the last 6 months, have not taken a course of antibiotics, are known cases of carcinoma lung or are pediatric cases were excluded. Patients unable to produce at least 5 mL of sputum were also excluded.
Radiologically, patients were considered to have active lesion if the CXR revealed diffuse parenchymal infiltration, consolidation, lymphadenopathy, pleural or pericardial effusion, miliary lesions, pneumothorax or cavitation. Normal radiographs and those showing scarring or calcification of lung, lymph nodes, or pleura were considered inactive. Concerning CD4 T-lymphocyte counts, patients were divided into two groups, those with 0–200 cells/μL and those with above 200 cells/μL.
A total of two sputum samples were collected on 2 days from each patient- Spot specimen on the first day, one early morning on the second day. Samples were collected in clean, sterile, leak-proof, wide-mouth, screw-capped containers. All microbiological processings were carried out in a biosafety cabinet. Each sample was processed by the Petroff's method  and subjected to the conventional Ziehl-Neelsen (ZN) staining, fluorescent Auramine-O (AO) staining,  and culture on modified Lowenstein-Jensen (LJ) medium.
For ZN smear reporting, smears were examined using a light microscope scanning at least 300 oil immersion fields before reporting a smear as negative. M. tuberculosis are seen as acid fast bacilli (AFB)-stained bright pink to red, in beaded or barred forms, while the tissues cells and other organisms are stained blue. For AO smear reporting, the fluorescence microscope used was the Axiostar plus Transmitted-Light Microscope with an attached Epifluorescence illuminator manufactured by Zeiss and it was fitted in a dark room. The film was examined with a 40× objective and a 10× eye piece. At least 70 high power fields were examined before reporting a smear as negative. The tubercle bacilli are seen as yellow luminous organisms in a dark field. Both types of sputum smear reporting were graded according to Forbes et al.,  In this study, ''No AFB seen''(NS) and ''Doubtful'' (D) are considered negative; 1+ as paucibacillary positive and 2+, 3+, and 4+ as multibacillary positive.
After processing, the early morning sputum specimen, 0.25 mL of the sediment was inoculated on to the surface of two modified LJ media prepared in-house.  The inoculum was spread over the surface of the slant, the tubes were incubated at 37°C and left in the slanted position for 7 days to permit even distribution of the inoculum over the entire surface of the medium. The tubes are then placed upright and incubation continued for 8 weeks. The caps of the bottles were loosened once weekly at the time of inspection for growth. Negative reports were given if no growth appeared after 8 weeks. Identification of M. tuberculosis was done on observation of slow rate of growth of nonpigmented, raised, rough, thick colonies with a nodular or wrinkled surface, positive ZN staining, and positive niacin production and nitrate reduction tests. A case was diagnosed as having PTB if the morning sputum sample grew M. tuberculosis on culture
At the time of sample collection, informed written consent was obtained from the patients and a proforma was used to collect data regarding each of them. On obtaining the study results, the data were completed and analyzed statistically using one-tailed (right tail) χ2 and P-value to compare binary data.
| Results|| |
Of the 147 eligible patients, 5 (3.4%) provided salivary sputum specimen, 13 (8.8 %) were unable to provide a morning sputum specimen, 3 (2%) did not have a culture performed, and 18 (12.2%) had a contaminated sputum culture giving a final sample size of 108 HIV-infected TB suspects. Patients excluded from the study did not differ from those included in terms of demographic characteristics.
Out of 108 patients studied, 72 (66.7%) were males and 36 (33.3%) were females. A total of 78 cases, 51 (65.4%) males and 27 (34.6%) females, were diagnosed as having PTB. Maximum number 45/78 (57.7%) of HIV-PTB cases were observed in the age group of 31-40 years. Among them 27/51 (52.9%) males and 13/27 (48.1%) females were matriculate and above. 9/27 (33.3%) females were housewives. 24/51 (47%) males and 10/27 (37%) females were unskilled workers. The rest were skilled workers. Loss of weight was observed among 64/78 (82%) patients, followed by difficulty in breathing at 50/78 (64.1%) and fever at 36/78 (46.2%). Hemoptysis was present in only 5/78 (6.4%).
Of 108 patients positivity rates for ZN, AO, and culture were 39 (36.1%), 80 (74.1%), and 78 (72.2%), respectively. All the isolates were identified as M. tuberculosis. 30 (27.8%) showed no growth and were culture-negatives. Additional yield of AO over ZN was 41 (38%). AO was able to detect all the culture positives.
Grade-wise comparison of the two methods of sputum microscopy is shown in [Table 1]. Results of ZN and AO are compared with that of culture in [Table 2] and [Table 3], respectively. Results of comparison of chest radiography findings with the microbiological methods of detection namely ZN, AO, and culture of M. tuberculosis are shown in [Table 4]. Comparison of absolute CD4 count T-lymphocyte counts with these microbiological methods is shown in [Table 5].
|Table 1: Comparison of conventional bright-field microscopy (Ziehl-Neelsen stain) and fluorescence microscopy (Auramine-O stain)|
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|Table 2: Comparison of Ziehl-Neelsen stain microscopy and culture results|
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|Table 3: Comparison of Auramine-O stain fluorescent microscopy and culture results|
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|Table 4: Correlation of radiological findings of disease with smear microscopy and culture findings|
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|Table 5: Correlation of CD4 counts with smear microscopy and culture findings|
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| Discussion|| |
In this study, the prevalence of HIV-PTB coinfection was high in the age group of 31-40 years. The prevalence was higher among the male patients at 51/78 (65.4%) as compared with the females at 27/78 (34.6%). Higher prevalence of HIV-TB coinfection have been reported among male patients by other such studies. 
In this study, positivity rates for ZN and AO were 39/108 (36.1%) and 80/108 (74.1%), respectively. The difference in case-yields between ZN stain and AO stain has been found to be highly significant (χ2 = 3.84, P value 0.05). AO results scored higher than ZN in several studies proving that fluorochrome staining of sputum smears in comparison to that of ZN staining is a better method of microscopy for the demonstration of the tubercle bacilli. , A study from India, of 200 patients diagnosed with TB by clinical and radiographic means in an area of high HIV prevalence, showed an incremental yield of 26% for FM over ZN microscopy for those coinfected with HIV.  Hence, fluorescence-based staining techniques show promise in increasing diagnostic yield over conventional staining in HIV-infected individuals. The added advantage of this technique is the reduced time taken by laboratory technicians to view the sample, 1 min compared with 4 min for conventional methods.
In the present study, it was observed that a total number of 41 (38%) sputum smears which were negative by ZN method were positive by AO stain. Among these, 27 (18 + 9) were paucibacillary and 14 (5 + 8 + 1) were multibacillary. This goes to prove that AO was more efficient in detecting paucibacillary cases. Patients with paucibacillary disease were more likely to be diagnosed by the fluorescence-based method.  HIV-PTB coinfected patients are likely to be paucibacillary cases due to the noncavitatory lesions. 
Culture of M. tuberculosis is the gold standard for diagnosis of TB. It is a much more sensitive and specific test than conventional smear examination. In this study, cultures were positive in 78/108 (72%) cases. Culture positivity rates in previous similar studies ranged from about 74% to 76%. , Sensitivity and specificity for ZN compared to culture were 48.7% and 96.6%, respectively, whereas sensitivity and specificity for AO were 100% and 93.3%, respectively. ZN method of staining is the most commonly employed technique of sputum microscopy. The major limitation of ZN microscopy is the lack of sensitivity, which varies from 20% to 78%, the lower figures are from ordinary laboratories, whereas the higher figures are from research laboratories, showing that adoption of the right method with sincerity is what is necessary for increasing the sensitivity of the method.  Sensitivity by ZN staining in case of HIV-PTB coinfection has been found to be even lower. , In a cross-sectional study of 1398 TB suspects in Nairobi using Löwenstein-Jensen culture as gold standard, the sensitivity of fluorescence microscopy (FM) was unaffected by HIV status and was twice that of ZN staining.  The specificity of FM was similarly unaffected.
A total of 18/147 (12.2%) eligible patients were excluded from the study due to a contaminated sputum culture. Such rates of contamination have been reported earlier.  In spite of the investments in infrastructure and training, inability to either rule in or rule out any given case as having PTB due to culture contamination is a major setback. Also, culture facilities are often inaccessible to patients in resource-limited TB endemic areas. Until such facilities become more widely available, FM may be started to improve diagnostic yield.
In relation to radiological extent of disease, analysis of sensitiveness of different bacteriological methods showed that ZN method was less sensitive in cases of inactive disease 4/21 (19%) as compared to both fluorescent microscopy: 12/21 (57%) and culture 11/21 (52.4%). The corresponding figures of sensitiveness in cases of active disease were 35/87 (40.2%) for ZN, 68/87 (78.2%) for AO and 67/87 (77%) for culture. It was evident from these results that sensitiveness of all three methods of detection were definitely higher in those showing active disease in their chest x-rays. In both categories of disease, sensitiveness of AO was found to be closer to that of culture which is the gold standard. In this study, CXR missed detection of 11/78 (14.1 %) culture-positive patients and diagnosed 20/30 (66.7%) of culture-negative patients. Among these 11 culture-positive patients, a majority of nine showed normal CXR and the other two showed scarring with calcified hilar lymph nodes. The ability of microbiological detection of PTB among those with inactive lesions in CXR is very important from the aspect of treatment of open cases in order to stop further transmission. CXRs can undoubtedly be very useful in localizing abnormalities in the lung. But, because TB is an infectious disease, diagnosis of TB should be by demonstration of M. tuberculosis on culture or AFB on smear microscopy. Besides, in HIV-PTB coinfection, evidence of the impaired tissue inflammatory response to infection is seen in the radiographic appearances of PTB, with reduced consolidation, fibrosis, and cavitation.  Lack of cavitation in turn results in low numbers of bacilli in sputum specimen. Thus, results of sputum smear microscopy for AFB are frequently negative. 
There were 16 patients in the CD4 0-200 cells/μL group and 92 in the CD4 above 200 cells/μL group. It has been reported that in patients with CD4 lower than 200, noncavitory infiltration and consolidation predominates.  Inability to produce sputum explains why there were much lesser number of patients in the former group. Unlike other opportunistic infections like cryptococcal meningitis or toxoplasmosis, which occur at very low CD4 counts, PTB is unique in that it can occur over a wide range of CD4 counts.  For AO and culture, the positivity to negativity ratio was around 1:1 for CD4 between 0 and 200 cells/μL, whereas it was 3:1 in cases of CD4 count above 200 cells/μL. This difference in the two groups with CD4 count cut-off value 200 cells/μL endorses the fact by the WHO that sputum smear negativity increases with increase in degree of immune-suppression. Since 2010, the WHO recommended that all HIV positive TB patients are to be rapidly started on antiretroviral therapy (ART) within the first 8 weeks of TB treatment.  After November 2011, NACO has started following this WHO recommendation, before which ART was advised for patients with CD4 <200 cells/μL. ART reduces the individual risk of TB disease by 65% irrespective of CD4 cell count.  Therefore, among the HIV-infected patients suspected of PTB, the detection of PTB is more important than doing the CD4 count in order to start ART and improve the prognosis and outcome.
In India, TB care and HIV care are increasingly being coordinated, but the full benefits have yet to be realized.  Sputum smear examination remains the routine standard of diagnosis. Other current diagnostic tests for TB, such as tuberculin skin test, immunodiagnostics, antigen-based detection assays, nucleic acid amplification tests are failing to help stem the tide of deaths from smear-negative disease in patients with HIV.  Since AO was able to detect all the culture positives and decrease the number of sputum smear negatives by detecting more of the paucibacillary cases, its use is highly recommended. The limitation for its widespread use has been the high cost for fluorescent microscopes. Now, low-cost, robust light-emitting diode (LED) microscopes have facilitated the expansion of FM into low-resource settings.  In 2010, the WHO recommended that LED FM be phased in to replace ZN microscopy for TB diagnosis. 
As a strength of this study, we believe the replacement of chronic cough with current cough as a screening question could have helped in PTB detection rates.  The collection of minimum 5 mL of sputum specimens is known to be associated with increased smear positivity.  We had a single experienced and dedicated research technician who was able to provide consistency of techniques. A larger sample size with blinding of smear examiners in order to rule out observer bias would have made the study even more conclusive.
In conclusion, FM can enhance detection of PTB among HIV positive patients, mostly with atypical CXR findings. This is important as more patients can be put on a regimen for smear-positive TB, contributing to improved treatment and reduced transmission. Once the TB bacilli are detected, ART may also be started irrespective of CD4 count to improve prognosis and outcome.
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[Table 1], [Table 2], [Table 3], [Table 4], [Table 5]