International Journal of Infectious Diseases
Volume 13, Issue 3 , Pages 374-379, May 2009

Urine as an adjunct specimen for the diagnosis of active pulmonary tuberculosis

Division of Clinical Microbiology, Department of Laboratory Medicine, All India Institute of Medical Sciences, Ansari Nagar, New Delhi 110 029, India

Received 5 March 2008; received in revised form 17 May 2008; accepted 14 July 2008. published online 03 November 2008.

Corresponding Editor: Sheldon Brown, New York, USA

Article Outline

Summary 

Background

The diagnosis of pulmonary tuberculosis (PTB) is conventionally established by examination of three Ziehl–Neelsen stained smears; however, negative results do not preclude active TB. Since tubercle bacilli or their nucleic acids are also expected to be excreted through the kidneys, we assessed spot urine as a supplementary specimen for diagnosing PTB.

Methods

A total of 164 respiratory specimens (147 sputum, 15 bronchoalveolar lavage, and two gastric lavage) from 81 suspected PTB cases were prospectively collected and processed. A total of 112 non-TB controls were also included in the study. For three consecutive days, morning urine specimens were collected from all patients and controls, and were processed for culture by BACTEC™ MGIT 960 (mycobacteria growth indicator tube) and Lowenstein–Jensen methods and for PCR by amplifying a 441-bp fragment of the hsp65 gene (Mycobacterium genus-specific) and a 786-bp fragment of the cfp32 gene (TB complex-specific).

Results

Of the 81 patients suspected of having PTB, 46 (56.8%) were sputum culture-positive. Of these, 12 (26.1%) were also urine culture-positive for Mycobacterium tuberculosis. Of the 35 sputum culture-negative cases, three (8.6%) were urine culture-positive. The TB complex specific PCR (cfp32) was positive in 52.2% (24/46) of the bacteriologically-confirmed and 28.6% (10/35) of the bacteriologically-negative PTB patients. In none of the control subjects were urine culture or PCR found to be positive for M. tuberculosis.

Conclusions

Specific PCR and culture examination of spot urine samples from suspected PTB patients significantly improved the detection rate of PTB and should be encouraged in resource-limited settings and where multiple pulmonary specimens are not feasible.

Keywords: Tuberculosis, Pulmonary tuberculosis, Diagnosis, Urine, PCR

 

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Introduction 

Tuberculosis (TB) is a major public health problem of global importance and it is the second leading cause of death worldwide, killing nearly 1.6 million people in 2005.1 Several studies have identified the clinical manifestations and symptomatology of sputum smear-positive individuals.2, 3, 4 The most common method for the diagnosis of pulmonary tuberculosis (PTB) is microscopic examination of Ziehl–Neelsen stained smears, which has a variable detection rate of 20–70%.5 However, approximately 20–50% of patients with PTB are smear-negative, and 10% of these patients remain culture-negative, even on three consecutive days.5 This phenomenon is more common in patients infected with multidrug-resistant Mycobacterium tuberculosis (MDR-TB).6 Several studies have also shown that most of the smear- and culture-negative patients will develop bacteriologically-positive disease in the course of time.2, 5, 7 In sputum-scarce PTB cases, bronchoalveolar lavage (BAL) fluid is a preferred clinical specimen in adults and gastric lavage in young children, but these specimens can be obtained only in a tertiary healthcare setting and have very low detection rates.5, 8

Colby postulated that tubercle bacilli could be excreted through the kidneys and that the organisms could be demonstrated in the urine of active TB patients who have no symptoms pertaining to the urinary tract.9 This hypothesis was confirmed by studies carried out in HIV-negative10, 11, 12 and HIV-positive cases.13, 14 These studies showed that urine could be used as an adjunct specimen due to the convenience and non-invasive nature of collection. However, in smear- and culture-positive cases it may not be necessary to include other samples, and such studies may be a matter for the records only. A study by Torrea et al.15 confirmed the utility of urine specimens for diagnosing PTB by nested PCR with a sensitivity of 64.3% in culture-negative PTB cases and 46.3% in extra-pulmonary tuberculosis (EPTB) cases. Though India harbors the majority of TB cases, no such study has yet been undertaken in this country. Therefore we carried out this study in order to evaluate the utility of urine as an adjunct or alternative specimen for diagnosing smear- and/or culture-negative PTB, using culture and PCR methods.

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Materials and methods 

Patients and specimens 

This prospective hospital-based study was conducted from July 2005 to June 2006 at the Department of Laboratory Medicine, All India Institute of Medical Sciences (AIIMS), New Delhi, India. AIIMS is a tertiary care medical center. During the study period, the clinical data of 215 patients with suspected PTB, referred from various clinics of this institute and other hospitals in and around Delhi for mycobacterial culture, were investigated. Only 81 of these fulfilled the inclusion criteria and were recruited into this study.16, 17 A total of 112 healthy volunteers, including 15 laboratory staff, with no present or past history of TB were also included in the study as control subjects. Cases of suspected genito-urinary TB with symptoms of burning micturition, unexplained sterile pyuria, or hematuria, and/or cases ultrasonographically suggestive of pyelonephritis were excluded from the study. Also only anti-tuberculosis treatment (ATT)-naïve patients were included in the study. The results of routine laboratory investigations such as erythrocyte sedimentation rate (ESR), total leukocyte count, and liver enzyme values were recorded for both the suspected TB and control subjects.

To establish the presence of PTB, at least two sputa (spot and early morning) were collected from each patient with a productive cough. BAL or gastric lavage specimens were taken from sputum-scarce patients. The sputum specimens were collected and brought to the laboratory by the patients themselves. The sputa received from the suspected PTB patients were first decontaminated by NALC-NaOH (modified Petroff’s method) as previously described.18

An early morning or spot urine sample (approximately 500ml) was also collected simultaneously from the patients and controls in a sterile wide-mouthed container for three consecutive days; the first two samples were stored in a refrigerator and all three samples were then pooled and processed on the third day. The pooled urine specimens from each patient were centrifuged at 3000×g for 20min. The resulting pellet was decontaminated with an equal amount of 4% NaOH. After incubation for 15minutes, the suspension was neutralized with phosphate-buffered saline (PBS; pH 6.8) and again centrifuged at 10 000rpm for 20min.

The pellets of decontaminated respiratory and urine specimens were resuspended in PBS; smears were made for Ziehl–Neelsen staining and 0.1ml was inoculated on Lowenstein–Jensen (L–J) slants while 0.5ml was inoculated in BACTEC™ MGIT 960 tubes (mycobacteria growth indicator tube; BD Diagnostics, Sparks, MD, USA) for culture isolation. The remaining aliquots of the suspended pellets were stored at −80°C and processed further for DNA isolation and analysis by PCR. Sputum and urine samples were processed separately to avoid possible cross-contamination.

The L–J slants were incubated at 37°C for 6 weeks.18 The inoculated MGIT 960 tubes were loaded into the BACTEC™ MGIT 960 system, and the growth was continuously monitored in fluorescence units, which flash positive after reaching a cut-off growth set by the manufacturer. Ziehl–Neelsen staining for acid-fast bacilli (AFB) was used to confirm positive BACTEC™ MGIT 960 tubes and any growth on L–J medium; Gram staining was also carried out to check for contamination. The specimens having AFB with contaminants were reprocessed as per the protocol above, while the others were discarded. Cultures were considered negative for mycobacterial culture only after 42 days, as per the manufacturer’s guidelines. The Mycobacterium species isolated from the clinical specimens were identified by phenotypic and biochemical tests, including heat stable catalase, nitrate, niacin, and arylsulfatase tests.18

DNA isolation and analysis by PCR 

The DNA from an aliquot of the decontaminated specimens was isolated as described previously by Ausubel et al.19 In brief, the decontaminated pellets were lysed with lysozyme and proteinase K–SDS, and DNA extracted by the phenol chloroform method followed by precipitation with 70% ice-cold ethanol. The resulting DNA pellets were solubilized in Tris–EDTA buffer and used for the PCR. Mycobacterium genus-specific PCR was done by amplifying a 441-bp fragment of the hsp65 gene20 and TB complex-specific PCR by amplifying a 786-bp fragment of the cfp32 gene.21 The PCR master mix was prepared by combining 500mM KCl, 100mM Tris HCl (pH 9.0), 0.2mM each deoxynucleoside triphosphate (dATP, dGTP, dTTP, and dCTP), 1.5mM MgCl2, 20μM cfp32 forward: 5′-ACCAACGATGGTGTGTCCAT-3′ and cfp32 reverse: 5′-CTTGTCGAACCGCATACCCT-3′ primers and 1U of Taq DNA polymerase (Promega, USA). To 20μl of this master mix, 5μl of template DNA was added. The reaction was carried out in an MTC-100 thermal cycler (MJ Research, USA). The genus- and TB complex-specific PCRs were run separately with a program of an initial denaturation at 96°C for 10min, followed by cycles of denaturation at 94°C for 1min, annealing at 60°C for 1min, and extension at 72°C for 1min, and a final extension at 72°C for 10min. The TB complex- and genus-specific PCRs were programmed with 30 and 45 cycles, respectively. DNA isolated from the standard strains of M. tuberculosis H37 Rv and Mycobacterium avium (kind gift from Dr V.M. Katoch, NJILOMD, Agra, India) were included as positive controls and sterile water as negative control, in each run; all precautions to prevent cross-contamination were taken. Amplified products were electrophoresed through a 2% agarose gel in Tris–acetate buffer. Target bands of 441bp for the genus-specific PCR and 786bp for the M. tuberculosis complex-specific PCR were visualized by staining with ethidium bromide. The samples found negative by genus- and TB-specific PCR were ruled out of PCR inhibition by amplifying the human γ-globulin gene with a product of 578bp as an internal control.22

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Results 

Clinical details 

The clinical data of 215 suspected cases of TB were available for analysis during this period, but only 81 cases could be included in the study after applying the inclusion criteria as described in the Materials and methods. Of the 81 cases, 58 (71.6%) were male and 23 (28.4%) female, with a mean age of 48±12.4 years. The most common symptoms observed in these patients were fever in 77 (95.1%), weight loss in 71 (87.7%), cough with expectoration in 64 (79.0%) and without expectoration in 10 (12.3%), raised ESR in 58 (71.6%), and hemoptysis in 10 (12.3%). Four (4.9%) patients had no fever but presented with cough and weight loss. All patients were ATT-naïve, but in 16 (19.8%) cases there was history of empiric fluoroquinolone treatment prescribed by the local physicians before the patient was referred to our hospital.

Pulmonary samples 

Of the 81 patients, 64 had a productive cough and provided sputum samples (n=147 samples). For the remaining 17 patients, BAL (n=15) and gastric lavage (n=2) samples were obtained. Therefore, a total of 164 samples were processed for mycobacterial smear, culture, and PCR tests. With the exception of sputum, samples were only taken once from each patient. Overall 46 of the 81 (56.8%) suspected PTB patients were confirmed to be positive by culture, either by sputum (n=43) or BAL (n=3) examination, while 35 remained smear- and culture-negative.

Smear, culture, and PCR findings 

Of the 64 patients who provided sputa (n=147), PTB could be confirmed bacteriologically (either by smear and/or culture) in 43 patients (n=98 sputa), while for 21 patients (49 sputum samples) PTB could not be confirmed by either of these methods. From the 43 bacteriologically-confirmed PTB patients, we obtained 98 sputum samples, and 42 (42.9%) of these sputa obtained from 21 (48.8%) patients were both smear- and culture-positive. The remaining 56 (57.1%) sputa from 22 (51.2%) patients were smear-negative but culture-positive. Of the 15 BAL samples none were smear-positive and only three were culture-positive, while 12 remained negative by both methods. No gastric lavage sample was detected positive by smear or culture examination (Table 1). Between the two culture systems, the mycobacterial isolation rate of BACTEC™ MGIT 960 was statistically significantly higher than that of the L–J culture method (Chi-square test, p=0.01). As expected, the mycobacterial detection rate of the PCR systems was comparatively higher than that of any of the culture methods (Chi-square test, p=0.001).

Table 1. Total number of respiratory samples collected and their positivity rate by different tests carried out in the study.
Nature of sampleNumber of casesMycobacterial detection rate from the respiratory specimens
Microscopy-positiveL–J-positiveMGIT-positiveGenus-specific PCR-positiveMTB complex-specific PCR-positive
Sputum (n=147)6442 (28.6%)62 (42.2%)98 (66.7%)121 (82.3%)117 (79.6%)
BAL (n=15)15003 (20%)6 (40%)6 (40%)
Gastric lavage (n=2)200011
Total (n=164)8142 (25.6%)62 (37.8%)101 (61.6%)128 (78.0%)124 (75.6%)

L–J, Lowenstein–Jensen; MGIT, mycobacteria growth indicator tube; PCR, polymerase chain reaction; MTB, Mycobacterium tuberculosis; BAL, bronchoalveolar lavage.

Urine specimens 

Urine samples from 81 suspected PTB patients were subjected to smear, culture, and PCR examinations. Of these, 15 (18.5%) were culture-positive by BACTEC™ MGIT 960. However, the culture isolation rate was significantly higher (38.1%) if the pulmonary samples from the same patient were also smear- and culture-positive as compared to those patients whose pulmonary samples were smear-, culture-, and PCR-negative (5.6%) (Table 2). This difference was highly significant (p<0.001). It is also important to note that approximately 12% of patients whose other investigations on pulmonary specimens were all negative could also be found urine culture-positive by MGIT 960. Of the 15 BAL samples, three were culture-positive, as mentioned above, but the urine culture from only one patient (33.3%) was positive. Nevertheless, urine cultures from two patients (13.3%) were MGIT 960 culture-positive. No urine sample from a sputum-scarce patient was smear- or L–J culture-positive. Therefore, the cumulative total of bacteriologically-confirmed cases among the sputum-scarce TB cases increased from 17.6% (3/17) to 29.4% (5/17) by using urine as an adjunct specimen.

Table 2. Mycobacteriuria detection rate of various in vitro diagnostic methods applied to urinary specimens from pulmonary tuberculosis patients.
Suspected PTB cases (N=81) (and results of pulmonary samples)Detection rate of mycobacteriuria
MicroscopyL–JMGITGenus-specific PCRMTB-specific PCR
Bacteriologically-confirmed PTB (N=46)a3 (6.5%)5 (10.9%)12 (26.1%)25 (54.3%)24 (52.2%)
Smear-, culture-, and PCR-positive (N=21)3 (14.3%)4 (19.0%)8 (38.1%)13 (61.9%)13 (61.9%)
Smear-negative, culture- and PCR-positive (N=25)01 (4%)4 (16%)12 (48%)11 (44%)
Bacteriologically-negative PTB (N=35)b003 (8.6%)11 (31.4%)10 (28.6%)
Smear- and culture-negative and PCR-positive (N=17)002 (11.8%)7 (41.2%)7 (41.2%)
Smear-, culture-, and PCR-negative (N=18)001 (5.6%)4 (22.2%)3 (16.7%)
Controls (N=112)001c2c0

PTB, pulmonary tuberculosis; L–J, Lowenstein–Jensen; MGIT, mycobacteria growth indicator tube; PCR, polymerase chain reaction; MTB, Mycobacterium tuberculosis.

aAt least one specimen from a patient was positive in smear and/or L–J or BACTEC MGIT 960 culture and/or PCR.

bSpecimen(s) collected from a patient were either positive or negative by PCR and negative by both smear and culture.

cNon-tuberculous mycobacteria detected.

With the application of genus-specific PCR, overall 25 of 46 (54.3%) bacteriologically-confirmed PTB patients were found PCR-positive suggesting mycobacteriuria. The PCR positivity could reach as high as 61.9% in sputum smear-positive cases. Even in those patients whose pulmonary samples were negative by all bacteriological methods, the urine PCR was positive in 31.4% of patients. Therefore, the application of PCR for urine samples was most rewarding in both bacteriologically-confirmed and -unconfirmed patients (Table 2).

Irrespective of the PCR results, all the cases were treated with ATT as per the RNTCP (Revised National Tuberculosis Control Programme), India guidelines. Of the 24 bacteriologically-confirmed cases using both respiratory and urine specimens, only six (25%) could be followed up and four responded to ATT. Among the 22 bacteriologically-confirmed but urine culture- and PCR-negative PTB cases, 13 (59.1%) could be followed up and nine (40.9%) of them responded to the treatment. Of the 11 urine PCR-positive but bacteriologically-negative PTB cases, all were followed up and nine (81.8%) of them responded to the treatment. Of the eight patients sputum PCR-positive but negative by all other tests, four were followed up and two responded to ATT. In those who responded to ATT, their fever and cough disappeared, they gained weight, and repeat respective samples after 3 months became negative by both culture and PCR methods.

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Discussion 

The African region, China, and India collectively account for 69% of undetected TB cases.1 Unfortunately, for the last few years the progress of PTB case detection has decelerated globally; it has almost stalled in India and China and fell short of the global planned milestone of 65%.1 The paucibacillary form of TB is a major diagnostic challenge. This is the most challenging task in TB endemic and resource-poor countries where infrastructure, the cost of equipment and reagents, and the lack of suitably trained staff are the major concerns. Therefore, rapid diagnostic methods like PCR could be the future hope. However, obtaining good quality sputum specimens or other pulmonary samples is always problematic. In the present study we attempted to apply the most sensitive diagnostic tool to the most easily available clinical sample, i.e., urine, with an improved detection rate. In our study, when urine samples were tested along with pulmonary specimens and when genus- and species-specific and sensitive PCR methods were applied to these samples, we were able to detect an additional 41.2% of PTB cases. These findings are highly significant and encouraging for at least tertiary healthcare level settings. Not only PCR but urine culture methods also improved the mycobacterial isolation rate.

Nevertheless, the bacteriological confirmation of TB from the appropriate clinical specimen cannot be undermined. It is of significance not only for effective management but also for speciation of the isolate, its characterization, and anti-mycobacterial drug susceptibility studies. Paucibacillary TB poses a challenge in establishing the correct diagnosis and management and in the containment of spread of drug-resistant PTB.6, 23 In sputum-scarce or sputum smear- and culture-negative cases, the usual alternative specimens are BAL and gastric lavage fluids, but collection of these specimens is often painful and awkward, and these specimens add insignificant advantage. Our study also shows a very limited improvement in detection rate.

There are only a few studies demonstrating the utility of urine as a clinical specimen in diagnosing TB in both HIV-positive and HIV-negative cases. Torrea et al.15 evaluated the utility of urine specimens using a nested PCR assay in suspected TB cases. However, the major limitation of their study was a lack of bacteriological confirmation of the urine specimens and true positivity rate of PCR. In addition, no analysis was made to correlate the positive predictive value of urine with the other site-specific specimens analyzed in their study. Another recent study by Rebollo et al. in 2006,24 dealt with this limitation and correlated the results of their PCR with bacteriologically-confirmed urine specimens. But in this study, only 14 smear-negative PTB cases were included. Therefore, the false positivity of PCR and cost-effectiveness of using urine as an adjunct specimen could not be evaluated in a group of smear- and culture-negative TB patients.

In the present study, an attempt was made to evaluate the feasibility of using urine as an adjunct specimen in diagnosing mycobacterial infection. The results show that more than a quarter (26.1%) of the smear- and/or culture-positive PTB cases also excreted M. tuberculosis in their urine specimens. Excretion of M. tuberculosis was reported as early as in 1975 by Bentz et al.10 Two recent studies also reproduced the findings of Bentz et al. using modern diagnostic tools.15, 24 However, no previous study has reported so high a culture isolation rate of M. tuberculosis from urine as reported in the present study. This could be because the earlier authors15, 24 centrifuged urine samples at 3000×g with an isolation rate of only 19%, while we in the present study used 10 000rpm for processing the urine samples, and we feel strongly that in future studies clinical pathologists should use 10 000rpm speed for urine samples. Centrifugation plays a very important role in the retrieval of living mycobacteria. A higher detection rate was also found while culturing respiratory6 and other non-respiratory specimens if a higher centrifugation speed was used (data not shown).25

In comparison with the smear and culture methods, PCR was more sensitive in our study, and in addition, PCR was positive in 31.4% of bacteriologically-negative samples using genus-specific primers and 28.6% using M. tuberculosis-complex-specific primers. A similar detection rate has been reported from Burkina Faso,15 Spain,24 and India.26

However, these studies also show that mycobacteriuria is not a continuous process. This is further evident from our results of 46 culture-proven PTB cases; even using the most sensitive culture system only 12 (26.1%) patients had cultivable mycobacteriuria. However, PCR detected mycobacterial DNA in as many as 54.3% of urine samples, suggesting that the excretion of DNA is a comparatively more continuous phenomenon. We ruled out positivity of our PCR system using all possible internal and patient controls (Table 2). In fact the PCR was not positive in all the urine samples from mycobacteriologically-confirmed cases, suggesting a high specificity but optimized sensitivity. The low positivity rate of PCR in proven PTB cases could be explained by the fact that not all of the patients with PTB will have circulating mycobacteria that will be filtered out of the kidneys through the urine. It is also possible that M. tuberculosis or its fragments may or may not have been excreted during the time of sample collection.27, 28, 29 Furthermore, the PCR reaction was found inhibited in four samples, which was evident from the amplification of internal controls. Endogenous PCR inhibitors like acidic polysaccharides, glycoproteins, urea, and unidentified non-proteinaceous DNA-associated substances cause chelation of free magnesium ions and are known to inhibit the PCR amplification.30

As in most developing and resource-poor countries, laboratory diagnosis of TB remains a problem because of a lack of laboratory facilities and increased workload, mostly due to high HIV seroprevalence. This high rate of undiagnosed or unconfirmed TB contributes significantly to treatment delays and often to the development of drug resistance. Therefore the development of sensitive and rapid diagnostic tools is urgently required, and our laboratory is working in that direction. The results of this study lead us to suggest that urine specimens along with the other respiratory specimens will possibly increase the chance of diagnosing active TB, especially in sputum smear-negative/scarce pulmonary TB cases. This is more important if we apply PCR on this easily available clinical material. It may minimize the use of more invasive techniques for collecting samples, such as BAL and gastric lavage. We also recommend that when other clinical specimens have failed to confirm the diagnosis, a urine sample be used for culture isolation and PCR tests.

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Acknowledgements 

We wish to thank Dr V.M. Katoch of National JALMA Institute for Leprosy and other Mycobacterial Diseases, Agra for providing us with the standard strains of mycobacteria.Funding: Financial support from the Indian Council of Medical Research, Government of India to SS is acknowledged. A senior research fellowship to KG is also acknowledged.Ethical approval: This work was conducted as part of the fulfillment of a PhD thesis (KG). The study protocol is routinely followed in the collection and processing of samples for diagnostic and clinical practice at the All India Institute of Medical Sciences, New Delhi, and hence no prior ethical clearance was required. An informed consent was obtained while recruiting the control subjects in the study, following an explanation of the objectives and the need to collect specimens from them. Links between the patient and control personal and/or clinical data and the sample information were removed to protect patient privacy.

Conflict of interest: No conflict of interest to declare.

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PII: S1201-9712(08)01495-1

doi:10.1016/j.ijid.2008.07.022

Refers to corrigendum:

  • Corrigendum to “Urine as an adjunct specimen for the diagnosis of active pulmonary tuberculosis” [International Journal of Infectious Diseases 13 (2009) 374-379] , 02 August 2010

    Krishnamoorthy Gopinath, Sarman Singh
    International Journal of Infectious Diseases September 2010 (Vol. 14, Issue 9, Page e826)

International Journal of Infectious Diseases
Volume 13, Issue 3 , Pages 374-379, May 2009

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