Occurrence of Overlooked Zoonotic Tuberculosis: Detection of Mycobacterium bovis in Human Cerebrospinal Fluid
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微生物临床杂志 2006年第4期
Departments of Biotechnology Neurology, All India Institute of Medical Sciences
Department of Pediatrics, Safdarjung Hospital, New Delhi 110029, India
ABSTRACT
The paucibacillary nature of the cerebrospinal fluid (CSF) has been a major obstacle in the diagnosis of human tuberculous meningitis (TBM). This study shows that with molecular techniques direct precise determination to the species level of mycobacterial pathogens can be made. The present report describes the utility of a nested PCR (N-PCR) assay (A. Mishra, A. Singhal, D. S. Chauhan, V. M. Katoch, K. Srivastava, S. S. Thakral, S. S. Bharadwaj, V. Sreenivas, and H. K. Prasad, J. Clin. Microbiol. 43:5670-5678, 2005) in detecting M. tuberculosis and M. bovis in human CSF. In 2.8% (6/212) of the samples, M. tuberculosis was detected, and in 17% (36/212), M. bovis was detected. Mixed infection was observed in 22 samples. Comparative analysis of clinical diagnosis, smear microscopy, and N-PCR in 69 patients (TBM, 25; non-TBM, 44) showed that the sensitivity of N-PCR (61.5%) was greater than that of smear microscopy (38.4%). Determination to the species level is important from the viewpoint of determining the prevalence of these mycobacteria in a community and would influence strategies currently adopted for the prevention of tuberculosis.
INTRODUCTION
Tuberculosis (TB) is a chronic, systemic infectious disease caused by Mycobacterium tuberculosis. The most common clinical manifestation is pulmonary TB. The inhaled bacilli can localize in alternate sites, leading to extrapulmonary TB (EPTB). Among the different manifestations of EPTB, tuberculous meningitis (TBM) has been considered to be a fatal form (41). Fatality rates in developing countries have been reported to range from 44 to 69% (15, 18, 30). In fact, delayed or erroneous diagnosis often results in serious long-term debilitating complications. Central nervous system involvement has frequently been found secondary to TB elsewhere in the body, particularly the lungs. The presence of TB elsewhere in the body favors the diagnosis, although its absence does not exclude it. The great majority of patients with neuro-TB are diagnosed on the basis of clinical criteria, imaging, and laboratory investigation of the cerebrospinal fluid (CSF). The clinical response to antituberculosis therapy in all forms of neuro-TB is excellent, provided the diagnosis is made early, before an irreversible neurological defect occurs.
Hence, precise and rapid clinical diagnosis of TBM is a critical component of the management of TBM patients (12, 24, 25). Culture and Ziehl-Neelsen staining of the CSF are specific but insensitive due to the paucity of bacilli. Nucleic acid amplification techniques have shown promising results in overcoming this drawback (20, 22, 30, 42).
M. tuberculosis is a member of the M. tuberculosis complex (MTC), which includes M. bovis, "M. canetti," M. microti, and M. africanum. M. bovis infection of the central nervous system may lead to EPTB, similar to M. tuberculosis infection, with identical symptomatologies (10, 16). M. bovis infection has been reported in immunocompetent, as well as in immunosuppressed, individuals (9, 21, 25, 33). Hence, the present study was undertaken to detect M. tuberculosis and M. bovis in human CSF by using an in-house nested PCR (N-PCR) assay (26). The assay targets the 27-bp difference in the C terminus of the hupB gene in M. tuberculosis (Rv2986c) and M. bovis (Mb3010c) to differentiate M. tuberculosis from M. bovis (32). The utility of the assay for direct detection of M. tuberculosis and M. bovis in bovine samples was confirmed in comparison with standard culture techniques (26).
MATERIALS AND METHODS
Patients. CSF samples from 212 patients were investigated. The patient distribution was as follows. For the first part of the study, 112 patients admitted to the neurology ward of the All India Institute of Medical Sciences (AIIMS), New Delhi, were investigated. Subsequently, for the second part of the study, CSF samples from 100 children (12 years old) admitted to the pediatric ward of Safdarjung Hospital, New Delhi, India, were investigated. The institutional ethical committee approved the study. At the time of data analysis, the AIIMS hospital records of the 69 patients were obtained. These 69 cases were separated into TBM and NTBM (nontubercular meningitis) groups on the basis of the criteria described by Ahuja et al. (1) (Table 1). CSF was collected under aseptic conditions by lumbar puncture. Five hundred microliters to 2.0 ml of CSF was available for the study. Samples were stored at –20°C, prior to processing for target DNA for N-PCR and smear microscopy for acid-fast bacilli (AFB; auramine O stain).
Mycobacterial strains. M. tuberculosis H37Rv DNA was obtained from TB research material, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. Lowenstein-Jensen slant cultures of M. tuberculosis H37Rv and M. bovis AN5 were obtained from the Central JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, India.
Preparation of mycobacterial cell lysates containing target DNA. (i) Mycobacterial cultures. One milliliter of a logarithmic-phase culture of M. tuberculosis or M. bovis grown in 7H9 Middlebrook broth was pelleted and resuspended in 100 μl of 0.1% of Triton X-100. The suspension was boiled (90°C for 40 min) and centrifuged (10,000 x g for 10 min). The supernatant was stored at –20°C and used as template DNA in a PCR.
(ii) CSF. Target DNA was prepared from CSF by a modification of the method of Chakravorty and Tyagi (6). CSF was filtered (0.22-μm pore size; Millipore). The CSF container was rinsed with 2 ml of inhibitory removing solution (25 M guanidinium isothiocyanate, 0.025 M EDTA, 0.05 M Tris, 0.5% Sarkosyl, 0.186 M -mercaptoethanol, pH 7.5) and filtered. The membrane was transferred to a 1.5-ml Eppendorf tube containing 500 μl of inhibitory removing solution, incubated for 15 min at room temperature, and centrifuged (10,000 x g for 10 min). The supernatant was discarded, and the membrane was washed with sterile water. Two hundred microliters of 0.1% Triton X-100 was added to the membrane and vortexed vigorously. The suspension was used to make smears and subsequently boiled in a dry bath (90°C for 40 min), followed by centrifugation (10,000 x g for 10 min). The supernatant was stored at –20°C and used as template DNA.
N-PCR assay for hupB DNA target (international patent application PCT/IN03/00302, 1 July 2004, L. S. Davar and Co., Calcutta, India). Primers N (5'-GGAGGGTTGGGATGAACAAAGCAG-3') and S (5'-GTATCCGTGTGTCTTGACCTATTTG-3') were used to amplify the hupB gene as previously described (N-S PCR, Fig. 1A) (32). The N-PCR assay for the C-terminal portion of the hupB gene was carried out with primers F (5'-CCAAGAAGGCGA CAAAGG-3') and R (5'-GACAGCTTTCTTGGCGGG-3') (Fig. 1A) (26). Four microliters of the amplified product of a PCR with primers N and S was used as the template DNA for the N-PCR. Each reaction mixture (40 μl) of the N-PCR contained 1.25 mM MgCl2, 200 μM deoxynucleoside triphosphates, 0.5 μM primers F and R, 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 0.08% Nonidet P-40, and 1.0 U of Taq DNA polymerase. The reaction mixture was subjected to initial denaturation at 94°C for 10 min and 35 cycles each of 1.0 min at 94°C and annealing and extension at 59°C for 0.30 min, followed by a final extension at 72°C for 7 min. The products were analyzed on a 10% polyacrylamide gel and stained with ethidium bromide. The expected sizes of amplicons for M. tuberculosis and M. bovis were 116 and 89 bp, respectively. The limit of detection was 50 fg, which is equivalent to five tubercle bacilli (26). Further, in an earlier report, the hupB-based PCR assay was shown to be specific for M. tuberculosis and M. bovis when tested with members of the MTC, as well as with other mycobacterial species (32). N-PCR products generated from standard strains of M. tuberculosis and M. bovis were included (positive control). A dual positive control was also included to discern mixed infection with M. tuberculosis and M. bovis. The N-PCR products in this case were generated from a mixture of the target DNAs of M. tuberculosis H37Rv and M. bovis AN5.
DNA sequencing. Primer F- and R-amplified N-PCR products from 10 samples that showed an 89-bp product of M. bovis were eluted from agarose gel with a mini Elute Gel Extraction kit (QIAGEN). The extracted DNA was lyophilized and sent for sequencing to the DNA Sequencing Resource Center at The Rockefeller University.
Alternatively, with CSF samples showing mixed infection, both of the bands (116 and 89 bp) were cut from a 10% polyacrylamide gel and DNA was extracted by the crush-and-soak method as previously described (37). These extracted N-PCR products were cloned into the pGEMT vector with a TA cloning kit (Promega) according to the manufacturer's instructions. The clones were sequenced at the DNA sequencing facility, South Campus Delhi University, New Delhi, India.
The sequences obtained were aligned with the sequences of M. tuberculosis and M. bovis with the CLUSTALW software (http://www.ebi.ac.uk/clustalw/).
Statistical analysis. The trend in detection of M. tuberculosis and M. bovis by N-PCR in CSF samples derived from patients clinically categorized was determined by the trend chi-square test.
RESULTS
Two hundred twelve CSF samples were processed for the detection of M. tuberculosis and M. bovis by the N-PCR assay. Determination to the species level of the mycobacterial pathogens, namely, M. tuberculosis and M. bovis, present in the human CSF samples was established by molecular size analysis of the N-PCR products electrophoresed on a 10% polyacrylamide gel with appropriate controls.
Detection and identification of M. tuberculosis and M. bovis in CSF. The detection and differentiation of M. tuberculosis and M. bovis in representative CSF samples are depicted in Fig. 1. Figure 1B illustrates the single-band N-PCR products obtained in four of the five CSF samples (lanes 2, 3, 7, and 8). The PCR products in these samples were found to align with the PCR product obtained in the case of the standard M. bovis (Fig. 1B, lane 6) and was distinctly different from the PCR product obtained in the case of standard M. tuberculosis (Fig. 1B, lane 4).
Figure 1C shows the dual amplified bands occasionally detected on polyacrylamide gel electrophoresis. Two bands of 116 and 89 bp were seen in samples 71 and 83 (Fig. 1C, lanes 11 and 17), which were identical in molecular size to those generated in the dual positive control (Fig. 1C, lane 16). The higher-molecular-weight PCR product in lanes 11 and 17 (Fig. 1C) corresponded to and aligned with the PCR product obtained for M. tuberculosis (Fig. 1C, lane 15). The lower band corresponded to and aligned with the standard M. bovis strain (Fig. 1C, lane 13). Single bands matching the standard M. bovis and M. tuberculosis strains were seen in lanes 12 and 18, respectively (Fig. 1C). These assorted patterns obtained for the 112 CSF samples from the AIIMS hospital investigated have been summarized in Table 2.
Of the 112 samples (neurology ward, AIIMS hospital) investigated, 37 (33%) were positive for M. tuberculosis and M. bovis by N-PCR (Table 2). A mixed pattern of amplified PCR products was seen in the CSF samples (Table 2). In 17% (19/112) of the samples, M. bovis was detected. Infection with M. tuberculosis alone was detected in 2.7% (3/112) of the samples investigated. Simultaneous infection with both pathogens was established in 15/112 samples (13.4%). In 12.5% (14/112) of the samples, AFB were detected microscopically (Table 2). In all samples positive for AFB, M. tuberculosis and/or M. bovis were detected by N-PCR. However, a limited number of the smear-negative samples (23/112) were positive by N-PCR.
Twenty-seven of the 100 CSF samples from the pediatric ward of Safdarjung Hospital were found to be positive for M. tuberculosis and/or M. bovis by N-PCR assay. The positive-case distribution was as follows: 3 samples were positive for M. tuberculosis, 17 were positive for M. bovis, and mixed infection was detected in 7 samples.
Clinical categorization of patients. The 69 patients from the AIIMS hospital were categorized into definite (n = 9), highly probable (n = 3), probable (n = 5), and possible (n = 8) TBM and NTBM (n = 44) (Table 3) on the basis of the criteria described in Table 1 (1).
Correlation of clinical categorization of 69 patients with smear microscopy and N-PCR results. The results of the N-PCR were matched with the clinical categorization and smear microscopy results (Table 3). Positivity in the case of smear microscopy was limited to the nine cases grouped as definite TBM. The trend in detection of M. tuberculosis and M. bovis in CSF corresponded to the clinical classification of the patients (Fig. 2). All patients classified as definite TBM were positive by N-PCR. However, the N-PCR positivity for M. tuberculosis and M. bovis decreased with the decreased probability of clinical assessment of TBM (Table 3). Five of eight (62.5%), 1/8 (12.5%), and 5/44 (11.4%) cases classified as highly probable-to-probable TBM, possible TBM, and NTBM, respectively, were N-PCR positive. Comparative scrutiny of three parameters, namely, clinical diagnosis, smear microscopy, and N-PCR, showed that the sensitivity of the N-PCR (60.0%) was greater than that of smear microscopy (36.0%, Table 4). The specificity was 88.6% compared to that of smear microscopy (Table 4).
Sequence analysis of single or dual N-PCR products in a representative CSF sample. The dual bands obtained from CSF samples were extracted and cloned individually into the pGEMT vector as described in Materials and Methods. The sequence analyses of the cloned inserts is represented in Fig. 3B to E. Each insert was compared with the sequence of the C-terminal end of the hupB gene of M. tuberculosis (tb-116) and M. bovis (bo-89). Figure 3A depicts the sequence alignment of the N-PCR products of the C-terminal parts of the hupB gene of M. tuberculosis and M. bovis, showing the 27-bp difference between the two products (32).
Figure 3B and C show a comparative sequence analysis of the clone containing the higher-molecular-weight N-PCR product (116 bp) as an insert. The sequence has been compared with those of M. tuberculosis (tb-116, panel C) and M. bovis (bo-89, panel B). Complete identity was seen with that of M. tuberculosis (Fig. 3C). Comparison with the sequence of M. bovis (Fig. 3B) revealed an additional 27 bp in the sequence of the cloned insert. Hence, it was inferred that the higher-molecular-weight N-PCR product was derived from M. tuberculosis.
Figure 3D and E show a comparative sequence analysis of the clone containing the lower-molecular-weight N-PCR product (89 bp) as an insert. Complete identity was seen with that of M. bovis (bo-89, Fig. 3D). However, comparison of the insert sequence with that of M. tuberculosis (tb-116, Fig. 3E) revealed a discrepancy in the alignment; this was due to the additional 27 bp in the larger hupB gene of M. tuberculosis. This additional stretch of 27 bp was absent in the insert sequence. Therefore, it was concluded that the lower-molecular-weight N-PCR product was derived from M. bovis.
Direct DNA sequencing of N-PCR products of 10 samples that gave an amplified product equivalent to that of M. bovis on a polyacrylamide gel was done. All sequences of these samples matched the hupB gene sequence of M. bovis (bo-89). Hence, it was concluded that the N-PCR products of these samples were derived from M. bovis.
DISCUSSION
Though human TB is caused mainly by M. tuberculosis, an unknown proportion of cases is due to M. bovis (10). Human TB due to M. bovis is rare in developed nations. This is mainly due to the practice of animal TB control and elimination programs, together with milk pasteurization (15). In developing countries, animal TB is widespread and therefore constitutes a potential infectious source for humans (zoonotic TB). In Africa, approximately 85% of cattle and 82% of the human population live in areas where the disease is prevalent (10). Human disease caused by M. bovis has been confirmed in several African countries (28), France (35), Australia (11), and England (14). Infection of humans with M. bovis occurs mainly by consumption of contaminated milk or other dairy or meat products. The oral route has been the most common route of infection. Besides the oral route, inhalation of aerosolized infectious pathogens from infected animals has been considered to be a potential port of entry into susceptible hosts (3). Due to differences in the routes of infection, M. bovis is more likely to cause nonpulmonary disease (15). There are limited reports in India (27, 39, 40) and in underdeveloped countries (10, 16) relating to the prevalence of infection of cattle with M. tuberculosis and/or M. bovis. In India, there are no surveys to date to assess the public health problems posed by zoonotic TB. Human infection with M. bovis in immunocompetent, as well as in immunocompromised, individuals has been reported (3, 9, 16, 21, 25, 33). The epidemic of human immunodeficiency virus in developing countries, particularly where M. bovis infection prevails in animals and conditions favor zoonotic transmission, constitutes a serious public health threat (2).
The present study focused on the diagnosis of TBM. The tests currently used for rapid diagnosis of TBM are limited to the detection of AFB in smears, followed by culture and biochemical tests. However, both of these approaches are limited in the ability to identify the mycobacterial pathogen to the species level. Moreover, the commonly used Lowenstein-Jensen medium for primary isolation of mycobacteria from clinical samples is not conducive to M. bovis isolation (17). Hence, the isolation of M. bovis from clinical samples has been scanty. Further, TB caused by M. tuberculosis in humans is clinically, radiologically, and histopathologically indistinguishable from TB caused by M. bovis (44).
The problems associated with the sensitivity of TBM diagnosis by smear microscopy and the prolonged time taken by culture have been overcome by molecular techniques (4, 7, 9, 20, 22, 42). However, most of these techniques have been limited to detection of pathogenic mycobacteria as belonging to the MTC. Therefore, they are incapable of differential identification of the members of the MTC. Unlike these targets, the hupB gene target used in the N-PCR assay has the potential advantage of detecting and identifying the closely related members of the MTC, viz., M. tuberculosis and M. bovis (32). The utility of the hupB gene target for differentiating M. tuberculosis and M. bovis not only in isolates but also directly in bovine specimens has been previously demonstrated (26). This is an advantage over the other assays, which target multiple loci and genes to differentiate M. tuberculosis from M. bovis (19, 34). Identification to the species level of members of the MTC present in a sample would not be important from the viewpoint of chemotherapy, as both M. tuberculosis and M. bovis have been shown to respond to standard chemotherapeutic regimens, with the exception of pyrazinamide (29). However, identification to the species level would be important in determining the transmission chain, reservoirs of infection, and prevalence of mycobacterial pathogens in suspected cases of TBM. This, in turn, would be useful in formulating preventive therapies and strategies in the interest of public health. The differential ability of BCG immunization to generate effective immunity against TBM among children in particular (23, 36) and its inability to provide protection against pulmonary TB in adults (8) indirectly indicate the role of M. bovis as a causative agent of pediatric TBM, as substantiated by the present study.
The notable aspect of the N-PCR assay has been the detection of M. tuberculosis and M. bovis infections together in CSF samples. Detection of mixed infection with more than one strain of M. tuberculosis in human clinical samples by molecular techniques has been reported (5, 31, 38, 43). Mixed infections in these reports were established by the definite patterns in DNA fingerprints observed in isolates from a single sample. Besides DNA fingerprinting, spoligotyping and amplification of specific gene targets have been used to identify M. tuberculosis and M. bovis in archival tissue (45). Kidane et al. (21) have detected mixed infection with M. tuberculosis and M bovis in human lymphadenitis. In this study, the patients in whom M. bovis or mixed infection was detected by the N-PCR assay were equally distributed in rural and urban areas. The increased risk of TBM in these individuals perhaps relates to not only their socioeconomic standards, reflecting their nutritional status, but also their poor living standards, overcrowding, and the prevailing unsanitary conditions. In our earlier study (26), we had demonstrated both by culture and N-PCR assay the prevalence of M. tuberculosis, M. bovis, and mixed infections in bovines. As both in the villages and cities no restriction is placed on the movement of dairy or stray cattle, owing to economic compulsions and religious norms prevalent in our country and the high frequency of individuals coming in contact with these animals, there is a potential for transmission of these pathogens from infected cattle to humans (zoonosis) and from infected humans to cattle (reverse zoonosis) (13).
In summary, this study shows that infection by members of the MTC can be determined with appropriate molecular techniques and defined gene targets. This would help in constituting appropriate strategies for prevention of human TB by mycobacterial pathogens other than the usual M. tuberculosis.
ACKNOWLEDGMENTS
A.J. was a post-MD trainee at AIIMS, a program supported by the Department of Biotechnology, Government of India. Senior research fellowships awarded to A.S. and P.K. from the University Grants Commission and the India Council of Medical Research are acknowledged. The financial support of DBT, Government of India, is acknowledged.
N.P.S. and A.S. contributed equally to this report.
Present address: Department of Biochemistry, Lady Hardinge Medical College, New Delhi 110001, India.
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Department of Pediatrics, Safdarjung Hospital, New Delhi 110029, India
ABSTRACT
The paucibacillary nature of the cerebrospinal fluid (CSF) has been a major obstacle in the diagnosis of human tuberculous meningitis (TBM). This study shows that with molecular techniques direct precise determination to the species level of mycobacterial pathogens can be made. The present report describes the utility of a nested PCR (N-PCR) assay (A. Mishra, A. Singhal, D. S. Chauhan, V. M. Katoch, K. Srivastava, S. S. Thakral, S. S. Bharadwaj, V. Sreenivas, and H. K. Prasad, J. Clin. Microbiol. 43:5670-5678, 2005) in detecting M. tuberculosis and M. bovis in human CSF. In 2.8% (6/212) of the samples, M. tuberculosis was detected, and in 17% (36/212), M. bovis was detected. Mixed infection was observed in 22 samples. Comparative analysis of clinical diagnosis, smear microscopy, and N-PCR in 69 patients (TBM, 25; non-TBM, 44) showed that the sensitivity of N-PCR (61.5%) was greater than that of smear microscopy (38.4%). Determination to the species level is important from the viewpoint of determining the prevalence of these mycobacteria in a community and would influence strategies currently adopted for the prevention of tuberculosis.
INTRODUCTION
Tuberculosis (TB) is a chronic, systemic infectious disease caused by Mycobacterium tuberculosis. The most common clinical manifestation is pulmonary TB. The inhaled bacilli can localize in alternate sites, leading to extrapulmonary TB (EPTB). Among the different manifestations of EPTB, tuberculous meningitis (TBM) has been considered to be a fatal form (41). Fatality rates in developing countries have been reported to range from 44 to 69% (15, 18, 30). In fact, delayed or erroneous diagnosis often results in serious long-term debilitating complications. Central nervous system involvement has frequently been found secondary to TB elsewhere in the body, particularly the lungs. The presence of TB elsewhere in the body favors the diagnosis, although its absence does not exclude it. The great majority of patients with neuro-TB are diagnosed on the basis of clinical criteria, imaging, and laboratory investigation of the cerebrospinal fluid (CSF). The clinical response to antituberculosis therapy in all forms of neuro-TB is excellent, provided the diagnosis is made early, before an irreversible neurological defect occurs.
Hence, precise and rapid clinical diagnosis of TBM is a critical component of the management of TBM patients (12, 24, 25). Culture and Ziehl-Neelsen staining of the CSF are specific but insensitive due to the paucity of bacilli. Nucleic acid amplification techniques have shown promising results in overcoming this drawback (20, 22, 30, 42).
M. tuberculosis is a member of the M. tuberculosis complex (MTC), which includes M. bovis, "M. canetti," M. microti, and M. africanum. M. bovis infection of the central nervous system may lead to EPTB, similar to M. tuberculosis infection, with identical symptomatologies (10, 16). M. bovis infection has been reported in immunocompetent, as well as in immunosuppressed, individuals (9, 21, 25, 33). Hence, the present study was undertaken to detect M. tuberculosis and M. bovis in human CSF by using an in-house nested PCR (N-PCR) assay (26). The assay targets the 27-bp difference in the C terminus of the hupB gene in M. tuberculosis (Rv2986c) and M. bovis (Mb3010c) to differentiate M. tuberculosis from M. bovis (32). The utility of the assay for direct detection of M. tuberculosis and M. bovis in bovine samples was confirmed in comparison with standard culture techniques (26).
MATERIALS AND METHODS
Patients. CSF samples from 212 patients were investigated. The patient distribution was as follows. For the first part of the study, 112 patients admitted to the neurology ward of the All India Institute of Medical Sciences (AIIMS), New Delhi, were investigated. Subsequently, for the second part of the study, CSF samples from 100 children (12 years old) admitted to the pediatric ward of Safdarjung Hospital, New Delhi, India, were investigated. The institutional ethical committee approved the study. At the time of data analysis, the AIIMS hospital records of the 69 patients were obtained. These 69 cases were separated into TBM and NTBM (nontubercular meningitis) groups on the basis of the criteria described by Ahuja et al. (1) (Table 1). CSF was collected under aseptic conditions by lumbar puncture. Five hundred microliters to 2.0 ml of CSF was available for the study. Samples were stored at –20°C, prior to processing for target DNA for N-PCR and smear microscopy for acid-fast bacilli (AFB; auramine O stain).
Mycobacterial strains. M. tuberculosis H37Rv DNA was obtained from TB research material, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, Md. Lowenstein-Jensen slant cultures of M. tuberculosis H37Rv and M. bovis AN5 were obtained from the Central JALMA Institute for Leprosy and Other Mycobacterial Diseases, Agra, India.
Preparation of mycobacterial cell lysates containing target DNA. (i) Mycobacterial cultures. One milliliter of a logarithmic-phase culture of M. tuberculosis or M. bovis grown in 7H9 Middlebrook broth was pelleted and resuspended in 100 μl of 0.1% of Triton X-100. The suspension was boiled (90°C for 40 min) and centrifuged (10,000 x g for 10 min). The supernatant was stored at –20°C and used as template DNA in a PCR.
(ii) CSF. Target DNA was prepared from CSF by a modification of the method of Chakravorty and Tyagi (6). CSF was filtered (0.22-μm pore size; Millipore). The CSF container was rinsed with 2 ml of inhibitory removing solution (25 M guanidinium isothiocyanate, 0.025 M EDTA, 0.05 M Tris, 0.5% Sarkosyl, 0.186 M -mercaptoethanol, pH 7.5) and filtered. The membrane was transferred to a 1.5-ml Eppendorf tube containing 500 μl of inhibitory removing solution, incubated for 15 min at room temperature, and centrifuged (10,000 x g for 10 min). The supernatant was discarded, and the membrane was washed with sterile water. Two hundred microliters of 0.1% Triton X-100 was added to the membrane and vortexed vigorously. The suspension was used to make smears and subsequently boiled in a dry bath (90°C for 40 min), followed by centrifugation (10,000 x g for 10 min). The supernatant was stored at –20°C and used as template DNA.
N-PCR assay for hupB DNA target (international patent application PCT/IN03/00302, 1 July 2004, L. S. Davar and Co., Calcutta, India). Primers N (5'-GGAGGGTTGGGATGAACAAAGCAG-3') and S (5'-GTATCCGTGTGTCTTGACCTATTTG-3') were used to amplify the hupB gene as previously described (N-S PCR, Fig. 1A) (32). The N-PCR assay for the C-terminal portion of the hupB gene was carried out with primers F (5'-CCAAGAAGGCGA CAAAGG-3') and R (5'-GACAGCTTTCTTGGCGGG-3') (Fig. 1A) (26). Four microliters of the amplified product of a PCR with primers N and S was used as the template DNA for the N-PCR. Each reaction mixture (40 μl) of the N-PCR contained 1.25 mM MgCl2, 200 μM deoxynucleoside triphosphates, 0.5 μM primers F and R, 10 mM Tris-HCl (pH 8.8), 50 mM KCl, 0.08% Nonidet P-40, and 1.0 U of Taq DNA polymerase. The reaction mixture was subjected to initial denaturation at 94°C for 10 min and 35 cycles each of 1.0 min at 94°C and annealing and extension at 59°C for 0.30 min, followed by a final extension at 72°C for 7 min. The products were analyzed on a 10% polyacrylamide gel and stained with ethidium bromide. The expected sizes of amplicons for M. tuberculosis and M. bovis were 116 and 89 bp, respectively. The limit of detection was 50 fg, which is equivalent to five tubercle bacilli (26). Further, in an earlier report, the hupB-based PCR assay was shown to be specific for M. tuberculosis and M. bovis when tested with members of the MTC, as well as with other mycobacterial species (32). N-PCR products generated from standard strains of M. tuberculosis and M. bovis were included (positive control). A dual positive control was also included to discern mixed infection with M. tuberculosis and M. bovis. The N-PCR products in this case were generated from a mixture of the target DNAs of M. tuberculosis H37Rv and M. bovis AN5.
DNA sequencing. Primer F- and R-amplified N-PCR products from 10 samples that showed an 89-bp product of M. bovis were eluted from agarose gel with a mini Elute Gel Extraction kit (QIAGEN). The extracted DNA was lyophilized and sent for sequencing to the DNA Sequencing Resource Center at The Rockefeller University.
Alternatively, with CSF samples showing mixed infection, both of the bands (116 and 89 bp) were cut from a 10% polyacrylamide gel and DNA was extracted by the crush-and-soak method as previously described (37). These extracted N-PCR products were cloned into the pGEMT vector with a TA cloning kit (Promega) according to the manufacturer's instructions. The clones were sequenced at the DNA sequencing facility, South Campus Delhi University, New Delhi, India.
The sequences obtained were aligned with the sequences of M. tuberculosis and M. bovis with the CLUSTALW software (http://www.ebi.ac.uk/clustalw/).
Statistical analysis. The trend in detection of M. tuberculosis and M. bovis by N-PCR in CSF samples derived from patients clinically categorized was determined by the trend chi-square test.
RESULTS
Two hundred twelve CSF samples were processed for the detection of M. tuberculosis and M. bovis by the N-PCR assay. Determination to the species level of the mycobacterial pathogens, namely, M. tuberculosis and M. bovis, present in the human CSF samples was established by molecular size analysis of the N-PCR products electrophoresed on a 10% polyacrylamide gel with appropriate controls.
Detection and identification of M. tuberculosis and M. bovis in CSF. The detection and differentiation of M. tuberculosis and M. bovis in representative CSF samples are depicted in Fig. 1. Figure 1B illustrates the single-band N-PCR products obtained in four of the five CSF samples (lanes 2, 3, 7, and 8). The PCR products in these samples were found to align with the PCR product obtained in the case of the standard M. bovis (Fig. 1B, lane 6) and was distinctly different from the PCR product obtained in the case of standard M. tuberculosis (Fig. 1B, lane 4).
Figure 1C shows the dual amplified bands occasionally detected on polyacrylamide gel electrophoresis. Two bands of 116 and 89 bp were seen in samples 71 and 83 (Fig. 1C, lanes 11 and 17), which were identical in molecular size to those generated in the dual positive control (Fig. 1C, lane 16). The higher-molecular-weight PCR product in lanes 11 and 17 (Fig. 1C) corresponded to and aligned with the PCR product obtained for M. tuberculosis (Fig. 1C, lane 15). The lower band corresponded to and aligned with the standard M. bovis strain (Fig. 1C, lane 13). Single bands matching the standard M. bovis and M. tuberculosis strains were seen in lanes 12 and 18, respectively (Fig. 1C). These assorted patterns obtained for the 112 CSF samples from the AIIMS hospital investigated have been summarized in Table 2.
Of the 112 samples (neurology ward, AIIMS hospital) investigated, 37 (33%) were positive for M. tuberculosis and M. bovis by N-PCR (Table 2). A mixed pattern of amplified PCR products was seen in the CSF samples (Table 2). In 17% (19/112) of the samples, M. bovis was detected. Infection with M. tuberculosis alone was detected in 2.7% (3/112) of the samples investigated. Simultaneous infection with both pathogens was established in 15/112 samples (13.4%). In 12.5% (14/112) of the samples, AFB were detected microscopically (Table 2). In all samples positive for AFB, M. tuberculosis and/or M. bovis were detected by N-PCR. However, a limited number of the smear-negative samples (23/112) were positive by N-PCR.
Twenty-seven of the 100 CSF samples from the pediatric ward of Safdarjung Hospital were found to be positive for M. tuberculosis and/or M. bovis by N-PCR assay. The positive-case distribution was as follows: 3 samples were positive for M. tuberculosis, 17 were positive for M. bovis, and mixed infection was detected in 7 samples.
Clinical categorization of patients. The 69 patients from the AIIMS hospital were categorized into definite (n = 9), highly probable (n = 3), probable (n = 5), and possible (n = 8) TBM and NTBM (n = 44) (Table 3) on the basis of the criteria described in Table 1 (1).
Correlation of clinical categorization of 69 patients with smear microscopy and N-PCR results. The results of the N-PCR were matched with the clinical categorization and smear microscopy results (Table 3). Positivity in the case of smear microscopy was limited to the nine cases grouped as definite TBM. The trend in detection of M. tuberculosis and M. bovis in CSF corresponded to the clinical classification of the patients (Fig. 2). All patients classified as definite TBM were positive by N-PCR. However, the N-PCR positivity for M. tuberculosis and M. bovis decreased with the decreased probability of clinical assessment of TBM (Table 3). Five of eight (62.5%), 1/8 (12.5%), and 5/44 (11.4%) cases classified as highly probable-to-probable TBM, possible TBM, and NTBM, respectively, were N-PCR positive. Comparative scrutiny of three parameters, namely, clinical diagnosis, smear microscopy, and N-PCR, showed that the sensitivity of the N-PCR (60.0%) was greater than that of smear microscopy (36.0%, Table 4). The specificity was 88.6% compared to that of smear microscopy (Table 4).
Sequence analysis of single or dual N-PCR products in a representative CSF sample. The dual bands obtained from CSF samples were extracted and cloned individually into the pGEMT vector as described in Materials and Methods. The sequence analyses of the cloned inserts is represented in Fig. 3B to E. Each insert was compared with the sequence of the C-terminal end of the hupB gene of M. tuberculosis (tb-116) and M. bovis (bo-89). Figure 3A depicts the sequence alignment of the N-PCR products of the C-terminal parts of the hupB gene of M. tuberculosis and M. bovis, showing the 27-bp difference between the two products (32).
Figure 3B and C show a comparative sequence analysis of the clone containing the higher-molecular-weight N-PCR product (116 bp) as an insert. The sequence has been compared with those of M. tuberculosis (tb-116, panel C) and M. bovis (bo-89, panel B). Complete identity was seen with that of M. tuberculosis (Fig. 3C). Comparison with the sequence of M. bovis (Fig. 3B) revealed an additional 27 bp in the sequence of the cloned insert. Hence, it was inferred that the higher-molecular-weight N-PCR product was derived from M. tuberculosis.
Figure 3D and E show a comparative sequence analysis of the clone containing the lower-molecular-weight N-PCR product (89 bp) as an insert. Complete identity was seen with that of M. bovis (bo-89, Fig. 3D). However, comparison of the insert sequence with that of M. tuberculosis (tb-116, Fig. 3E) revealed a discrepancy in the alignment; this was due to the additional 27 bp in the larger hupB gene of M. tuberculosis. This additional stretch of 27 bp was absent in the insert sequence. Therefore, it was concluded that the lower-molecular-weight N-PCR product was derived from M. bovis.
Direct DNA sequencing of N-PCR products of 10 samples that gave an amplified product equivalent to that of M. bovis on a polyacrylamide gel was done. All sequences of these samples matched the hupB gene sequence of M. bovis (bo-89). Hence, it was concluded that the N-PCR products of these samples were derived from M. bovis.
DISCUSSION
Though human TB is caused mainly by M. tuberculosis, an unknown proportion of cases is due to M. bovis (10). Human TB due to M. bovis is rare in developed nations. This is mainly due to the practice of animal TB control and elimination programs, together with milk pasteurization (15). In developing countries, animal TB is widespread and therefore constitutes a potential infectious source for humans (zoonotic TB). In Africa, approximately 85% of cattle and 82% of the human population live in areas where the disease is prevalent (10). Human disease caused by M. bovis has been confirmed in several African countries (28), France (35), Australia (11), and England (14). Infection of humans with M. bovis occurs mainly by consumption of contaminated milk or other dairy or meat products. The oral route has been the most common route of infection. Besides the oral route, inhalation of aerosolized infectious pathogens from infected animals has been considered to be a potential port of entry into susceptible hosts (3). Due to differences in the routes of infection, M. bovis is more likely to cause nonpulmonary disease (15). There are limited reports in India (27, 39, 40) and in underdeveloped countries (10, 16) relating to the prevalence of infection of cattle with M. tuberculosis and/or M. bovis. In India, there are no surveys to date to assess the public health problems posed by zoonotic TB. Human infection with M. bovis in immunocompetent, as well as in immunocompromised, individuals has been reported (3, 9, 16, 21, 25, 33). The epidemic of human immunodeficiency virus in developing countries, particularly where M. bovis infection prevails in animals and conditions favor zoonotic transmission, constitutes a serious public health threat (2).
The present study focused on the diagnosis of TBM. The tests currently used for rapid diagnosis of TBM are limited to the detection of AFB in smears, followed by culture and biochemical tests. However, both of these approaches are limited in the ability to identify the mycobacterial pathogen to the species level. Moreover, the commonly used Lowenstein-Jensen medium for primary isolation of mycobacteria from clinical samples is not conducive to M. bovis isolation (17). Hence, the isolation of M. bovis from clinical samples has been scanty. Further, TB caused by M. tuberculosis in humans is clinically, radiologically, and histopathologically indistinguishable from TB caused by M. bovis (44).
The problems associated with the sensitivity of TBM diagnosis by smear microscopy and the prolonged time taken by culture have been overcome by molecular techniques (4, 7, 9, 20, 22, 42). However, most of these techniques have been limited to detection of pathogenic mycobacteria as belonging to the MTC. Therefore, they are incapable of differential identification of the members of the MTC. Unlike these targets, the hupB gene target used in the N-PCR assay has the potential advantage of detecting and identifying the closely related members of the MTC, viz., M. tuberculosis and M. bovis (32). The utility of the hupB gene target for differentiating M. tuberculosis and M. bovis not only in isolates but also directly in bovine specimens has been previously demonstrated (26). This is an advantage over the other assays, which target multiple loci and genes to differentiate M. tuberculosis from M. bovis (19, 34). Identification to the species level of members of the MTC present in a sample would not be important from the viewpoint of chemotherapy, as both M. tuberculosis and M. bovis have been shown to respond to standard chemotherapeutic regimens, with the exception of pyrazinamide (29). However, identification to the species level would be important in determining the transmission chain, reservoirs of infection, and prevalence of mycobacterial pathogens in suspected cases of TBM. This, in turn, would be useful in formulating preventive therapies and strategies in the interest of public health. The differential ability of BCG immunization to generate effective immunity against TBM among children in particular (23, 36) and its inability to provide protection against pulmonary TB in adults (8) indirectly indicate the role of M. bovis as a causative agent of pediatric TBM, as substantiated by the present study.
The notable aspect of the N-PCR assay has been the detection of M. tuberculosis and M. bovis infections together in CSF samples. Detection of mixed infection with more than one strain of M. tuberculosis in human clinical samples by molecular techniques has been reported (5, 31, 38, 43). Mixed infections in these reports were established by the definite patterns in DNA fingerprints observed in isolates from a single sample. Besides DNA fingerprinting, spoligotyping and amplification of specific gene targets have been used to identify M. tuberculosis and M. bovis in archival tissue (45). Kidane et al. (21) have detected mixed infection with M. tuberculosis and M bovis in human lymphadenitis. In this study, the patients in whom M. bovis or mixed infection was detected by the N-PCR assay were equally distributed in rural and urban areas. The increased risk of TBM in these individuals perhaps relates to not only their socioeconomic standards, reflecting their nutritional status, but also their poor living standards, overcrowding, and the prevailing unsanitary conditions. In our earlier study (26), we had demonstrated both by culture and N-PCR assay the prevalence of M. tuberculosis, M. bovis, and mixed infections in bovines. As both in the villages and cities no restriction is placed on the movement of dairy or stray cattle, owing to economic compulsions and religious norms prevalent in our country and the high frequency of individuals coming in contact with these animals, there is a potential for transmission of these pathogens from infected cattle to humans (zoonosis) and from infected humans to cattle (reverse zoonosis) (13).
In summary, this study shows that infection by members of the MTC can be determined with appropriate molecular techniques and defined gene targets. This would help in constituting appropriate strategies for prevention of human TB by mycobacterial pathogens other than the usual M. tuberculosis.
ACKNOWLEDGMENTS
A.J. was a post-MD trainee at AIIMS, a program supported by the Department of Biotechnology, Government of India. Senior research fellowships awarded to A.S. and P.K. from the University Grants Commission and the India Council of Medical Research are acknowledged. The financial support of DBT, Government of India, is acknowledged.
N.P.S. and A.S. contributed equally to this report.
Present address: Department of Biochemistry, Lady Hardinge Medical College, New Delhi 110001, India.
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