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RIGINAL
ARTICLE
Establishment of a New Zealand Rabbit Model of Spinal Tuberculosis Guangqi Geng, MD,* Qian Wang, MD,w Jiandang Shi, MD,* Junfa Yan, MS,z Ningkui Niu, MD,* and Zili Wang, MD*
Study Design: This was an experimental study. Objective: To investigate and evaluate the experimental method of establishing a New Zealand rabbit model of spinal tuberculosis. Summary of Background Data: Establishing animal models of tuberculosis is critical to the experimental and clinical study of tuberculosis, especially spinal tuberculosis. However, the rapid spread of Mycobacterium tuberculosis and subsequent high mortality thwarted their effort. Since then, no animal models have been established of spinal tuberculosis. Methods: Forty-two New Zealand rabbits were randomly divided into experimental (n = 20), control (n = 20), and blank groups (n = 2). Experimental animals were sensitized by complete Freund’s adjuvant. A hole drilled under the upper endplate of the L4 vertebral body was filled with a gelfoam sponge infused with 0.1 mL H37Rv standard M. tuberculosis suspension (in controls, culture medium, and saline). Blank animals received no treatment. Results: Survival 8 weeks after surgery was 89.5%, 94.7%, and 100% in experimental, control, and blank groups, respectively. The model was successfully established in all surviving experimental rabbits. In experimental animals, vertebral body destruction at 4 weeks was 50% by x-ray; 83.3% by computed tomography reconstruction and magnetic resonance imaging; at 8 weeks, 58.8% by x-ray and 100% by computed tomograph reconstruction and magnetic resonance imaging. At 8 weeks, experimental animals developed vertebral destruction, granulation, and necrosis and 17.6% had psoas abscess. Histopathology revealed numerous lymphocytes and epithelioid cells, trabecular bone fracture, and coagulative necrosis in the vertebrae of ex-
Received for publication July 10, 2014; accepted September 26, 2014. From the *Department of Spinal Surgery, General Hospital of Ningxia Medical University, Yinchuan, China; wUniversity of South Florida, Morsani College of Medicine,Tampa, FL; and zNingxia Medical University, Yinchuan, China. G.G., Q.W., and J.S. contributed equally to this work and should be considered as co-first authors. Supported by National Natural Science Foundation of China (Item Number: 81060149) and Ningxia Natural Science Foundation (Item Number: NZ10117). These funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. The authors declare no conflict of interest. Reprints: Zili Wang, MD, Department of Spinal Surgery, General Hospital of Ningxia Medical University, 804 Shengli Street, Yinchuan 750004, China (e-mail:
[email protected]). Copyright r 2014 Wolters Kluwer Health, Inc. All rights reserved.
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perimental animals; bacterium culture was 52.9% positive. Control and blank animals showed no such changes. Conclusions: A New Zealand rabbit of spinal tuberculosis model can be successfully established by drilling a hole in the upper endplate of the vertebral body, filling with gelfoam sponge infused with H37Rv standard M. tuberculosis suspension after sensitization by complete Freund’s adjuvant. Key Words: spinal tuberculosis, animal model, complete Freund’s adjuvant, New Zealand rabbit, Mycobacterium tuberculosis (J Spinal Disord Tech 2015;28:E140–E145)
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stablishing animal models of tuberculosis is critical to the experimental and clinical study of tuberculosis,1–3 especially spinal tuberculosis. As early as 1969, Hodgson et al4 injected Mycobacterium tuberculosis into the paraspinal tissue of unsensitized experimental animals to establish an animal model. However, the rapid spread of M. tuberculosis and subsequent high mortality thwarted their effort. Since then, no animal models have been established of spinal tuberculosis. However, the resurgence of tuberculosis, the increased incidence of spinal tuberculosis, and the rising rate of reoperation all require the basic research that rests on animal models.5 In the current study, we successfully established a New Zealand rabbit spinal tuberculosis model after sensitizing the rabbits with complete Freund’s adjuvant and carried out comprehensive evaluation of the model by imaging, histopathologic, and bacteriological studies.
METHODS All animals received humane care in compliance with the Guide for the Care and Use of Laboratory Animals, published by the US National Institutes of Health (Publication No. 85-23, revised 1996). The experiment protocol and animal welfare was approved by the Animal Ethics Committee at Ningxia Medical University. All surgery was performed under sodium pentobarbital anesthesia, and all efforts were made to minimize suffering.
Experimental M. tuberculosis, Main Reagents, and Instruments Human M. tuberculosis strain H37Rv was obtained from the China Institute of Biological Products (Wuhan, J Spinal Disord Tech
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China). Main reagents included complete Freund’s adjuvant (Sigma Aldrich, St. Louis, MO), modified Sauton medium, and modified Lowenstein-Jensen medium. Model establishment was completed in a P3 laboratory. Instruments included a Computed Radiography Digital Imaging System (Neu-Alpine, Shenyang, China), a Brilliance 64-slice CT scanner (Philips, Amsterdam, the Netherlands), a MAGNETOM Symphony 1.5 T MRI system (Siemens AG, Munich, Germany), and a digital microscope color camera (Leica Camera AG, Solms, Germany).
Management of M. tuberculosis Human M. tuberculosis strain H37Rv was cultured for 2 to 3 weeks. Well-grown colonies were selected and placed in 0.05% Tween-80 normal saline. Five mg/mL suspension was produced by McFarland nephelometry (presumably an OD 600), preserved in a 41C refrigerator (no >24 h), and shaken well before application. Medium and normal saline were mixed to prepare 5 mg/mL suspension for the control group.
Experimental Animals and Grouping Forty-two adults (8–12 weeks) New Zealand white rabbits, regardless of sex, 3 months age, weighing 2.5–3.0 kg, were provided by Dilepu Experimental Animal Center (Xian, Shaanxi, China) [License No. of medical experimental animal production: SCXK (Shaanxi) 2008001, permit No. 20090235]. The experimental protocol was approved by the appropriate ethics committee of the university. Tuberculin purified protein derivative 5 IU tests were carried out before the experiment and animals with negative results were used for the current study. The rabbits were divided randomly into 3 groups using a random table: 20 in the experimental group, 20 in the control group, and 2 in the blank group. During experiment, all rabbits were raised separately in individual cages with a standardized living environment and feeding patterns. The animal house was sterilized by ultraviolet radiation for 1 hour per day. At the end of the experiment, the rabbits were killed, sealed, and cremated.
Preparation of Animal Model Sensitization With Complete Freund’s Adjuvant One month before surgery, the 20 New Zealand rabbits in the experimental group received 0.1 mL complete Freund’s adjuvant (a mixture of lanolin and liquid paraffin containing 4.5 mg/mL bovine M. tuberculosis) through an intradermal injection in the nape of the lower neck; rabbits in the control group and blank group did not receive this procedure.
Anesthesia Rabbits were injected with 3% sodium pentobarbital (30 mg/kg) via the auricular vein. During surgery, local anesthesia was applied, using 5–10 mL 0.5% lidocaine. Copyright
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Rabbit Model of Spinal Tuberculosis
Implantation of M. tuberculosis After skin preparation, rabbits were placed in the right lateral decubitus position and routine skin sterilization and draping were carried out. A longitudinal incision of 6 cm was made along the end of the left 12th rib inferior to the iliac crest. The L3–4 disk space and adjacent vertebrae were exposed via the lateral approach. A cavity 3 mm in diameter and 5 mm in depth was drilled anteriorly from the left to the right posteriorly and 5 mm inferior to the upper endplate of the L4 vertebra at an angle of 30 degrees to the coronal plane. After hemostasis, the cavity was filled with gelfoam sponge, then 0.1 mL suspension was slowly infused into the gelfoam sponge. The same procedure was carried out in the control group, injecting normal saline mixed with broth into the gelfoam instead of M. tuberculosis. No specific treatment was performed in the blank group. The surgery was carried out in strict compliance with the principles of aseptic technique, standard precaution, and animal ethics.
Study Parameters Observation of General Conditions Survival, daily activities, mental states, eating activity, and wound infection of the animals were observed twice per day (approximately at 10 AM and 4 PM).
Imaging Studies Imaging studies were completed 8 weeks after injection of M. tuberculosis x-ray, computed tomography (CT) reconstruction, and magnetic resonance imaging (MRI) were performed the day M. tuberculosis was administered and at 4 and 8 weeks after surgery to observe the extent of destruction of the intervertebral disk and vertebral body and formation of sequestrum and abscess. All these checking procedures were performed under anesthesia.
Histopathologic Observation Rabbits in different groups were then dissected after anesthesia for gross observation of the target vertebra and adjacent normal vertebrae. Routine H&E staining was carried out for the affected intervertebral disk, its upper and lower endplates, and some parts of the vertebral body to observe histopathologic changes.
M. tuberculosis Culture During the 8 weeks operation, 0.5 g of granulation tissue was harvested from the operation site of all surviving rabbits in the experimental group and the control group. After homogenization, each specimen was separately placed in modified Lowenstein-Jensen medium, cultured at 361C, and continuously observed for 6 weeks. The results were analyzed in accordance with the standard of tuberculosis diagnosis and bacteriological testing published in 1995 by the Chinese Tuberculosis Prevention and Treatment Association.6 www.jspinaldisorders.com |
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RESULTS One rabbit in the experimental group died due to diarrhea 3 weeks after sensitization. Other rabbits in the experimental group had 0.8–2.0 cm induration on the nape of the neck without ulceration at 1 month after sensitization.
Animal Survival All rabbits were euthanized pharmacology, which consisted of the application of prior anesthesia, as mentioned above, associated with a dose of 2 mL of potassium chloride intravascularly. Animals were euthanized upon reaching a certain clinical condition (eg, a certain level of weight loss, anorexia, paraplegia, or other diseases), and all surviving animals were euthanized at the end of the 8-week study period. Survival of the animals after model establishment is provided in Table 1. In the experimental group, model establishment was completed in 19 rabbits. Among these, 1 rabbit died due to poor eating 4 weeks after surgery. The rabbit did not live for a long time and the tuberculosis focus was not found in the dead body; so it was considered that the main reason was related to postoperative trauma. The external environment was also taken as an unimportant part in the experiment. Another rabbit died 49 days after surgery; a large amount of pus was found within the chest cavity and diffuse nodules of varying sizes were observed on the lung surface. It was considered that the cause of death was as a result of multi-organ failure due to disseminated M. tuberculosis. The rabbit was not included in the result of this experiment because it did not complete all the experimental procedure. Hence, 17 rabbits survived to the end of the experiment; the model was successfully established in these rabbits. In the control group, one rabbit died due to anesthesia accident and another died on the 13th postoperative day due to poor eating. The remaining 18 rabbits recovered satisfactorily after surgery without significant changes in body weight. Both rabbits in the blank group ate well and survived to 8 weeks.
Imaging Findings The results of x-ray, CT, and MRI images at 8 weeks are shown in Figure 1. This was observed by 3 imaging experts. The number of animals with changes is recorded in Table 2. X-ray revealed that, in the experimental group, the intervertebral space appeared blurred and became narrowed with newly formed osteophytes. This intervertebral narrowing was found in 9 of 18 rabbits at 4 weeks and 10 of 17 at 8 weeks (Table 2). CT reconstruction showed sclerotic destruction, including irregular cotton-wool-like
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changes within the vertebral body, new bone formation at the margins of the vertebral body, and a narrowed or absent intervertebral space. Moreover, abscesses and soft tissue calcification could be observed. There was no obvious sequestrum or porosis (Figs. 1A, B). Intervertebral space narrowing and cotton-wool-like destruction of the vertebral body were both observed in 15 of 18 animals at 4 weeks and in all 17 animals at 8 weeks (Table 2). T1weighted imaging (T1WI) MRI showed low signals in the upper end and anterior margin of the vertebral body, which showed up as mixed high signals on T2-weighted imaging (T2WI). The intervertebral disk was shown as low signals (Figs. 1C–E). At 8 weeks, all 17 surviving experimental animals showed abnormal intervertebral spaces and cottonwool-like destruction of the vertebral body by CT reconstruction and abnormal intervertebral space/vertebral body on T1W1 and T2W1 MRI (Table 2). These x-ray and CT changes were not observed in the control group and blank group. MRI T1WI showed slightly low signals in the endplate, which were shown as slightly high signals in T2WI. There was no abnormal signal in the intervertebral disk. Detailed data are presented in Table 2.
Gross Observation The intervertebral body of normal adult New Zealand rabbits is thin and long with bulky ends and a narrow center. In the upper and lower ends, the maximum length of the anteroposterior diameter and mediolateral diameter are 8 and 10 mm, respectively. However, the anteroposterior diameter in the center of the vertebrae for rabbits is only about 2 mm. The vertebral body in the rabbits is predominantly composed of cortical bone. In all 17 surviving rabbits of the experimental group, at 8 weeks after surgery, we grossly observed varying degrees of intervertebral disk destruction, destruction of 2 adjacent endplates, formation of caseous necrotic substances, and granulation tissue. Psoas abscess was found in three rabbits. Only 1 rabbit died in the experimental group due to disseminated M. tuberculosis. However, no disseminated tuberculosis was found in the chest or other organs of the remaining 17 rabbits. In the control or blank groups, no destruction of the intervertebral disk or vertebral body and no abscess were found.
Histopathologic Observation Histopathologic study of the 17 rabbits in the experimental group showed trabecular destruction and small sequestrum formation in the affected vertebral bodies. This was discovered by 2 pathologist. Coagulative
TABLE 1. Animal Survival in the 3 Groups After Model Establishment Groups (n) Experimental group (20) Control group (20) Blank group (2)
Day of Sensitization
Day of Model Establishment
4 Weeks Post Model Establishment
8 Weeks Post Model Establishment
Survival Rate (%)
19
19
18
17
89.5
20 2
19 2
18 2
18 2
94.7 100
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Rabbit Model of Spinal Tuberculosis
FIGURE 1. The experimental group, 8 weeks after surgery, as shown by computed tomography (CT) reconstruction (A and B) and magnetic resonance imaging (MRI) (C–E). A, CT reconstruction in a sagittal plane shows a loss of the involved intervertebral space, prevertebral abscess, and calcification (the arrow shows prevertebral abscess); (B) CT reconstruction in the axial plane shows vertebral destruction with peripheral osteosclerosis (the arrow shows focus). C, T2-weighted imaging (T2WI) MRI of the sagittal view presenting a low-signal intervertebral disk with fusiform low-signal abscess at the anterior margin of the vertebral body (the arrow shows prevertebral abscess). D, T1-weighted imaging (T1WI) MRI of the sagittal view of (C). E, T2-weighted imaging (T2WI) MRI of the axial view presenting low-signal abscess at the anterior margin of the vertebral body (the arrows shows prevertebral abscess). The high-signal circular image is the abscess wall (the arrow shows prevertebral abscess).
necrosis and numerous lymphocytes and scant epithelial cells were present in the area of destruction. However, no multinucleated giant cells or typical tuberculosis nodules were observed. In the control group, trabecular bone structure was normal with mild inflammatory cell infiltration and no sequestrum or epithelioid cell formation.
Culture of M. tuberculosis The result of culture was positive in 9 of 17 rabbits in the experimental group, in which slightly yellow colonies were evenly and firmly attached to the medium. One specimen was contaminated. No mycobacterial growth was found in 7 specimens. The rate of positive culture Copyright
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results was 52.9%. No mycobacterial growth was observed in the 18 rabbits of the control group.
DISCUSSION New Zealand rabbits are sensitive to M. tuberculosis, commonly used to establish models for lung tuberculosis.7–11 In the current study, the survival rate of rabbits in the experimental group was 89.5% and model establishment succeeded in all surviving rabbits. We attribute the successful establishment of a spinal tuberculosis model in the current study to the following reasons. First, complete Freund’s adjuvant was used for sensitization. Complete Freund’s adjuvant is an oily emulsion enriched with bovine www.jspinaldisorders.com |
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TABLE 2. Positive Changes in the Survived Rabbits in the Experimental Group 4 and 8 Weeks After Surgery
X-ray Time after Surgery (wk) 4 8
Computed Tomography (CT) Reconstruction
No. Intervertebral Intervertebral Rabbits Space Bony Space Surviving Narrowing Spur Narrowing 18 17
9 10
2 6
Cotton-wool-like Destruction of the Vertebral Body
15 17
M. tuberculosis killed with high temperature. The slow release and continuous stimulation of immunogenic substances such as polysaccharides, lipids, or proteins from the cell wall components of M. tuberculosis inside the oil droplets enhanced the systemic resistance of the host to M. tuberculosis,12 and activation of the cellular immune response. Moreover, the local lesion of the vertebral body was aggravated by a delayed allergic reaction, which made the bone destruction model more complete.12 This method was recently used to establish an autoimmune arthritis animal model.13 Lindberg14 and Wu et al12 successfully achieved sensitization by utilizing complete Freund’s adjuvant to establish a knee tuberculosis model and found the spread of M. tuberculosis to be extremely rare in the experimental animals. Second, we used the standard M. tuberculosis strain, H37Rv, in the current study. This strain is highly invasive and infectious, with an antigenicity similar to tuberculosis commonly seen in clinical practice. However, its toxicity is moderate and the amount of bacteria can be more easily controlled than wild bacteria. It has been successfully used to establish a pulmonary tuberculosis model.15 Third, the location of implantation was appropriate. The site for implanting M. tuberculosis by drilling a cavity in New Zealand rabbits should be 5 mm from the upper or lower endplates of the vertebral body, with a depth of drilling of 5 mm. Implanting M. tuberculosis at this site is consistent with the principle of spinal tuberculosis development and this site provides the maximum area for establishing a model in the vertebral body. Finally, we used an appropriate implantation method by which to introduce the bacterium. M. tuberculosis suspension can be infused throughout a gelfoam sponge to reduce or avoid the overflow of bacteria with bleeding. Tuli16 established a femur tuberculosis model in guinea pigs and found that the rate of tuberculosis lesion increased 100% in the group in which gelfoam sponges were placed after drilling compared with the group without gelfoam sponge. Imaging examination is an important method for evaluating the rabbit tuberculosis model. In the current study, we found that the typical x-ray presentations were blurring and narrowing of the intervertebral space, with uneven vertebral density and new bone formation at the margin of the vertebral body. However, the positive rate was low and it was likely to be found relatively late. CT reconstruction shows better definition than x-ray.17 At the adjacent endplates, high-density osseous hyperplasia was mixed with low-density osteolytic destruction, and cotton-wool-like changes and osteophyte formation were
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Magnetic Resonance Imaging (MRI) (Abnormal Signals in T1WI and T2WI)
15 17
Bony Intervertebral Vertebral Spur Abscess Space Body Abscess 2 6
1 3
15 17
13 17
1 3
observed. These changes may be due to the large amount of cortical bone in the rabbit vertebral body. Hence, bone destruction is mainly characterized by new bone formation of cortical bone tuberculosis. MRI is more sensitive than other imaging modalities.18 On the MRI, the rabbit model manifested similarly to human spinal tuberculosis.19 MRI can reveal early inflammatory changes in the upper end and anterior margin of the vertebral body, together with intervertebral disk damage. Histopathologic examination showed that the spinal tuberculosis mainly presented as trabecular breakage and tiny sequestrum formation in the vertebral body. Tuberculoid inflammatory changes observed within the lesion included necrosis, massive numbers of lymphocytes, and few epithelial cells. However, neither multinucleated giant cell reaction nor typical tuberculous nodules or tuberculous cavity could be observed. This result may be related to the short disease course observed, the relatively mild destruction that occurred, and the material selected. Although mycobacterial culture is the “gold standard” for diagnosing spinal tuberculosis,19 it cannot be used to obtain an accurate diagnosis in a short-term study, due to its long duration and low positive rate.20 In the current study, the M. tuberculosis strain H37Rv was implanted into the vertebral body of New Zealand rabbits sensitized by complete Freund’s adjuvant and the results evaluated by x-ray, CT reconstruction, MRI, histopathologic examination, and bacterial culture. The result was a successful animal model of spinal tuberculosis. Our method is simple, the amount of bacteria can be controlled easily, and individual differences within groups are trivial. However, further studies should be carried out to strengthen the management and operation of the links during model establishment and increase the survival rate and success rate of experimental animals. ACKNOWLEDGMENTS The authors thank MedCom Asia Inc. for her help with proofreading and editing the manuscript. REFERENCES 1. Walker R, Pumell G, McLaren SG, et al. Early detection of bone infection and differentiationfrom post-surgical inflammation using 2deoxy-2-[18F-fluoro-D-glucose positron emission tomography (FDGPET) in an animal model [J]. J Orthopaed Res. 2005;23:1484–1489. 2. Lu X, Xiong Z, Li J, et al. Metabonomic study on’Kidney-Yang Deficiency syndrome’ and intervention effects of Rhizoma Drynariae extracts in rats using ultra performance liquid chromatography coupled with mass spectrometry[J]. Talanta. 2011;83:700–708.
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3. Marho¨fer RJ, Oellien F, Selzer PM. Drug discovery and the use of computational approaches for infectious diseases. Future Med Chem. 2011;3:1011–1025. 4. Hodgson AR, Wong W, Yau A. X-ray Appemvznces of Tuberculosis of the Spine. Springfield, IL: Charles C. Thomas; 1969. 5. Wang Z, Wang Q. Surgical strategy for spinal tuberculosis. Chin J Orthop. 2010;30:717–723. 6. Chinese Anti-tuberculosis Association. The laboratory science procedure of diagnostic bacteriology in tuberculosis. Bull Chinese Antitubercul Assoc. 1996;18:80–83. 7. Dannenberg AM Jr. Perspectives on clinical and preclinical testing of new tuberculosis vaccines. Clin Microbiol Rev. 2010;23:781–794. 8. Mendez S, Hatem CL, Kesavan AK, et al. Susceptibility to tuberculosis: composition of tuberculous granulomas in Thorbecke and outbred New Zealand White rabbits. Vet Immunol Immunopathol. 2008;122:167–174. 9. Patel K, Jhamb SS, Singh PP. Models of latent tuberculosis: their salient features, limitations, and development. J Lab Physicians. 2011;3:75–79. 10. Manabe YC, Kesavan A, Lopez-Molina J, et al. The aerosol rabbit model of TB latency, reactivation and immune reconstitution inflammatory syndrome. Tuberculosis (Edinb). 2008;88:187–196. 11. Tsenova L, Harbacheuski R, Sung N, et al. BCG vaccination confers poor protection against M. tuberculosis HN878-induced central nervous system disease. Vaccine. 2007;25:5126–5132.
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12. Wu QQ, Duan LS, Liang GF, et al. A model for tuberculosis of the knee joint in rabbit and its application. Tuber& Thor Tumor. 2002;9:174–176. 13. Pragasam SJ, Murunikkara V, Sabina EP, et al. Ameliorative effect of p-coumaric acid, a common dietary phenol, on adjuvant-induced arthritis in rats. Rheumatol Int. 2013;33:325–334. 14. Lindberg L. Experimental skeletal tuberculosis in the guinea pig. Acta Orthop Scand. 1967;suppl 98:11–80. 15. Whelan AO, Coad M, Cockle PJ, et al. Revisiting host preference in the Mycobacterium tuberculosis complex: experimental infection shows M. tuberculosis H37Rv to be avirulent in cattle. PLoS One. 2010;5:e8527. 16. Tuli SM, Brighton CT, Morton HE, et al. The experimental induction of localised skeletal tuberculous lesions and their accessibility to streptomycin. J Bone Joint Surg Br. 1974;56B:551–559. 17. Jain AK. Tuberculosis of the spine: a fresh look at an old disease. J Bone Joint Surg Br. 2010;92:905–913. 18. Zaidi H, Akram MH, Wala MS. Frequency and magnetic resonance imaging patterns of tuberculous spondylitis lesions in adults. J Coll Physicians Surg Pak. 2010;20:303–306. 19. Lalvani A. Diagnosing tuberculosis infection in the 21st century: new tools to tackle an old enemy. Chest. 2007;131:1898–1906. 20. Ge JZ, Wang ZL, Qin SB, et al. Mycobacterium tuberculosis culture and identification in pus of the spinal tuberculosis. J Ningxia Med Coll. 2004;26:251–252.
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