Apoptosis in peripheral blood lymphocytes in intestinal tuberculosis and Crohn's disease: Implications to diagnostic differentiation.

AIM: Intestinal tuberculosis (ITb) and Crohn's disease (CD) mimic each other often leading to misdiagnosis. We evaluated the difference between ITb and CD using the extent of apoptosis in peripheral blood lymphocytes. METHODS: CD4+ cells as a percentage of the lymphocytes and viable, dead, total apoptotic, early apoptotic, and late apoptotic CD4+ cells were assessed in the peripheral blood by flow cytometry in healthy controls and patients with confirmed active ITb and CD prior to initiating therapy. Early apoptotic and total apoptotic cells were further expressed as a proportion of the percentage of CD4+ cells. RESULTS: The percentages of CD4+ cells (6.5 [3.0, 8.7] vs. 13.40 [10.15, 13.40]; p < 0.001), total apoptotic cells (0.13 [0.0, 0.22] vs. 0.08 [0.0, 0.21]; p = 0.045), early apoptotic (1.24 [0.55, 2.54] vs. 0.71 [0.40, 1.30]; p = 0.037), and the proportion of the latter two parameters (17.18 [5.61, 57.33] vs. 4.84 [2.71, 9.83]; p-value 0.039) and (17.18 [7.4, 67.50] vs. 5.51 [3.10, 11.03]; p-value 0.036) were significantly different between patients with ITb and CD. The best sensitivity, specificity, and positive and negative predictive values for the diagnosis of ITb were seen with the CD4+ cell percentage (82.6%, 82.4%, 86.4%, 77.8%, respectively) and the proportion of early apoptotic cells (73.9%, 70.6%, 77.3%, 66.7%, respectively). CONCLUSION: CD4+ cells as a percentage of peripheral blood lymphocytes and the proportion of early apoptotic CD4+ cells show promise to diagnostic differentiation between ITb and CD.

Interleukin-22 receptor 1 is expressed in multinucleated giant cells: A study on intestinal tuberculosis and Crohn's disease.

BACKGROUND: It is challenging to distinguish intestinal tuberculosis from Crohn's disease due to dynamic changes in epidemiology and similar clinical characteristics. Recent studies have shown that polymorphisms in genes involved in the interleukin (IL)-23/IL-17 axis may affect intestinal mucosal immunity by affecting the differentiation of Th17 cells. AIM: To investigate the specific single-nucleotide polymorphisms (SNPs) in genes involved in the IL-23/IL-17 axis and possible pathways that affect susceptibility to intestinal tuberculosis and Crohn's disease. METHODS: We analysed 133 patients with intestinal tuberculosis, 128 with Crohn's disease, and 500 normal controls. DNA was extracted from paraffin-embedded specimens or whole blood. Four SNPs in the IL23/Th17 axis (IL22 rs2227473, IL1ß rs1143627, TGFß rs4803455, and IL17 rs8193036) were genotyped with TaqMan assays. The transcriptional activity levels of different genotypes of rs2227473 were detected by dual luciferase reporter gene assay. The expression of IL-22R1 in different intestinal diseases was detected by immunohistochemistry. RESULTS: The A allele frequency of rs2227473 (P = 0.030, odds ratio = 0.60, 95% confidence interval: 0.37-0.95) showed an abnormal distribution between intestinal tuberculosis and healthy controls. The presence of the A allele was associated with a higher IL-22 transcriptional activity (P < 0.05). In addition, IL-22R1 was expressed in intestinal lymphoid tissues, especially under conditions of intestinal tuberculosis, and highly expressed in macrophage-derived Langhans giant cells. The results of immunohistochemistry showed that the expression of IL-22R1 in patients with Crohn's disease and intestinal tuberculosis was significantly higher than that in patients with intestinal polyps and colon cancer (P < 0.01). CONCLUSION: High IL-22 expression seems to be a protective factor for intestinal tuberculosis. IL-22R1 is expressed in Langhans giant cells, suggesting that the IL-22/IL-22R1 system links adaptive and innate immunity.

CD4+ CD25+ FOXP3+ T cell frequency in the peripheral blood is a biomarker that distinguishes intestinal tuberculosis from Crohn's disease.

BACKGROUND: Distinguishing between Crohn's Disease (CD) and Intestinal Tuberculosis (ITB) has been a challenging task for clinicians due to their similar presentation. CD4+FOXP3+ T regulatory cells (Tregs) have been reported to be increased in patients with pulmonary tuberculosis. However, there is no such data available in ITB. The aim of this study was to investigate the differential expression of FOXP3+ T cells in patients with ITB and CD and its utility as a biomarker. METHODS: The study prospectively recruited 124 patients with CD, ITB and controls: ulcerative colitis (UC) and patients with only haemorrhoidal bleed. Frequency of CD4+CD25+FOXP3+ Tregs in peripheral blood (flow cytometry), FOXP3 mRNA expression in blood and colonic mucosa (qPCR) and FOXP3+ T cells in colonic mucosa (immunohistochemistry) were compared between controls, CD and ITB patients. RESULTS: Frequency of CD4+CD25+FOXP3+ Treg cells in peripheral blood was significantly increased in ITB as compared to CD. Similarly, significant increase in FOXP3+ T cells and FOXP3 mRNA expression was observed in colonic mucosa of ITB as compared to CD. ROC curve showed that a value of >32.5% for FOXP3+ cells in peripheral blood could differentiate between CD and ITB with a sensitivity of 75% and a specificity of 90.6%. CONCLUSION: Phenotypic enumeration of peripheral CD4+CD25+FOXP3+ Treg cells can be used as a non-invasive biomarker in clinics with a high diagnostic accuracy to differentiate between ITB and CD in regions where TB is endemic.

Chronic infections of the small intestine.

PURPOSE OF REVIEW: Chronic infections of the small intestine cause significant morbidity and mortality globally. This review focuses on the recent advances in the field of our understanding of selected intestinal infections. RECENT FINDINGS: Primary and secondary immunodeficiency increase the susceptibility to many chronic intestinal infections. Endoscopy and intestinal biopsies are central to establishing a diagnosis of these conditions. Tuberculosis (TB) remains a major global health challenge. Emerging therapeutic agents to counteract multidrug-resistant strains have shown clinical efficacy, but concerns regarding mortality remain. PCR-based diagnostic TB tests have the potential to reduce diagnostic delays, but remain to be validated for intestinal infections. Adjunctive diagnostic imaging modalities can differentiate infections from Crohn's disease with increasing accuracy. Whipple's disease remains rare, but there have been substantial advances in our understanding of the causative organism Tropheryma whipplei. Extended treatment with broad-spectrum antibiotics is effective in most cases. The narrow therapeutic window and limited armamentarium for treating invasive filamentous fungal infections contribute to their significant morbidity and high rates of mortality. SUMMARY: The speed and accuracy of diagnosing chronic intestinal infections have improved with recent imaging and laboratory methodologies. Significant research opportunities remain for clinicians and scientists to improve the diagnostic accuracy and clinical outcomes of chronic intestinal infections.

Immune thrombocytopenic purpura induced by intestinal tuberculosis in a liver transplant recipient.

A variety of clinical manifestations are associated directly or indirectly with tuberculosis. Among them, haematological abnormalities can be found in both the pulmonary and extrapulmonary forms of the disease. We report a case of immune thrombocytopenic purpura (ITP) associated with intestinal tuberculosis in a liver transplant recipient. The initial management of thrombocytopenia, with steroids and intravenous immunoglobulin, was not successful, and the lack of tuberculosis symptoms hampered a proper diagnostic evaluation. After the diagnosis of intestinal tuberculosis and the initiation of specific treatment, a progressive increase in the platelet count was observed. The mechanism of ITP associated with tuberculosis has not yet been well elucidated, but this condition should be considered in cases of ITP that are unresponsive to steroids and intravenous immunoglobulin, especially in immunocompromised patients and those from endemic areas.

Effectiveness of interferon-gamma release assays for differentiating intestinal tuberculosis from Crohn's disease: a meta-analysis.

AIM: To investigate the clinical usefulness of interferon-gamma release assays (IGRAs) in the differential diagnosis of intestinal tuberculosis (ITB) from Crohn's disease (CD) by meta-analysis. METHODS: A systematic search of English language studies was performed. We searched the following databases: Medline, Embase, Web of Science and the Cochrane Library. The Standards for Reporting Diagnostic Accuracy initiative and Quality Assessment for Studies of Diagnostic Accuracy tool were used to assess the methodological quality of the studies. Sensitivity, specificity, and other measures of the accuracy of IGRAs in the differential diagnosis of ITB from CD were pooled and analyzed using random-effects models. Receiver operating characteristic curves were applied to summarize overall test performance. Two reviewers independently judged study eligibility while screening the citations. RESULTS: Five studies met the inclusion criteria. The average inter-rater agreement between the two reviewers for items in the quality checklist was 0.95. Analysis of IGRAs for the differential diagnosis of ITB from CD produced summary estimates as follows: sensitivity, 0.74 (95%CI: 0.68-0.80); specificity, 0.87 (95%CI: 0.82-0.90); positive likelihood ratio, 5.98 (95%CI: 3.79-9.43); negative likelihood ratio, 0.28 (95%CI: 0.18-0.43); and diagnostic odds ratio, 26.21 (95%CI: 14.15-48.57). The area under the curve was 0.92. The evaluation of publication bias was not significant (P = 0.235). CONCLUSION: Although IGRAs are not sensitive enough, they provide good specificity for the accurate diagnosis of ITB, which may be helpful in the differential diagnosis of ITB from CD.

Cytokine gene expression in intestinal tuberculosis and Crohn's disease.

BACKGROUND: Intestinal tuberculosis (TB) and Crohn's disease closely resemble each other clinically and morphologically. Little is known of cytokine regulation in intestinal TB. OBJECTIVE: To compare cytokine gene expression in colonic mucosa and peripheral blood mononuclear cells (PBMC) in TB with that in Crohn's disease. METHODS: Biopsies were obtained from normal and ulcerated colonic mucosa of 12 intestinal TB and 11 Crohn's disease patients, and PBMC from 15 intestinal TB and 12 Crohn's disease patients and 11 healthy volunteers. RNA was extracted, and the expression of selected cytokines, chemokines and pattern recognition receptors quantified by reverse transcriptase real-time polymerase chain reaction using SYBR green. RESULTS: The mRNA expression of interleukin-8 (IL-8), induced protein-10, tumour necrosis factor-alpha, IL-23 p19 and IL-12 p40, and Toll-like receptors (TLR) 1 and 2 in the ulcerated mucosa was increased in both intestinal TB and Crohn's disease. Expression of growth-related oncogene-alpha was increased in intestinal TB, while expression of interferon-gamma (IFN-) and TLR 4, 5 and 9 was increased in Crohn's disease. Expression of RANTES (regulated upon activation, normal T-cell expressed and secreted) was decreased in Crohn's disease. Secretion of IFN- or IL-10 from PBMC was not significantly altered in either disease. PBMC mRNA expression of IL-1, IL-6 and IL-8 mRNA was upregulated in Crohn's disease, while that of IL-17 was upregulated in intestinal TB. CONCLUSIONS: Cytokine gene expression patterns in intestinal mucosa and PBMC of intestinal TB were remarkably similar to Crohn's disease, and demonstrated innate immune activation and T-helper 1 polarisation.

[The symptoms and surgical tactics for complicated forms of the abdominal cavity tuberculosis].

The results of treatment of 12 patients, suffering complicated forms of abdominal tuberculosis and external intestinal fistulas, were presented. Late diagnosis of abdominal tuberculosis in the patients, suffering the complications phase of the disease, is caused by unclear symptoms presence in early stages of the disease. Clinical and laboratory indices in peritonitis of a phthisis origin are nonspeciphic. In 91% of patients, admitted to the hospital for complicated forms of abdominal tuberculosis and external intestinal fistulas, the operative treatment was indicated. Surgical intervention (more frequently right-sided hemicolectomy, enterostomy, the abscesses opening, the caseously-changed lymph nodes excision, formation of anastomosis) was performed in 11 patients for peritonitis and external intestinal fistulas. The method of a secure invagination anastomoses formation was elaborated, permitting to perform primary restoration operations. An early diagnosis, early effective therapy and radical surgical intervention conduction for complicated abdominal tuberculosis promote the patients to survive.

Intestinal tuberculosis: a diagnostic challenge--case report and review of the literature.

Even though tuberculosis is considered rare in developed countries, its rising incidence, especially in high-risk populations, places intestinal tuberculosis in the differential diagnosis of patients with atypical abdominal symptoms or signs. We, herein, report the case of an immunocompetent woman, from a nonendemic area, who developed intestinal tuberculosis, emphasizing the diagnostic challenges caused due to nonspecific symptoms, inconclusive clinical, laboratory, and imaging findings, which could not rule in or rule out tuberculosis. Antituberculosis treatment was administered based on endoscopic findings and histological features of mucosal biopsies, which were indicative of intestinal tuberculosis, and the patient showed a marked clinical and laboratory improvement. We also review the evidence with regard to the diagnostic accuracy of the different available tests for intestinal tuberculosis.

Can an immunohistochemistry method differentiate intestinal tuberculosis from Crohn's disease in biopsy specimens?

BACKGROUND: It is sometimes difficult to diagnose whether a patient has intestinal tuberculosis or Crohn's disease because both have similar clinical, pathologic, and endoscopic features. However, their therapies are completely different and a mistake in diagnosis can result with deterioration. Many laboratory methods for the diagnosis of tuberculosis require considerable time to receive a diagnostic result. We wanted to evaluate whether an immunohistochemical tuberculosis staining method can be helpful for faster differentiation of biopsy materials. METHODS: We used formalin-fixed paraffin-embedded histologically diagnosed small intestine (n=1), colon (n=7), skin (n=8), lung (n=5), lymph node (n=24) tuberculosis and Crohn's disease (n = 28) biopsy materials only with granulomas. Demographic characteristics like age and gender were also obtained. Pathology specimens were stained immunohistochemically with an antibody to VP-M660, targeting the 38-kDa antigen of Mycobacterium tuberculosis. RESULTS: In the M. tuberculosis group, 33/45 of patients have positive immunohistochemistry (IHC) staining (73% sensitivity, 93% specificity), whereas only two of 28 patients have positive staining in the Crohn's group (p<0.001). The positive staining with IHC was detected as 85.7, 75, 75, and 60% in colon, lymph node, skin, and lung granulomas, respectively, in M. tuberculosis patients. CONCLUSIONS: Immunohistochemical staining of biopsy specimens with anti-VP-M660 seems to be a simple and fast technique with 73% sensitivity and 93% specificity for establishing an earlier differentiation of M. tuberculosis from Crohn's disease.

Experience with two cases of intestinal tuberculosis: utility of the QuantiFERON-TB Gold test for diagnosis.

BACKGROUND: Intestinal tuberculosis is rare in Western countries, with incidence rates of 35.7 and 0.43 per 100,000 per year for the immigrant and native populations, respectively. Despite a clear increase in the frequency of extrapulmonary tuberculosis in immunosuppressed patients, the clinical features of intestinal tuberculosis are seen rarely. A typical clinical presentation includes abdominal pain, weight loss, fever, weakness, nausea, vomiting, obstruction, and bleeding. Intestinal tuberculosis often mimics inflammatory bowel disease or malignant neoplasia, and its preoperative diagnosis is a challenge. Microbiologic diagnosis often is difficult because the causative microorganism requires a long incubation period. METHODS: Two case reports and review of the pertinent literature. RESULTS: We report two cases of colonic tuberculosis mimicking cecal carcinoma in one patient and periappendiceal abscess in the other. A 75 year-old man underwent right hemicolectomy for a right colon mass. Preoperative laboratory, radiologic, and endoscopic evaluations were negative for tuberculosis and carcinoma. The QuantiFERON-TB Gold test was positive. Surgical specimen histologic review showed non-caseating granulomas and rare Ziehl-Neelsen-positive bacilli. A 35 year-old man, born in Sri Lanka but living in Italy for 10 years, came to our attention for a periappendiceal abscess. Multiple peritoneal micro-nodules were found at laparotomy. Their extemporaneous histologic examination showed granulomas and giant-cell inflammation. A right hemicolectomy was performed. The QuantiFERON-TB Gold test, performed on peritoneal fluid and blood, was positive in both. CONCLUSIONS: The QuantiFERON-TB Gold test may hold promise for use in intestinal inflammatory diseases when tuberculosis is suspected but conventional workup is not diagnostic.

Anti-Saccharomyces cerevisiae antibody is not useful to differentiate between Crohn's disease and intestinal tuberculosis in India.

CONTEXT: Clinical, endoscopic, radiological and histological parameters of intestinal tuberculosis (IT) and Crohn's disease (CD) are so similar that differentiation between these two diseases, which require different treatment, is difficult. Anti- Saccharomyces cerevisiae antibody (ASCA), which is often present in the sera of patients with CD, may be potentially useful to differentiate CD from IT. AIM: To evaluate the role of enzyme-linked immunosorbent assay test for ASCA in serum in differentiating CD from intestinal tuberculosis. SETTINGS AND DESIGN: Prospective case-control study. MATERIALS AND METHODS: Sixteen patients with IT, 16 CD, 36 UC diagnosed using standard parameters and 12 controls (11 healthy subjects and one with colonic carcinoma) were tested for IgG ASCA in serum. STATISTICAL ANALYSIS USED: Categorical variables were analyzed using Chi-square test with Yates' correction, as applicable. Continuous variables were analyzed using Mann-Whitney U test. RESULTS: Eight of 16 (50%) patients with IT, 10 of 16 with CD (62%), nine of 35 with UC (26%) and one of 12 controls tested positive for ASCA in serum. Though the frequency of ASCA in serum was comparable among patients with IT and CD (8/16 vs. 10/16, P = ns), IT and UC (8/16 vs. 9/35, P =ns), CD and UC (10/16 vs. 9/35, P =ns), its frequency in CD or IT but not in UC was higher than healthy controls (P Conclusions: Serum ASCA is unlikely to be useful to differentiate between CD and IT in India.

Anti-Saccharomyces cerevisiae antibody does not differentiate between Crohn's disease and intestinal tuberculosis.

The clinical, morphological, and histological features of intestinal tuberculosis (IT) and Crohn's disease (CD) mimic so much, that it becomes difficult to differentiate between them. The sensitivity of anti-Saccharomyces cerevisiae antibody (ASCA) IgG and ASCA IgA in CD is 60%-80%, whereas the specificity is almost 90%. There are no reports of study of ASCA in patients with IT, nor has it ever been used to differentiate CD from IT. Patients with ulcerative colitis (UC; n=25), CD (n=59), and IT (n=30) and 21 healthy controls were included in this study. The location and behavior of CD were classified according to the Modified Montreal classification. Five milliliters of blood was taken from them and serum was stored at -70 degrees C. ASCA antibodies (both IgG and IgA) were estimated using commercially available ELISA kits (AESKU Diagnostics, Germany). Anti-neutrophilic cytoplasmic antibody was measured by indirect immunofluorescence test. ASCA IgA was positive in 4.7%, 28%, 33.9%, and 43.3% and ASCA IgG was positive in 4.7%, 24%, 50.8%, and 46.6% of healthy controls and patients with UC, CD, and IT, respectively. Either ASCA IgG or ASCA IgA was positive in 9.5%, 40%, 61% and 66.6% of healthy controls, UC, CD, and IT, respectively. ANCA was positive in 0%, 32%, 10.1%, and 6.6% of healthy controls, UC, CD, and IT, respectively. ASCA IgG was positive in a significantly higher number of patients with CD (P<0.0001) and IT (P<0.0001) in comparison to healthy controls. ASCA IgA was positive in a significantly higher number of patients with UC (P<0.04), CD (P<0.013), and IT (P<0.006) in comparison to healthy controls. In comparisons between diseases, ASCA IgG was positive in significantly more patients with CD (P<0.001) and IT (P<0.001) in comparison to UC. There was no significant difference in ASCA IgA (33.9% vs. 43.3%), ASCA IgG (50.86% vs. 46.6%), or ANCA (10.7%, 7.4%) in patients with CD and IT, respectively. There was no correlation between ASCA and duration, location and behavior of CD, and IT. We conclude that ASCA IgG and ASCA IgA do not help to differentiate between IT and CD.

Clinical presentation of abdominal tuberculosis in HIV seronegative adults.

BACKGROUND: The accurate diagnosis of abdominal tuberculosis usually takes a long time and requires a high index of suspicion in clinic practice. Eighty-eight immune-competent patients with abdominal tuberculosis were grouped according to symptoms at presentation and followed prospectively in order to investigate the effect of symptomatic presentation on clinical diagnosis and prognosis. METHODS: Based upon the clinical presentation, the patients were divided into groups such as non-specific abdominal pain & less prominent in bowel habit, ascites, alteration in bowel habit, acute abdomen and others. Demographic, clinical and laboratory features, coexistence of pulmonary tuberculosis, diagnostic procedures, definitive diagnostic tests, need for surgical therapy, and response to treatment were assessed in each group. RESULTS: According to clinical presentation, five groups were constituted as non-specific abdominal pain (n = 24), ascites (n = 24), bowel habit alteration (n = 22), acute abdomen (n = 9) and others (n = 9). Patients presenting with acute abdomen had significantly higher white blood cell counts (p = 0.002) and abnormalities in abdominal plain radiographs (p = 0.014). Patients presenting with alteration in bowel habit were younger (p = 0.048). The frequency of colonoscopic abnormalities (7.5%), and need for therapeutic surgery (12.5%) were lower in patients with ascites, (p = 0.04) and (p = 0.001), respectively. There was no difference in gender, disease duration, diagnostic modalities, response to treatment, period to initial response, and mortality between groups (p > 0.05). Gastrointestinal tract alone was the most frequently involved part (38.5%), and this was associated with acid-fast bacteria in the sputum (p = 0.003). CONCLUSION: Gastrointestinal tract involvement is frequent in patients with active pulmonary tuberculosis. Although different clinical presentations of patients with abdominal tuberculosis determine diagnostic work up and need for therapeutic surgery, evidence based diagnosis and consequences of the disease does not change.

Cytologic diagnosis of pancreatic tuberculosis in immunocompetent and immunocompromised patients: a report of 2 cases.

BACKGROUND: Pancreatic tuberculosis (PT) is a very rare occurrence in the setting of extrapulmonary tuberculosis. It usually occurs as a complication of miliary tuberculosis in immunodeficient individuals, particularly with HIV infections, but isolated involvement in an immunocompetent patient is extremely rare. Pancreatic involvement by tuberculosis in immunocompromised states, such as AIDS, and isolated involvement in immunocompetent patients require a high index of suspicion. Fine needle aspiration cytology under imaging guidance is highly conclusive. CASES: A patient presented with diabetes mellitus and a pancreatic mass clinically diagnosed as pancreatic carcinoma. A second patient, who was HIV seropositive, presented with generalized lymphadenopathy and a pancreatic mass, clinically diagnosed as lymphoma. These were conclusively diagnosed by fine needle aspiration cytology (FNAC) as PT under imaging guidance. After antituberculous therapy the first patient showed a dramatic improvement, whereas the second died; an autopsy was performed. Because of the atypical presentation of PT in immunocompromised and immunocompetent patients, FNAC can be used as a first-line diagnostic intervention. FNAC under imaging guidance is sensitive, specific, rapid and inexpensive. It resolves the diagnostic dilemma and thus avoids major surgery for a clinically diagnosed neoplasm.

Massive gastrointestinal tuberculosis in a young patient without immunosuppression.

Although the lung is the major site for Mycobacterium tuberculosis infection, gastrointestinal involvement can be present as part of multiorgan disease process or, less commonly, can be seen as primary gastrointestinal tuberculosis. In the cases where the culture is negative, it can be difficult to differentiate tuberculosis from Crohn's disease based on both the clinical and histological features. When side effects of classic antimycobacteria are encountered, we can initially add ciprofloxacin to the treatment of tuberculosis. We reported a case of 19-yr-old patient, who was treated as Crohn's disease and worsen. We began to tuberculosis treatment, and the patient improved clinically and histologically. The main point in this case is that widespread involvement of gastrointestinal tract can be brought about by non resistant strains of Mycobacterium tuberculosis even in immunocompetent patients.

[An unusual manifestation of tuberculosis in a female patient after kidney transplantation. Case report]. / Neobvyklá manifestace tuberkulózy u nemocné po transplantaci ledviny. Kazuistika.

Organ transplant recipients are at the increased risk of infection complications. Herein, we present a case of unusual localisation of tuberculosis in renal allograft recipient treated by combination of cyclosporine A, mycophenolate mofetil and steroids. After the non specific prodromes, surgery due to ileus discovered ileocaecal tumour. Microscopically tuberculosis inflammatory process with ulcerations of the intestinal mucosa and classification of the lymphatic nodes, instead of the expected lymphoma was proved to be present. We are discussing the incidence of tuberculosis in relation to immunosuppressive therapy as well as possible pathways of tuberculosis transmission.