Study On Early Diagnosis Of Tuberculosis-associated Renal Injury

ObjectiveTuberculosis (TB) is a major global public health problem. The World Health Organization estimates that one-third of the world's population is infected with Mycobacterium tuberculosis (MTB), which is responsible for 8.7 million new TB cases and approximately 3 million deaths annually. Genitourinary TB is commonly a late manifestation of an earlier symptomatic or asymptomatic pulmonary TB infection. As one of the most common sites of involvement of extrapulmonary TB, genitourinary TB accounts for 15%-20% of infections outside of the lungs. Among genitourinary TB, diagnosis of renal tuberculosis (RTB) remains difficult, especially in the early stage due to the vagueness of chronic, intermittent, and non-specific urinary symptoms. As a result, many patients are not diagnosed or misdiagnosed and lose the chance of early treatment and progress to end-stage renal disease (ESRD), a life-threatening condition. Therefore, the early diagnosis of renal TB is very important to prevent progressive destruction of the kidney.However, traditional laboratory techniques, such as direct microscopic observation and mycobacterial culture on semisolid or liquid medium, are far from being sufficiently sensitive and specific for rapid MTB identification. To test positive, an acid-fast bacilli (AFB) smear requires 5000-10000 AFB/ml. Although urine MTB culture is more sensitive than AFB smear, culture growth is slow; most samples do not show visible colonies of MTB before one month, thus delaying the diagnosis.Genitourinary TB accounts for approximately15% of all extrapulmonary cases, and may involve any portion of the genitourinary tract. Local symptoms predominate. However, in the early stage of RTB, most patients may be asymptomatic and abnormal urinalyses are detected only as part of routine clinical evaluations or incidentally in the hospital while undergoing treatment for unrelated disorders. The urinalysis may reveal mild proteinuria, microscopic hematuria, and leucocyturia in acidic urine. Recently, due to some symptomatic treatment, the major clinical manifestation of some RTB patients gives priority to renal disease instead of cystitis. Several cases of glomerulonephritis associated with active TB have been described in the literature.MTB can induce both cellular immune and humoral immune responses when bacilli invade the body. Studies have shown that MTB infection can lead to the disturbances of Thl/Th2 cells, which may give rise to nephritis. On the other hand, type III allergic reactions induced by immune complexes can cause tissue injury and may serve as the pathogenesis of TB. The role of disseminated tuberculosis in the pathogenesis of glomerulonephritis has been presumed to be dependent on humoral immunity and immune complexes have been reported detectable at high levels in the active phase of disseminated tuberculosis. As a result, some patients with early RTB show clinical features of chronic nephritis, such as hematuria, proteinuria, edema, and hypertension. Very often such patients are misdiagnosed and the disease is discovered only after severe destructive lesions of the kidneys have developed. In this study, urinalyses of all patients in the CS-RTB group showed proteinuria, hematuria, and leucocyturia without any symptoms of cystitis, such as urinary frequency, dysuria, and flank pain. We did not detect deposition of immune complexes in the renal biopsy. This may be because of the low level of immune complexes in the early stages of RTB.Therefore, diagnostic tests devoted to the rapid, sensitive, and specific identification of MTB infections has been the key element for early diagnosis of RTB.Our investigation included four aspects:①To detect MTB DNA in the renal biopsy specimens by real time PCR.②To study the expression of secretory antigen 85 (Ag85) of MTB and interferon-gamma (IFN-γ) in the renal tissues of patients with RTB; to discuss the relationship between the expression of Ag85 and IFN-γ.③To study the expression of early secretory antigen target-6 (ESAT-6) of MTB in the renal tissues of patients with RTB.④To discuss the sensitivity and specificity of Ag85, ESAT-6 and TB-DNA in the early diagnosis of renal tuberculosis.Materials and MethodsThe patients were divided into the following groups:renal tuberculosis (RTB); non-renal tuberculosis (N-RTB); and clinically suspected renal tuberculosis (CS-RTB).Thirty patients were selected for the RTB group who underwent unilateral nephrectomy because of RTB, which was confirmed by pathologic diagnosis. Morning urine samples were collected from the patients for MTB culture before surgery.The N-RTB group consisted of 30 patients, including seven patients with minimal change nephritic syndrome,13 patients with mesangioproliferative glomerulonephritis, five patients with membranous nephropathy, one patient with glomerulosclerosis, and four patients with focal segmental glomerulonephritis. No patients had pulmonary or extrapulmonary TB. The tuberculin skin tests and serum TB antibody titers were negative. The patients were diagnosed with primary glomerulonephritis and the condition improved after routine therapy.The CS-RTB group was comprised of 30 patients who had extranephric TB, hematuria, and proteinuria. The tuberculin skin tests and serum TB antibody titers were positive, while the kidney ultrasonic examination and radiologic tests did not show any abnormalities.Patients in the N-RTB and CS-RTB groups underwent renal biopsy and morning urine samples were collected for MTB culture.All of the patients (52 males and 38 females with a mean age of 42.5±19.5 years) were selected from patients who were admitted to our hospital between 2004 and 2007.Urine MTB cultureUrine samples were pre-treated by decontamination with 4%(w/v) NaOH and centrifugation at 1500 g for 10 min. The sediment was used for MTB culture. MTB culture was performed using in-house made Lowenstein-Jensen (LJ) solid medium with a maximum incubation period of 8 weeks. The results were read and reported weekly. Ziehl Neelsen (ZN) staining was performed to confirm the presence of mycobacteria when colonies were noted in the LJ medium.1. Part 1(1)DNA isolationTwo renal biopsies were obtained in all the cases and the length of each biopsy was approximately 12 mm. One specimen was divided into three parts for three different histopathologic diagnostic procedures. The other specimen was used for DNA extraction. DNA was extracted with the QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. The DNA concentration was measured and 100 ng of DNA was used for a real time PCR reaction, according to the instructions of the real time PCR kit. (2) Real-time PCRThe primers were synthesized by TaKaRa Company (Dalian, China). The recommended concentration ranges were from 0.2-1.0μM. In this study, the final concentration of primers was 0.4μM, which has been shown to be a suitable concentration in preliminary experiments.All PCRs were performed in the ABI PRISM 7900HT Real-Time PCR System (Applied Biosystems, Foster City, CA, USA) with a 384-well plate. The thermal conditions were as follows:stage 1 (initial denaturation),95℃for 1 min,1 cycle; stage 2 (PCR),95℃for 5s and 60℃for 30s,40 cycles; and stage 3 (dissociation),95℃for 15s,60℃for 1 min, and 95℃for 15s,1 cycle. Every well was loaded with a PCR mixture containing SYBR Premix Ex TaqⅡ(2×; 10μl), PCR forward primer (10μM; 0.8μl), PCR reverse primer (10μM; 0.8μl), ROX reference dye (50×; 0.4μl), DNA template (2.0μl), and dH2O (6.0μl) in a total volume of 20μl. Each sample was tested in triplicate and data were collected in the last step for analysis with the SDS software version 2.0a23 (Applied Biosystems). Thirty-five and 40 were used as cycle threshold (CT) cut-off values. For the real-time experiments, post-PCR DNA melt curve analysis was performed to assess amplification specificity. In case of ambiguity, DNA gel electrophoresis was performed.MTB DNA amplification was performed using insertion element IS6110 as a multi-copy target for molecular detection of the MTB complex.-actin was used as a housekeeper gene to control the DNA purification procedure and the existence of possible PCR inhibitors. The amplification of TB-DNA and-actin was performed in the same reaction. For each sample, we prepared two microtubes (one for TB-DNA amplification and one for-actin). The reaction system was the same, with the exception of the primers.2. Part 2To study the expression of Ag85 of MTB and IFN-yin renal tissues with immunohistochemistry method and discuss the relationship between the expression of Ag85 and IFN-y.1. Deparaffinize and rehydrate sections as follows:3×3 min with xylene; 3×2 min with 100% ethanol; 2 min with 95% ethanol,2 min with 80% ethanol,2 min with 70% ethanol, and 5 min with PBS.2. Retrieve antigen using one the antigen retrieval methods described below.3. Block endogenous peroxidases by soaking slides in a solution of 90% methanol/3% H2O2 for 15 min at room temperature (RT). Then wash 3×5 min with PBS.4. Shake and wipe off excess PBS. Circle all sections with a pap pen. Add 75 ul of Blocking buffer to each section immediately. Do not touch sections with tip.5. Incubate 1 hr to overnight at RT in a humidified chamber. Do not let the slides touch each other.6. Dilute primary antibody in Blocking buffer (dilutions will vary depending on tested antibody). Add 75 ml per section and incubate 1 hr to overnight at RT in a humidified chamber.7. Drain primary antibody off section. Wash slides 3 x 10 min in PBS. You may have to wash slides in PBS+0.1%-0.5% Tween-20 for some primary antibodies.8. Dilute secondary antibody 1:1000 in Blocking buffer without Tween-20. Add 75 ml per section and incubate for 1 hr at RT in a humidified chamber.9. Drain secondary antibody and wash slides 5 x 10 min in PBS+0.1% Tween-20.(For secondary antibodies that are peroxidase conjugated, go to step 11.)10. Make ABC according to Vector protocol 30 min before time of use (mix 5 ml of PBS with 2 drops of solution A and 2 drops of solution B). Incubate samples for 45 min at RT. Wash 5 min in PBS.11. Make DAB according to Vector protocol in ddH2O. WEAR GLOVES:Mix 5 ml ddH2O with 2 drops of buffer,4 drops of DAB and 2 drops of H2O2. (If you want a gray-black stain, add 2 drops of the Nickel solution, and mix). Add immediately to slides and wait for color change (approximately 2-10 min). Drain slides and place into ddH2O for 5 min. Dispose of DAB waste with bleach.12. Counterstain with methyl green (1 min) or hematoxylin (3 sec). Wash 3 times with ddH2O.13. Immediately dehydrate in 70% ethanol,80% ethanol, and 100% ethanol (one dip each).14. Mount with Permount and seal coverslip with nail polish.3. Part 3 To study the expression of ESAT-6 of MTB in renal tissues with immunohistochemistry method.The same protocol as above.Results1. Part 1In the RTB group, seven patients were urine MTB culture-positive. For real-time PCR,25 samples tested positive by using 35 as the CT cut-off value and 28 samples tested positive by using 40 as the CT cut-off value.In the N-RTB group, no patients were urine MTB culture-positive. Four patients were CT35 positive (two patients with focal segmental glomerulonephritis, one patient with glomerulosclerosis, and one patient with membranous nephropathy). Thirteen patients were CT40-positive (besides the four patients above, five patients with mesangioproliferative glomerulonephritis, two patients with minimal change nephritis syndrome, one patient with membranous nephropathy, and one patient with focal segmental glomerulonephritis). Neither granulomas nor evidence of acid-fast bacilli were noted in any of the positive biopsies. We followed those patients for 12 months and no patients developed RTB.The sensitivity and specificity of the urine MTB culture were 23.3% and 100%, respectively. The sensitivity and specificity of real-time PCR (CT 40) were 93.3% and 56.7%, respectively. When using 35 as the CT cut off value, the sensitivity and specificity were 83.3% and 86.7%, respectively. Compared with CT 40, the specificity was significantly increased (Table 3).In the CS-RTB group, when using 35 as the CT cut-off value of real-time PCR,9 samples tested positive. While using 40 as the CT cut-off value,14 samples tested positive.2. Part 2The expression of Ag85 and IFN-gamma were significantly increased in renal tuberculosis group,3 cases in non-renal tuberculosis group and 18 cases in experimental group. Compared with positive group, there was no significant difference in the optical density in experimental group. While compared with renal tuberculosis group, there was significant difference in experimental group. There was a positive linear correlation between ag85 and IFN-gamma. The sensitivity of ag85 was 100% and the specificity was 75%.3. Part 3The expression of ESAT-6 were significantly increased in renal tuberculosis group, 4 cases in non-renal tuberculosis group and 15 cases in experimental group. Compared with renal tuberculosis group, there was no significant difference in the optical density in experimental group. While compared with non-renal tuberculosis group, there was significant difference in experimental group. The sensitivity of ESAT-6 was 100% and the specificity was 80%.ConclusionThese data suggested that:1. Detection of MTA DNA in renal biopsy tissues by real-time PCR is highly sensitive. Moreover, it can increase the diagnostic accuracy and provide valuable information which would complete other clinical data for the early diagnosis of RTB. Although urine MTB culture is still the gold standard for RTB diagnosis, we speculate that if real-time PCR is widely adopted within clinical practice, it will be a powerful tool for the rapid and accurate diagnosis of RTB.2. The expression of Ag85 and IFN-gamma were significantly increased in renal tuberculosis group. There was a positive linear correlation between Ag85 and IFN-gamma. The sensitivity of Ag85 was 100% and the specificity was 75% which demonstrated that Ag85 played an important role in the early diagnosis of renal tuberculosis.3. The expression of ESAT-6 were significantly increased in renal tuberculosis group, The sensitivity of ESAT-6 was 100% and the specificity was 80% which is higher than that of Ag85. It showed that ESAT-6 played an important role in the early diagnosis of renal tuberculosis.