Detection of mycobacterial DNA in pleural fluid from patients with tuberculous pleurisy by means of the polymerase chain reaction: comparison of two protocols.

BACKGROUND: The detection of mycobacterial DNA in clinical samples on the basis of the polymerase chain reaction is a promising approach for the rapid diagnosis of tuberculous infections. No consensus exists, however, about which protocols are most sensitive, and the usefulness of this approach in the diagnosis of tuberculous effusions has been assessed in few patients. METHODS: The sensitivity of two protocols was compared for the detection of DNA from Mycobacterium tuberculosis in samples containing known amounts of mycobacterial DNA and in DNA extracted from 15 tuberculous pleural effusions. The results obtained for pleural fluid have been compared with cytological findings and with results obtained by standard microbiological techniques. RESULTS: Mycobacteria could be detected by acid fast staining in none and by culture in three of the 15 pleural fluid samples. A protocol based on the detection of the IS6110 insertion element (which could detect one mycobacterial genome/sample reproducibly) gave a positive result in nine of the 15 tuberculous effusions, though some samples were only intermittently positive (p less than 0.05 compared with culture). In contrast, a protocol based on the detection of the gene coding for the 65 kD mycobacterial antigen (which could detect mycobacterial genomes only if there were at least 10/sample) gave a positive result in three of the 15 tuberculous effusions. Pleural fluid that was always positive with the amplification procedure detecting the IS6110 sequence contained more neutrophils (30% (SD 27%)) than samples that were intermittently positive or always negative (3% (3%)); mycobacterial DNA was never detected in the four samples containing less than 1% neutrophils. CONCLUSIONS: The amplification of the IS6110 insertion element represents a rapid and sensitive means of detecting M tuberculosis in tuberculous effusions. The enrichment of cells containing mycobacteria (possibly neutrophils) before DNA extraction may be required to improve the sensitivity of this approach.     

Comparison of polymerase chain reaction for IS6110 and Amplicor in the diagnosis of tuberculosis.

BACKGROUND: Polymerase chain reaction (PCR) amplification of Mycobacterium tuberculosis DNA offers the potential of a sensitive and specific diagnostic test for tuberculosis. To evaluate this technique from the clinician's perspective, samples were collected from patients with chronic respiratory disease and the sensitivity and specificity of a newly introduced commercially available PCR kit (Amplicor) was compared with that of an established method to detect the target sequence IS6110. METHODS: Sputum or bronchial washings from patients with active tuberculosis, previously treated tuberculosis or other selected respiratory illnesses were analysed by both techniques and their sensitivity and specificity determined. RESULTS: Amplicor was more specific than IS6110 in the diagnosis of active infection (98% versus 79%). Both techniques were equally sensitive (92%). CONCLUSION: These results suggest that analysis of respiratory samples by Amplicor PCR in inner city populations of patients has greater specificity for a diagnosis of active tuberculosis than PCR for IS6110, and thus Amplicor PCR may aid the clinician in making a diagnosis of active tuberculosis.     

术前短期化疗对脊柱结核手术安全性的实验研究

目的通过巢式聚合酶链式反应（polymerase chain reaction,PCR）动态检测脊柱结核患者围手术期外周血的MTB-DNA的IS6110基因的含量,评估术前短期化疗对脊柱结核患者的手术安全性。方法根据IS6110基因设计两对特异性硫化修饰引物,结合高效保真聚合酶建立的巢式PCR,动态检测25例术前短期化疗的脊柱结核患者术前1天、术后1天、术后7天、术后14天外周血MTB-DNA的IS6110基因的含量变化,对外周血扩增产物IS6110基因含量进行比较分析。结果脊柱结核患者围手术期外周血IS6110基因含量变化的两种趋势：（1）15例患者伴随术后结核病灶的清除,外周血中的结核杆菌IS6110基因的从术后1天就开始逐渐减少;（2）10例患者术后1天时外周血中结核杆菌IS6110基因有短暂回升,但与术前1天外周血中结核杆菌IS6110基因含量比较无显著性差异（P〉0.05）,随着术后持续抗痨治疗,结核杆菌IS6110基因含量在术后1～2周均减少,均未发生结核杆菌血行播散并发症。结论如果脊柱结核患者一般情况好,术前短期化痨后行手术治疗安全有效。     

Mycobacterium tuberculosis DNA in tissues affected by sarcoidosis

BACKGROUND: Although some studies have reported the presence of Mycobacterium tuberculosis (MTb) DNA in tissues affected by sarcoidosis, the data are conflicting. The aim of this study was to collect prospectively tissue from patients with sarcoidosis in whom tuberculosis had been excluded, and to use polymerase chain reaction (PCR) to search for DNA sequences specific for MTb. METHODS: Fresh tissue samples (node or lung biopsy) taken from 23 patients with newly diagnosed sarcoidosis, 10 with other respiratory disease, and four patients with culture positive tuberculosis were analysed using PCR to amplify a 123 bp fragment of IS6110, the insertion element present in MTb, and nested PCR to further amplify an 85 bp sequence within the 123 bp product. DNA was also extracted from formalin fixed tissue from eight additional patients with sarcoidosis. RESULTS: MTb DNA was not detected in any of the tissue samples from patients with sarcoidosis or other respiratory disease but was found in all four patients with tuberculosis. CONCLUSIONS: This study has shown the absence of MTb DNA in lymph node and lung biopsy samples from patients with sarcoidosis. MTb is therefore unlikely to be a factor in the pathogenesis of this disease.     

Rapid diagnosis of genitourinary tuberculosis by polymerase chain reaction and non-radioactive DNA hybridization

OBJECTIVE: To establish a polymerase chain reaction (PCR) assay for the rapid detection and identification of mycobacteria in urine, and to assess the value of such assay in routine laboratory diagnosis of genitourinary tuberculosis. MATERIALS AND METHODS: Urine specimens from 1000 patients with clinical suspicion of urinary tuberculosis were examined. Two assays for the detection and identification of Mycobacterium tuberculosis (M. tuberculosis) complex and mycobacteria other than tuberculosis (MOTT) by non-radioactive DNA hybridization of PCR-product were applied. The first assay used PCR primers and probe derived from M. tuberculosis species-specific DNA insertion sequence, IS6110. The second utilized mycobacterium genus-specific sequence encoding ribosomal ribonucleic acid (16S rRNA). The results obtained by PCR were compared with those obtained by standard microbiological methods, acid-fast bacilli (AFB) stain and culture. RESULTS: Compared with cultures, the sensitivity of AFB staining was 52.07% and the specificity was 96.7%. In comparison to the results of culture, the overall sensitivity and specificity of the IS6110-PCR assay was 95.59% and 98.12% respectively. While the corresponding results for the 16S rRNA gene-PCR were 87.05% and 98. 9%. CONCLUSION: The high sensitivity and specificity in addition to the potential for rapid detection of mycobacteria, makes this test a useful tool in the clinical management of mycobacterial infection in urine. Urine specimens may contain M. tuberculosis and/or other mycobacteria; therefore, there are advantages in using genus-specific primers in parallel with species-specific primers in PCR assay.     

Clinical value of the measurement of Mycobacterium tuberculosis specific antibody in pulmonary tuberculosis.

BACKGROUND: A serological test that could help to diagnose tuberculosis, especially smear negative disease, would contribute to patient management. METHODS: Levels of antibody to distinct antigens of Mycobacterium tuberculosis were assessed for their value in the diagnosis and management of pulmonary tuberculosis. Serum was taken from 52 patients who were smear positive, from 27 patients who were smear negative but with evidence of active tuberculosis (sputum culture positive in 16, response to antituberculosis chemotherapy in 11), from 11 patients with old healed tuberculosis (pre-antibiotic era), and from 39 healthy subjects vaccinated with BCG. RESULTS: In smear positive tuberculosis an enzyme linked immunosorbent assay using a single 38 kDa antigen gave a diagnostic sensitivity of 80% with a 100% specificity. In smear negative pulmonary tuberculosis, however, combination of the 19 kDa antigen, lipoarabinomannan (ML 34 epitope), and hsp 65 (TB 78 epitope) was needed to achieve a sensitivity of 64% with a specificity of 95%. Recurrent and extensive radiographic disease with a poor prognosis was associated with high anti-38 kDa and low anti-14 kDa antibody levels in patients with active disease. Patients with less pulmonary cavitation had high anti-19 kDa titres. Bacteriological relapse during treatment was indicated by a rise in anti-14 kDa (TB68 epitope) antibodies. Four patients with non-tuberculous mycobacterial infection showed no anti-38 kDa antibody. CONCLUSION: Antigen or epitope specific serology may help in the diagnosis, assessment of prognosis, and monitoring of chemotherapy in patients with pulmonary tuberculosis.     

Diagnostic potential of multi‐targeted LAMP (loop‐mediated isothermal amplification) for osteoarticular tuberculosis

Delay in diagnosing osteoarticular tuberculosis (OATB) contributes significantly to morbidity by causing disfiguration and neurological sequelae. The delay caused by conventional culture and the expertise and expense involved in other nucleic acid based tests, make LAMP (loop‐mediated isothermal amplification) assay a favorable middle path. We evaluated LAMP assay using IS6110 and MPB64 for rapid diagnosis of OATB by comparing with IS6110 PCR and culture. LAMP assay was performed on 140 synovial fluid and pus samples (10 culture‐positive proven cases, 80 culture‐negative probable cases, and 50 negative controls) using three set of primer pairs each for IS6110 and MPB64. LAMP assay, using two‐target approach, had an overall sensitivity and specificity of 90% and 100% in detecting OATB. Sensitivity of IS6110 PCR, IS6110 LAMP, and MPB64 LAMP was 80%, 100%, and 100%, respectively, for confirmed cases and 72.5%, 81.75%, and 86.25%, respectively, for probable cases. Six additional cases were picked using two‐target approach. LAMP assay utilizing IS6110 and MPB64 is a cost‐effective technique for an early and reliable diagnosis of OATB. © 2016 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 35:361–365, 2017.

     

PCR diagnosis on formalin-fixed, paraffin-embedded tissues with acid-fast stain and culture negativity in chronic dialysis patients of cervico-mediastinal tuberculous lymphadenitis

Background: Bacteriologic studies often provide negative results in tuberculous infection, and do not favour early diagnosis. Polymerase chain reaction (PCR) is known to diagnose tuberculosis quickly. With this in mind, we used PCR to detect mycobacterial DNA on formalin-fixed, paraffin-embedded tissues with acid-fast stain and culture negativity in two dialysis patients with cervico-mediastinal lymphadenopathy. Methods: Sections of neck lymph nodes were cut at two different levels. At each level, two semi-adjacent sections with a thickness of 5 &mgr;m each were cut using standard microtomes with disposable blades. The first section mounted on a glass slide was stained b Ziehl-Neelsen, and the second section was examined by PCR based on a 123 bp fragment of IS6110 that is specific for the Mycobacterium tuberculosis complex. Results: The histology of lymph nodes disclosed inflammatory necrotizing granulomas, but acid-fast stain for M. tuberculosis was negative in the two patients. DNA of M. tuberculosis was detected in lymph node samples from each patient by PCR on the IS6110 element and by dot-blot hybridization. Conclusions: PCR assay is a potentially useful approach for early and rapid diagnosis of tuberculous lymphadenitis in chronic dialysis patients, since mycobacterial staining and culture often provide negative results.     

Identification of HIV patients with active pulmonary tuberculosis using urine based polymerase chain reaction assay

BACKGROUND: Despite the increased dissemination of tuberculosis among HIV infected patients, the diagnosis is difficult to establish. Traditional microbiological methods lack satisfactory sensitivity. We have developed a highly sensitive and specific nested polymerase chain reaction (PCR) capable of detecting Mycobacterium tuberculosis DNA in urine specimens and have used this test to examine urine specimens from HIV patients with active pulmonary tuberculosis. METHODS: Urine specimens from 13 HIV infected patients with microbiologically proven active pulmonary tuberculosis, 10 AIDS patients with non-tuberculous mycobacterial infection (documented by blood culture), 53 AIDS patients with no evidence of mycobacterial disease, and 80 healthy subjects (25 with positive skin test to purified protein derivative) were tested for M tuberculosis using PCR, acid fast staining (AFS), and culture. RESULTS: Of the urine specimens from patients with active tuberculosis, all tested positive by PCR, two by culture, and none by AFS. No reactivity was observed in urine specimens from patients with non-tuberculous mycobacterial infection. Of the 53 AIDS patients without mycobacterial infection, one had a positive urine PCR. Normal subjects were all negative. CONCLUSIONS: Urine based nested PCR for M tuberculosis may be a useful test for identifying HIV patients with pulmonary tuberculosis.     

Epidemiology of tuberculosis on Gran Canaria: a 4 year population study using traditional and molecular approaches

BACKGROUND: In recent years several population based studies using restriction fragment length polymorphism (RFLP) analysis have shown a higher rate of recent transmission of tuberculosis than previously thought. This study was undertaken to determine the transmission patterns of tuberculosis and the potential causes of recent transmission on the island of Gran Canaria (Spain). METHODS: The strains of all patients diagnosed with tuberculosis confirmed by culture between 1 January 1993 and 31 December 1996 were typed by RFLP using the insertion sequence IS6110. A cluster was defined as two or more isolates with an identical RFLP pattern. Epidemiological linkage through contact tracing was investigated. RESULTS: Of the total of 719 patients, 153 (21.3%) were excluded because there was inadequate bacterial DNA for genotyping (n=129) or the isolates of Mycobacterium tuberculosis had less than five copies of IS6110 (n=24). The isolates from 409 patients (72.3%) were grouped into 78 different clusters with an estimated 58.5% of the cases being due to recent transmission. Young age was the only significant predictor of clustering. Only in 147 (35.9%) of the 409 patients belonging to a cluster could an epidemiological link be found. 111 patients (19.6%) were identified as having had previous contact with a tuberculosis patient and 81 of them (72.9%) belonged to a cluster. The three largest clusters included 75, 49 and 20 patients, respectively. CONCLUSION: Recent transmission is frequent among patients with tuberculosis on Gran Canaria and could be associated with certain aspects of control measures. Some of the clusters described in the study could be due to the prevalence of particular strains of M tuberculosis on the island.     

Associate investigations: detection of tuberculosis infections in children resulting in discovery of undiagnosed tuberculosis in adults

The authors present the design and implementation of associate investigations of young children with positive tuberculin skin test results. Case study analysis of an associate investigation was done using epidemiologic surveillance techniques, medical interviewing, sociogram mapping, tuberculin skin testing, radiographic evidence, and bacteriologic analysis. Deoxyribonucleic acid fingerprinting of the Mycobacterium tuberculosis isolates using a standardized IS6110-based restriction fragment length polymorphism analysis and IS6110-independent DNA spoligotyping methods was done to track and identify specific bacterial strains. Deoxyribonucleic acid fingerprinting and spoligotyping done on isolates obtained from family members demonstrated same-strain transmission of M. tuberculosis. Three adults with active pulmonary disease and six individuals with latent tuberculosis (TB) were discovered during this investigation. The arrival of a family member from Mexico who had the same strain suggests that the source case lives in Mexico. A child with positive tuberculin skin test results indicates recent and potentially ongoing transmission of TB in the community. Targeted tuberculin skin testing performed on high-risk groups by primary care physicians allows for detection of TB infections. When TB infections are discovered in children, associate investigations can result in the discovery of undiagnosed adult cases and prevent further transmission within the community.     

Polymerase chain reaction for the detection of Mycobacterium tuberculosis in synovial fluid, tissue samples, bone marrow aspirate and peripheral blood

Extrapulmonary tuberculosis accounts for approximately 10% of tuberculous infections; the musculoskeletal system is involved in a small number of these (10%). Skeletal tuberculosis is an indolent disease, and diagnosis may be delayed. Conventional methods are time consuming and have a low sensitivity rate. In recent years PCR-based protocols raised hopes as a reliable and fast diagnostic tool for extrapulmonary tuberculosis. The authors report the detection of Mycobacterium tuberculosis complex DNA in specimens from six patients using a nested PCR protocol specific for IS6110 insertion element of Mycobacterium tuberculosis complex. Three men and three women are reported with ages ranging from 42 to 68 years. The sites of infection were the knee and shoulder in one case each, the hip in two cases, and the thoracic spine in two cases. Diagnosis was established within three days, and treatment was initiated promptly. PCR is a technically easy approach that can be used as a first step diagnostic tool for early recognition and treatment of bone and joint tuberculosis.     

Molecular epidemiology of tuberculosis in London 1995-7 showing low rate of active transmission

BACKGROUND: Tuberculosis notification rates for London have risen dramatically in recent years. Molecular typing of Mycobacterium tuberculosis has contributed to our understanding of the epidemiology of tuberculosis throughout the world. This study aimed to assess the degree of recent transmission of M tuberculosis in London and subpopulations of the community with high rates of recent transmission. METHODS: M tuberculosis isolates from all persons from Greater London diagnosed with culture positive tuberculosis between 1 July 1995 and 31 December 1997 were genetically fingerprinted using IS6110 restriction fragment length polymorphism (RFLP) typing. A structured proforma was used during record review of cases of culture confirmed tuberculosis. Cluster analysis was performed and risk factors for clustering were examined in a univariate analysis followed by a logistic regression analysis with membership of a cluster as the outcome variable. RESULTS: RFLP patterns were obtained for 2042 isolates with more than four copies of IS6110; 463 (22.7%) belonged to 169 molecular clusters, which ranged in size from two (65% of clusters) to 12 persons. The estimated rate of recent transmission was 14.4%. Young age (0-19 years) (odds ratio (OR) 2.65, 95% confidence interval (CI) 1.59 to 4.44), birth in the UK (OR 1.55, 95% CI 1.04 to 2.03), black Caribbean ethnic group (OR 2.19, 95% CI 1.15 to 4.16), alcohol dependence (OR 2.33, 95% CI 1.46 to 3.72), and streptomycin resistance (OR 1.82, 95% CI 1.15 to 2.88) were independently associated with an increased risk of clustering. CONCLUSIONS: Tuberculosis in London is largely caused by reactivation or importation of infection by recent immigrants. Newly acquired infection is also common among people with recognised risk factors. Preventative interventions and early diagnosis of immigrants from areas with a high incidence of tuberculosis, together with thorough contact tracing and monitoring of treatment outcome among all cases of tuberculosis (especially in groups at higher risk of recent infection), remains most important.     

Empirical treatment with a fluoroquinolone delays the treatment for tuberculosis and is associated with a poor prognosis in endemic areas

BACKGROUND: A study was conducted to evaluate the effect of the empirical use of fluoroquinolones on the timing of antituberculous treatment and the outcome of patients with tuberculosis in an endemic area. METHODS: All patients with culture confirmed tuberculosis aged > or =14 years diagnosed between July 2002 and December 2003 were included and their medical records were reviewed. RESULTS: Seventy nine (14.4%) of the 548 tuberculosis patients identified received a fluoroquinolone (FQ group), 218 received a non-fluoroquinolone antibiotic (AB group), and 251 received no antibiotics before antituberculous treatment. Fifty two (65.8%) experienced clinical improvement after fluoroquinolone use. In the FQ group the median interval from the initial visit to starting antituberculous treatment was longer than in the AB group and in those who received no antibiotics (41 v 16 v 7 days), and the prognosis was worse (hazard ratio 6.88 (95% CI 1.84 to 25.72)). More patients in the FQ and AB groups were aged >65 years (53.2% and 61.0% v 31.5%), had underlying disease (53.2% and 46.8% v 34.3%), and were hypoalbuminaemic (67.2% and 64.9% v 35.1%). Of the nine mycobacterial isolates obtained after fluoroquinolone use from nine patients whose initial isolates were susceptible to ofloxacin, one (11.1%) was resistant to ofloxacin (after fluoroquinolone use for 7 days). Independent factors for a poor prognosis included empirical fluoroquinolone use, age >65, underlying disease, hypoalbuminaemia, and lack of early antituberculous treatment. CONCLUSIONS: 14.4% of our patients with tuberculosis received a fluoroquinolone before the diagnosis. With a 34 day delay in antituberculous treatment and more frequent coexistence of underlying disease and hypoalbuminaemia, empirical fluoroquinolone treatment was associated with a poor outcome. Mycobacterium tuberculosis isolates could obtain ofloxacin resistance within 1 week.     

Tuberculosis in contacts need not indicate disease transmission

BACKGROUND: Traditional contact investigation is an important tool for controlling tuberculosis. It may also help to indicate drug susceptibility patterns when Mycobacterium tuberculosis cultures are not available. Such investigations often underestimate the degree of transmission found by genotyping, but overestimation may also occur. This report is the result of a routine successive DNA restriction fragment length polymorphism (RFLP) analysis of M tuberculosis isolated in Norway. METHOD: Fifteen immigrants belonging to the same community were notified with tuberculosis during February to September 2003. The mycobacterial isolates were analysed by RFLP. RESULTS: All 15 patients had social contact with each other and 13 belonged to the same church community. A total of 14 cultures were positive for M tuberculosis. Among these isolates, six different genotypes were found. Five patients had not acquired the infection from the putative source. CONCLUSIONS: Reactivation of tuberculosis may occur in contacts during the development of an outbreak. In such situations, traditional contact investigations may overestimate the rate of transmission found by genotyping of M tuberculosis. When cultures are unavailable and presumed drug susceptibility patterns are based on that of contacts, such overestimation may lead to incorrect treatment of a patient. Contact investigations must be combined with genotyping of M tuberculosis to conclude how tuberculosis is transmitted. This is especially important in persons with several risk factors for infection.     

Detection of Mycobacterium tuberculosis DNA in the sclerotic spinal wall

Recent studies have shown that the major spinal lesion in spinal tuberculosis is predominantly sclerotic and accounts for >70% of the lesion. In this type of sclerosis, apart from spinal reactive hyperplasia and increased bone density, the most severe lesion is the formation of a hard outer osteoid shell (the sclerotic wall) around the cheese-like substances and granulated tissues. In the current study, polymerase chain reaction detection of Mycobacterium tuberculosis in the sclerotic wall was performed. Surgical specimens were obtained from 18 patients with spinal tuberculosis with peripheral sclerotic wall (as shown by computed tomography) and included the sclerotic wall, subnormal bone tissue outside the sclerotic wall, and iliac bone tissue (control). The IS986 gene in the samples was amplified by polymerase chain reaction followed by DNA sequencing. The obtained sequences were then compared with the published sequences in GenBank using DNATools version 5.1 software (International Centre for Genetic Engineering and Biotechnology, Trieste, Italy). The polymerase chain reaction results showed that 16 specimens from the sclerotic spinal wall, 3 from the subnormal bone, and 0 from the controls were positive for M tuberculosis, indicating a statistically significant difference (P<.05). These results indicated that M tuberculosis was present in the spinal sclerotic wall. Combined with our previous studies, we conclude that the sclerotic wall should be considered a lesion in patients with spinal tuberculosis.     

Mycobacterial infection in HIV seropositive and seronegative populations, 1987-93.

BACKGROUND--Although the causes of the worldwide resurgence of tuberculosis are multifactorial, the HIV epidemic is believed to have had a central role. Control is further threatened by the emergence of multidrug-resistant tuberculosis. METHODS--A retrospective evaluation was undertaken of trends in pulmonary and extrapulmonary culture positive mycobacterial pathology, and the prevalence of drug-resistant tuberculosis in both HIV seropositive and, presumptively, HIV seronegative patients receiving their clinical care at St Mary's Hospital, London. Five hundred and thirty eight patients (188 of whom were known to be HIV seropositive) with positive mycobacterial isolates between January 1987 and March 1993 were identified from laboratory records. These were cross referenced with drug surveillance records. RESULTS--Overall, between 1987 and 1992 there was a progressive 3.5 fold increase in positive mycobacterial isolates and a 2.5 fold increase in patients with proven mycobacterial infection. This increase was greater within the HIV seropositive population. A total of 663 positive mycobacterial isolates was evaluated; the major pathogen identified was Mycobacterium tuberculosis (379 isolates, 57%). Three hundred and fourteen patients were diagnosed as having M tuberculosis, 49 of whom were HIV seropositive. M tuberculosis was predominantly isolated from the lung. Of 358 positive cultures for M tuberculosis (68 HIV seropositive, 290 presumptively HIV seronegative), only 27 isolates (7.6%), almost exclusively derived from presumed HIV seronegative patients, were resistant to either isoniazid, rifampicin, or both drugs together. No increases in drug-resistant isolates were observed over this period. CONCLUSIONS--There has been a considerable increase in the incidence of tuberculosis in both HIV seronegative and seropositive populations during the study period. The emergence of drug-resistant tuberculosis was not observed.     

Quantitative polymerase chain reaction assessment of chimerism in non-human primates after sex-mismatched islet and bone marrow transplantation

BACKGROUND: Accurate assessment of chimerism in recipients of islet and bone marrow transplantation (BMT) may allow for a clearer assessment of the role of chimerism in islet engraftment or rejection. A quantitative polymerase chain reaction (PCR) assay was developed for the detection of the sex-determining region of the Y chromosome (SRY) in peripheral blood samples from female non-human primate recipients of allogeneic male islets and vertebral body marrow (VBM) from the same donor. METHODS: The assay incorporates a synthetic internal standard (IS) containing the same primer template sequences as the target to compete for primer annealing and amplification. Each DNA sample was coamplified with a constant amount of IS. The concentration of male DNA in the test samples was calculated from the regression equation of a standard curve that was generated by plotting the logarithm of the ratio of the intensities of SRY to IS PCR products versus the logarithm of known percentages of input male DNA. RESULTS: This method allows for a correction of the variability of efficiency of the PCR technique and also overcomes the drawback of time-consuming competitive PCR. Using this assay, we quantitated the amount of male DNA in samples taken from female baboon recipients of male islets and VBM. There was detectable male donor DNA in the samples taken one day after BMT; pre-BMT samples were negative. This technique works well for samples obtained from rhesus and cynomogus monkeys as well. CONCLUSIONS: It is a practical method for accurately evaluation of chimerism after sex-mismatched allogeneic BMT in non-human primate models.     

Use of amplified Mycobacterium tuberculosis direct test (Gen-probe Inc., San Diego, CA, USA) in the diagnosis of tubercular synovitis and early arthritis of knee joint

Background:

The diagnosis of knee joint tuberculosis, especially in early stages of synovial disease, has more often been based on clinicoradiological suspicion, with no single test claiming to be a dependable rapid diagnostic test with high sensitivity and specificity. Nuclear amplification tests in vogue like the polymerase chain reaction have shown variable sensitivity and false positivity rates in various studies. We evaluated the role of Amplified Mycobacterium tuberculosis Direct Test (AMTDT) or Genprobe in the diagnosis of knee joint tuberculosis in early, especially, early synovitis and arthritis cases.

Patients and Methods:

Thirty two patients of suspected knee joint tuberculosis were subjected to diagnostic arthroscopy during the study period. The synovial fluid and tissue were subjected to mycobacterial culture, histopathology, and AMTDT. A comparative analysis of the sensitivity and specificity of this new test with culture and histopathology was done and the time taken for reporting was calculated for each test.

Results:

Out of 32 tissue samples, 8 were found to be positive with mycobacterial culture [Lowenstein Jensen (LJ)/Bactec], 11 were positive with histopathology, and 5 were found to positive with AMTDT. The sensitivity of AMTDT was found to be 62.5% and specificity was 100% with a P value of 0.083. The results were obtained earliest with AMTDT with a mean reporting time of 1.2 days, while the results of histopathology were obtained in a mean time of 6.8 days, BacT alert in 22.5 days, and conventional LJ medium culture results in 48.6 days.

Conclusion:

AMTDT or Genprobe is a rapid diagnostic test for early diagnosis of tubercular arthritis, but has low sensitivity in knee joint tuberculosis. Nuclear amplification tests are still far from being a single promising alternative to conventional tests in cases of joint tuberculosis. Routine use of arthroscopic biopsies in all suspected cases is helpful in the early diagnosis of knee joint tuberculosis.     

Utility of cytomegalovirus viral load in renal transplant patients in Argentina

BACKGROUND: Cytomegalovirus (CMV) is the most prevalent viral disease in solid organ transplantation. Detection of CMV DNA in peripheral blood mononuclear cells (PBMC) by polymerase chain reaction (PCR) frequently occurs in renal allograft recipients, yielding false positive results in seropositive patients free of CMV disease. We evaluated the clinical utility of a quantitative PCR-enzyme-linked immunosorbent assay (ELISA) for identifying patients with CMV disease. METHODS: Three hundred and fifty samples from 65 consecutive renal transplant recipients were studied. DNA was extracted from PBMC weekly up to the day of discharge and after any further admission. Samples were tested by a qualitative PCR method, and all positive samples were further studied by a quantitative PCR-ELISA method. The quantitative PCR-ELISA method used an internal standard (IS) that contained the primer sequences used in the qualitative CMV PCR. Detection and quantification was performed in 96-well plates coated with IS or CMV specific probes. RESULTS: Forty-one of 65 patients (63.1%) showed positive results by the qualitative PCR, but only 8 of these patients were diagnosed with CMV disease. Positive samples were re-analyzed by the quantitative assay. The 8 patients with CMV disease had a mean CMV viral load of 1,438+/-687 viral copies (VC)/10(6) PBMC, and the 33 without CMV disease had a mean value of 219.6+/-117.2 VC/10(6) PBMC (P<0.01). None of the 33 patients without CMV disease had viral loads higher than 500 VC/10(6) PBMC. Using 500 VC/10(6) PBMC as a cut-off value for CMV disease, the quantitative PCR showed a sensitivity and specificity of 100% compare to clinical diagnosis. CONCLUSION: CMV viral load may be useful in the diagnosis of CMV disease in renal transplant patients.