Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning.

Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39-31.27% for rpoB-PCR, 36.44-60.83% for IS6110- PCR, 75.29-92.93% for nested-rpoB PCR, and 87.98-99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100% specificity of each PCR method were calculated as 69.15-100%. Our results indicated that nested-rpoB PCR combined with TA cloning and sequencing is a preferred method for the detection of MTB DNA in EPTB samples with high sensitivity and specificity which confirm the histopathology results.

Synchronously diagnosed eosinophilic granuloma and Hodgkin's disease in a 12-year-old boy: a case report.

INTRODUCTION: Synchronous composite tumors are uncommon. Simultaneous, rather than metachronous or consecutive, occurrences of eosinophilic granuloma and Hodgkin's lymphoma in children are very rare. This is the first report of this kind in the medical literature. CASE PRESENTATION: We report the case of a 12-year-old Iranian boy with eosinophilic granuloma localized in his leg around the knee and Hodgkin's lymphoma in a cervical lymph node. The two tumours occurred synchronously before the patient had received any treatment. CONCLUSION: Several cases of an association between eosinophilic granuloma and lymphoproliferative disorder have been reported. Some of these cases involve Hodgkin's lymphoma and Langerhans cell histiocytosis occurring in the same patient. Genetic or environmental etiologies have been postulated for eosinophilic granulomas which occur following Hodgkin's lymphomas, but have as yet not been proven. To our knowledge, synchronous occurrence of these two malignant processes in a patient who has not received any prior treatment is rare in children.