Bacterial dissemination in Mycobacterium tuberculosis by CD+ T-cells & proinflammatory cytokines.

Background & objectives: As CD4+ and CD8+ T lymphocyte numbers decline, the conventional, localized forms of tuberculosis shift to the atypical, disseminated forms. Variations in lymphocyte and immune cell expression levels affect how tuberculosis manifests in disseminated forms. Understanding the relationship between lymphocyte counts (CD4+ and CD8+) and pro-inflammatory cytokines such as tumour necrosis factor-alpha, interleukin-12 and interferon, we may therefore be able to shed light on how infections spread and suggest potential biomarkers for these immune factors. Methods: In this study, 15 guinea pigs were infected with Mycobacterium tuberculosis (M.tb) H37Rv strain and grouped into three groups of five each for further investigation. Serum samples and bronchoalveolar lavage (BAL) fluid were examined for the expression of pro-inflammatory cytokines and T-cell subsets in guinea pigs infected with pulmonary tuberculosis and disseminated tuberculosis. Results: We found that M.tb escapes macrophages due to pro-inflammatory cytokine dysregulation. Despite the protective immunity created by T-cells and cytokines, M.tb bacilli may spread to other organs due to inflammation induced by these immune components. A high number of T-cells and stimulated cytokine production are involved in triggering inflammation after necrotic tissue develops and tuberculosis spreads. Interpretation & conclusions: Our findings imply that increased bacilli in the spleen at the 8th wk of infection may be caused by the overexpression of CD4+ T-cell lymphocyte subsets and cytokines that generated inflammation during the 4th wk of infection. This is a pilot study with a small sample size and less assertive inference. Larger studies would be helpful to validate the results of the present investigation.

Spoligotyping of Mycobacterium tuberculosis isolates from Pulmonary Tuberculosis patients from North Karnataka, India.

Tuberculosis (TB) is a leading cause of morbidity and mortality in low income countries. Multi-drug resistant (MDR-TB) is seen as the reason for many TB outbreaks globally and is also a threat to control programmes. India accounts for 27% TB cases worldwide. Our study was undertaken to understand the outbreaks related to MTB. All the sputum samples were subjected to microscopy and smear positive samples were cultured on Lowenstein-Jensen (L-J) media. Identification was carried by biochemical analysis. A total of 57 isolates were subjected to Drug Susceptibility testing (DST) and spoligotyping, where eleven MDR-TB isolates were confirmed, of which ten were SIT1/Beijing and one SIT53/T1. Spoligotyping results showed that the predominant lineage in this region was SIT1/Beijing followed by SIT124/U and the strains which did not match spoligodatabase were named as orphans. In this study, MDR-TB was associated with SIT1/Beijing and mono resistance belonged to CAS1_DEL.

Omental desmoplastic small round cell tumor with metastasis.

Desmoplastic small round cell tumor (DSRCT) is a very rare diagnosis with about 200 cases reported in literature. DSRCT is a recently described histopathological entity by Gerald and Rosai in 1989. Abdominopelvic cavity especially peritoneum is the most common site. We report a case of a huge omental DSRCT with lymph node metastasis which was initially misdiagnosed as gastrointestinal stromal tumor on radiology. A 26-year-old male presented with complaints of upper abdominal swelling associated with constant dull pain. On examination there was a large 15 × 12 cm intraabdominal mass in the epigastric and umbilical region. Imaging studies were suggestive of neoplastic mesenchymal etiology. Image-guided fine-needle aspiration cytology (FNAC) was suggestive of mesenchymal neoplastic etiology. On laparotomy, there was a huge 20 × 15 cm mass arising from omentum with multiple omental and mesenteric seedlings and mesenteric, peripancreatic and perigastric lymphadenopathy. The patient underwent debulking surgery with uneventful post-operative recovery. Histopathological examination with immunohistochemistry revealed a diagnosis of DSRCT of omentum and small bowel mesentery with lymph node metastasis. Patient then received adjuvant chemotherapy with multiple chemotherapeutic drugs as per P6 protocol and has stable disease at 1 year follow up.

A clinicopathological study of peripheral schwannomas.

AIM AND OBJECTIVE: Schwannomas are benign neoplasms of neural origin with sporadic or syndromic occurence. They are commonly seen in cranial nerves. Peripheral schwannomas occur rarely and may have unique presentations. The aim of this study is to evaluate the clinico-pathological characteristics of peripheral schwannomas. METHODS: A retrospective cross sectional study of peripheral schwannomas excluding head neck region was conducted. The study group consisted of 18 cases which were recorded over a period of seven years. The corresponding data were collected from the archives of the Department of Pathology. RESULTS: Male to female ratio was 1:1. The average age of the cases was 47 years. The most common site was the upper limbs (55.55%) followed by lower limbs, chest and penis. The lesions mostly presented as painless swellings (62%). Histopathological examination revealed classic features of schwannoma. Secondary changes included cystic degeneration, foam cells, epitheloid cells, hyalinization, microcystic change and collection of plasma cells. All cases were confirmed by positive S100 staining. CONCLUSION: Peripheral schwannomas may be missed due to its rarity and atypical presentations. Both clinicians and pathologists should be aware of this common entity at unusual sites for the proper management of the patients. Surgery is usually the treatment of choice.

Potential of a metabolic gene (accA3) of M. leprae as a marker for leprosy reactions.

Understanding the mechanism(s) of reactions in leprosy remains a challenging task for both clinicians and basic scientists. While there is some understanding of host processes associated with different type of lepra reactions, there is very little information about bacterial factors triggering these inflammatory processes. This study is continuation of our earlier research programme on leprosy genomics in which significant transcription of 11 genes was observed during active disease and these included accA3 gene. In present study, we have investigated the potential of this gene or its gene product as molecular and or immunological marker for studying the reactions. Using quantitative Real-Time RT-PCR significant higher expression (mean log2 ratio=3.39) of accA3 was observed in specimens from leprosy reaction cases compared with cases without reactions. in silico homology model of this protein was analyzed for hydrophilic and B-cell epitope regions. Peptides with maximum antigenecity were selected, cloned, expressed and used to study sero-reactivity across the disease spectrum by indirect ELISA. While sero-reactivity was observed in leprosy cases the antibody levels did not vary significantly between the patient/s of same clinical type with and without reaction thereby indicating the limitation of this approach for this purpose. Measurement of transcription of this gene has, thus, potential as a molecular marker for monitoring the reactions.

Association of tuberculous endometritis with infertility and other gynecological complaints of women in India.

Endometrial biopsy samples derived from 393 patients with assorted gynecological complaints were investigated for mycobacterial infection. By employment of four different techniques, mycobacterial pathogens were detected irrespective of the nature/type of clinical complaint. Mycobacterium tuberculosis was the predominant pathogen detected among the samples investigated.

Isolation of Mycobacterium bovis & M. tuberculosis from cattle of some farms in north India--possible relevance in human health.

BACKGROUND & OBJECTIVE: Infection due to Mycobacterium bovis typically occurs in cattle and animals transmit infection to each other. The choice of appropriate clinical specimen is very important for isolation of M. bovis and M. tuberculosis from cattle. The present study reports the isolation of M. tuberculosis and M. bovis from different types of specimens from cattle suspected to be suffering from tuberculosis in certain organized cattle farms in north India. METHODS: A total of 768 specimens (heparinized or EDTA containing blood (162), fine needle aspirates from prescapular lymph gland (PSLG,160), milk (154), pharyngeal swab (PhS, 98), rectal pinch (RP, 97) and faecal sample (97) from 161 cattle of organized cattle farms in north India suspected to be suffering from tuberculosis were analyzed. After decontamination by modified Petroff's method isolation of M.tuberculosis complex was done on Lowenstein-Jensen medium (with and without pyruvate). The culture isolates were identified as M. tuberculosis and M. bovis on the basis of biochemical tests. RESULTS: A total of 54 M. tuberculosis complex isolates were obtained, of them 40 were identified as M.bovis and 14 as M. tuberculosis. M.bovis were isolated from 12 of 38 animals in group A (Tuberculin +ve with signs of tuberculosis), 7 of 37 animals in group B (Tuberculin +ve and apparently healthy), 9 of 21 group C animals in (Tuberculin -ve with clinical signs of tuberculosis), 4 of 26 animals in group D (Tuberculin -ve and apparently healthy), 4 of 27 group E animals (having non-mycobacterial infection) and 4 of 12 animals in group F (having clinical signs such as debilitated condition, cough, decreasing milk production, etc). Maximum number of M. bovis (19/40, 47.5%) and M. tuberculosis (5/14, 35.7%) isolates were grown from prescapular lymph gland biopsy (PSLG) followed by blood from which 9/40 (22.5%) M. bovis and 4/14 (28.5%) M. tuberculosis were isolated. M. bovis [6/40(15%)] and M. tuberculosis [4/14(28.5%)] were also isolated from milk. Only 3/40 (7.5%) isolates of M.bovis could be isolated from 97 rectal pinch followed by 98 pharyngeal swab 2/40 (5%) and 97 fecal samples 1/40 (2.5%) while 1/14 (7.1%) M.tuberculosis isolates were obtained from pharyngeal swab. INTERPRETATION & CONCLUSION: Among the samples analyzed, PSLG was found to be most suitable specimen for isolation of M. tuberculosis complex from cattle and is thus of diagnostic importance. M. bovis in milk indicates the need to investigate the transmission to human in such settings. Isolation of M. bovis and/or M. tuberculosis from apparently healthy cattle indicates sub-clinical infection in the herd. Further, isolation of a significant number of M. tuberculosis from cattle suggests possible human-to-cattle transmission which need to be confirmed by prospective studies including tools like DNA fingerprinting.

Development and evaluation of real-time RT-PCR assay for quantitative estimation of viable Mycobacterium leprae in clinical samples.

Detection of live organisms by molecular methods has special significance in leprosy where causative organism can not be cultivated in vitro. Such techniques would be especially important for monitoring the progress of the disease. While real-time RT- PCR technology will be appropriate for this purpose, there is very little experience of use of such tools in leprosy. This study describes the development of a quantitative RT-PCR targeting 16S rRNA based on primers used in a semi quantitative RT-PCR and its application on clinical samples including slit scraping and biopsies. RNA was extracted from biopsies from 3 lepromatous leprosy (LL) cases and standard curve was generated by plotting crossing over point against the dilutions of input RNA quantity (number of bacilli used for RNA extraction). Real-time RT-PCR was performed for quantitative detection of live M. leprae in 28 slit (13/28 smear positive) scrappings and 32 biopsies (22/32 smear positive). Number of viable bacteria as estimated by solid stained bacilli and real-time PCR correlated (no difference p>0.05). The test achieved a theoretical analytical sensitivity limit of up to single live bacillus even considering 11.3% efficiency of RNA preparation which was calculated by spiking of known number of leprosy bacilli in non leprosy skin biopsies (PCR negative). All smear positive cases were positive by this assay. This assay appears to be a promising tool for detection and quantification of viable bacilli in selected clinical situations and should be of use even in smear negative cases also.

Estimation of efflux mediated multi-drug resistance and its correlation with expression levels of two major efflux pumps in mycobacteria.

Multidrug resistance has been posing an increasing problem in the treatment of tuberculosis. Mutations in the genomic targets of drugs have been identified as the major mechanism behind this resistance. However, high degree of resistance in some isolates towards major drugs like rifampicin, isoniazid, ethambutol and streptomycin can not be explained solely on the basis of mutations. Besides this, certain other mechanisms like efflux pumps have also been considered as alternative mechanisms in the drug resistant isolates where there is no mutation and these mechanisms are specially important for drug resistance in non-tuberculous mycobacteria (NTM). In this study, we have estimated efflux pump mediated drug resistance in different mycobacterial species with the help of efflux pump inhibitors. All major anti-tuberculous drugs have been shown to be extruded by efflux pumps and the degree to which these drugs are extruded, vary in different mycobacterial species and isolates. The correlation of this resistance with functional activity of two major efflux pump genes pstB and Rv1258c was also assessed by reverse transcription PCR. Besides the significant role of these pumps observed, other efflux pumps, present in mycobacteria, may also be involved in drug resistance and need to be investigated.

Detection of M. leprae by reverse transcription- PCR in biopsy specimens from leprosy cases: a preliminary study.

A reverse transcription (RT)-PCR assay targeting 16S rRNA of Mycobacterium leprae has been used to detect M.leprae specific nucleic acids. This study has been initiated to gain experience about detection of RNA from seven biopsy specimens by RT-PCR assay using species- specific primers described earlier. These biopsy specimens were from clinically confirmed and untreated leprosy cases belonging to BB and BL types. The earlier reported method was established in our laboratory. 171 bp fragment by RT-PCR was amplified from 4/7 cases. The positives results by RT-PCR were from the biopsies from fresh or short term treated cases whereas negative results were from specimens from long term treated cases showing clinical features of relapse. DNA targeting PCR (36 KDa) showed positivity in both groups. These results suggest that RT-PCR positivity possibly reflect the presence of viable organisms. Thus as earlier predicted RT-PCR assay may be useful for viability determinations for assessing the response to chemotherapy as well as presence of persisters in relapse cases.

Analysis of quantitative relationship between viability determination in leprosy by MFP, ATP bioluminescence and gene amplification assay.

Two hundred twenty-one untreated, borderline lepromatous/lepromatous (BL/LL) leprosy patients have been investigated for viability by the mouse foot pad method (MFP), adenosine triphosphate (ATP) and polymerase chain reaction (PCR). The biopsies were collected at the beginning of and 12/24 months after treatment. The patient group was treated with a) immunotherapy (BCG/Mw) + MDT; b) MDT + pyrazinamide; c) control MDT; d) MDT + minocycline 100 mg once a month supervised + ofloxacin 400 mg once a month supervised. Biopsies were divided in three parts for use in the mouse foot pad, molecular and ATP investigations. In untreated and treated patients (at 12 and 24 months), there was a general agreement among all three techniques, and PCR and ATP showed higher positivity as compared to MFP. Further, there was good correlation among the viable biomass estimated by bacillary ATP levels, PCR assay and growth in mouse foot pads. The positivity was observed by MFP as well as PCR assay (18-kDa and 36-kDa) from all of the specimens when the ATP content was more than 3.6 pg/million. When the ATP content was below 3.5 pg/million, the positive takes in MFP decreased but the PCR positivity correlated with ATP bioluminescence up to 0.04 pg/million. When the ATP content was even lower, the uptake in the MFP was possibly a matter of chance, while PCR positivity was observed in 96% of the cases. For specimens with undetectable ATP, positivity was seen in 1% of the cases, showing the inability of ATP bioluminescence method to detect low background due to host ATP. PCR signals in some cases could be due to the higher sensitivity of the method or persistence of DNA after bacterial death in some cases. On the whole, the PCR methods even though targeting DNA have shown good correlations with biomass which confirm their usefulness in monitoring therapeutic responses in leprosy.

Comparison between optical coherence tomography and fundus fluorescein angiography for the detection of cystoid macular edema in patients with uveitis.

PURPOSE: To compare optical coherence tomography (OCT) with fundus fluorescein angiography (FFA) for the detection of cystoid macular edema (CME) in patients with uveitis. DESIGN: Prospective comparative observational series. PARTICIPANTS: One hundred twenty-one eyes of 58 patients with uveitis of varied causes (seven patients were studied twice). TESTING: Patients with suspected CME underwent OCT scanning followed by FFA at the same visit. MAIN OUTCOME MEASURES: Detection and distribution of macular edema. RESULTS: One hundred eight eyes had similar results on both OCT and FFA in that 67 eyes had CME and 41 eyes had no CME. In 10 eyes subretinal fluid was detected on OCT but not FFA. Five of these eyes had CME on FFA but not OCT. Three other eyes had CME that was detected by FFA but not by OCT. Compared with FFA, the OCT sensitivity for detecting CME was 96% (including the eyes with subretinal fluid), and the OCT specificity was 100%. CONCLUSIONS: OCT is as effective at detecting CME as is FFA but is superior in demonstrating axial distribution of fluid.

Papillofoveal traction in macular hole formation: the role of optical coherence tomography.

OBJECTIVES: To determine the validity of the assumption that optical coherence tomographic scans of macular holes have a discrete linear signal (DLS) that represents a detached posterior vitreous face, and to analyze the DLS in macular hole pathogenesis. METHODS: Optical coherence tomographic scans were taken of 3 situations in which the vitreous conditions were known: (1) dissected intact vitreous, (2) clinically evident Weiss rings, and (3) maculae before and after saccades in eyes without a biomicroscopic posterior vitreous detachment. In addition, 70 eyes of 35 patients with macular holes underwent clinical examination and optical coherence tomographic scanning that passed through the optic disc and the fovea or macular hole. RESULTS: Spatial properties of the DLS matched those of the posterior vitreous face in the situations examined. Of the 70 eyes, 16 (23%) had a biomicroscopic posterior vitreous detachment, whereas a DLS was demonstrated in 40 (57%). Of the 54 eyes without a biomicroscopic posterior vitreous detachment, 18 (33%) had a DLS attached focally to the optic disc margin and the fovea or macular hole. All 7 of the "can opener" holes examined had a nasally "hinged" central flap, 6 with a focally attached DLS. CONCLUSIONS: The DLS corresponds to the posterior vitreous face. Anteronasal papillofoveal traction may generate some macular holes.

Effect of treatment on PCR positivity in multibacillary leprosy patients treated with conventional and newer drugs ofloxacin and minocycline.

In order to develop objective criteria to monitor trends of therapeutic responses positivity of PCR signals and ATP assay methods has been compared in multibacillary (MB) leprosy patients. Biopsies from lesions of 95 BL/LL patients before and after one year of treatment with a new drug regimen comprising of conventional and newer drugs ofloxacin and minocycline have been studied. These biopsies were processed for bacillary ATP assay and PCR positivity for a 36 kDa gene target by earlier published methods. In the untreated patients bacillary ATP levels were detectable in all specimens and ranged from 0.02 to more than 36 pg/millions organisms. After one year of treatment ATP levels were not detectable in any of the 57 biopsies specimens available for analysis. However, PCR signals were detectable in 3 out of 57 biopsies. In two specimens signals were very weak detectable only by hybridization. It may be concluded that DNA based PCR assay may be useful in monitoring the trends of therapeutic responses in MB patients under treatment.