IS6110 Polymerase Chain Reaction Research Articles

First Genotyping of Mycobacterium avium Subspecies Paratuberculosis from Large Ruminant Population at Satna District in Madhya Pradesh in India to Bison Type

Background: Paratuberculosis or Johne’s disease in large ruminants is caused due to Mycobacterium avium subspecies paratuberculosis. The bacterial disease in large ruminants is characterized by chronic diarrhea, weakness and emaciation. There are no reports on Paratuberculosis in this area’s large ruminant population and its genotyping. Methods: A total of 76 samples (26 fecal and 50 milk) of large ruminants from Satna district in Madhya Pradesh were investigated (microscopical; for the presence of acid-fast organisms and molecular methods; for gene-specific amplification) for the presence of Mycobacterium avium subspecies paratuberculosis infection. Result: Microscopical examinations of fecal samples revealed 11.54% positivity and milk samples 14% positivity of Mycobacterium avium subspecies paratuberculosis infection. A single fecal and 2 milk samples were found positive in the IS900 polymerase chain reaction test (413 bp product). Specific amplification of 608 bp IS1311 product followed by restriction endonuclease analysis proved the genotype of Mycobacterium avium subspecies paratuberculosis as Bison type.

Comparison of Protein B Polymerase Chain Reaction (PCR) With IS6110 PCR for Diagnosis of Tuberculous Meningitis Patients.

Purpose Tuberculous meningitis (TBM) is a diagnostic challenge. With the conventional staining and culture techniques being too insensitive and time-consuming, and the commercial detection systems being costly, polymerase chain reaction (PCR) seems lucrative for routine laboratories. The purpose of this study was to evaluate the diagnostic potential of protein b antigen polymerase chain reaction (Pab PCR) for TBM, in comparison to IS6110. Another purpose was to compute a cut-off at which adenosine deaminase (ADA) could diagnose TBM. Material and methods This is a prospective case-control study to measure the diagnostic accuracy of PCR, BACTEC culture, Lowenstein-Jensen (LJ) culture, ADA, and acid-fast bacilli (AFB) smear tests in TBM. CSF from 50 TBM patients (10 confirmed, 40 clinically suspected) and 40 controls was subjected to Pab PCR and IS6110 PCR, and performance was compared against culture and composite reference standards. Results The overall sensitivity, specificity, positive predictive value (PPV), and negative predictive value (NPV) of Pab PCR in diagnosing TBM were 82%, 100%, 100%, and 81.63%, respectively, and that of IS6110 PCR were 74%, 100%, 100%, and 75.47%, respectively. Both PCRs outperformed culture (p<0.001). Though performance of both PCRs was comparable (p=0.335) with excellent agreement (k=0.86), Pab PCR detected four additional cases, one culture-positive and three culture-negative clinically suspected. ADA of 6.5 IU/L was able to differentiate between TBM and non-TBM with 86% sensitivity and 63% specificity. Conclusions Molecular tools such as PCR have the potential to increase the clinician's ability to diagnose tuberculous meningitis. Pab PCR is a rapid and reliable method for diagnosing TBM in routine microbiology laboratories.

First identification of Mycobacterium avium subsp. paratuberculosis in wild ruminants in a zoo in Mexico.

Background and Aim:Paratuberculosis (PTB) is an infectious disease that induces chronic enteritis in ruminants. It is caused by Mycobacterium avium subsp. paratuberculosis (MAP). In this study, we evaluated the presence of MAP using bacteriological, molecular, and anatomopathological studies, based on the clinical suspicion of PTB in a zoo, in an area housing 10 scimitar-horned oryx (Oryx dammah), five giraffes (Giraffa camelopardalis), and three blue wildebeests (Connochaetes taurinus).Materials and Methods:From November 2016 to June 2017, fecal samples were collected from individuals of the three species on four occasions, resulting in a total of 56 fecal samples. In addition, five small intestine samples were collected from the necropsies of three adult scimitar-horned oryx females and two oryx calves. MAP identification was performed through isolation in Herrold’s medium with egg yolk, mycobactin, and sodium pyruvate, Ziehl–Neelsen staining, IS900 polymerase chain reaction (IS900 PCR), and anatomopathological examination of intestine samples.Results:Diffuse granulomatous enteritis with abundant acid-fast bacilli was found in two out of five intestine samples from adult scimitar-horned oryx females. MAP was isolated in 7/56 (12.5%) of the fecal samples from four scimitar-horned oryx, one giraffe, and two wildebeest samples. Two out of 5 (40%) samples obtained from scimitar-horned oryx tested positive. IS900 PCR yielded five positive samples (two fecal samples and three small intestine samples). MAP isolates were classified as Type C (Cattle) using type-specific PCR.Conclusion:These results demonstrated the presence of MAP in the area evaluated and indicated the importance of both sampling live animals and conducting postmortem examinations. The use of bacteriological and histopathological diagnostic techniques demonstrated in this study will provide insight into the health status and prevalence of paratuberculosis in wild ruminants under human care.

Comparison of Polymerase Chain Reaction With Fluorescent Microscopy for Detecting Pulmonary Tuberculosis in Smear-Negative Sputum Samples in Quetta, Pakistan

Background: One of the common clinical manifestations of extra-pulmonary tuberculosis is tuberculous meningitis (TBM). New methods, which are simple and effective, are necessary for early diagnosis of TBM. Objective: This study aimed to assess the use of fluorescence microscopy and polymerase chain reaction (PCR) techniques targeting pncA and IS6110. PCR is a fast and reliable technique for diagnosing tuberculosis (TB), particularly in paucibacillary specimens such as smear-negative sputum for diagnosing patients with Mycobacterium tuberculosis. Materials and Methods: The pncA and IS6110 multiplex-PCR methods were applied to analyze sputum using a specific pair of primers designed to amplify the insertion sequence, pncA, and IS6110 in the M. tuberculosis genome. A total of 200 sputum samples were collected from patients with TB. Out of 200 patients, 54.0% (n=108) and 46.0% (n=92) were males and females at Fatima Jinnah General and Chest Hospital, Quetta, respectively. Results: M. tuberculosis was detected as 15 (16.3%) and 18 (16.7%) for pncA and IS6110 using PCR and fluorescence microscopy, respectively. M. tuberculosis DNA detected by PCR and smear-negative fluorescence microscopy was 16.50% (n=33) and 14.70% (n=18), respectively. In other words, PCR assay detected the increased prevalence of M. tuberculosis. Risk factors revealed high exposure to receiving TB infection which was associated with urban areas (OR=1.07, 95% CI: 0.34–3.35), TB in family (OR=1.22, 95% CI: 0.38-3.88), over crowdedness (OR=2.93, 95% CI: 0.37-23.7), malnutrition (OR: 1.39; 95% CI: 0.52-3.74), and rural areas (OR: 1.54, 95% CI: 0.72-3.31). Conclusion: A wide range of molecular assays are now being developed and reviewed for detecting TB. We conclude that the use of pncA and IS6110 PCR assays is beneficial in the quick diagnosis of TB meningitis.

Genetic diversity of Mycobacterium avium sp. paratuberculosis by mycobacterial interspersed repetitive Unit-Variable number tandem repeat and multi-locus short-sequence repeat one-sentence summary: Genetic diversity of Mycobacterium avium sp. paratuberculosis isolates.

Paratuberculosis is an enteric disease caused by Mycobacterium avium sp. paratuberculosis (MAP) that affects mainly ruminant producing losses to the livestock industry. Many molecular epidemiological methods have been used to discriminate MAP isolates. The aim of this study was to describe the genetic diversity of the Argentinean MAP isolates using a combination of two molecular systems, the mycobacterial interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) ("automated and "non-automated") and the multi-locus short-sequence repeat (MLSSR) system. Thirty-two isolates were identified as MAP of C type by IS900 polymerase chain reaction (PCA) and IS1311 PCA-restriction enzyme analysis. The main patterns found by both MIRU-VNTR systems were INMV1 (54.5%), INMV2 (24.2%) and INMV11 (9.1%). The INMV5, INMV8 and INMV16 were represented with one isolate each (3.0%). Only 4 MIRU-VNTR loci were polymorphic. Those isolates sharing the same INMV patterns were analyzed by MLSSR, being locus 2 the most polymorphic one showing isolates with 9, 10, 11, and more than 11 "G" repeats. Besides, the global discriminatory power among isolates could be increased using both techniques. Based on these results, a short version of the "automated" MIRU-VNTR could be used as a screening tool to group isolates genetically related and subsequently perform the SSR using locus 2 on those isolates sharing the same INMV pattern.

Molecular diagnostics for verification of pleural tuberculosis in Morocco

Pleural tuberculosis (pTB) is a very common form of extrapulmonary tuberculosis (TB). pTB diagnostics represents a major burning challenge worldwide due to the limitations of available conventional diagnostic tools. These latter include microscopic examination of the pleural fluid for acid-fast bacilli, mycobacterial culture of pleural fluid in solid or liquid media, sputum or pleural tissue, and histopathological examination of pleural tissue; these tests have recognized limitations for clinical use. Hence, to overcome these limitations, attention has been devoted to new nucleic acid amplification (NAA) diagnostic tests such as the polymerase chain reaction (PCR) and real-time PCR (RT-PCR), owing to their accuracy, rapidity, high sensitivity and specificity. Within this context, this prospective study was conducted to evaluate the performance of molecular diagnosis methods for differentiation between tuberculosis and non-tuberculosis pleural effusions. Fifty patients with pleural effusion were enrolled in this prospective study in Rabat, Morocco. The efficacy of conventional polymerase chain reaction (PCR) in the diagnostics of tuberculous pleurisy by targeting IS6110 and mycobacterial internal transcribed spacer (MYITS) was evaluated compared to histopathologic examination and culture data. Our results showed that IS6110 PCR could “rule in” pTB, the sensitivity and specificity being 41.6% and 85.7%, respectively. Therefore, the findings confirmed that molecular tests exert a relatively high specificity in EPTB but lower sensitivity, thus a positive test is considered as a pTB case whereas negative one cannot exclude the disease. Although the study was limited by a small sample size, it adds to the body of evidence of usefulness of molecular testing as adjuncts to histopathologic examination for accurate diagnosis of pTB, to treat timely and to avoid the emergence and spread of drug resistant pTB. However, further efforts should be made to increase the sensitivity of NAA methods and to identify the best molecular targets to be useful in clinical practice.

Multi-targeted loop mediated amplification PCR for diagnosis of extrapulmonary tuberculosis

AimsThe present study was conceived to evaluate multi-targeted loop mediated amplification (MLAMP) for the rapid diagnosis of extrapulmonary tuberculosis (EPTB). Methods and resultsA total of 700 patients were included who were classified into 2 groups: Group 1 (n = 400) included a 100 culture confirmed EPTB patients and 300 culture negative, suspected EPTB patients. Group 2 (n = 300) included negative controls from non-tubercular patients. All samples were subjected to Ziehl-Neelsen microscopy, solid culture on Lowenstein Jensen media, Polymerase chain reaction (PCR) targeting IS6110 gene and LAMP targeting both IS6110 and MPB64 individually and as MLAMP. The overall sensitivity of microscopy, culture, IS6110 PCR, IS6110 LAMP, MPB64 LAMP and the MLAMP assay were 12%, 25%, 72.5%, 80% and 86.6% respectively and the specificity of all the tests was 100%. ConclusionMLAMP is a rapid robust tool for the diagnosis of EPTB and utilizing two targets for M. tuberculosis can improve the overall sensitivity and increase the yield of detection from extrapulmonary samples. The rapidity, ease of performance and low cost make MLAMP an excellent alternative in low-income, resource limited settings.

Investigation of Mycobacterium paratuberculosis in Arabian dromedary camels (Camelus dromedarius).

Aim:Mycobacterium avium subspecies paratuberculosis (MAP) causes Johne’s disease in ruminants. This study aimed to investigate Mycobacterium paratuberculosis infection in clinically infected camels on the immunological, conventional bacteriological, and molecular biological basis.Materials and Methods:A total of 30 Arabian camels (Camelus dromedarius) were examined in this study. The camels were suffering from signs ranging from mild to severe infections (that did not respond to antibiotic treatment) to chronic or intermittent diarrhea. Camels were grouped into three groups based on their age, sex, and breed. Detection of anti-MAP antibodies in camels’ serum, Ziehl–Neelsen (ZN) staining technique on rectal scraps, direct recognition of MAP in stool and tissue specimens by IS900 polymerase chain reaction (PCR) assay, and finally isolation and molecular description of MAP from fecal and tissue samples were carried out.Results:Five MAP isolates were recovered from these investigated camel samples giving an isolation rate of 16.6%, while eight camels were identified by PCR (26.6%). Five camels yielded MAP in their feces by ZN fecal staining (16.6%), whereas ELISA detected anti-MAP antibodies in nine camels only (30%).Conclusion:From the obtained results, we concluded that the gold standard for the diagnosis of MAP is the culture method despite its limitations. Molecular diagnosis (PCR) could be a useful tool in the identification of truly positive and negative camels; however, great care should be given regarding the primers specificity and sensitivity.

Utility of PCR targeting IS6110 and MPT64 genes in the diagnosis of extrapulmonary tuberculosis

Abstract Introduction Extrapulmonary tuberculosis (EPTB) is one of the major diagnostic challenges in clinical practice. The aim of the study was to compare the utility of Polymerase Chain Reaction (PCR) with conventional techniques in the diagnosis of EPTB. Materials and methods Specimens from 150 patients with a presumptive diagnosis of EPTB were examined by Zeihl-Neelsen staining and mycobacterial culture on Lowenstein Jensen medium. Histopathology was done for lymph node aspirates and endometrial biopsies. DNA from all the samples was amplified by PCR targeting 123 bp and 240 bp fragments of IS6110 and MPT64 genes respectively. Results IS6110 PCR was more sensitive (82.1%) as compared to PCR for MPT64 gene (81%) though the difference was not found to be statistically significant. Four (2.6%) and 2 (1.3%) samples were positive by IS6110-PCR only and MPT64-PCR only respectively.Conclusion: PCR with multiple targets can have potentially important role in improving diagnostic accuracy of EPTB.

Exploring the potential hazard of Mycobacterium avium subspecies paratuberculosis as a cause for Crohn's disease.

Background:Mycobacterium avium subspecies paratuberculosis (MAP) is the causative agent of Johne’s disease (JD), or paratuberculosis in ruminants has been suspected to be implicated in the pathogenesis of Crohn’s disease (CD) in humans with chronic inflammatory intestinal changes. As the hypothesis is now fast being recognized that MAP could possibly be the etiological agent of CD which is found to be excreted in milk of dairy animals subclinically or terminally ill with JD.Aim:The present study was aimed to detect MAP in milk by polymerase chain reaction (PCR) targeting IS900 and to describe the excretion pattern of MAP in milk from asymptomatic lactating cows and does with relevance to the public health significance.Materials and Methods:A total of 77 milk samples were collected randomly from lactating animals which include cows (45) and does (32). All the 77 milk samples were processed to identify the presence of MAP by employing the direct IS900 PCR as per the standard protocol.Results:Out of 77 milk samples from asymptomatic lactating animals, 12 (15.58%) were showed positivity for IS900 PCR in which 5 (11.11%) were from lactating cows and 7 (21.87%) were from lactating does.Conclusion:In our study, 15.58% of milk samples showed IS900 positivity which indicates the presence of subclinical MAP infection in lactating animals. Hence, there is a possibility for excretion of MAP through milk which can be a potential threat for CD in humans by raw milk consumption. Therefore, the prevention of MAP in the food chain need to be assured by sourcing raw products from animal herds free of MAP infection.

Faecal Culture and IS900 PCR Assay for the Detection of Mycobacterium avium subsp. paratuberculosis in Bovine Faecal Samples

Mycobacterium avium subsp. paratuberculosis is a pathogen that causes johne’s disease in animals and is implicated in Crohn’s disease in humans. Culture of Mycobacterium avium subsp. paratuberculosis (MAP) from faeces has been considered the gold standard for the diagnosis of paratuberculosis for many years. However, direct faecal polymerase chain reaction (PCR) is becoming more widely used, demonstrating similar sensitivity and specificity to culture. In the present study, faecal culture and IS900 Polymerase chain reaction (PCR) assay of faecal samples was done on 200 clinically suspected cases of Johne’s disease in dairy cattle. One isolates appeared only on the mycobactin J supplemented media at 8–16 weeks post-inoculation. A total of 7 faecal samples out of 200 samples were detected positive by IS900 PCR assay for Mycobacterium avium subsp. paratuberculosis (MAP) yielding an expected product of size 229 bp. The sensitivity of the IS900 PCR was assessed by making ten fold serial dilutions of the known concentration (5 ng/µl) of the standard genomic DNA of MAP. The detection limit of the IS900 PCR was upto 5 pg/µl.

Comparative Diagnostic Utility of IS6110 PCR Assay in CSF and Peripheral Blood Samples of Tuberculous Meningitis Patients: A Pilot Study from Central India.

Tuberculous Meningitis (TBM) is the most severe form of Central Nervous System Tuberculosis (CNS-TB) and constitutes about 6% of all Extrapulmonary TB (EPTB) cases. Most guidelines for the diagnosis and management of TBM agree on the use of simple Cerebrospinal Fluid (CSF) analysis using molecular tools like Polymerase Chain Reaction (PCR). However, the sensitivity of PCR varies while using a CSF sample. In the present study, we have compared the diagnostic utility of PCR assay in both peripheral blood and CSF sample collected from TBM cases. To evaluate the application of the peripheral blood PCR assay as an alternate tool for TBM diagnosis compared to conventional CSF-PCR based system. A total of 50 TBM patients were prospectively recruited from in patient department wards of Central India Institute of Medical Sciences (CIIMS) between January 2014 - Feburary 2015. Mycobacterium tuberculosis (MTB) specific IS6110 PCR and BactT liquid culture were performed in 20 of recruited cases classified as Stage 1, 2 and 3 based on British Medical Research Council (BMRC) contemporary clinical criteria for the severity of TBM. Clinical characteristics were summarised in terms of percentages for categorical variables, i.e., age groups, gender, signs and symptoms. All statistical analysis was carried out using MedCalc software version 11.6. Overall IS6110 PCR positivity in CSF was around 80% (16/20), which was higher than culture (29.3%) and peripheral blood (39%). Out of 8 positive cases, stage wise positivity of peripheral blood PCR assay in three TBM stages was 0% (stage1) 50% (stage 2) and 67% (Stage 3) respectively. Positivity of peripheral blood PCR was significantly more (86%) in patients with CSF culture/ IS6110 PCR positive for MTB infection with sensitivity and specificity of 50 and 100% respectively. Increased positivity rates of peripheral blood PCR was observed with decreased CSF/Blood sugar ratio in stage 3 cases, suggesting enhanced probability of mycobacterial blood dissemination in cases of TBM severity. Our results suggest that although the molecular diagnosis of TBM infection in CSF remains the method of choice, peripheral blood based PCR can be used as a good alternative to CSF in case of TBM severity where the repeated CSF collection may be needed. However, study demands further validation in large cohorts to justify the present hypothesis.

Detection of Mycobacterium avium subsp. paratuberculosis in kidney samples of red deer (Cervus elaphus) in Portugal: Evaluation of different methods.

Paratuberculosis or Johne’s disease, caused by Mycobacterium avium subsp. paratuberculosis (Map), is a chronic granulomatous enteritis affecting both domestic and wild ruminants. The present work is part of a wider set of studies designed to assess the prevalence of paratuberculosis in free ranging red deer (Cervus elaphus). With that purpose, 877 free-ranging red deer legally hunted in the Centre-eastern Portugal were submitted to necropsy and sampled for molecular methods, microbiology and histopathology. Thirty-seven (4.2%) kidneys revealed acid-fast bacilli when screened with the Ziehl-Neelsen technique. Map was detected by IS900 polymerase chain reaction (PCR) in thirty (81.1%) of the Ziehl-Neelsen positive kidneys. Subsequent PCR and/or culture from the different organs of the 37 examined animals allowed us to detect 86.4% (32 animals) infected red deer. Our results suggest that renal involvement in Map infected deer may be underdiagnosed and thus the routine examination of this organ and its inclusion in PCR techniques designed for Map detection could substantially improve the diagnostic of paratuberculosis in red deer.

Evaluation of multiplex PCR using MPB64 and IS6110 primers for rapid diagnosis of tuberculous meningitis

Tuberculous meningitis (TBM) is one of those most serious manifestations of extra-pulmonary tuberculosis and prompt diagnosis and treatment is required for better clinical outcome. It is difficult to diagnose due to lack of rapid, sensitive, and specific tests. Newer methods, which are easy and reliable, are required to diagnose TBM at an early stage. Thus our aim was to evaluate the Multiplex polymerase chain reaction (PCR) technique, using primers directed against the insertion sequence IS6110 and MPB64 gene for the detection of Mycobacterium tuberculosis in Cerebrospinal fluid (CSF), for rapid diagnosis of TBM patients. 102 CSF samples were analyzed from patients suspected with TBM along with a control group of 10 patients having other neurological disorders. CSF sediments were analyzed individually for M.tuberculosis DNA by Multiplex PCR using two set of primers targeting insertion sequence IS6110 and gene MBp64, which is very specific for MTBC. Out of 37 patients diagnosed with TBM clinically, MPB64 PCR was positive in 22, IS6110 PCR was positive in 28, both PCR using Multiplex were positive in 34 and Microscopy was positive in one. Thus Sensitivity of MPB64 PCR, IS6110 PCR, Multiplex PCR and Microscopy were found to be 62.3%, 75.4%, 91.8% and 2.7% respectively. In non TBM group PCR was negative in all cases hence, the specificity was 100%. Multiplex PCR system using primers targeting IS6110 and MPB64, for the detection of M.tuberculosis DNA in CSF samples, has high sensitivity than any one of them alone, and could be used for the early detection of TBM in CSF samples.

Molecular and immunohistochemical detection ofMycobacterium bovisin formalin-fixed tissues from animals with spontaneous bovine tuberculosis

Bovine tuberculosis (TB) is an infectious and emerging but neglected disease of cattle, caused by Mycobacterium bovis. Diagnostic techniques including histopathology, acid fast staining, immunohistochemistry, polymerase chain reaction (PCR) and real-time PCR were compared for extent of accurate diagnosis of bovine TB. Study was conducted on 16 archival tissue samples collected from suspected cases of bovine tuberculosis. Histopathological studies revealed both well-organized and poorly-organized granulomas in the lungs and lymph nodes. Acid fast bacilli were detected by Ziehl Neelsen's staining in 13 (81.25%) of the 16 suspected cases. Immunohistochemistry using specific polyclonal antibody against M. bovis detected mycobacterial antigen extracellularly in caseous areas as well as intracellularly in macrophages and giant cells. DNA was extracted from formalin-fixed, paraffin-embedded tissues and subjected to IS6110 PCR using T4/T5 and INS1/INS2 primers specific for Mycobacterium tuberculosis complex (MTC) with an expected band size of 123 bp and 245 bp, respectively. The IS6110 PCR assay was positive in 12 out of 16 cases (75%). IS6110 real-time PCR using IS6110_T primers confirmed 6 (37.5%) samples positive for M. bovis. The present investigation indicated that real-time PCR using IS6110_T primers had increased specificity over conventional IS6110 PCR assay and can be used for detecting specific M. bovis infection in animals suspected for bovine tuberculosis.

Comparative Evaluation of Indirect-Elisaand IS900 PCR Assay for Diagnosis of Ovine Johne's Disease

Ovine Johne's disease (OJD) is caused by Mycobacterium avium sub sp. paratuberculosis (MAP). The disease presents diverse bacteriological, immunological and pathological spectra leading to variable efficacies of diagnostic methods at different times during the course of infection. The study was carried out to evaluate the sensitivity and specificity of indirect-enzymelinked immunosorbent assay (ELISA) using IS900 faecal polymerase chain reaction (PCR) assay as gold standard. A total of 100 clinical samples, serum (n = 50) and faeces (n = 50), were collected from 50 adult Bannur sheep from Livestock Research and Information Centre farm Nagamangala, Mandya district of Karnataka. Of 100 total samples, 23 (23%) serum and 44 (44%) faecal samples were positive by indirect-ELISA and IS900 PCR assay, respectively. The sensitivity and specificity of indirect-ELISA compared with IS900 PCR assay was 50% and 98.21%, respectively. Kappa and area under curve value indicates moderate agreement between indirect-ELISA and IS900 faecal PCR assay. The present study indicated that IS900 faecal PCR was more sensitive and specific test than indirect-ELISA in detecting for MAP infection and for early diagnosis of JD in sheep. Indirect-ELISA was screening test and IS900 PCR assay was an individual confirmatory test. Combination of indirect-ELISA with IS900 faecal PCR assay may be adopted as a model strategy for screening and diagnosis of JD in sheep.

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.

Comparitive study on the efficacy of ELISA and IS900 PCR for the diagnosis of Paratuberculosis in goats

Paratuberculosis, one of the chronic granulomatous enteritis that predominantly affects ruminants worldwide, is caused by Mycobacterium avium subsp. paratuberculosis (MAP). It is most efficiently diagnosed by MAP from faeces by Polymerase Chain Reaction (PCR). Serological tests like Enzyme Linked Immuno Sorbent Assay (ELISA) also provides a rapid and cost-effective alternative diagnostic tool. Present study was carried out to directly evaluate the sensitivity and specificity of ELISA (ID vet innovative diagnostics; France) using IS900 PCR as a gold standard. Serum and faecal samples were collected from 180 adult goats of either sex, from Malappuram and Thrissur districts of Kerala with unknown paratuberculosis status. Faecal samples were processed for direct IS900 PCR and serum samples were tested for MAP antibodies using Indirect ELISA kit. IS900 PCR detected 38 out of 180 confirmed to be shedding MAP. ELISA detected 22 out of 180 animals as positive. Overall, ELISA was 50 % sensitive and 97.9 % specific in comparison to IS900 PCR. The IS900 PCR outperformed ELISA in detecting animals potentially infected with MAP and is more sensitive than ELISA at detecting animals suspected of paratuberculosis. But, for early diagnosis of paratuberculosis in goats, ELISA can be done as easy and rapid farm level identification and IS900 PCR as individual confirmatory test.

Goat milk as a non-invasive sample for confirmation of Mycobacterium avium subspecies paratuberculosis by IS900 PCR

Mycobacterium avium subsp. paratuberculosis (MAP) causes Johne's disease (JD) in cattle, sheep, goats and other ruminants, and Crohn’s disease in humans. MAPs are shed to external environment through feces and milk. The present study was aimed to evaluate the utility of milk as a non-invasive sample in stage II MAP infections in goats using IS900 polymerase chain reaction (PCR) and sequencing analysis. A total of 32 milk samples from lactating does were collected. Within these 32 milk samples, 15 were collected from pre-confirmed JD positive goats. By IS900 PCR, all the 15 (100%) known JD positive goat milk samples revealed the presence of MAP. However, no unknown goat was identified as MAP positive. The results of this study established the usefulness of milk as a non-invasive sample in screening and confirmation of stage II MAP infection in goats.

Isolation and molecular characterization of Mycobacterium bovis from Kafue lechwe (Kobus leche kafuensis) from Zambia

Bovine tuberculosis (BTB) is a chronic bacterial disease caused by Mycobacterium bovis. Infections due to M. bovis, which serves as a stable reservoir, can pose serious challenge to control and eradicate in both wildlife and livestock at the interface. This study aimed at isolating and characterizing M. bovis from Kafue lechwe (Kobus leche kafuensis) and black lechwe (Kobus leche smithemani) at the animal/human interface in Zambia. The samples with lesions compatible with BTB collected during the hunting seasons of 2009 and 2010 were cultured for isolation of mycobacteria using Stonebrink with pyruvate (BD Diagnostics, MD, USA) and Middlebrook 7H10 (BD Diagnostics) slants. Isolated mycobacteria were identified using IS6110 polymerase chain reaction and deletion analysis. Molecular characterization of the isolates was performed using spoligotyping and mycobacteria interspersed repetitive unit-variable number tandem repeat (MIRU-VNTR) with nine loci. Data was analyzed using BioNumerics software 6.1. Out of the 39 samples, acid fast bacilli were detected in 27 (69.2%) based on smear microscopy. Seven isolates were found to belong to Mycobacterium tuberculosis complex, and all were identified as M. bovis based on deletion analysis. All seven isolates were identical on spoligotyping as belonging to the SB0120 (SIT 482). MIRU-VNTR differentiated the isolates into five different patterns. This study has confirmed that M. bovis circulates in the Kafue lechwe, and non-tuberculous mycobacteria were detected in the black lechwe in Zambia which represents a wildlife reservoir, with a potential to spillover to cattle and humans. Isolates of M. bovis from lechwe antelopes are much conserved as only one spoligotype was detected. The study has shown that three loci differentiated fairly well. This option is cheap and less laborious, and hence a better option in resource-strained country like Zambia. The study further showed that some of the loci recommended by the European Reference Laboratory are not suitable for typing M. bovis in Zambia.