A rapid and sensitive system for recovery of nucleic acids from Mycobacteria sp. on archived glass slides

The field of diagnostics continues to advance rapidly with a variety of novel approaches, mainly dependent upon high technology platforms. Nonetheless much diagnosis, particularly in developing countries, still relies upon traditional methods such as microscopy. Biological material, particularly nucleic acids, on archived glass slides is a potential source of useful information both for diagnostic and epidemiological purposes. There are significant challenges faced when examining archived samples in order that an adequate amount of amplifiable DNA can be obtained. Herein, we describe a model system to detect low numbers of bacterial cells isolated from glass slides using (laser capture microscopy) LCM coupled with PCR amplification of a suitable target. Mycobacterium smegmatis was used as a model organism to provide a proof of principle for a method to recover bacteria from a stained sample on a glass slide using a laser capture system. Ziehl-Neelsen (ZN) stained cells were excised and catapulted into tubes. Recovered cells were subjected to DNA extraction and pre-amplified with multiple displacement amplification (MDA). This system allowed a minimum of 30 catapulted cells to be detected following a nested real-time PCR assay, using rpoB specific primers. The combination of MDA and nested real-time PCR resulted in a 30-fold increase in sensitivity for the detection of low numbers of cells isolated using LCM. This study highlights the potential of LCM coupled with MDA as a tool to improve the recovery of amplifiable nucleic acids from archived glass slides. The inclusion of the MDA step was essential to enable downstream amplification. This platform should be broadly applicable to a variety of diagnostic applications and we have used it as a proof of principle with a Mycobacterium sp. model system

Multi-centre evaluation of real-time multiplex PCR for detection of carbapenemase genes OXA-48, VIM, IMP, NDM and KPC

Background: Resistance to carbapenem antibiotics is emerging worldwide among Enterobacteriaceae. To prevent hospital transmission due to unnoticed carriage of carbapenemase producing micro-organisms in newly admitted patients, or follow-up of patients in an outbreak setting, a molecular screening method was developed for detection of the most prevalent carbapenemase genes; bla(OXA-48), bla(VIM), bla(IMP), bla(NDM), and bla(KPC). Methods: A real-time multiplex PCR assay was evaluated using a collection of 86 Gram negative isolates, including 62 carbapenemase producers. Seven different laboratories carried out this method and used the assay for detection of the carbapenemase genes on a selection of 20 isolates. Results: Both sensitivity and specificity of the multiplex PCR assay was 100%, as established by results on the strain collection and the inter-laboratory comparisons. Conclusions: In this study, we present a multiplex real-time PCR that is a robust, reliable and rapid method for the detection of the most prevalent carbapenemases bla(OXA-48), bla(VIM), bla(IMP), bla(NDM), and bla(KPC), and is suitable for screening of broth cultured rectal swabs and for identification of carbapenemase genes in cultures

Mycobacterium avium ssp. paratuberculosis als mogelijke oorzaak van de ziekte van Crohn: resultaten van een patientenstudie in Nederland.

A case control study was performed to investigate the possible role of Mycobacterium avium ssp. paratuberculosis (Map) in the aetiology of Crohn's disease (CD). Biopsy samples were collected from the ileum and colon of CD patients, Ulcerative Colitis (UC) patients and control persons. The biopsy samples were either cultured in MGIT and BACTEC medium, formaline-fixed, or immediately snap-frozen in liquid N2. The presence of Map bacteria in the cultures was determined using a nested PCR assay targeted to the conserved IS900 DNA-sequence of Map. 27% of CD patients, 6% of UC patients, and 28% of control persons were positive for MGIT-PCR. The BACTEC cultures resulted in a slightly smaller percentage of PCR positive patients, 22% for CD patients, 7% for UC patients and 25% for control persons. The presence of Map was further studied applying the IS900-PCR directly on DNA from frozen biopsy samples. 7% of CD patients, 8% of UC patients and 5% of control persons were PCR positive for Map. The presence of Map was also investigated in formalin-fixed biopsies samples using a hyperimmune antiserum to the M. avium-complex. With immunoperoxidase (IP) staining, M. avium-complex antigens were observed in biopsy samples from 20% of CD patients, 13% of UC patients and 29% of control persons. Surprisingly, these data show even a 50% higher presence of Map-antigens in control persons (29%) compared to CD patients (20%). Our data clearly show that Map is present both in IBD patients (CD and UC) and in control persons. The results of culturing and IP staining show a good correlation; with both techniques, an average of 23% of CD patients and 27% of control persons is positive for Map. Remarkably, the results of culturing and IP staining show an even higher prevalence of Map in control persons compared to CD patients. Moreover, only in a few instances patients were positive in two different tests (PCR, IP, MGIT and BACTEC), and even with both culturing assays almost no overlap in positive results is observed. This result indicates that the number of Map cells in human tissue is very low, and that the prevalence of Map in the population of IBD patients and of control persons is difficult to estimate. Unexpectedly, we obtained evidence that members of the Chlamydiales family are present in high amounts in most of the CD and UC patients, whereas they were of low incidence in control persons.In conclusion, our data do not support the hypothesis that Map is directly involved in Crohn's disease, but does not exclude that Map infection may play a role in the aetiology of Crohn's Disease for a susceptible subset of the population.De bacterie Mycobacterium avium ssp. paratuberculosis (Map) wordt beschouwd als een mogelijke oorzaak van de ziekte van Crohn (morbus Crohn, MC). In samenwerking met Gelre ziekenhuizen heeft het RIVM een onderzoek uitgevoerd naar het voorkomen van Map in darmbiopten van patienten met MC, patienten met Ulcerative Colitis (UC) en controlepersonen zonder darmontsteking. De aanwezigheid van Map in de darmbiopten werd onderzocht met behulp van kweekmethoden, een DNA amplificatiemethode (PCR) en een immunologische detectiemethode (immunoperoxidase kleuring, IP-kleuring). Met de PCR-methode werd Map aangetoond in 7% van de MC-patienten, 8% van de UC-patienten en 5% van de controlepersonen. Met de gecombineerde kweek- en PCR-methoden waren gemiddeld 25% van de MC-patienten, 7% van de UC patienten en 27% van de controlepersonen positief. Met behulp van de IP-kleuring werd Map aangetoond in 20% van de MC-patienten, 13% van de UC-patienten en 29% van de controlepersonen. De resultaten van de kweek- en PCR-methoden tonen geen significant verschil tussen de aanwezigheid van Map in MC-patienten vergeleken met controlepersonen. De resultaten van de IP-kleuring tonen zelfs een hoger percentage Map-positieve controlepersonen vergeleken bij MC-patienten. Onze resultaten tonen duidelijk aan dat Map zowel bij MC-patienten als bij UC-patienten en controlepersonen voorkomt. Naast de aanwezigheid van Map is ook gekeken naar de aanwezigheid van Chlamydia in de biopten. Op grond van IP en in situ hybridisatie kleuringen bleken Chlamydiae in grote aantallen aanwezig te zijn in de biopten van MC- en UC-patienten en nauwelijks aanwezig in de biopten van controle patienten.In conclusie: onze resultaten ondersteunen niet de hypothese dat Map direct betrokken is bij de ziekte van Crohn, maar sluiten ook niet uit dat Map een rol speelt bij het ontstaan van de ziekte van Crohn in een gevoelig deel van de populatie

Mycobacterium avium ssp. paratuberculosis als mogelijke oorzaak van de ziekte van Crohn: resultaten van een patientenstudie in Nederland.

De bacterie Mycobacterium avium ssp. paratuberculosis (Map) wordt beschouwd als een mogelijke oorzaak van de ziekte van Crohn (morbus Crohn, MC). In samenwerking met Gelre ziekenhuizen heeft het RIVM een onderzoek uitgevoerd naar het voorkomen van Map in darmbiopten van patienten met MC, patienten met Ulcerative Colitis (UC) en controlepersonen zonder darmontsteking. De aanwezigheid van Map in de darmbiopten werd onderzocht met behulp van kweekmethoden, een DNA amplificatiemethode (PCR) en een immunologische detectiemethode (immunoperoxidase kleuring, IP-kleuring). Met de PCR-methode werd Map aangetoond in 7% van de MC-patienten, 8% van de UC-patienten en 5% van de controlepersonen. Met de gecombineerde kweek- en PCR-methoden waren gemiddeld 25% van de MC-patienten, 7% van de UC patienten en 27% van de controlepersonen positief. Met behulp van de IP-kleuring werd Map aangetoond in 20% van de MC-patienten, 13% van de UC-patienten en 29% van de controlepersonen. De resultaten van de kweek- en PCR-methoden tonen geen significant verschil tussen de aanwezigheid van Map in MC-patienten vergeleken met controlepersonen. De resultaten van de IP-kleuring tonen zelfs een hoger percentage Map-positieve controlepersonen vergeleken bij MC-patienten. Onze resultaten tonen duidelijk aan dat Map zowel bij MC-patienten als bij UC-patienten en controlepersonen voorkomt. Naast de aanwezigheid van Map is ook gekeken naar de aanwezigheid van Chlamydia in de biopten. Op grond van IP en in situ hybridisatie kleuringen bleken Chlamydiae in grote aantallen aanwezig te zijn in de biopten van MC- en UC-patienten en nauwelijks aanwezig in de biopten van controle patienten.In conclusie: onze resultaten ondersteunen niet de hypothese dat Map direct betrokken is bij de ziekte van Crohn, maar sluiten ook niet uit dat Map een rol speelt bij het ontstaan van de ziekte van Crohn in een gevoelig deel van de populatie.A case control study was performed to investigate the possible role of Mycobacterium avium ssp. paratuberculosis (Map) in the aetiology of Crohn's disease (CD). Biopsy samples were collected from the ileum and colon of CD patients, Ulcerative Colitis (UC) patients and control persons. The biopsy samples were either cultured in MGIT and BACTEC medium, formaline-fixed, or immediately snap-frozen in liquid N2. The presence of Map bacteria in the cultures was determined using a nested PCR assay targeted to the conserved IS900 DNA-sequence of Map. 27% of CD patients, 6% of UC patients, and 28% of control persons were positive for MGIT-PCR. The BACTEC cultures resulted in a slightly smaller percentage of PCR positive patients, 22% for CD patients, 7% for UC patients and 25% for control persons. The presence of Map was further studied applying the IS900-PCR directly on DNA from frozen biopsy samples. 7% of CD patients, 8% of UC patients and 5% of control persons were PCR positive for Map. The presence of Map was also investigated in formalin-fixed biopsies samples using a hyperimmune antiserum to the M. avium-complex. With immunoperoxidase (IP) staining, M. avium-complex antigens were observed in biopsy samples from 20% of CD patients, 13% of UC patients and 29% of control persons. Surprisingly, these data show even a 50% higher presence of Map-antigens in control persons (29%) compared to CD patients (20%). Our data clearly show that Map is present both in IBD patients (CD and UC) and in control persons. The results of culturing and IP staining show a good correlation; with both techniques, an average of 23% of CD patients and 27% of control persons is positive for Map. Remarkably, the results of culturing and IP staining show an even higher prevalence of Map in control persons compared to CD patients. Moreover, only in a few instances patients were positive in two different tests (PCR, IP, MGIT and BACTEC), and even with both culturing assays almost no overlap in positive results is observed. This result indicates that the number of Map cells in human tissue is very low, and that the prevalence of Map in the population of IBD patients and of control persons is difficult to estimate. Unexpectedly, we obtained evidence that members of the Chlamydiales family are present in high amounts in most of the CD and UC patients, whereas they were of low incidence in control persons.In conclusion, our data do not support the hypothesis that Map is directly involved in Crohn's disease, but does not exclude that Map infection may play a role in the aetiology of Crohn's Disease for a susceptible subset of the population.LNV/VW

Population structure of mixed Mycobacterium tuberculosis infection is strain genotype and culture medium dependent

Contains fulltext : 125779.pdf (publisher's version ) (Open Access)BACKGROUND: Molecular genotyping methods have shown infection with more than one Mycobacterium tuberculosis strain genotype in a single sputum culture, indicating mixed infection. AIM: This study aimed to develop a PCR-based genotyping tool to determine the population structure of M. tuberculosis strain genotypes in primary Mycobacterial Growth Indicator Tubes (MGIT) and Lowenstein-Jensen (LJ) cultures to identify mixed infections and to establish whether the growth media influenced the recovery of certain strain genotypes. METHOD: A convenience sample of 206 paired MGIT and LJ M. tuberculosis cultures from pulmonary tuberculosis patients resident in Khayelitsha, South Africa were genotyped using an in-house PCR-based method to detect defined M. tuberculosis strain genotypes. RESULTS: The sensitivity and specificity of the PCR-based method for detecting Beijing, Haarlem, S-family, and LAM genotypes was 100%, and 75% and 50% for detecting the Low Copy Clade, respectively. Thirty-one (15%) of the 206 cases showed the presence of more than one M. tuberculosis strain genotype. Strains of the Beijing and Haarlem genotypes were significantly more associated with a mixed infection (on both media) when compared to infections with a single strain (Beijing MGIT p = 0.02; LJ, p<0.01) and (Haarlem: MGIT p<0.01; LJ, p = 0.01). Strains with the Beijing genotype were less likely to be with "other genotype" strains (p<0.01) while LAM, Haarlem, S-family and LCC occurred independently with the Beijing genotype. CONCLUSION: The PCR-based method was able to identify mixed infection in at least 15% of the cases. LJ media was more sensitive in detecting mixed infections than MGIT media, implying that the growth characteristics of M. tuberculosis on different media may influence our ability to detect mixed infections. The Beijing and Haarlem genotypes were more likely to occur in a mixed infection than any of the other genotypes tested suggesting pathogen-pathogen compatibility