Molecular analysis of Mycobacterium tuberculosis DNA from a family of 18th century Hungarians

The naturally mummified remains of a mother and two daughters found in an 18th century Hungarian crypt were analysed, using multiple molecular genetic techniques to examine the epidemiology and evolution of tuberculosis. DNA was amplified from a number of targets on the Mycobacterium tuberculosis genome, including DNA from IS6110, gyrA, katG codon 463, oxyR, dnaA–dnaN, mtp40, plcD and the direct repeat (DR) region. The strains present in the mummified remains were identified as M. tuberculosis and not Mycobacterium bovis, from katG and gyrA genotyping, PCR from the oxyR and mtp40 loci, and spoligotyping. Spoligotyping divided the samples into two strain types, and screening for a deletion in the MT1801–plcD region initially divided the strains into three types. Further investigation showed, however, that an apparent deletion was due to poor DNA preservation. By comparing the effect of PCR target size on the yield of amplicon, a clear difference was shown between 18th century and modern M. tuberculosis DNA. A two-centre system was used to confirm the findings of this study, which clearly demonstrate the value of using molecular genetic techniques to study historical cases of tuberculosis and the care required in drawing conclusions. The genotyping and spoligotyping results are consistent with the most recent theory of the evolution and spread of the modern tuberculosis epidemic

Evaluation of yield of currently available diagnostics by sample type to optimize detection of respiratory pathogens in patients with a community-acquired pneumonia

Background: For the detection of respiratory pathogens, the sampling strategy may influence the diagnostic yield. Ideally, samples from the lower respiratory tract are collected, but they are difficult to obtain. Objectives: In this study, we compared the diagnostic yield in sputum and oropharyngeal samples (OPS) for the detection of respiratory pathogens in patients with community-acquired pneumonia (CAP), with the objective to optimize our diagnostic testing algorithm. Methods: Matched sputum samples, OPS, blood cultures, serum, and urine samples were taken from patients (>18 years) with CAP and tested for the presence of possible respiratory pathogens using bacterial cultures, PCR for 17 viruses and five bacteria and urinary antigen testing. Results: When using only conventional methods, that is, blood cultures, sputum culture, urinary antigen tests, a pathogen was detected in 49·6% of patients (n = 57). Adding molecular detection assays increased the yield to 80%. A pathogen was detected in 77 of the 115 patients in OPS or sputum samples by PCR. The sensitivity of the OPS was lower than that of the sputum samples (57% versus 74%). In particular, bacterial pathogens were more often detected in sputum samples. The sensitivity of OPS for the detection of most viruses was higher than in sputum samples (72% versus 66%), except for human rhinovirus and respiratory syncytial virus. Conclusion: Addition of PCR on both OPS and sputum samples significantly increased the diagnostic yield. For molecular detection of bacterial pathogens, a sputum sample is imperative, but for detection of most viral pathogens, an OPS is sufficient

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; blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC.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 blaOXA-48, blaVIM, blaIMP, blaNDM and blaKPC, and is suitable for screening of broth cultured rectal swabs and for identification of carbapenemase genes in cultures