Development of two real-time PCR assays for the detection of Mycoplasma hyopneumoniae in clinical samples

In order to improve the diagnosis of enzootic pneumonia (EP) in pigs two real-time polymerase chain reaction (rtPCR) assays for the detection of Mycoplasma hyopneumoniae in bronchial swabs from lung necropsies were established and validated in parallel. As a gold standard, the current "mosaic diagnosis" was taken, including epidemiological tracing, clinical signs, macro- and histopathological lesions of the lungs and immunofluorescence. One rtPCR is targeting a repeated DNA element of the M. hyopneumoniae genome (REP assay), the other a putative ABC transporter gene (ABC assay). Both assays were shown to be specific for M. hyopneumoniae and did not cross react with other bacteria and mollicutes from pig. With material from pigs of defined EP-negative farms the two assays showed to be 100% specific. When testing lungs from pig farms with EP, the REP assay detected 50% and the ABC assay 90% of the farms as positive. Both tests together detected all positive farms. Within a positive herd the two assays tested similarly with on average over 90% of the lung samples analysed from a single farm showing positive scores. A series of samples with suspicion of EP and samples from pigs with diseases other than respiratory taken from current routine diagnostic was assayed. None of the assays showed false positive results. The sensitivities in this sample group were 50% for the REP and 70% for the ABC assays and for both assays together 85%. The two assays run in parallel are therefore a valuable tool for the improvement of the current diagnosis of EP.

Performance of HBsAg point-of-care tests for detection of diagnostic escape-variants in clinical samples

BACKGROUND Hepatitis B viruses (HBV) harboring mutations in the a-determinant of the Hepatitis B surface antigen (HBsAg) are associated with reduced reactivity of HBsAg assays. OBJECTIVES To evaluate the sensitivity and specificity of three HBsAg point-of-care tests for the detection of HBsAg of viruses harboring HBsAg mutations. STUDY DESIGN A selection of 50 clinical plasma samples containing HBV with HBsAg mutations was used to evaluate the performance of three HBsAg point-of-care tests (Vikia(®), bioMérieux, Marcy-L'Étoile, France. Alere Determine HBsAg™, Iverness Biomedical Innovations, Köln, Germany. Quick Profile™, LumiQuick Diagnostics, California, USA) and compared to the ARCHITECT HBsAg Qualitative(®) assay (Abbott Laboratories, Sligo, Ireland). RESULTS The sensitivity of the point-of-care tests ranged from 98% to 100%. The only false-negative result occurred using the Quick Profile™ assay with a virus harboring a D144A mutation. CONCLUSIONS The evaluated point-of-care tests revealed an excellent sensitivity in detecting HBV samples harboring HBsAg mutations.

The use of proteomics for the assessment of clinical samples in research

Proteomics, the analysis of expressed proteins, has been an important developing area of research for the past two decades [Anderson, NG, Anderson, NL. Twenty years of two-dimensional electrophoresis: past, present and future. Electrophoresis 1996;17:443-53]. Advances in technology have led to a rapid increase in applications to a wide range of samples; from initial experiments using cell lines, more complex tissues and biological fluids are now being assessed to establish changes in protein expression. A primary aim of clinical proteomics is the identification of biomarkers for diagnosis and therapeutic intervention of disease, by comparing the proteomic profiles of control and disease, and differing physiological states. This expansion into clinical samples has not been without difficulties owing to the complexity and dynamic range in plasma and human tissues including tissue biopsies. The most widely used techniques for analysis of clinical samples are surface-enhanced laser desorption/ionisation mass spectrometry (SELDI-MS) and 2-dimensional gel electrophoresis (2-DE) coupled to matrix-assisted laser desorption ionisation [Person, MD, Monks, TJ, Lau, SS. An integrated approach to identifying chemically induced posttranslational modifications using comparative MALDI-MS and targeted HPLC-ESI-MS/MS. Chem. Res. Toxicol. 2003;16:598-608]-mass spectroscopy (MALDI-MS). This review aims to summarise the findings of studies that have used proteomic research methods to analyse samples from clinical studies and to assess the impact that proteomic techniques have had in assessing clinical samples. © 2004 The Canadian Society of Clinical Chemists. All rights reserved.

UK epidemic strains of meticillin-resistant Staphylococcus aureus in clinical samples from Malta

Since 1999, the European Antimicrobial Resistance Surveillance System (EARSS) has monitored the rise in infection due to a number of organisms, including meticillin-resistant Staphylococcus aureus (MRSA). The EARSS reported that MRSA infections within intensive care units account for 25-50% of infections in many central and southern European countries, these included France, Spain, Great Britain, Malta, Greece and Italy. Each country has defined epidemic MRSA (EMRSA) strains; however, the method of spread of these strains from one country to another is unknown. In this current study, DNA profiles of 473 isolates of MRSA collected from the UK and Malta were determined by PFGE. Analysis of the data showed that two countries separated by a large geographical distance had a similar DNA profile pattern. Additionally it was demonstrated that strains of EMRSA normally found in the UK were also found in the Maltese cohort (EMRSA 15 and 16). A distinct DNA profile was found in the Maltese cohort, which may be a local EMRSA, and accounted for 14.4% of all Maltese isolates. The appearance of the same MRSA and EMRSA profiles in two separate countries suggests that MRSA can be transferred out of their country of origin and potentially establish in a new locality or country.

Colorimetric Assessment of BCR-ABL1 Transcripts in Clinical Samples Via Gold Nanoprobes

Gold nanoparticles functionalized with thiolated oligonucleotides (Au-nanoprobes) have been used in a range of applications for the detection of bioanalytes of interest, from ions to proteins and DNA targets. These detection strategies are based on the unique optical properties of gold nanoparticles, in particular, the intense color that is subject to modulation by modification of the medium dieletric. Au-nanoprobes have been applied for the detection and characterization of specific DNA sequences of interest, namely pathogens and disease biomarkers. Nevertheless, despite its relevance, only a few reports exist on the detection of RNA targets. Among these strategies, the colorimetric detection of DNA has been proven to work for several different targets in controlled samples but demonstration in real clinical bioanalysis has been elusive. Here, we used a colorimetric method based on Au-nanoprobes for the direct detection of the e14a2 BCR-ABL fusion transcript in myeloid leukemia patient samples without the need for retro-transcription. Au-nanoprobes directly assessed total RNA from 38 clinical samples, and results were validated against reverse transcription-nested polymerase chain reaction (RT-nested PCR) and reverse transcription-quantitative polymerase chain reaction (RT-qPCR). The colorimetric Au-nanoprobe assay is a simple yet reliable strategy to scrutinize myeloid leukemia patients at diagnosis and evaluate progression, with obvious advantages in terms of time and cost, particularly in low- to medium-income countries where molecular screening is not routinely feasible. Graphical abstract Gold nanoprobe for colorimetric detection of BCR-ABL1 fusion transcripts originating from the Philadelphia chromosome.

A more reliable PCR for detection of Mycobacterium tuberculosis in clinical samples

Diagnostic techniques based on PCR have two major problems: false-positive reactions due to contamination with DNA fragments from previous PCRs (amplicons) and false-negative reactions caused by inhibitors that interfere with the PCR. We have improved our previously reported PCR based on the amplification of a fragment of the Mycobacterium tuberculosis complex-specific insertion element IS6110 with respect to both problems. False-positive reactions caused by amplicon contamination were prevented by the use of uracil-N-glycosylase and dUTP instead of dTTP. We selected a new set of primers outside the region spanned by the formerly used primers to avoid false-positive reactions caused by dTTP-containing amplicons still present in the laboratory. With this new primer set, 16 copies of the IS6110 insertion element, the equivalent of two bacteria, could be amplified 10(10) times in 40 cycles, resulting in a mean efficiency of 77% per cycle. To detect the presence of inhibitors of the Taq polymerase, which may cause false-negative reactions, part of each sample was spiked with M. tuberculosis DNA. The DNA purification method using guanidinium thiocyanate and diatoms effectively removed most or all inhibitors of the PCR. However, this was not suitable for blood samples, for which we developed a proteinase K treatment followed by phenol-chloroform extraction. This method permitted detection of 20 M. tuberculosis bacteria per ml of whole blood. Various laboratory procedures were introduced to reduce failure or inhibition of PCR and avoid DNA cross contamination. We have tested 218 different clinical specimens obtained from patients suspected of having tuberculosis. The samples included sputum (n=145), tissue biopsy samples (n=25), cerebrospinal fluid (n=15), blood (n=14), pleural fluid (n=9), feces, (n=7), fluid from fistulae (n=2), and pus from a wound (n=1). The results obtained by PCR were consistent with those obtained with culture, which is the "gold standard." We demonstrate that PCR is a useful technique for the rapid diagnosis of tuberculosis at various sites.

Genetic diversity of Cryptosporidium identified in clinical samples from cities in Brazil and Argentina

The identification and characterisation of Cryptosporidium genotypes and subtypes are fundamental to the study of cryptosporidiosis epidemiology, aiding in prevention and control strategies. The objective was to determine the genetic diversity of Cryptosporidium in samples obtained from hospitals of Rio de Janeiro, Brazil, and Buenos Aires, Argentina. Samples were analysed by microscopy and TaqMan polymerase chain reaction (PCR) assays for Cryptosporidium detection, genotyped by nested-PCR-restriction fragment length polymorphism (RFLP) analysis of the 18S rRNA gene and subtyped by DNA sequencing of the gp60 gene. Among the 89 samples from Rio de Janeiro, Cryptosporidium spp were detected in 26 by microscopy/TaqMan PCR. In samples from Buenos Aires, Cryptosporidium was diagnosed in 15 patients of the 132 studied. The TaqMan PCR and the nested-PCR-RFLP detected Cryptosporidium parvum , Cryptosporidium hominis , and co-infections of both species. In Brazilian samples, the subtypes IbA10G2 and IIcA5G3 were observed. The subtypes found in Argentinean samples were IbA10G2, IaA10G1R4, IaA11G1R4, and IeA11G3T3, and mixed subtypes of Ia and IIa families were detected in the co-infections. C. hominis was the species more frequently detected, and subtype family Ib was reported in both countries. Subtype diversity was higher in Buenos Aires than in Rio de Janeiro and two new subtypes were described for the first time.

Utility of a panviral microarray for detection of swine respiratory viruses in clinical samples

Several factors have recently converged, elevating the need for highly parallel diagnostic platforms that have the ability to detect many known, novel, and emerging pathogenic agents simultaneously. Panviral DNA microarrays represent the most robust approach for massively parallel viral surveillance and detection. The Virochip is a panviral DNA microarray that is capable of detecting all known viruses, as well as novel viruses related to known viral families, in a single assay and has been used to successfully identify known and novel viral agents in clinical human specimens. However, the usefulness and the sensitivity of the Virochip platform have not been tested on a set of clinical veterinary specimens with the high degree of genetic variance that is frequently observed with swine virus field isolates. In this report, we investigate the utility and sensitivity of the Virochip to positively detect swine viruses in both cell culture-derived samples and clinical swine samples. The Virochip successfully detected porcine reproductive and respiratory syndrome virus (PRRSV) in serum containing 6.10 × 10(2) viral copies per microliter and influenza A virus in lung lavage fluid containing 2.08 × 10(6) viral copies per microliter. The Virochip also successfully detected porcine circovirus type 2 (PCV2) in serum containing 2.50 × 10(8) viral copies per microliter and porcine respiratory coronavirus (PRCV) in turbinate tissue homogenate. Collectively, the data in this report demonstrate that the Virochip can successfully detect pathogenic viruses frequently found in swine in a variety of solid and liquid specimens, such as turbinate tissue homogenate and lung lavage fluid, as well as antemortem samples, such as serum.

Genetic diversity of Mycobacterium avium isolates recovered from clinical samples and from the environment: molecular characterization for diagnostic purposes

Isolation of Mycobacterium avium complex (MAC) organisms from clinical samples may occur in patients without clinical disease, making the interpretation of results difficult. The clinical relevance of MAC isolates from different types of clinical samples (n = 47) from 39 patients in different sections of a hospital was assessed by comparison with environmental isolates (n = 17) from the hospital. Various methods for identification and typing (commercial probes, phenotypic characteristics, PCR for detection of IS1245 and IS901, sequencing of the hsp65 gene, and pulsed-field gel electrophoresis) were evaluated. The same strain was found in all the environmental isolates, 21 out of 23 (91.3%) of the isolates cultured from urine samples, and 5 out of 19 (26.3%) isolates from respiratory specimens. This strain did not cause disease in the patients. Testing best characterized the strain as M. avium subsp. hominissuis, with the unusual feature that 81.4% of these isolates lacked the IS1245 element. Contamination of certain clinical samples with an environmental strain was the most likely event; therefore, characterization of the environmental mycobacteria present in health care facilities should be performed to discard false-positive isolations in nonsterile samples, mainly urine samples. Molecular techniques applied in this study demonstrated their usefulness for this purpose.

Survey of Listeria spp. in matched clinical, food and refrigerator samples at home level in Brazil

Listeria monocytogenes is a bacterial pathogen that represents a serious threat during pregnancy and several cases of listeriosis have been linked to the consumption of contaminated foods worldwide. In Brazil, there is no report of foodborne listeriosis, despite some sporadic cases of infection by this bacterium occur. In general in our country, there is no awareness of medical personnel to instruct moms-to-be to avoid high risk foods. In the present study, a total of 141 samples were surveyed for the presence of Listeria spp., including cervicovaginal samples of patients, foods and home refrigerators. No clinical sample was positive for Listeria spp., but it was isolated from two refrigerators. L. monocytogenes was detected in two food samples out of five positive ones for Listeria spp. In conclusion, it was shown the presence of contaminated food items at home level and the lack of information on the risks of listeriosis, indicating the need of implementation of food safety education programs. (C) 2007 Elsevier Ltd. All rights reserved.

Molecular method for the characterization of Coxiella burnetii from clinical and environmental samples: variability of genotypes in Spain

Background: Coxiella burnetii is a highly clonal microorganism which is difficult to culture, requiring BSL3 conditions for its propagation. This leads to a scarce availability of isolates worldwide. On the other hand, published methods of characterization have delineated up to 8 different genomic groups and 36 genotypes. However, all these methodologies, with the exception of one that exhibited limited discriminatory power (3 genotypes), rely on performing between 10 and 20 PCR amplifications or sequencing long fragments of DNA, which make their direct application to clinical samples impracticable and leads to a scarce accessibility of data on the circulation of C. burnetii genotypes. Results: To assess the variability of this organism in Spain, we have developed a novel method that consists of a multiplex (8 targets) PCR and hybridization with specific probes that reproduce the previous classification of this organism into 8 genomic groups, and up to 16 genotypes. It allows for a direct haracterization from clinical and environmental samples in a single run, which will help in the study of the different genotypes circulating in wild and domestic cycles as well as from sporadic human cases and outbreaks. The method has been validated with reference isolates. A high variability of C. burnetii has been found in Spain among 90 samples tested, detecting 10 different genotypes, being those adaA negative associated with acute Q fever cases presenting as fever of intermediate duration with liver involvement and with chronic cases. Genotypes infecting humans are also found in sheep, goats, rats, wild boar and ticks, and the only genotype found in cattle has never been found among our clinical samples. Conclusions: This newly developed methodology has permitted to demonstrate that C. burnetii is highly variable in Spain. With the data presented here, cattle seem not to participate in the transmission of C. burnetii to humans in the samples studied, while sheep, goats, wild boar, rats and ticks share genotypes with the human population.