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Liu J.,University of Virginia | Gratz J.,Kilimanjaro Clinical Research Institute | Amour C.,Haydom Lutheran Hospital | Kibiki G.,Kilimanjaro Clinical Research Institute | And 8 more authors.
Journal of Clinical Microbiology

The TaqMan Array Card (TAC) system is a 384-well singleplex real-time PCR format that has been used to detect multiple infection targets. Here we developed an enteric TaqMan Array Card to detect 19 enteropathogens, including viruses (adenovirus, astrovirus, norovirus GII, rotavirus, and sapovirus), bacteria (Campylobacter jejuni/C. coli, Clostridium difficile, Salmonella, Vibrio cholerae, diarrheagenic Escherichia coli strains including enteroaggregative E. coli [EAEC], enterotoxigenic E. coli [ETEC], enteropathogenic E. coli [EPEC], and Shiga-toxigenic E. coli [STEC]), Shigella/enteroinvasive E. coli (EIEC), protozoa (Cryptosporidium, Giardia lamblia, and Entamoeba histolytica), and helminths (Ascaris lumbricoides and Trichuris trichiura), as well as two extrinsic controls to monitor extraction and amplification efficiency (the bacteriophage MS2 and phocine herpesvirus). Primers and probes were newly designed or adapted from published sources and spotted onto microfluidic cards. Fecal samples were spiked with extrinsic controls, andDNAand RNAwere extracted using the QiaAmp StoolDNAminikit and the QuickGeneRNATissue kit, respectively, and then mixed with Ag- Path-ID One Step real-time reverse transcription-PCR (RT-PCR) reagents and loaded into cards. PCR efficiencies were between 90% and 105%, with linearities of 0.988 to 1. The limit of detection of the assays in the TAC was within a 10-fold difference from the cognate assays performed on plates. Precision testing demonstrated a coefficient of variation of below5%within a run and 14% between runs. Accuracy was evaluated for 109 selected clinical specimens and revealed an average sensitivity and specificity of 85% and 77%, respectively, compared with conventional methods (including microscopy, culture, and immunoassay) and 98% and 96%, respectively, compared with our laboratory-developed PCR-Luminex assays. This TAC allows fast, accurate, and quantitative detection of a broad spectrum of enteropathogens and is well suited for surveillance or clinical purposes. Copyright © 2013, American Society for Microbiology. Source

Verweij J.J.,Laboratory for Medical Microbiology and Immunology

For many years PCR-and other DNA-based methods of pathogen detection have been available in most clinical microbiology laboratories; however, until recently these tools were not routinely exploited for the diagnosis of parasitic infections. Laboratories were initially reluctant to implement PCR as incorporation of such assays within the algorithm of tools available for the most accurate diagnosis of a large variety of parasites was unclear. With regard to diagnosis of intestinal parasitic infections, the diversity of parasites that one can expect in most settings is far less than the parasitological textbooks would have you believe, hence developing a simplified diagnostic triage is feasible. Therefore the classical algorithm based on population, patient groups, use of immuno-suppressive drugs, travel history etc. is also applicable to decide when to perform and which additional techniques are to be used, if a multiplex PCR panel is used as a first-line screening diagnostic. Copyright © 2014 Cambridge University Press. Source

Van Rijen M.M.L.,Laboratory for Microbiology and Infection Control | Bosch T.,National Institute for Public Health and the Environment | Verkade E.J.M.,Laboratory for Microbiology and Infection Control | Verkade E.J.M.,Laboratory for Medical Microbiology and Immunology | And 3 more authors.

Background: Livestock-associated MRSA (MC398) has emerged and is related to an extensive reservoir in pigs and veal calves. Individuals with direct contact with these animals and their family members are known to have high MC398 carriage rates. Until now it was assumed that MC398 does not spread to individuals in the community without pig or veal calf exposure. To test this, we identified the proportion of MC398 in MRSA positive individuals without contact with pigs/veal calves or other known risk factors (MRSA of unknown origin; MUO). Methods: In 17 participating hospitals, we determined during two years the occurrence of MC398 in individuals without direct contact with livestock and no other known risk factor (n = 271) and tested in a post analysis the hypothesis whether hospitals in pig-dense areas have higher proportions of MC398 of all MUO. Results: Fifty-six individuals (20.7%) without animal contact carried MC398. In hospitals with high pig-densities in the adherence area, the proportion of MC398 of all MUO was higher than this proportion in hospitals without pigs in the surroundings. Conclusions: One fifth of the individuals carrying MUO carried MC398. So, MC398 is found in individuals without contact to pigs or veal calves. The way of transmission from the animal reservoir to these individuals is unclear, probably by human-tohuman transmission or by exposure to the surroundings of the stables. Further research is needed to investigate the way of transmission. © 2014 van Rijen et al. Source

Van Der Auwera G.,Institute of Tropical Medicine | Ravel C.,Montpellier University | Verweij J.J.,Leiden University | Verweij J.J.,Laboratory for Medical Microbiology and Immunology | And 3 more authors.
Journal of Clinical Microbiology

Several genetic markers have been described for discriminating Leishmania species. In most reported cases, one or a few polymorphisms are the basis of species identification, and the methods were validated on a limited number of strains from a particular geographical region. Therefore, most techniques may underestimate the global intraspecies variability and are applicable only in certain areas. In addition, interlaboratory standardization is mostly absent, complicating comparisons among different studies. Here, we compared species typing results from all sequence polymorphisms found in four popular markers that can be applied directly on clinical samples: the miniexon or spliced leader, the internal transcribed spacer of the ribosomal DNA array, the 7SL RNA gene, and the heat shock protein 70 gene. Clustering was evaluated among 74 Leishmania strains, selected to represent a wide geographic distribution and genetic variability of the medically relevant species of the genus. Results were compared with a multilocus sequence typing (MLST) approach using 7 single-copy household genes and with multilocus enzyme electrophoresis (MLEE), still considered the gold standard by some. We show that strain groupings are highly congruent across the four different single-locus markers, MLST, and MLEE. Overall, the heat shock protein 70 gene and the miniexon presented the best resolutions for separating medically relevant species. As gene sequence analysis is validated here on a global scale, it is advocated as the method of choice for use in genetic, clinical, and epidemiological studies and for managing patients with unknown origins of infection, especially in Western infectious disease clinics dealing with imported leishmaniasis. Copyright © 2014 American Society for Microbiology. All Rights Reserved. Source

Verkade E.,Laboratory for Microbiology and Infection Control | Verkade E.,Laboratory for Medical Microbiology and Immunology | Kluytmans J.,Laboratory for Microbiology and Infection Control | Kluytmans J.,Laboratory for Medical Microbiology and Immunology | Kluytmans J.,VU University Amsterdam
Infection, Genetics and Evolution

Over the past 15. years the epidemiology of methicillin-resistant Staphylococcus aureus (MRSA) has changed significantly. Being initially a nosocomial pathogen, other clones have been detected in the community, leading to infections in relatively young and healthy individuals lacking contact with healthcare. More recently, a specific clone of MRSA CC398 emerged, which has spread extensively in livestock animals and is also found in retail meat. People in contact with food production animals are at high risk of colonization. The ways in which MRSA CC398 can be transmitted to humans are direct contact with animals, environmental contamination, and eating or handling contaminated meat. The role of MRSA CC398 as a food pathogen needs further research. Recently, whole genome sequencing and other genetic analyses have shown that livestock-associated strains are distinct from human-derived strains. However, there is also an exchange of strains between the reservoirs. Livestock-associated and human-associated strains of CC398 share some virulence factors, but there are also distinct virulence factors that appear to be important in host adaptation. Exchange of genes encoding these virulence factors between strains may expand the host range and thereby threaten public health. Since the emergence of MRSA CC398 in humans, approximately 10. years ago, this clone has shown a remarkable evolution, which is described in this review. © 2013 Elsevier B.V. Source

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