A Novel Semiquantitative Fluorescence-Based Multiplex Polymerase Chain Reaction Assay for Rapid Simultaneous Detection of Bacterial and Parasitic Pathogens from Blood

Abstract

A multiplex polymerase chain reaction assay was developed for the rapid simultaneous detection of category A select bacterial agents (Bacillus anthracis and Yersinia pestis) and parasitic pathogens (Leishmania species) in blood using the Cepheid Smart Cycler platform. B. anthracis (Sterne) and Yersinia. pseudotuberculosis were used in the assay for optimization for B. anthracis and Y. pestis, respectively. The specificity of the target amplicons [protective antigen gene of B. anthracis and rRNA genes of other pathogens or human (internal control)] was evaluated by staining the amplicons with SYBR Green I and determining their individual melting temperatures (T(m)). As a novel approach for pathogen semiquantitation, the Tm peak height of the amplicon was correlated with a known standard curve of pathogen-spiked samples. This assay was able to detect DNA in blood spiked with less than 50 target cells/ml for all of the pathogens. The sensitivity of this assay in blood was 100% for the detection of Leishmania donovani from leishmaniasis patients and B. anthracis (Sterne) from symptomatic mice. The time necessary for performing this assay including sample preparation was less than 1.5 hours, making this a potentially useful method for rapidly diagnosing and monitoring the efficacy of drugs or vaccines in infected individuals.

Collection characteristics of a batch-type wetted wall bioaerosol sampling cyclone

“…6 suggests that the GeneXpert does not allow accurate determination of BG in the hydrosol state of a BWWC. Similar observations regarding the use of the Genexpert for quantitative purposes, emphasizing careful optimization of the conditions and primer selection, have been offered by other researchers (Ulrich et al 2006;Ferre 1992;Selvapandiyan et al 2005).…”

Mediterranean visceral leishmaniasis associated with acute lymphoblastic leukemia (ALL)

“…The PCR technique has several advantages including the ability to work with small amounts of target material, the follow-up of treatment as well as the assessment of the successful cure of visceral leishmaniasis and fast detection of Leishmania in symptomatic patients and asymptomatic carriers as well as Leishmania /HIV co-infected patients (Bossolasco et al 2003;Mary et al 2004;Selvapandiyan et al 2005). The most suitable target for the DNA-based diagnosis is kinetoplast DNA minicircles.…”

Transfusion transmitted leishmaniasis. What to do with blood donors from endemic areas?

“…Selvapandiyan et al developed a multiplex polymerase chain reaction assay able to simultaneously identify the presence of both bacterial (B. anthracis and Y. pestis) and parasitic (Trypanosomatid species, including Leishmania) pathogens in blood specimens. To obtain higher sensitivity of the assay, the researchers chose to specifically amplify genes that can be found in multiple copies in the microorganisms [102]. Finally, and in contrast to the above-mentioned consideration on PCR efficacy, other authors argue that little experience with Leishmania primers may lead to falsepositive results, which can be a real concern [103].…”