Analytical Performance of a Multiplex Real-Time PCR Assay Using TaqMan Probes for Quantification of Trypanosoma cruzi Satellite DNA in Blood Samples

Yersinia pestis, the aetiological agent of the plague, causes sporadic disease in endemic areas of the world and is classified as a National Institute of Allergy and Infectious Diseases Category A Priority Pathogen because of its potential to be used as a bioweapon. Health departments, hospitals and government agencies need the ability to rapidly identify and characterize cultured isolates of this bacterium. Assays have been developed to perform this function; however, they are limited in their ability to distinguish Y. pestis from Yersinia pseudotuberculosis. This report describes the creation of a real-time PCR assay using Taqman probes that exclusively identifies Y. pestis using a unique target sequence of the yihN gene on the chromosome. As with other Y. pestis PCR assays, three major genes located on each of the three virulence plasmids were included: lcrV on pCD1, caf1 on pMT1 and pla on pPCP1. The quadruplex assay was validated on a collection of 192 Y. pestis isolates and 52 near-neighbour isolates. It was discovered that only 72 % of natural plague isolates from the states of New Mexico and Utah harboured all three virulence plasmids. This quadruplex assay proved to be 100 % successful in differentiating Y. pestis from all near neighbours tested and was able to reveal which of the three virulence plasmids a particular isolate possessed.

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Detection of Aspergillus species in bone marrow transplant patients

The Journal of Infection in Developing Countries ◽

10.3855/jidc.807 ◽

2010 ◽

Vol 4 (08) ◽

pp. 511-516 ◽

Cited By ~ 12

Author(s):

Parisa Badiee ◽

Abdolvahab Alborzi

Keyword(s):

Bone Marrow ◽

Real Time ◽

Bone Marrow Transplant ◽

Real Time Pcr ◽

Clinical Signs ◽

Marrow Transplant ◽

Aspergillus Species ◽

Pcr Assay ◽

Blood Samples ◽

Transplant Patients

Introduction: Invasive aspergillosis is a severe complication of cytotoxic chemotherapies and bone marrow transplantation (BMT). The aim of this study was to assess the utility of a real-time PCR assay for the early diagnosis of Aspergillus species in blood samples from BMT patients. Methodology: Blood specimens (n = 993) from patients (n = 82) scheduled for BMT were collected prior to transplant and for 100 days post transplantation. The specimens were later tested using an Aspergillus-specific real-time PCR assay. Cultures of clinical samples, along with sonography and computerized tomographic scans, were performed as standard of care. Results: Aspergillus DNA was positive in 94 sequential blood samples from 13 patients with clinical and radiological signs of infection. Samples from three of these patients were PCR-positive for Aspergillus in the first week of admission, prior to transplantation. Four patients with aspergillosis were cured with antifungal agents and nine died. An additional 12 patients without clinical signs of infection were PCR-positive on one occasion each, while two patients with clinical signs of infection were PCR-negative. Compared to routine methods of aspergillosis diagnosis, the respective sensitivity, specificity, negative, and positive predictive values of the PCR method by patient were 86.6%, 82%, 96.5% and 52%. Conclusions: The results show that Aspergillus infections in the blood of bone marrow transplant patients can be dectected by PCR methods. Early detection of Aspergillus infections by PCR has the potential to positively impact patient mortality rate and provide cost savings to hospitals.

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Evaluating Performance of Multiplex Real Time PCR for the Diagnosis of Malaria at Elimination Targeted Low Transmission Settings of Ethiopia.

10.21203/rs.3.rs-572301/v1 ◽

2021 ◽

Author(s):

Mahlet Belachew ◽

Mistire Wolde ◽

Desalegn Nega ◽

Bokretsion Gidey ◽

Legessie Negash ◽

...

Keyword(s):

Light Microscopy ◽

Real Time ◽

Malaria Elimination ◽

Real Time Pcr ◽

Nucleic Acid Amplification ◽

Molecular Testing ◽

Pcr Assay ◽

Blood Samples ◽

Low Transmission ◽

Highly Sensitive

Abstract Background: Malaria incidence has declined in Ethiopia in the past ten years. Current malaria diagnostic tests, including light microscopy and antigen-detecting rapid tests (RDTs) cannot reliably detect low-density infections. Studies have shown that nucleic acid amplification tests are highly sensitive and specific in detecting malaria infection. Thus, this study took place with the aim of evaluating the performance of multiplex real time PCR for the diagnosis of malaria using patient samples collected from health facilities located at malaria elimination targeted low transmission settings in Ethiopia. Methods: A health facility based cross sectional survey was conducted in selected malaria sentinel sites. Malaria suspected febrile outpatients referred to laboratory for malaria testing between December 2019 and March 2020 were enrolled into this study. Socio demographic information and capillary blood samples were collected from the study participants and tested at spot with RDTs. Additionally, five circles of dry blood sample (DBS) samples on Whatman filter paper and thick and thin smear were prepared for molecular testing and microscopic examination, respectively. Multiplex real time PCR assay was performed at EPHI malaria laboratory. The performance of multiplex real time PCR assay, microscopy and RDT for the diagnosis of malaria was compared and evaluated against each other.Results: Out of 271 blood samples, multiplex real time PCR identified 69 malaria cases as P. falciparum infection, 16 as P. vivax and 3 as mixed infections. Of the total samples, light microscopy detected 33 as Pf, 18 as PV and RDT detected 43 as Pf, 17 as PV, and one mixed infection. Using light microscopy as reference test, the sensitivity and specificity of multiplex real time PCR were 100% (95% CI [93-100]) and 83.2% (95% CI [77.6-87.9]), respectively. Using multiplex real time PCR as a reference, light microscopy and RDT had sensitivity of 58% (95% CI [46.9-68.4] and 67% (95% CI [56.2-76.7]); and 100% (95% CI [98-100] and 98.9 (95% CI (96-99.9), respectively. Substantial level of agreement was reported between microscopy and multiplex real time PCR results with kappa value of 0.65. Conclusions: Multiplex real time PCR had an advanced performance in parasite detection and species identification on febrile patients’ samples than did microscopy and RDT in low malaria transmission settings. It is highly sensitive malaria diagnostic method that can be used in malaria elimination program, particularly for community based epidemiological samples. Although microscopy and RDT had reduced performance when compared to multiplex real time PCR, still had an acceptable performance in diagnosis of malaria cases on patient samples at clinical facilities.

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Evaluation of a novel real-time PCR assay for the detection, identification and quantification of Plasmodium species causing malaria in humans

Malaria Journal ◽

10.1186/s12936-021-03842-8 ◽

2021 ◽

Vol 20 (1) ◽

Author(s):

Kim van Bergen ◽

Toon Stuitje ◽

Robert Akkers ◽

Eric Vermeer ◽

Rob Castel ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Limit Of Detection ◽

False Negative ◽

Performance Characteristics ◽

Analytical Performance ◽

Melting Curve Analysis ◽

Mixed Infections ◽

Plasmodium Ovale ◽

Pcr Assay

Abstract Background The entry of PCR-based techniques into malaria diagnostics has improved the sensitivity and specificity of the detection of Plasmodium infections. It has been shown that humans are regularly infected by at least six different Plasmodium species. The MC004 real-time PCR assay for malaria diagnosis is a novel single-tube assay that has been developed for the purpose of simultaneously detecting all Plasmodium species known to infect humans, and discrimination between Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale wallikeri, Plasmodium ovale curtisi, Plasmodium knowlesi (including differentiation of three strains) and Plasmodium cynomolgi (including differentiation of three strains). Detection and identification of Plasmodium species relies on molecular beacon probe-based melting curve analysis. In addition, this assay might be used to quantify the parasitaemia of at least P. falciparum by calculating the level of parasitaemia directly from the Cq-value. Methods The samples used in this study comprised reference samples, patient samples, and synthetic controls. The following analytical performance characteristics of the MC004 assay were determined: analytical specificity, limit of detection, the ability to detect mixed infections, and the potential to determine the level of parasitaemia of P. falciparum, including assessment of within-run and between-run precisions. Results No false positive or false negative results were observed. The limit of detection of P. falciparum was 1 × 10–3 IU/mL (WHO standard). Mixed infections with P. falciparum and non-falciparum species were correctly identified. A calibration curve could be established to quantify the parasitaemia of at least P. falciparum. The within-run and between-run precisions were less than 20% CV at the tested parasitaemia levels of 0.09%, 0.16%, 2.15% and 27.27%. Conclusion Based upon the analytical performance characteristics that were determined, the MC004 assay showed performance suitable for use in clinical settings, as well as epidemiological studies.

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