Quantification of Leishmania infantum DNA by a Real-Time PCR Assay with High Sensitivity

A total of 1204 meticillin-resistant Staphylococcus aureus (MRSA) screens (3340 individual swabs) were tested to evaluate a staphylococcal cassette chromosome mec (SCCmec) real-time PCR. In total, 148 (12.3 %) of the screens were MRSA-positive, where 146 (12.1 %) were MRSA-positive by the SCCmec real-time PCR assay. In contrast, 128 (10.6 %) screens were MRSA-positive by culture. One hundred and twenty-six (10.5 %) of the screens were positive by both culture and PCR. Twenty of the 1204 screens (1.66 %) were negative by culture but positive by PCR; these samples were sequenced. In 14 of the cases, a homology search confirmed the sequence as SCCmec, indicating that these samples could be considered true positives. Two of the 1204 (0.2 %) screens were positive by culture and negative by PCR. The mean turnaround time (TAT) for PCR-negative swabs was 6 h 12 min and for PCR-positive swabs was 6 h 48 min. In comparison, for culture-negative swabs the mean TAT was 29 h 30 min and for culture-positive swabs was 69 h. The cost per swab for routine culture was £0.41 (€0.48) and that of the real-time PCR assay was £2.35 (€2.75). This optimized, in-house, inexpensive, real-time PCR test maintained a very high sensitivity and specificity when evaluated under real-time laboratory conditions. The TAT of this real-time PCR assay was substantially lower than that of chromogenic culture. It was also maintained throughout the entire process, which can be taken as an indirect measure of test performance. This study showed that implementation of a molecular test can be achieved with limited resources in a standard microbiology laboratory.

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Toward point-of-care diagnostics of Candida auris

10.1101/2020.02.10.20021683 ◽

2020 ◽

Cited By ~ 1

Author(s):

Geoffrey Mulberry ◽

Sudha Chaturvedi ◽

Vishnu Chaturvedi ◽

Brian N. Kim

Keyword(s):

New York ◽

Real Time ◽

Real Time Pcr ◽

Positive Impact ◽

Point Of Care ◽

Low Cost ◽

High Sensitivity ◽

Clinical Samples ◽

Pcr Assay ◽

Candida Auris

AbstractCandida auris is a multidrug-resistant yeast that presents global health threat for the hospitalized patients. Early diagnostic of C. auris is crucial in control, prevention, and treatment. Candida auris is difficult to identify with standard laboratory methods and often can be misidentified leading to inappropriate management. A newly-devised real-time PCR assay played an important role in the ongoing investigation of the C. auris outbreak in New York metropolitan area. The assay can rapidly detect C. auris DNA in surveillance and clinical samples with high sensitivity and specificity, and also useful for confirmation of C. auris cultures. Despite its positive impact, the real-time PCR assay is difficult to deploy at frontline laboratories due to high-complexity set-up and operation. Using a low-cost handheld real-time PCR device, we show that the C. auris can potentially be identified in a low-complexity assay without the need for high-cost equipment. An implementation of low-cost real-time PCR device in hospitals and healthcare facilities is likely to accelerate the diagnosis of C. auris and for control of the global epidemic.

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The development of a real-time PCR assay for the quantification of Leishmania infantum DNA in canine blood

The Veterinary Journal ◽

10.1016/j.tvjl.2008.05.018 ◽

2009 ◽

Vol 182 (2) ◽

pp. 356-358 ◽

Cited By ~ 43

Author(s):

Milena de Paiva Cavalcanti ◽

Maria Edileuza Felinto de Brito ◽

Wayner Vieira de Souza ◽

Yara de Miranda Gomes ◽

Frederico G.C. Abath

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Leishmania Infantum ◽

Pcr Assay

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A Real-Time PCR Assay for Bat SARS-Like Coronavirus Detection and Its Application to Italian Greater Horseshoe Bat Faecal Sample Surveys

The Scientific World JOURNAL ◽

10.1100/2012/989514 ◽

2012 ◽

Vol 2012 ◽

pp. 1-8 ◽

Cited By ~ 20

Author(s):

Andrea Balboni ◽

Laura Gallina ◽

Alessandra Palladini ◽

Santino Prosperi ◽

Mara Battilani

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Screening Tool ◽

High Sensitivity ◽

Pcr Assay ◽

Viral Ecology ◽

Epidemiological Surveys ◽

Epidemiological Evaluation ◽

Polymerase Chain ◽

Horseshoe Bat

Bats are source of coronaviruses closely related to the severe acute respiratory syndrome (SARS) virus. Numerous studies have been carried out to identify new bat viruses related to SARS-coronavirus (bat-SARS-like CoVs) using a reverse-transcribed-polymerase chain reaction assay. However, a qualitative PCR could underestimate the prevalence of infection, affecting the epidemiological evaluation of bats in viral ecology. In this work an SYBR Green-real time PCR assay was developed for diagnosing infection with SARS-related coronaviruses from bat guano and was applied as screening tool in a survey carried out on 45 greater horseshoe bats (Rhinolophus ferrumequinum) sampled in Italy in 2009. The assay showed high sensitivity and reproducibility. Its application on bats screening resulted in a prevalence of 42%. This method could be suitable as screening tool in epidemiological surveys about the presence of bat-SARS-like CoVs, consequently to obtain a more realistic scenario of the viral prevalence in the population.

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Development of a real-time PCR assay with fluorophore-labelled hybridization probes for detection of Schistosoma mekongi in infected snails and rat feces

Parasitology ◽

10.1017/s0031182012000649 ◽

2012 ◽

Vol 139 (10) ◽

pp. 1266-1272 ◽

Cited By ~ 5

Author(s):

O. SANPOOL ◽

P. M. INTAPAN ◽

T. THANCHOMNANG ◽

P. SRI-AROON ◽

V. LULITANOND ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Melting Curve ◽

High Sensitivity ◽

Curve Analysis ◽

Melting Curve Analysis ◽

Intermediate Hosts ◽

Pcr Assay ◽

Fecal Samples ◽

Schistosoma Mekongi

SUMMARYSchistosoma mekongi, a blood-dwelling fluke, is a water-borne parasite that is found in communities along the lower Mekong River basin, i.e. Cambodia and Lao People's Democratic Republic. This study developed a real-time PCR assay combined with melting-curve analysis to detect S. mekongi in laboratory setting conditions, in experimentally infected snails, and in fecal samples of infected rats. The procedure is based on melting-curve analysis of a hybrid between an amplicon from S. mekongi mitochondrion sequence, the 260 bp sequence specific to S. mekongi, and specific fluorophore-labelled probes. This method could detect as little as a single cercaria artificially introduced into a pool of 10 non-infected snails, a single cercaria in filtered paper, and 2 eggs inoculated in 100 mg of non-infected rat feces. All S. mekongi-infected snails and fecal samples from infected rats were positive. Non-infected snails, non-infected rat feces, and genomic DNA of other parasites were negative. The method gave high sensitivity and specificity, and could be applied as a fast and reliable tool for cercarial location in water environments in endemic areas and for epidemiological studies and eradication programmes for intermediate hosts.

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Development of a two-step nucleic acid amplification test for accurate diagnosis of the Mycobacterium tuberculosis complex

Scientific Reports ◽

10.1038/s41598-021-85160-2 ◽

2021 ◽

Vol 11 (1) ◽

Author(s):

Chien-Ru Lin ◽

Hsin-Yao Wang ◽

Ting-Wei Lin ◽

Jang-Jih Lu ◽

Jason Chia-Hsun Hsieh ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Nucleic Acid ◽

Real Time ◽

Sensitivity And Specificity ◽

Real Time Pcr ◽

Disease Transmission ◽

High Sensitivity ◽

Nucleic Acid Amplification ◽

Amplification Efficiency ◽

Pcr Assay

AbstractThe Mycobacterium tuberculosis complex (MTBC) remains one of the top 10 leading causes of death globally. The early diagnosis of MTBC can reduce mortality and mitigate disease transmission. However, current nucleic acid amplification diagnostic test methods are generally time-consuming and show suboptimal diagnostic performance, especially in extrapulmonary MTBC samples or acid-fast stain (AFS)-negative cases. Thus, development of an accurate assay for the diagnosis of MTBC is necessary, particularly under the above mentioned conditions. In this study, a single-tube nested real-time PCR assay (N-RTP) was developed and compared with a newly in-house-developed high-sensitivity real-time PCR assay (HS-RTP) using 134 clinical specimens (including 73 pulmonary and 61 extrapulmonary specimens). The amplification efficiency of HS-RTP and N-RTP was 99.8% and 100.7%, respectively. The sensitivity and specificity of HS-RTP and N-RTP for the diagnosis of MTBC in these specimens were 97.5% (77/79) versus 94.9% (75/79) and 80.0% (44/55) versus 89.1% (49/55), respectively. The sensitivity and specificity of HS-RTP and N-RTP for the diagnosis of MTBC in pulmonary specimens were 96.3% (52/54) versus 96.3% (52/54) and 73.7.0% (14/19) versus 89.5% (17/19), respectively; in extrapulmonary specimens, the sensitivity and specificity of HS-RTP and N-RTP were 100% (25/25) versus 92% (23/25) and 83.3% (30/36) versus 88.9% (32/36), respectively. Among the AFS-negative cases, the sensitivity and specificity of HS-RTP and N-RTP were 97.0% (32/33) versus 90.9% (30/33) and 88.0% (44/50) versus 92.0% (46/50), respectively. Overall, the sensitivity of HS-RTP was higher than that of N-RTP, and the performance was not compromised in extrapulmonary specimens and under AFS-negative conditions. In contrast, the specificity of the N-RTP assay was higher than that of the HS-RTP assay in all types of specimens. In conclusion, the HS-RTP assay would be useful for screening patients suspected of exhibiting an MTBC infection due to its higher sensitivity, while the N-RTP assay could be used for confirmation because of its higher specificity. Our results provide a two-step method (screen to confirm) that simultaneously achieves high sensitivity and specificity in the diagnosis of MTBC.

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A duplex real-time PCR assay for the detection and differentiation of Leishmania infantum and Leishmania tarentolae in vectors and potential reservoir hosts

10.22541/au.159665095.58124878 ◽

2020 ◽

Author(s):

Maria Stefania Latrofa ◽

Jairo Mendoza Roldan ◽

Ranju Manoj ◽

Filipe Dantas Torres ◽

Domenico Otranto

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Leishmania Infantum ◽

Pcr Assay ◽

Leishmania Tarentolae ◽

Potential Reservoir ◽

Reservoir Hosts

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Leishmania-FAST15: A rapid, sensitive and low-cost real-time PCR assay for the detection of Leishmania infantum and Leishmania braziliensis kinetoplast DNA in canine blood samples

Molecular and Cellular Probes ◽

10.1016/j.mcp.2016.08.006 ◽

2017 ◽

Vol 31 ◽

pp. 65-69 ◽

Cited By ~ 19

Author(s):

Filipe Dantas-Torres ◽

Kamila Gaudêncio da Silva Sales ◽

Lidiane Gomes da Silva ◽

Domenico Otranto ◽

Luciana Aguiar Figueredo

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Leishmania Infantum ◽

Low Cost ◽

Leishmania Braziliensis ◽

Kinetoplast Dna ◽

Pcr Assay ◽

Blood Samples

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Molecular Diagnosis of Leishmaniasis: Quantification of Parasite Load by a Real-Time PCR Assay with High Sensitivity

Pathogens ◽

10.3390/pathogens10070865 ◽

2021 ◽

Vol 10 (7) ◽

pp. 865

Author(s):

Germano Castelli ◽

Federica Bruno ◽

Stefano Reale ◽

Simone Catanzaro ◽

Viviana Valenza ◽

...

Keyword(s):

Real Time ◽

Quantitative Pcr ◽

Real Time Pcr ◽

High Sensitivity ◽

Parasite Load ◽

Clinical Samples ◽

Pcr Assay ◽

Predictive Values ◽

Low Sensitivity

Real-time PCR was developed to quantify Leishmania infantum kinetoplast DNA and optimized to achieve a sensitivity of 1 parasite/mL. For this purpose, we cloned the conserved kDNA fragment of 120 bp into competent cells and correlated them with serial dilutions of DNA extracted from reference parasite cultures calculating that a parasite cell contains approximately 36 molecules of kDNA. This assay was applied to estimate parasite load in clinical samples from visceral, cutaneous leishmaniasis patients and infected dogs and cats comparing with conventional diagnosis. The study aimed to propose a real-time PCR for the detection of Leishmania DNA from clinical samples trying to solve the diagnostic problems due to the low sensitivity of microscopic examination or the low predictive values of serology and resolve problems related to in vitro culture. The quantitative PCR assay in this study allowed detection of Leishmania DNA and quantification of considerably low parasite loads in samples that had been diagnosed negative by conventional techniques. In conclusion, this quantitative PCR can be used for the diagnosis of both human, canine and feline Leishmaniasis with high sensitivity and specificity, but also for evaluating treatment and the endpoint determination of leishmaniasis.

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