Incidence of Pulmonary Aspergillosis and Correlation of Conventional Diagnostic Methods with Nested PCR and Real-Time PCR Assay Using BAL Fluid in Intensive Care Unit Patients

Background and objectives: Human papillomavirus (HPV) is the most prevalent etiological agent of viral sexually-transmitted infection. This study retrospectively evaluated the impact of a switch to a real-time PCR assay in the HPV prevalence and genotypes distribution by a quasi-experimental before-and-after approach. Materials and Methods: In total, 1742 samples collected from 1433 patients were analyzed at the UOC Microbiology and Virology of Policlinico of Bari, Italy. HPV DNA detection was performed using initially nested PCR and subsequently multiplex real-time PCR assay. Results: Statistically significant difference in HPV overall prevalence after the introduction of the real-time assay was not detected (48.97% vs. 50.62%). According to different extraction-DNA amplification methods, differences were observed in the prevalence rates of HPV-45, 68, 40, 42, and 43. The lowest prevalence for HPV-45 was observed in the Magna Pure-Real Time PCR group, while HPV-68, 40, 42, and 43 were less observed in the Qiagen-Real Time PCR group. After, a multivariate logistic regression, an increase in the prevalence of HPV-42 (aOR: 4.08, 95% CI: 1.71–9.73) was associated with the multiplex real-time PCR assay. Conclusions: Although this study is a not a direct comparison between two diagnostic methods because it has a sequential structure, it serves to verify the impact of a new molecular assay on HPV distribution. Moreover, the stability of HPV prevalence over time suggests that the population composition and the behavioral variables did not likely change during the observation period. Our study proposes that the introduction of a molecular test for HPV detection may be related to changes of HPV genotypes distribution.

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Real-Time PCR Assay Compared to Nested PCR and Antigenemia Assays for Detecting Cytomegalovirus Reactivation in Adult T-Cell Leukemia-Lymphoma Patients

Journal of Clinical Microbiology ◽

10.1128/jcm.41.9.4382-4387.2003 ◽

2003 ◽

Vol 41 (9) ◽

pp. 4382-4387 ◽

Cited By ~ 18

Author(s):

J. Ikewaki ◽

E. Ohtsuka ◽

R. Kawano ◽

M. Ogata ◽

H. Kikuchi ◽

...

Keyword(s):

T Cell ◽

Real Time ◽

Real Time Pcr ◽

Nested Pcr ◽

T Cell Leukemia ◽

Cell Leukemia ◽

Pcr Assay ◽

Adult T Cell Leukemia ◽

Cytomegalovirus Reactivation

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Roles of the multiplex real-time PCR assay and β-D-glucan in a high-risk population for intra-abdominal candidiasis (IAC)

Medical Mycology ◽

10.1093/mmy/myz123 ◽

2019 ◽

Vol 58 (6) ◽

pp. 789-796 ◽

Cited By ~ 1

Author(s):

J Fortún ◽

M J Buitrago ◽

F Gioia ◽

E Gómez-Gª de la Pedrosa ◽

M E Alvarez ◽

...

Keyword(s):

High Risk ◽

Real Time ◽

Real Time Pcr ◽

Volume Overload ◽

Diagnostic Methods ◽

Practice Volume ◽

High Risk Population ◽

Pcr Assay ◽

European Consensus ◽

Abdominal Candidiasis

Abstract Multiplex quantitative real-time PCR (MRT-PCR) using blood can improve the diagnosis of intra-abdominal candidiasis (IAC). We prospectively studied 39 patients with suspected IAC in the absence of previous antifungal therapy. Blood cultures, MRT-PCR, and β-D-glucan (BDG) in serum were performed in all patients. IAC was defined according to the 2013 European Consensus criteria. For MRT-PCR, the probes targeted the ITS1 or ITS2 regions of ribosomal DNA. Candidaemia was confirmed only in four patients (10%), and IAC criteria were present in 17 patients (43.6%). The sensitivity of MRT-PCR was 25% but increased to 63.6% (P = .06) in plasma obtained prior to volume overload and transfusion; specificity was above 85% in all cases. BDG performance was improved using a cutoff > 260 pg/ml, and improvement was not observed in samples obtained before transfusion. In this cohort of high risk of IAC and low rate of bloodstream infection, the performance of non-culture-based methods (MRT-PCR or BDG) was moderate but may be a complementary tool given the limitations of diagnostic methods available in clinical practice. Volume overload requirements, in combination with other factors, decrease the accuracy of MRT-PCR in patients with IAC.

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P1437 Real time PCR for monitoring herpes simplex virus load in tracheal aspirates of intensive care unit patients

International Journal of Antimicrobial Agents ◽

10.1016/s0924-8579(07)71276-1 ◽

2007 ◽

Vol 29 ◽

pp. S401

Author(s):

N. De Vos ◽

A. Vankeerberghen ◽

A. Boel ◽

I. Demeyer ◽

L. Van Hoovels ◽

...

Keyword(s):

Intensive Care Unit ◽

Intensive Care ◽

Herpes Simplex Virus ◽

Herpes Simplex ◽

Real Time ◽

Real Time Pcr ◽

Virus Load ◽

Simplex Virus ◽

Tracheal Aspirates

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Using whole-genome sequencing and a pentaplex real-time PCR to characterize third-generation cephalosporin-resistant Enterobacteriaceae from Southeast Queensland, Australia

10.1101/2020.02.20.958728 ◽

2020 ◽

Author(s):

AG Stewart ◽

EP Price ◽

K Schabacker ◽

M Birikmen ◽

PNA Harris ◽

...

Keyword(s):

Intensive Care Unit ◽

Intensive Care ◽

Whole Genome Sequencing ◽

Real Time ◽

Genome Sequencing ◽

Real Time Pcr ◽

Generation Cephalosporin ◽

Whole Genome ◽

Third Generation ◽

Sensitivity Testing

AbstractThird-generation cephalosporin-resistant (3GC-R) Enterobacteriaceae represent a major threat to human health. Here, we captured 288 3GC-R Enterobacteriaceae clinical isolates from 258 patients presenting at a regional Australian hospital over a 14-month period. Alongside routine mass spectrometry speciation and antibiotic sensitivity testing, isolates were examined using a rapid (~40 min) pentaplex real-time PCR assay targeting the most common extended spectrum β-lactamases (ESBLs; CTX-M-1 and CTX-M-9 groups, plus TEM, SHV, and an internal 16S ribosomal DNA control). Additionally, AmpC CMY β-lactamase prevalence was examined using a singleplex PCR. A subset of isolates, including all 3GC-R isolates obtained from the intensive care unit, were subjected to whole-genome sequencing (WGS) to assess transmission dynamics, the presence of unidentified resistance determinants, and genotyping accuracy. Escherichia coli (80.2%) and Klebsiella pneumoniae (17.0%) were dominant, with Klebsiella oxytoca, Klebsiella aerogenes and Enterobacter cloacae infrequently identified. Ceftriaxone and cefoxitin resistance was identified in 97% and 24.5% of E. coli and K. pneumoniae isolates, respectively. Consistent with global findings in Enterobacteriaceae, the majority (98.3%) of isolates harbored at least one β-lactamase gene, with 144 (50%) encoding blaCTX-M-1 group, 92 (31.9%) blaCTX-M-9 group, 48 (16.7%) blaSHV, 133 (46.2%) blaTEM, and 34 (11.8%) blaCMY. WGS of β-lactamase negative or carbapenem-resistant isolates identified uncommon ESBLs and carbapenemases, including blaNDM and blaIMP, and confirmed all PCR-positive genotypes. No evidence of transmission among intensive care unit patients was identified. We demonstrate that our PCR assays enable the rapid and cost-effective identification of ESBLs in the hospital setting, which has important infection control and therapeutic implications.

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Evaluation of the Multiplex Real-Time PCR DermaGenius® Assay for the Detection of Dermatophytes in Hair Samples from Senegal

Journal of Fungi ◽

10.3390/jof8010011 ◽

2021 ◽

Vol 8 (1) ◽

pp. 11

Author(s):

Mouhamadou Ndiaye ◽

Rosalie Sacheli ◽

Khadim Diongue ◽

Caroline Adjetey ◽

Rajae Darfouf ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Melting Curve ◽

Diagnostic Methods ◽

Melting Curve Analysis ◽

Microsporum Canis ◽

Molecular Tools ◽

Pcr Assay ◽

Epidermophyton Floccosum ◽

Hair Samples

For the successful treatment of dermatophytoses, especially tinea capitis, there is a need for accurate and rapid diagnostic methods. A lot of recent literature has focused on the detection of dermatophytes directly on sample material such as nails, hair and skin scrapings. Molecular tools offer the ability to rapidly diagnose dermatophytosis within 48 h. This study aimed to compare the results of a commercial real-time PCR (real-time PCR) assay DermaGenius®(DG) 2.0 complete multiplex kit with those of conventional diagnostic methods (direct microscopy and culture). A total of 129 hair samples were collected in Dakar (Senegal) from patients suspected of dermatophytosis. DG was applied for the molecular detection of Candida albicans, Trichophyton rubrum/soudanense, T. interdigitale, T. tonsurans, T. mentagrophytes, T. violaceum, Microsporum canis, M. audouinii, Epidermophyton floccosum, T. benhamiae and T. verrucosum. Dermatophytes species and C. albicans were differentiated by melting curve analysis. The sensitivity and specificity of the PCR assay were 89.3% and 75.3%, respectively. DG PCR was significantly more sensitive than culture (p < 0.001). DG PCR is fast and robust to contamination. In this paper, the main questions discussed were the replacement of culture by a broad-spectrum fungal real-time PCR and the implementation of DG PCR into a routine laboratory in Senegal.

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Rapid Detection and Differentiating of the Predominant Salmonella Serovars in Chicken Farm by TaqMan Multiplex Real-Time PCR Assay

Frontiers in Cellular and Infection Microbiology ◽

10.3389/fcimb.2021.759965 ◽

2021 ◽

Vol 11 ◽

Author(s):

Suhua Xin ◽

Hong Zhu ◽

Chenglin Tao ◽

Beibei Zhang ◽

Lan Yao ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Epidemiologic Study ◽

Clinical Diagnostics ◽

Diagnostic Methods ◽

Molecular Diagnostic ◽

Clinical Samples ◽

Analytical Sensitivity ◽

Pcr Assay ◽

Chicken Farm

Salmonella has been known as an important zoonotic pathogen that can cause a variety of diseases in both animals and humans. Poultry are the main reservoir for the Salmonella serovars Salmonella Pullorum (S. Pullorum), Salmonella Gallinarum (S. Gallinarum), Salmonella Enteritidis (S. Enteritidis), and Salmonella Typhimurium (S. Typhimurium). The conventional serotyping methods for differentiating Salmonella serovars are complicated, time-consuming, laborious, and expensive; therefore, rapid and accurate molecular diagnostic methods are needed for effective detection and prevention of contamination. This study developed and evaluated a TaqMan multiplex real-time PCR assay for simultaneous detection and differentiation of the S. Pullorum, S. Gallinarum, S. Enteritidis, and S. Typhimurium. In results, the optimized multiplex real-time PCR assay was highly specific and reliable for all four target genes. The analytical sensitivity corresponded to three colony-forming units (CFUs) for these four Salmonella serovars, respectively. The detection limit for the multiplex real-time PCR assay in artificially contaminated samples was 500 CFU/g without enrichment, while 10 CFU/g after pre-enrichment. Moreover, the multiplex real-time PCR was applied to the poultry clinical samples, which achieved comparable results to the traditional bacteriological examination. Taken together, these results indicated that the optimized TaqMan multiplex real-time PCR assay will be a promising tool for clinical diagnostics and epidemiologic study of Salmonella in chicken farm and poultry products.

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Detection and molecular characterization of Cryptosporidium spp. in captive canaries (Serinus canaria) using different diagnostic methods

Revista Brasileira de Parasitologia Veterinária ◽

10.1590/s1984-296120180010 ◽

2018 ◽

Vol 27 (1) ◽

pp. 60-65 ◽

Cited By ~ 3

Author(s):

Vinícius da Silva Camargo ◽

Bruna Nicoleti Santana ◽

Elis Domingos Ferrari ◽

Alex Akira Nakamura ◽

Walter Bertequini Nagata ◽

...

Keyword(s):

Real Time ◽

Malachite Green ◽

Real Time Pcr ◽

Nested Pcr ◽

18S Rrna ◽

Diagnostic Methods ◽

Negative Staining ◽

Cryptosporidium Spp ◽

Genotype Iii ◽

Pcr Targeting

Abstract This study used several diagnostic methods to examine the occurrence of and molecularly characterize Cryptosporidium spp. in captive canaries (Serinus canaria) in southern and southeastern Brazil. A total of 498 fecal samples were purified by centrifugal-flotation using Sheather's solution. Cryptosporidium spp. diagnosis was performed using three diagnostic methods: malachite green negative staining, nested PCR targeting the 18S rRNA gene, followed by sequencing the amplified fragments, and duplex real-time PCR targeting the 18S rRNA specific to detect Cryptosporidium galli and Cryptosporidium avian genotype III. The overall positivity for Cryptosporidium spp. (total samples positive in at least one protocol) from the microscopic analysis, nested PCR and duplex real-time PCR protocol results was 13.3% (66/498). The positivity rates were 2.0% (10/498) and 4.6% (23/498) for Cryptosporidium spp. by microscopy and nested PCR, respectively. Sequencing of 20 samples amplified by nested PCR identified C. galli (3.0%; 15/498), Cryptosporidium avian genotype I (0.8%; 4/498) and Cryptosporidium avium (0.2%; 1/498). Duplex real-time PCR revealed a positivity of 7.8% (39/498) for C. galli and 2.4% (12/498) for avian genotype III. Malachite green negative staining differed significantly from nested PCR in detecting Cryptosporidium spp. Duplex real-time PCR was more sensitive than nested PCR/sequencing for detecting gastric Cryptosporidium in canaries.

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