scholarly journals Sensitive Detection of Thirteen Bacterial Vaginosis-Associated Agents Using Multiplex Polymerase Chain Reaction

2015 ◽  
Vol 2015 ◽  
pp. 1-10 ◽  
Author(s):  
Natália Malaguti ◽  
Larissa Danielle Bahls ◽  
Nelson Shozo Uchimura ◽  
Fabrícia Gimenes ◽  
Marcia Edilaine Lopes Consolaro
Keyword(s):  
Polymerase Chain Reaction ◽  
Bacterial Vaginosis ◽  
Multiplex Polymerase Chain Reaction ◽  
Diagnostic Methods ◽  
Type I ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Predictive Values ◽  
Validation Parameters ◽  
Polymerase Chain

Bacterial vaginosis (BV) is characterized by a polymicrobial proliferation of anaerobic bacteria and depletion of lactobacilli, which are components of natural vaginal microbiota. Currently, there are limited conventional methods for BV diagnosis, and these methods are time-consuming, expensive, and rarely allow for the detection of more than one agent simultaneously. Therefore, we conceived and validated a multiplex polymerase chain reaction (M-PCR) assay for the simultaneous screening of thirteen bacterial vaginosis-associated agents (BV-AAs) related to symptomatic BV:Gardnerella vaginalis,Mobiluncus curtisii,Mobiluncus mulieris,Bacteroides fragilis,Mycoplasma hominis,Atopobium vaginae,Ureaplasma urealyticum,Megasphaeratype I, Clostridia-like bacteria vaginosis-associated bacteria (BVABs) 1, 2, and 3,Sneathia sanguinegens, andMycoplasma genitalium. The overall validation parameters of M-PCR compared to single PCR (sPCR) were extremely high, including agreement of 99.1% and sensitivity, specificity, and positive predictive values of 100.0%, negative predictive value of 97.0%, accuracy of 99.3%, and agreement with Nugent results of 100.0%. The prevalence of BV-AAs was very high (72.6%), and simultaneous agents were detected in 53.0%, which demonstrates the effectiveness of the M-PCR assay. Therefore, the M-PCR assay has great potential to impact BV diagnostic methods in vaginal samples and diminish associated complications in the near future.

scholarly journals Application of a Multiplex Polymerase Chain Reaction Assay for the Simultaneous Confirmation of Listeria monocytogenes and Other Listeria Species in Turkey Sample Surveillance†

2002 ◽  
Vol 65 (5) ◽  
pp. 780-785 ◽  
Author(s):  
IRENE V. WESLEY ◽  
KAREN M. HARMON ◽  
JAMES S. DICKSON ◽  
ANN RAMOS SCHWARTZ
Keyword(s):  
Polymerase Chain Reaction ◽  
Listeria Monocytogenes ◽  
Multiplex Pcr ◽  
Multiplex Polymerase Chain Reaction ◽  
Rrna Gene ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Listeria Species ◽  
Multiplex Pcr Assay ◽  
Polymerase Chain

A multiplex polymerase chain reaction was developed to simultaneously identify Listeria monocytogenes and species of the genus Listeria. Two sets of primers were used, with the first amplifying a 938-bp region of the 16S rRNA gene that is highly conserved in all Listeria species and the second amplifying a 174-bp region of the listeriolysin (hlyA) gene of L. monocytogenes. Thus, isolates of Listeria spp. yield a single 938-bp product, whereas L. monocytogenes isolates yield both the 938-bp product and a 174-bp product. The specificity of the assay was verified with all six Listeria species and 11 serotypes of L. monocytogenes, as well as nonrelated bacteria. The multiplex PCR assay was used to determine the incidence of Listeria spp., especially L. monocytogenes, in mechanically separated turkey samples (n = 150 samples). L. monocytogenes strains were selected by using the University of Vermont two-step enrichment protocol and plating to selective Palcam agar. The multiplex PCR assay was used for verification of presumptive Listeria colonies. Approximately 38% of mechanically separated turkey samples (57 of 150) yielded L. monocytogenes; an additional 18% of these samples (27 of 150) harbored other Listeria spp. Fifty-one percent (29 of 57) of the L. monocytogenes isolates were of serogroup 1, 44% (25 of 57) were of serogroup 4, and 2% (1 of 57) were assigned to serogroups other than 1 and 4.

Multiplex polymerase chain reaction as a mastitis screening test for Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis in bulk milk samples

2003 ◽  
Vol 70 (2) ◽  
pp. 149-155 ◽  
Author(s):  
Patchara Phuektes ◽  
Glenn F Browning ◽  
Garry Anderson ◽  
Peter D Mansell
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Streptococcus Dysgalactiae ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Milk Samples ◽  
Streptococcus Uberis ◽  
Bulk Milk ◽  
Polymerase Chain ◽  
Total Bacteria

Effective diagnostic tools for screening herds for mastitis pathogens are important in development and monitoring of mastitis control programmes. A multiplex polymerase chain reaction (PCR) assay for simultaneous detection of Staphylococcus aureus, Streptococcus agalactiae, Streptococcus dysgalactiae and Streptococcus uberis was used in preliminary studies to assess its applicability as an alternative method for monitoring mastitis caused by these organisms at the herd level. PCR was used to detect the presence of these organisms in bulk milk samples. Correlations with bulk milk somatic cell counts (BMCC), total bacteria counts and thermoduric bacteria counts were evaluated. A total of 176 bulk milk samples were collected from 42 herds on five consecutive occasions at approx. 10-d intervals. Str. uberis was the most common organism in these bulk milk samples. There was no relationship between presence of either Staph. aureus, Str. dysgalactiae or Str. uberis and BMCC, total bacteria counts or thermoduric bacteria counts. However, presence of Str. agalactiae was associated with high BMCC and total bacteria counts. The results of this study show that regular analysis of bulk milk using this multiplex PCR assay may be a useful tool for monitoring herd status with respect to Str. agalactiae, but is of less value for monitoring occurrence of Staph. aureus, Str. dysgalactiae and Str. uberis. Further investigations are needed to clarify the relationship between positive PCR results and the prevalence of infected cows in the herd.

scholarly journals Detecting Plasmodium falciparum in community surveys: a comparison of Paracheck Pf® Test and ICT Malaria Pf® Cassette Test to polymerase chain reaction in Mutasa District, Zimbabwe

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Nobert Mudare ◽  
Zvifadzo Matsena-Zingoni ◽  
Aramu Makuwaza ◽  
Edmore Mamini ◽  
Shungu S. Munyati ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Plasmodium Falciparum ◽  
Mitochondrial Gene ◽  
Dried Blood Spots ◽  
Diagnostic Methods ◽  
Chain Reaction ◽  
Cross Sectional ◽  
Predictive Values ◽  
Polymerase Chain ◽  
Pcr Targeting

Abstract Background Microscopy and rapid diagnostic tests (RDTs) are the main techniques used to diagnose malaria. While microscopy is considered the gold standard, RDTs have established popularity as they allow for rapid diagnosis with minimal technical skills. This study aimed to compare the diagnostic performance of two Plasmodium falciparum histidine-rich protein 2 (PfHRP2)-based RDTs (Paracheck Pf® Test (Paracheck) and Malaria Pf™ ICT (ICT)) to polymerase chain reaction (PCR) in a community survey. Methods A cross-sectional study was conducted between October 2012 and December 2014 in Mutasa District, Manicaland Province, eastern Zimbabwe. Households were randomly selected using satellite imagery, and 224 households were visited. Residents present in the household on the date of the visit were recruited for the study. Participants of all age groups from the selected households were screened with Paracheck and ICT RDTs in parallel. Dried blood spots (DBS) and thin and thick smears were collected. Parasite DNA extracted from the DBS was subjected to nested PCR targeting the Plasmodium cytochrome b mitochondrial gene. Data analysis was performed using the Cohen’s Kappa test to determine the interrater agreement and the sensitivity and specificity of the diagnostic test were reported. Results Results from a total of 702 participants were analysed. Most were females, 397 (57%), and the median age of participants was 21 years with an interquartile range of 9–39 years. Of those who were screened, 8 (1.1%), 35 (5.0%), and 21 (2.9%) were malaria parasite positive by microscopy, RDT and PCR, respectively. Paracheck and ICT RDTs had a 100% agreement. Comparing RDT and PCR results, 34 participants (4.8%) had discordant results. Most of the discordant cases were RDT positive but PCR negative (n = 24). Half of those RDT positive, but PCR negative individuals reported anti-malarials to use in the past month, which is significantly higher than reported anti-malarial drug use in the population (p < 0.001). The participant was febrile on the day of the visit, but relying on PfHRP2-based RDT would miss this case. Among the diagnostic methods evaluated, with reference to PCR, the sensitivity was higher with the RDT (52.4%) while specificity was higher with the microscopy (99.9%). The positive predictive value (PPV) was higher with the microscopy (87.5%), while the negative predictive values were similar for both microscopy and RDTs (98%). Overall, a strong correlated agreement with PCR was observed for the microscopy (97.9%) and the RDTs (95.2%). Conclusions Paracheck and ICT RDTs showed 100% agreement and can be used interchangeably. As malaria transmission declines and Zimbabwe aims to reach malaria elimination, management of infected individuals with low parasitaemia as well as non-P. falciparum infection can be critical.

scholarly journals Evaluation of a multiplex polymerase chain reaction for early diagnosis of ventriculostomy-related infections

2015 ◽  
Vol 123 (6) ◽  
pp. 1586-1592 ◽  
Author(s):  
Claire L. Gordon ◽  
Rafal Tokarz ◽  
Thomas Briese ◽  
W. Ian Lipkin ◽  
Komal Jain ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Pathogen Detection ◽  
High Sensitivity ◽  
Chain Reaction ◽  
Pcr Assay ◽  
Clinical Value ◽  
Parallel Testing ◽  
Diagnostic Approaches ◽  
Polymerase Chain

OBJECT Diagnosis of ventriculostomy-related infections (VRIs) is challenging due to the lack of rapid, sensitive assays for pathogen detection. The authors report the development of a multiplex polymerase chain reaction (PCR) assay for differential diagnosis of common VRI pathogens. METHODS MassTag PCR was used to develop a multiplex assay for detection of 11 VRI pathogens. The assay was established and optimized using cloned template standards and spiked samples and was then evaluated on CSF specimens from ventricular drains. Subjects were grouped into definite VRI, possible VRI, or no VRI based on conventional microbiology, CSF evaluation, and clinical parameters. RESULTS CSF specimens were obtained from 45 subjects (median age 49 years, interquartile range 32–63 years; 51% were male). The assay detected 10–100 genome copies. It detected a pathogen in 100% (6 of 6) of definite VRI cases in which a pathogen targeted by the assay was present; these represented 67% of all definite VRIs (6 of 9). Among subjects with a possible VRI, the assay detected a pathogen in 29% (5 of 17). In subjects without overt infection the presence of a pathogen was detected in 32% of subjects (6 of 19), albeit with lower signal compared with the VRI group. CONCLUSIONS MassTag PCR enabled parallel testing of CSF specimens for 11 pathogens of VRI. The high sensitivity of PCR combined with possible device colonization, specimen contamination, and concurrent antibiotic treatments limit the clinical value of the assay, similar to other current diagnostic approaches. With further optimization, multiplex PCR may provide timely identification of multiple possible VRI pathogens and guide management, complementing classic culture approaches.

Diagnostic determination of Norovirus infection as one of the major causes of infectious diarrhea in HIV patients using a multiplex polymerase chain reaction assay

2019 ◽  
Vol 30 (6) ◽  
pp. 550-556 ◽  
Author(s):  
Siyuan Yang ◽  
Min Li ◽  
Jingwei Cheng ◽  
Gang Wan ◽  
Yunao Zhou ◽  
...  
Keyword(s):  
Polymerase Chain Reaction ◽  
Multiplex Polymerase Chain Reaction ◽  
Diagnostic Methods ◽  
Enterotoxigenic Escherichia Coli ◽  
Rt Pcr ◽  
Infectious Diarrhea ◽  
Chain Reaction ◽  
Hiv Patients ◽  
Stool Specimens ◽  
Polymerase Chain

Although infectious diarrhea is one of the most common complications in human immunodeficiency virus (HIV)-infected patients, robust diagnostic methods for determining potential pathogens are still limited in the clinic. Norovirus, a type of calicivirus, has been shown to be the most common cause of gastroenteritis. Here, we used multiplex polymerase chain reaction as a diagnostic tool to verify Norovirus as the diarrhea-related pathogen in HIV-infected patients with unknown etiological information. Stool specimens obtained from 81 HIV-infected patients with diarrhea were analyzed by BioFire FilmArray Gastrointestinal (GI) panel. Among 26 HIV-infected patients with unknown etiological information, we detected Norovirus in 14 stool specimens of these patients with 100% sensitivity and specificity as confirmed by reverse transcription polymerase chain reaction (RT-PCR), and one specimen showed both Norovirus and enterotoxigenic Escherichia coli infection. Among the remaining 55 patients with verified Clostridium difficile infection, nine patients also detected positive for Norovirus infection. In conclusion, using FilmArray GI panel and RT-PCR, we determined that Norovirus infection as one of the main pathogens responsible for diarrhea in HIV patients.

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