scholarly journals Circulating Gut-Homing (α4β7+) Plasmablast Responses against Shigella Surface Protein Antigens among Hospitalized Patients with Diarrhea

2016 ◽  
Vol 23 (7) ◽  
pp. 610-617 ◽  
Author(s):  
Anuradha Sinha ◽  
Ayan Dey ◽  
Giulietta Saletti ◽  
Pradip Samanta ◽  
Partha Sarathi Chakraborty ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Surface Protein ◽  
Protein Antigens ◽  
Magnetic Cell Sorting ◽  
Content Type ◽  
Recent Onset ◽  
Microbiological Methods ◽  
Stool Specimens ◽  
Antibody Secreting Cells

Developing countries are burdened withShigelladiarrhea. Understanding mucosal immune responses associated with naturalShigellainfection is important to identify potential correlates of protection and, as such, to design effective vaccines. We performed a comparative analysis of circulating mucosal plasmablasts producing specific antibodies against highly conserved invasive plasmid antigens (IpaC, IpaD20, and IpaD120) and two recently identified surface protein antigens,pan-Shigellasurfaceprotein antigen 1 (PSSP1) and PSSP2, common to all virulentShigellastrains. We examined blood and stool specimens from 37 diarrheal patients admitted to the Infectious Diseases & Beliaghata General Hospital, Kolkata, India. The etiological agent of diarrhea was investigated in stool specimens by microbiological methods and real-time PCR. Gut-homing (α4β7+) antibody-secreting cells (ASCs) were isolated from patient blood by means of combined magnetic cell sorting and two-color enzyme-linked immunosorbent spot (ELISPOT) assay. Overall, 57% (21 of 37) and 65% (24 of 37) of the patients were positive forShigellainfection by microbiological and real-time PCR assays, respectively. The frequency of α4β7+IgG ASC responders against Ipas was higher than that observed against PSSP1 or PSSP2, regardless of theShigellaserotype isolated from these patients. Thus, α4β7+ASC responses to Ipas may be considered an indirect marker ofShigellainfection. The apparent weakness of ASC responses to PSSP1 is consistent with the lack of cross-protection induced by naturalShigellainfection. The finding that ASC responses to IpaD develop in patients with recent-onset shigellosis indicates that such responses may not be protective or may wane too rapidly and/or be of insufficient magnitude.

scholarly journals Comparison of Loop-Mediated Isothermal Amplification and Real-Time PCR Assays for Detection ofStrongyloidesLarvae in Different Specimen Matrices

2019 ◽  
Vol 57 (4) ◽  
Author(s):  
Matthew R. Watts ◽  
Rady Kim ◽  
Vishal Ahuja ◽  
Gemma J. Robertson ◽  
Yasmin Sultana ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Chronic Infection ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Diagnostic Methods ◽  
Percentage Agreement ◽  
Content Type ◽  
Loop Mediated Isothermal Amplification ◽  
Stool Specimens

ABSTRACTStrongyloides stercoraliscan cause disease that ranges from asymptomatic chronic infection to fatal hyperinfection. Diagnosis from stool can be challenging because the most sensitive conventional tests require live larvae to be effective and there can be low larval output in chronic infection. Nucleic acid amplification tests (NAAT) have been developed to complement existing diagnostic methods. We compared a recently developed loop-mediated isothermal amplification (LAMP) assay with a real-time PCR that has previously been validated with larval microscopy. The limits of detection—quantified using serial dilutions of DNA extracts from singleStrongyloides rattithird-stage (L3) larvae spiked into approximately 250 µl of 5 differentS. stercoralis-negative stool specimens—were 10−3(1/5 replicates) and 10−2(1/5 replicates) dilutions for PCR and LAMP, respectively. PCR was positive for 4/5 replicates at 10−2. LAMP was compared to PCR using extracts from 396 stool specimens collected in Bangladesh and Australia, of which 53 were positive and 343 were negative by PCR. The positive percentage agreement of LAMP was 77.3% (95% score confidence interval [CI], 64.5 to 86.6). The negative percentage agreement was 100% (95% CI, 98.9 to 100). In a preliminary investigation, PCR and LAMP assays were positive using DNA extracted from serum (PCR, 3/16 extracts; LAMP, 2/16 extracts) and bronchoalveolar lavage fluid (PCR and LAMP, 2/2 extracts), demonstrating proof of concept. Compared to PCR, the lower number of positive results using the LAMP assay may have been due to reaction inhibitors and DNA degradation, and strategies to improve the LAMP assay are discussed.

scholarly journals Evaluation of the New BD Max GC Real-Time PCR Assay, Analytically and Clinically as a Supplementary Test for the BD ProbeTec GC Qx Amplified DNA Assay, for Molecular Detection of Neisseria gonorrhoeae

2015 ◽  
Vol 53 (12) ◽  
pp. 3935-3937 ◽  
Author(s):  
Daniel Golparian ◽  
Stina Boräng ◽  
Martin Sundqvist ◽  
Magnus Unemo
Keyword(s):  
Neisseria Gonorrhoeae ◽  
Real Time ◽  
Sensitivity And Specificity ◽  
Real Time Pcr ◽  
Molecular Detection ◽  
Analytical Sensitivity ◽  
Pcr Assay ◽  
Content Type ◽  
Dna Assay ◽  
Supplementary Test

The new BD Max GC real-time PCR assay showed high clinical and analytical sensitivity and specificity. It can be an effective and accurate supplementary test for the BD ProbeTec GC Qx amplified DNA assay, which had suboptimal specificity, and might also be used for initial detection ofNeisseria gonorrhoeae.

scholarly journals Evaluation of StrepBSelectChromogenic Medium and the Fast-Track Diagnostics Group B Streptococcus (GBS) Real-Time PCR Assay Compared to Routine Culture for Detection of GBS during Antepartum Screening

2017 ◽  
Vol 55 (7) ◽  
pp. 2137-2142 ◽  
Author(s):  
Deirdre L. Church ◽  
Heather Baxter ◽  
Tracie Lloyd ◽  
Oscar Larios ◽  
Daniel B. Gregson
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Fast Track ◽  
Group B Streptococcus ◽  
Culture Method ◽  
Predictive Values ◽  
Content Type ◽  
Standard Culture ◽  
Group B ◽  
The Cost

ABSTRACTLife-threatening infection in neonates due to group BStreptococcus(GBS) is preventable by screening of near-term pregnant women and treatment at delivery. A total of 295 vaginal-rectal swabs were collected from women attending antepartum clinics in Calgary, Alberta, Canada. GBS colonization was detected by the standard culture method (Strep B Carrot Broth subcultured to blood agar with a neomycin disk) and compared to recovery with Strep Group B Broth (Dalynn Biologicals) subcultured to StrepBSelectchromogenic medium (CM; Bio-Rad Laboratories) and the Fast-Track Diagnostics GBS real-time PCR (quantitative PCR [qPCR]) assay (Phoenix Airmid Biomedical Corp.) performed with broth-enriched samples and the Abbottm2000sp/m2000rt system. A total of 62/295 (21%) women were colonized with GBS; 58 (19.7%) cases were detected by standard culture, while CM and qPCR each found 61 (20.7%) cases. The qPCR and CM were similar in performance, with sensitivities, specificities, and positive and negative predictive values of 98.4 and 98.4%, 99.6 and 99.6%, 98.4 and 98.4%, and 99.6 and 99.6%, respectively, compared to routine culture. Both qPCR and CM would allow more rapid reporting of routine GBS screening results than standard culture. Although the cost per test was similar for standard culture and CM, the routine use of qPCR would cost approximately four times as much as culture-based detection. Laboratories worldwide should consider implementing one of the newer methods for primary GBS testing, depending on the cost limitations of different health care jurisdictions.

Improved diagnosis of gastrointestinal infections using a semi-automated multiplex real-time PCR for detection of enteropathogens

2021 ◽  
Vol 70 (9) ◽  
Author(s):  
Berta Fidalgo ◽  
Elisa Rubio ◽  
Victor Pastor ◽  
Marta Parera ◽  
Clara Ballesté-Delpierre ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Enteric Pathogens ◽  
Pcr Analysis ◽  
Culture Methods ◽  
Gastrointestinal Infections ◽  
Content Type ◽  
Link Type ◽  
Microbiological Culture ◽  
Micro Organisms

Introduction. The identification of enteropathogens is critical for the clinical management of patients with suspected gastrointestinal infection. The FLOW multiplex PCR system (FMPS) is a semi-automated platform (FLOW System, Roche) for multiplex real-time PCR analysis. Hypothesis/Gap Statement. FMPS has greater sensitivity for the detection of enteric pathogens than standard methods such as culture, biochemical identification, immunochromatography or microscopic examination. Aim.The diagnostic performance of the FMPS was evaluated and compared to that of traditional microbiological procedures. Methodology. A total of 10 659 samples were collected and analysed over a period of 7 years. From 2013 to 2018 (every July to September), samples were processed using standard microbiological culture methods. In 2019, the FMPS was implemented using real-time PCR to detect the following enteropathogens: Shigella spp., Salmonella spp., Campylobacter spp., Giardia intestinalis, Entamoeba histolytica, Blastocystis hominis, Cryptosporidum spp., Dientamoeba fragilis, adenovirus, norovirus and rotavirus. Standard microbiological culture methods (2013–2018) included stool culture, microscopy and immunochromatography. Results. A total of 1078 stool samples were analysed prospectively using the FMPS from July to September (2019): bacterial, parasitic and viral pathogens were identified in 15.3, 9.71 and 5.29 % of cases, respectively. During the same period of 6 years (2013–2018), the proportion of positive identifications using standard microbiological methods from 2013 to 2018 was significantly lower. A major significant recovery improvement was observed for all bacteria species tested: Shigella spp./enteroinvasive Escherichia coli (EIEC) (P <0.05), Salmonella spp. (P <0.05) and Campylobacter spp. (P <0.05). Marked differences were also observed for the parasites G. intestinalis, Cryptosporidium spp. and D. fragilis. Conclusion. These results support the value of multiplex real-time PCR analysis for the detection of enteric pathogens in laboratory diagnosis with outstanding performance in identifying labile micro-organisms. The identification of unsuspected micro-organisms for less specific clinical presentations may also impact on clinical practice and help optimize patient management.

scholarly journals A Novel, Multiple-Antigen Pneumococcal Vaccine Protects against LethalStreptococcus pneumoniaeChallenge

2018 ◽  
Vol 87 (3) ◽  
Author(s):  
Win-Yan Chan ◽  
Claire Entwisle ◽  
Giuseppe Ercoli ◽  
Elise Ramos-Sevillano ◽  
Ann McIlgorm ◽  
...  
Keyword(s):  
Heat Shock ◽  
Protein A ◽  
Surface Protein ◽  
Surface Proteins ◽  
Rabbit Serum ◽  
Protein Antigens ◽  
Content Type ◽  
Multiple Antigen ◽  
Bacterial Lysates ◽  
Chromatography Step

ABSTRACTCurrent vaccination againstStreptococcus pneumoniaeuses vaccines based on capsular polysaccharides from selected serotypes and has led to nonvaccine serotype replacement disease. We have investigated an alternative serotype-independent approach, using multiple-antigen vaccines (MAV) prepared fromS. pneumoniaeTIGR4 lysates enriched for surface proteins by a chromatography step after culture under conditions that induce expression of heat shock proteins (Hsp; thought to be immune adjuvants). Proteomics and immunoblot analyses demonstrated that, compared to standard bacterial lysates, MAV was enriched with Hsps and contained several recognized protective protein antigens, including pneumococcal surface protein A (PspA) and pneumolysin (Ply). Vaccination of rodents with MAV induced robust antibody responses to multiple serotypes, including nonpneumococcal conjugate vaccine serotypes. Homologous and heterologous strains ofS. pneumoniaewere opsonized after incubation in sera from vaccinated rodents. In mouse models, active vaccination with MAV significantly protected against pneumonia, while passive transfer of rabbit serum from MAV-vaccinated rabbits significantly protected against sepsis caused by both homologous and heterologousS. pneumoniaestrains. Direct comparison of MAV preparations made with or without the heat shock step showed no clear differences in protein antigen content and antigenicity, suggesting that the chromatography step rather than Hsp induction improved MAV antigenicity. Overall, these data suggest that the MAV approach may provide serotype-independent protection againstS. pneumoniae.

scholarly journals Abilities of the mCP Agar Method and CRENAME Alpha Toxin-Specific Real-Time PCR Assay To Detect Clostridium perfringens Spores in Drinking Water

2013 ◽  
Vol 79 (24) ◽  
pp. 7654-7661 ◽  
Author(s):  
Andrée F. Maheux ◽  
Ève Bérubé ◽  
Dominique K. Boudreau ◽  
Romain Villéger ◽  
Philippe Cantin ◽  
...  
Keyword(s):  
Drinking Water ◽  
Water Sample ◽  
Real Time ◽  
Clostridium Perfringens ◽  
Real Time Pcr ◽  
Potable Water ◽  
Alpha Toxin ◽  
Pcr Assay ◽  
Content Type ◽  
The Public

ABSTRACTWe first determined the analytical specificity and ubiquity (i.e., the ability to detect all or most strains) of aClostridium perfringens-specific real-time PCR (rtPCR) assay based on thecpagene (cpartPCR) by using a bacterial strain panel composed ofC. perfringensand non-C. perfringens Clostridiumstrains. All non-C. perfringens Clostridiumstrains tested negative, whereas allC. perfringensstrains tested positive with thecpartPCR, for an analytical specificity and ubiquity of 100%. ThecpartPCR assay was then used to confirm the identity of 116 putativeC. perfringensisolates recovered after filtration of water samples and culture on mCP agar. Colonies presenting discordant results between the phenotype on mCP agar andcpartPCR were identified by sequencing the 16S rRNA andcpagenes. Four mCP−/rtPCR+colonies were identified asC. perfringens, whereas 3 mCP+/rtPCR−colonies were identified as non-C. perfringens. ThecpartPCR was negative with all 51 non-C. perfringensstrains and positive with 64 of 65C. perfringensstrains. Finally, we compared mCP agar and a CRENAME (concentration andrecovery of microbial particles,extraction ofnucleicacids, andmolecularenrichment) procedure pluscpartPCR (CRENAME +cpartPCR) for their abilities to detectC. perfringensspores in drinking water. CRENAME +cpartPCR detected as few as oneC. perfringensCFU per 100 ml of drinking water sample in less than 5 h, whereas mCP agar took at least 25 h to deliver results. CRENAME +cpartPCR also allows the simultaneous and sensitive detection ofEscherichia coliandC. perfringensfrom the same potable water sample. In itself, it could be used to assess the public health risk posed by drinking water potentially contaminated with pathogens more resistant to disinfection.

scholarly journals A New Highly Sensitive and Specific Real-Time PCR Assay Targeting the Malate Dehydrogenase Gene ofKingella kingaeand Application to 201 Pediatric Clinical Specimens

2018 ◽  
Vol 56 (8) ◽  
Author(s):  
Nawal El Houmami ◽  
Guillaume André Durand ◽  
Janek Bzdrenga ◽  
Anne Darmon ◽  
Philippe Minodier ◽  
...  
Keyword(s):  
Real Time ◽  
Malate Dehydrogenase ◽  
Real Time Pcr ◽  
Detection Sensitivity ◽  
Rt Pcr ◽  
Pcr Assay ◽  
Content Type ◽  
Clinical Specimens ◽  
Specificity And Sensitivity ◽  
Pcr Assays

ABSTRACTKingella kingaeis a significant pediatric pathogen responsible for bone and joint infections, occult bacteremia, and endocarditis in early childhood. Past efforts to detect this bacterium using culture and broad-range 16S rRNA gene PCR assays from clinical specimens have proven unsatisfactory; therefore, by the late 2000s, these were gradually phased out to explore the benefits of specific real-time PCR tests targeting thegroELgene and the RTX locus ofK. kingae. However, recent studies showed that real-time PCR (RT-PCR) assays targeting theKingellasp. RTX locus that are currently available for the diagnosis ofK. kingaeinfection lack specificity because they could not distinguish betweenK. kingaeand the recently describedKingella negevensisspecies. Furthermore,in silicoanalysis of thegroELgene from a large collection of 45K. kingaestrains showed that primers and probes fromK. kingaegroEL-based RT-PCR assays display a few mismatches withK. kingae groELvariations that may result in decreased detection sensitivity, especially in paucibacillary clinical specimens. In order to provide an alternative togroEL- and RTX-targeting RT-PCR assays that may suffer from suboptimal specificity and sensitivity, aK. kingae-specific RT-PCR assay targeting the malate dehydrogenase (mdh) gene was developed for predicting no mismatch between primers and probe and 18 variants of theK. kingae mdhgene from 20 distinct sequence types ofK. kingae. This novelK. kingae-specific RT-PCR assay demonstrated high specificity and sensitivity and was successfully used to diagnoseK. kingaeinfections and carriage in 104 clinical specimens from children between 7 months and 7 years old.

scholarly journals Extended-Interval Dosing of Rezafungin against Azole-Resistant Aspergillus fumigatus

2019 ◽  
Vol 63 (10) ◽  
Author(s):  
Nathan P. Wiederhold ◽  
Laura K. Najvar ◽  
Rosie Jaramillo ◽  
Marcos Olivo ◽  
Brian L. Wickes ◽  
...  
Keyword(s):  
Invasive Aspergillosis ◽  
Aspergillus Fumigatus ◽  
Real Time ◽  
Real Time Pcr ◽  
Quantitative Real Time Pcr ◽  
Content Type ◽  
Fungal Burden

ABSTRACT We evaluated extended-interval dosing of the investigational echinocandin rezafungin (1, 4, and 16 mg/kg on days 1, 4, and 7 postinoculation) for the treatment of disseminated invasive aspergillosis caused by azole-resistant Aspergillus fumigatus. Survival was significantly improved in mice treated with each dose of rezafungin and supratherapeutic posaconazole (20 mg/kg twice daily). Kidney fungal burden, as measured by quantitative real-time PCR, was also significantly reduced in mice treated with rezafungin although variability was observed.

scholarly journals A Real-Time PCR Assay for Simultaneous Detection and Differentiation of Four Common Entamoeba Species That Infect Humans

2020 ◽  
Vol 59 (1) ◽  
pp. e01986-20
Author(s):  
Ibne Karim M. Ali ◽  
Shantanu Roy
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Simultaneous Detection ◽  
Small Subunit ◽  
Clinical Samples ◽  
Pcr Assay ◽  
Content Type ◽  
Rdna Sequences ◽  
Appropriate Treatment

ABSTRACTThere are over 40 species within the genus Entamoeba, eight of which infect humans. Of these, four species (Entamoeba histolytica, E. dispar, E. moshkovskii, and E. bangladeshi) are morphologically indistinguishable from each other, and yet differentiation is important for appropriate treatment decisions. Here, we developed a hydrolysis probe-based tetraplex real-time PCR assay that can simultaneously detect and differentiate these four species in clinical samples. In this assay, multicopy small-subunit (SSU) ribosomal DNA (rDNA) sequences were used as targets. We determined that the tetraplex real-time PCR can detect amebic DNA corresponding to as little as a 0.1 trophozoite equivalent of any of these species. We also determined that this assay can detect E. histolytica DNA in the presence of 10-fold more DNA from another Entamoeba species in mixed-infection scenarios. With a panel of more than 100 well-characterized clinical samples diagnosed and confirmed using a previously published duplex real-time PCR (capable of detecting E. histolytica and E. dispar), our tetraplex real-time PCR assay demonstrated levels of sensitivity and specificity comparable with those demonstrated by the duplex real-time PCR assay. The advantage of our assay over the duplex assay is that it can specifically detect two additional Entamoeba species and can be used in conventional PCR format. This newly developed assay will allow further characterization of the epidemiology and pathogenicity of the four morphologically identical Entamoeba species, especially in low-resource settings.

Sign in / Sign up

Export Citation Format

Share Document