scholarly journals Co-detection of the measles vaccine and wild-type virus by real-time PCR: public health laboratory protocol

2021 ◽  
Vol 3 (11) ◽  
Author(s):  
Kamelia R. Stanoeva ◽  
Robert H. G. Kohl ◽  
Rogier Bodewes
Keyword(s):  
Public Health ◽  
Real Time ◽  
Measles Virus ◽  
Clinical Signs ◽  
Control Measures ◽  
Wild Type Virus ◽  
Clinical Samples ◽  
Wild Type ◽  
Characteristic Analysis ◽  
Genotype A

In rare cases vaccination with the measles virus vaccine genotype A (MeVA) may cause a vaccine reaction with clinical signs similar to infection with wild-type measles virus (MeVwt). Rapid differentiation between MeVA and MeVwt infection is important for taking adequate public health measures. Recently, a few MeVA real-time reverse-transcription quantitative PCR methods (RT-qPCRs) were described that can distinguish between MeVA and MeVwt. However, detection of MeVA does in theory not exclude infection with MeVwt. In the present study, we established a protocol for determination of co-infections with MeVA and MeVwt. To this end, MeVA RT-qPCRs were used in combination with the routine measles virus (MeV) RT-qPCR, and the results suggested that the differences between the RT-qPCR Ct values (delta Ct, ∆Ct) could be used as criteria. Subsequently, we tested samples from vaccine-associated measles cases that were confirmed by genotyping. In addition, experimental mixtures of MeVA and MeVwt were tested in different concentrations. All tested MeVA clinical samples had ∆Ct ≤3.6. The results of experimental mixtures showed a mean ∆Ct ≤2.8 for genotype A alone and >3.2 when combined with either genotype B3 or D8. The results of a receiver operator characteristic analysis indicated that the optimum ∆Ct for use as a cut-off value was 3.5, while with ∆Ct values of 2.9 and 3.7 sensitivity and specificity were respectively 1.00. Thus, ∆Ct could be used to exclude the presence of MeVwt if MeVA is detected and ∆Ct is <2.9, while ∆Ct >3.7 were highly suggestive of co-infection and ≥2.9 ∆Ct <3.7 warranted additional confirmation, such as next-generation sequencing. This RT-qPCR-based protocol could be used for the exclusion of infection with MeVwt in cases with vaccine-associated measles reaction, crucial for the timely implementation of public health prevention and control measures.

scholarly journals A Brazilian glycoprotein E-negative bovine herpesvirus type 1.2a (BHV-1.2a) mutant is attenuated for cattle and induces protection against wild-type virus challenge

2002 ◽  
Vol 22 (4) ◽  
pp. 135-140 ◽  
Author(s):  
Ana Cláudia Franco ◽  
Fernando Rosado Spilki ◽  
Paulo Augusto Esteves ◽  
Marcelo de Lima ◽  
Rudi Weiblen ◽  
...  
Keyword(s):  
Clinical Signs ◽  
Bovine Herpesvirus ◽  
Vaccine Virus ◽  
Parental Virus ◽  
Wild Type Virus ◽  
Wild Type ◽  
Glycoprotein E ◽  
Virus Shedding ◽  
Virus Challenge ◽  
Herpesvirus Type

The authors previously reported the construction of a glycoprotein E-deleted (gE-) mutant of bovine herpesvirus type 1.2a (BHV-1.2a). This mutant, 265gE-, was designed as a vaccinal strain for differential vaccines, allowing the distinction between vaccinated and naturally infected cattle. In order to determine the safety and efficacy of this candidate vaccine virus, a group of calves was inoculated with 265gE-. The virus was detected in secretions of inoculated calves to lower titres and for a shorter period than the parental virus inoculated in control calves. Twenty one days after inoculation, the calves were challenged with the wild type parental virus. Only mild signs of infection were detected on vaccinated calves, whereas non-vaccinated controls displayed intense rhinotracheitis and shed virus for longer and to higher titres than vaccinated calves. Six months after vaccination, both vaccinated and control groups were subjected to reactivation of potentially latent virus. The mutant 265gE- could not be reactivated from vaccinated calves. The clinical signs observed, following the reactivation of the parental virus, were again much milder on vaccinated than on non-vaccinated calves. Moreover, parental virus shedding was considerably reduced on vaccinated calves at reactivation. In view of its attenuation, immunogenicity and protective effect upon challenge and reactivation with a virulent BHV-1, the mutant 265gE- was shown to be suitable for use as a BHV-1 differential vaccine virus.

scholarly journals Progress towards Rapid Detection of Measles Vaccine Strains: a Tool To Inform Public Health Interventions

2016 ◽  
Vol 55 (3) ◽  
pp. 686-689 ◽  
Author(s):  
Jill K. Hacker
Keyword(s):  
Public Health ◽  
Measles Virus ◽  
Measles Vaccine ◽  
Wild Type ◽  
Pcr Assay ◽  
Public Health Response ◽  
The Public ◽  
Health Response ◽  
Epidemiological Tool ◽  
Type Strains

ABSTRACT Rapid differentiation of vaccine from wild-type strains in suspect measles cases is a valuable epidemiological tool that informs the public health response to this highly infectious disease. Few public health laboratories sequence measles virus-positive specimens to determine genotype, and the vaccine-specific real-time reverse transcriptase PCR (rRT-PCR) assay described by F. Roy et al. (J. Clin. Microbiol. 55:735–743, 2017, https://doi.org/10.1128/JCM.01879-16 ) offers a rapid, easily adoptable method to identify measles vaccine strains in suspect cases.

scholarly journals Cytokine Imbalance after Measles Virus Infection Has No Correlation with Immune Suppression

2009 ◽  
Vol 83 (14) ◽  
pp. 7244-7251 ◽  
Author(s):  
Mary Carsillo ◽  
Kay Klapproth ◽  
Stefan Niewiesk
Keyword(s):  
Virus Infection ◽  
Immune Suppression ◽  
Recombinant Virus ◽  
Measles Virus ◽  
Vaccine Virus ◽  
Gamma Interferon ◽  
Wild Type Virus ◽  
Wild Type ◽  
Spleen Cells ◽  
Th2 Response

ABSTRACT Measles virus infection leads to immune suppression. A potential mechanism is the reduction of interleukin 12 (IL-12) secretion during acute measles, resulting in a TH2 response. Studies in humans have reported conflicting results, detecting either a TH2 or a TH1 response. We have investigated the correlation between a TH2 response and immune suppression in specific-pathogen-free inbred cotton rats which were infected with measles vaccine and wild-type viruses. After infection of bone marrow-derived macrophages with wild-type virus, IL-12 secretion was reduced in contrast to the level for vaccine virus infection. In bronchoalveolar lavage cells, IL-12 secretion was suppressed after infection with both wild-type and vaccine virus on days 2, 4, and 6 and was detectable on days 8 and 10. After stimulation of mediastinal lymph node and spleen cells with UV-inactivated measles virus at various time points after infection, gamma interferon but no IL-4 was found. After stimulation with phorbol myristate acetate-ionomycin, high gamma interferon and low IL-4 levels were detected. To investigate whether the secretion of IL-4 contributes to immune suppression, a recombinant vaccine virus was created which secretes cotton rat IL-4. After infection with this recombinant virus, IL-4 secretion was enhanced. However, neither inhibition of concanavalin A-stimulated spleen cells nor keyhole limpet hemocyanin-specific proliferation of spleen cells was altered after infection with the recombinant virus in comparison to the levels with the parental virus. Our data indicate that measles virus infection leads to a decrease in IL-12 secretion and an increase in IL-4 secretion, but this does not seem to correlate with immune suppression.

scholarly journals Rapid Identification of Measles Virus Vaccine Genotype by Real-Time PCR

2016 ◽  
Vol 55 (3) ◽  
pp. 735-743 ◽  
Author(s):  
Felicia Roy ◽  
Lillian Mendoza ◽  
Joanne Hiebert ◽  
Rebecca J. McNall ◽  
Bettina Bankamp ◽  
...  
Keyword(s):  
Real Time ◽  
Real Time Pcr ◽  
Measles Virus ◽  
Rapid Identification ◽  
Rt Pcr ◽  
Outbreak Response ◽  
Reverse Transcription Pcr ◽  
Pcr Method ◽  
Measles Outbreaks ◽  
Genotype A

ABSTRACT During measles outbreaks, it is important to be able to rapidly distinguish between measles cases and vaccine reactions to avoid unnecessary outbreak response measures such as case isolation and contact investigations. We have developed a real-time reverse transcription-PCR (RT-PCR) method specific for genotype A measles virus (MeV) (MeVA RT-quantitative PCR [RT-qPCR]) that can identify measles vaccine strains rapidly, with high throughput, and without the need for sequencing to determine the genotype. We have evaluated the method independently in three measles reference laboratories using two platforms, the Roche LightCycler 480 system and the Applied Biosystems (ABI) 7500 real-time PCR system. In comparison to the standard real-time RT-PCR method, the MeVA RT-qPCR showed 99.5% specificity for genotype A and 94% sensitivity for both platforms. The new assay was able to detect RNA from five currently used vaccine strains, AIK-C, CAM-70, Edmonston-Zagreb, Moraten, and Shanghai-191. The MeVA RT-qPCR assay has been used successfully for measles surveillance in reference laboratories, and it could be readily deployed to national and subnational laboratories on a wide scale.

scholarly journals Rapid and real-time identification of fungi up to the species level with long amplicon Nanopore sequencing from clinical samples

2020 ◽  
Author(s):  
Sara D’Andreano ◽  
Anna Cuscó ◽  
Olga Francino
Keyword(s):  
Real Time ◽  
Clinical Signs ◽  
Amplicon Sequencing ◽  
Species Level ◽  
Clinical Samples ◽  
Microsporum Canis ◽  
Mock Community ◽  
Malassezia Pachydermatis ◽  
Long Read ◽  
Real Time Identification

ABSTRACTThe availability of long-read technologies, like Oxford Nanopore Technologies, provides the opportunity to sequence longer fragments of the fungal ribosomal operon, up to 6 Kb (18S-ITS1-5.8S-ITS2-28S), and to improve the taxonomy assignment of the communities up to the species level and in real-time. We assess the taxonomy skills of amplicons targeting a 3.5 Kb region (V3 18S-ITS1-5.8S-ITS2-28S D2) and a 6 Kb region (V1 18S-ITS1-5.8S-ITS2-28S D12) with the What’s in my pot (WIMP) classifier. We used the ZymoBIOMICS™ mock community and different microbiological fungal cultures as positive controls. Long amplicon sequencing correctly identified Saccharomyces cerevisiae and Cryptococcus neoformans from the mock community and Malassezia pachydermatis, Microsporum canis, and Aspergillus fumigatus from the microbiological cultures. Besides, we identified Rhodotorula graminis in a culture mislabeled as Candida spp.We applied the same approach to external otitis in dogs. Malassezia was the dominant fungal genus in dogs’ ear skin, whereas M. pachydermatis was the main species in the healthy sample. Conversely, we identified a higher representation of M. globosa and M. sympodialis in otitis affected samples. We demonstrate the suitability of long ribosomal amplicons to characterize the fungal community of complex samples, either healthy or with clinical signs of infection.

scholarly journals A Study in a Regional Hospital of a Mid-Sized Spanish City Indicates a Major Increase in Infection/Colonization by Carbapenem-Resistant Bacteria, Coinciding with the COVID-19 Pandemic

Antibiotics ◽  
2021 ◽  
Vol 10 (9) ◽  
pp. 1127
Author(s):  
Estefanía Cano-Martín ◽  
Inés Portillo-Calderón ◽  
Patricia Pérez-Palacios ◽  
José María Navarro-Marí ◽  
María Amelia Fernández-Sierra ◽  
...  
Keyword(s):  
Real Time ◽  
Bacterial Resistance ◽  
Control Measures ◽  
Clinical Samples ◽  
Resistant Bacteria ◽  
Major Advance ◽  
Prevention Measures ◽  
Cross Sectional ◽  
Effective Manner ◽  
Major Increase

Bacterial resistance to antibiotics has proven difficult to control over the past few decades. The large group of multidrug-resistant bacteria includes carbapenemase-producing bacteria (CPB), for which limited therapeutic options and infection control measures are available. Furthermore, carbapenemases associate with high-risk clones that are defined by the sequence type (ST) to which each bacterium belongs. The objectives of this cross-sectional and retrospective study were to describe the CPB population isolated in a third-level hospital in Southern Spain between 2015 and 2020 and to establish the relationship between the ST and the epidemiological situation defined by the hospital. CPB were microbiologically studied in all rectal and pharyngeal swabs and clinical samples received between January 2015 and December 2020, characterizing isolates using MicroScan and mass spectrometry. Carbapenemases were detected by PCR and Sanger sequencing, and STs were assigned by multilocus sequence typing (MLST). Isolates were genetically related by pulsed-field gel electrophoresis using Xbal, Spel, or Apal enzymes. The episodes in which each CPB was isolated were recorded and classified as involved or non-involved in an outbreak. There were 320 episodes with CPB during the study period: 18 with K. pneumoniae, 14 with Klebisella oxytoca, 9 with Citrobacter freundii, 11 with Escherichia coli, 46 with Enterobacter cloacae, 70 with Acinetobacter baumannii, and 52 with Pseudomonas aeruginosa. The carbapenemase groups detected were OXA, VIM, KPC, and NDM with various subgroups. Synchronous relationships were notified between episodes of K. pneumoniae and outbreaks for ST15, ST258, ST307, and ST45, but not for the other CPB. There was a major increase in infections with CPB over the years, most notably during 2020, coinciding with the COVID-19 pandemic. This study highlights the usefulness of gene sequencing techniques to control the spread of these microorganisms, especially in healthcare centers. These techniques offer faster results, and a reduction in their cost may make their real-time application more feasible. The combination of epidemiological data with real-time molecular sequencing techniques can provide a major advance in the transmission control of these CPB and in the management of infected patients. Real-time sequencing is essential to increase precision and thereby control outbreaks and target infection prevention measures in a more effective manner.

scholarly journals Harmonization of Bordetella pertussis Real-Time PCR Diagnostics in the United States in 2012

2014 ◽  
Vol 53 (1) ◽  
pp. 118-123 ◽  
Author(s):  
Margaret M. Williams ◽  
Thomas H. Taylor ◽  
David M. Warshauer ◽  
Monte D. Martin ◽  
Ann M. Valley ◽  
...  
Keyword(s):  
Public Health ◽  
Real Time ◽  
Bordetella Pertussis ◽  
Real Time Pcr ◽  
The United States ◽  
Extraction Methods ◽  
Clinical Samples ◽  
Acid Extraction ◽  
Rt Pcr ◽  
Content Type

Real-time PCR (rt-PCR) is an important diagnostic tool for the identification ofBordetella pertussis,Bordetella holmesii, andBordetella parapertussis. Most U.S. public health laboratories (USPHLs) target IS481, present in 218 to 238 copies in theB. pertussisgenome and 32 to 65 copies inB. holmesii. The CDC developed a multitarget PCR assay to differentiateB. pertussis,B. holmesii, andB. parapertussisand provided protocols and training to 19 USPHLs. The 2012 performance exercise (PE) assessed the capability of USPHLs to detect these threeBordetellaspecies in clinical samples. Laboratories were recruited by the Wisconsin State Proficiency Testing program through the Association of Public Health Laboratories, in partnership with the CDC. Spring and fall PE panels contained 12 samples each of viableBordetellaand non-Bordetellaspecies in saline. Fifty and 53 USPHLs participated in the spring and fall PEs, respectively, using a variety of nucleic acid extraction methods, PCR platforms, and assays. Ninety-six percent and 94% of laboratories targeted IS481in spring and fall, respectively, in either singleplex or multiplex assays. In spring and fall, respectively, 72% and 79% of USPHLs differentiatedB. pertussisandB. holmesiiand 68% and 72% identifiedB. parapertussis. IS481cycle threshold (CT) values forB. pertussissamples had coefficients of variation (CV) ranging from 10% to 28%. Of the USPHLs that differentiatedB. pertussisandB. holmesii, sensitivity was 96% and specificity was 95% for the combined panels. The 2012 PE demonstrated increased harmonization of rt-PCRBordetelladiagnostic protocols in USPHLs compared to that of the previous survey.

scholarly journals Attenuation of V- or C-Defective Measles Viruses: Infection Control by the Inflammatory and Interferon Responses of Rhesus Monkeys

2008 ◽  
Vol 82 (11) ◽  
pp. 5359-5367 ◽  
Author(s):  
Patricia Devaux ◽  
Gregory Hodge ◽  
Michael B. McChesney ◽  
Roberto Cattaneo
Keyword(s):  
Immune Responses ◽  
Measles Virus ◽  
Mononuclear Cells ◽  
Interferon Response ◽  
Wild Type Virus ◽  
Type Virus ◽  
Wild Type ◽  
Adaptive Immune Responses ◽  
C Protein ◽  
Adaptive Immune

ABSTRACT Patients recruited in virus-based cancer clinical trials and immunocompromised individuals in need of vaccination would profit from viral strains with defined attenuation mechanisms. We generated measles virus (MV) strains defective for the expression of either the V protein, a modulator of the innate immune response, or the C protein, which has multiple functions. The virulence of these strains was compared with that of the parental wild-type MV in a natural host, Macaca mulatta. Skin rash, viremia, and the strength of the innate and adaptive immune responses were characterized in groups of six animals. Replication of V- or C-protein-defective viruses was short-lived and reached lower levels in peripheral blood mononuclear cells and lymphatic organs compared to the wild-type virus; none of the mutants reverted to the wild type. The neutralizing antibody titers and MV-specific T-cell responses were equivalent in monkeys infected with the viral strains tested, documenting strong adaptive immune responses. In contrast, the inflammatory response was better controlled by wild-type MV, as revealed by inhibition of interleukin-6 and tumor necrosis factor alpha transcription. The interferon response was also better controlled by the wild-type virus than by the defective viruses. Since V- and C-defective MVs induce strong adaptive immune responses while spreading less efficiently, they may be developed as vaccines for immunocompromised individuals. Moreover, MV unable to interact with single innate immunity proteins may be developed for preferential replication in tumors with specific contexts of vulnerability.

scholarly journals Real-time loop-mediated isothermal amplification for rapid detection of Enterocytozoon hepatopenaei

PeerJ ◽  
2018 ◽  
Vol 6 ◽  
pp. e5993 ◽  
Author(s):  
Shao-Xin Cai ◽  
Fan-De Kong ◽  
Shu-Fei Xu ◽  
Cui-Luan Yao
Keyword(s):  
Real Time ◽  
Clinical Signs ◽  
Lamp Assay ◽  
Isothermal Amplification ◽  
Clinical Samples ◽  
Lamp Method ◽  
The Real ◽  
Loop Mediated Isothermal Amplification ◽  
Enterocytozoon Hepatopenaei ◽  
Time Loop

Background Enterocytozoon hepatopenaei (EHP) is a newly emerged microsporidian parasite that causes retarded shrimp growth in many countries. But there are no effective approaches to control this disease to date. The EHP could be an immune risk factor for increased dissemination of other diseases. Further, EHP infection involves the absence of obvious clinical signs and it is difficult to identify the pathogen through visual examination, increasing the risk of disease dissemination. It is urgent and necessary to develop a specific, rapid and sensitive EHP-infected shrimp diagnostic method to detect this parasite. In the present study, we developed and evaluated a rapid real-time loop-mediated isothermal amplification (real-time LAMP) for detection of EHP. Methods A rapid and efficient real-time LAMP method for the detection of EHP has been developed. Newly emerged EHP pathogens in China were collected and used as the sample, and three sets of specificity and sensitivity primers were designed. Three other aquatic pathogens were used as templates to test the specificity of the real-time LAMP assay. Also, we compared the real-time LAMP with the conventional LAMP by the serial dilutions of EHP DNA and their amplification curves. Application of real-time LAMP was carried out with clinical samples. Results Positive products were amplified only from EHP, but not from other tested species, EHP was detected from the clinical samples, suggesting a high specificity of this method. The final results of this assay were available within less than 45 min, and the initial amplification curve was observed at about 6 min. We found that the amplification with an exponential of sixfold dilutions of EHP DNA demonstrated a specific positive signal by the real-time LAMP, but not for the LAMP amplicons from the visual inspection. The real-time LAMP amplification curves demonstrated a higher slope than the conventional LAMP. Discussion In this study, pathogen virulence impacts have been increased in aquaculture and continuous observation was predominantly focused on EHP. The present study confirmed that the real-time LAMP assay is a promising and convenient method for the rapid identification of EHP in less time and cost. Its application greatly aids in the detection, surveillance, and prevention of EHP.

scholarly journals The pandemic of social media panic travels faster than the COVID-19 outbreak

2020 ◽  
Vol 27 (3) ◽  
Author(s):  
Anneliese Depoux ◽  
Sam Martin ◽  
Emilie Karafillakis ◽  
Raman Preet ◽  
Annelies Wilder-Smith ◽  
...  
Keyword(s):  
Public Health ◽  
Social Media ◽  
Real Time ◽  
Control Measures ◽  
Public Health Officials ◽  
The Creation ◽  
And Control

We need to rapidly detect and respond to public rumours, perceptions, attitudes and behaviours around COVID-19 and control measures. The creation of an interactive platform and dashboard to provide real-time alerts of rumours and concerns about coronavirus spreading globally would enable public health officials and relevant stakeholders to respond rapidly with a proactive and engaging narrative that can mitigate misinformation.

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