Optical #DNA #Mapping Combined with #Cas9-Targeted #Resistance #Gene #Identification for Rapid #Tracking of Resistance #Plasmids in a #NICU #Outbreak (mBio, abstract)

[Source: mBio, full page: (LINK). Abstract, edited.]

Optical DNA Mapping Combined with Cas9-Targeted Resistance Gene Identification for Rapid Tracking of Resistance Plasmids in a Neonatal Intensive Care Unit Outbreak

Santosh K. Bikkarolla, Viveka Nordberg, Fredrika Rajer, Vilhelm Müller, Muhammad Humaun Kabir, Sriram KK, Albertas Dvirnas, Tobias Ambjörnsson, Christian G. Giske, Lars Navér,Linus Sandegren, Fredrik Westerlund

Spyros Pournaras, Invited Editor, Karen Bush, Editor

DOI: 10.1128/mBio.00347-19



The global spread of antibiotic resistance among Enterobacteriaceae is largely due to multidrug resistance plasmids that can transfer between different bacterial strains and species. Horizontal gene transfer of resistance plasmids can complicate hospital outbreaks and cause problems in epidemiological tracing, since tracing is usually based on bacterial clonality. We have developed a method, based on optical DNA mapping combined with Cas9-assisted identification of resistance genes, which is used here to characterize plasmids during an extended-spectrum β-lactamase (ESBL)-producing Enterobacteriaceae outbreak at a Swedish neonatal intensive care unit. The outbreak included 17 neonates initially colonized with ESBL-producing Klebsiella pneumoniae (ESBL-KP), some of which were found to carry additional ESBL-producing Escherichia coli (ESBL-EC) in follow-up samples. We demonstrate that all ESBL-KP isolates contained two plasmids with the blaCTX-M-15 gene located on the smaller one (~80 kbp). The same ESBL-KP clone was present in follow-up samples for up to 2 years in some patients, and the plasmid carrying the blaCTX-M-15 gene was stable throughout this time period. However, extensive genetic rearrangements within the second plasmid were observed in the optical DNA maps for several of the ESBL-KP isolates. Optical mapping also demonstrated that even though other bacterial clones and species carrying blaCTX-M group 1 genes were found in some neonates, no transfer of resistance plasmids had occurred. The data instead pointed toward unrelated acquisition of ESBL-producing Enterobacteriaceae (EPE). In addition to revealing important information about the specific outbreak, the method presented is a promising tool for surveillance and infection control in clinical settings.


This study presents how a novel method, based on visualizing single plasmids using sequence-specific fluorescent labeling, could be used to analyze the genetic dynamics of an outbreak of resistant bacteria in a neonatal intensive care unit at a Swedish hospital. Plasmids are a central reason for the rapid global spread of bacterial resistance to antibiotics. In a single experimental procedure, this method replaces many traditional plasmid analysis techniques that together provide limited details and are slow to perform. The method is much faster than long-read whole-genome sequencing and offers direct genetic comparison of patient samples. We could conclude that no transfer of resistance plasmids had occurred between different bacteria during the outbreak and that secondary cases of ESBL-producing Enterobacteriaceae carriage were instead likely due to influx of new strains. We believe that the method offers potential in improving surveillance and infection control of resistant bacteria in hospitals.

Keywords: Antibiotics; Drugs Resistance; Enterobacteriaceae; Beta-lactams; Nosocomial Outbreaks; Diagnostic tests.



Ensuring On-site #Ebola #Patient #Monitoring and Follow-up: #Development of a #Laboratory Structure Embedded in an #ETC (Disaster Med Public Health Prep., abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Disaster Med Public Health Prep. 2019 Jun 24:1-7. doi: 10.1017/dmp.2019.39. [Epub ahead of print]

Ensuring On-site Ebola Patient Monitoring and Follow-up: Development of a Laboratory Structure Embedded in an Ebola Treatment Center.

Williams A1, Amand M2, Van den Bergh R1, De Clerck H3, Antierens A3, Chaillet P3.

Author information: 1 Médecins Sans Frontières,Operational Centre Brussels, Operational Research Unit LuxOR,Luxembourg, Luxembourg. 2 Médecins Sans Frontières,Operational Centre Brussels,Guinea Mission, Conakry,Guinea. 3 Médecins Sans Frontières,Operational Centre Brussels,Medical Department, Brussels,Belgium.



The capacity to rapidly distinguish Ebola virus disease from other infectious diseases and to monitor biochemistry and viremia levels is crucial to the clinical management of suspected Ebola virus disease cases. This article describes the design and practical considerations of a laboratory straddling the high- and low-risk zones of an Ebola treatment center to produce timely diagnostic and clinical results for informed case management of Ebola virus disease in real-life conditions. This innovation may be of relevance for actors requiring flexible laboratory implementation in contexts of high-communicability, high-lethality disease outbreaks.

KEYWORDS: Ebola virus; disease outbreaks; laboratories; outbreak response; patient monitoring

PMID: 31232266 DOI: 10.1017/dmp.2019.39

Keywords: Ebola; Diagnostic tests.


Development of a Specific #CHIKV-E2 #Monoclonal #Antibody for #Chikungunya #Diagnosis (Virol Sin., abstract)

[Source: Virologica Sinica, full page: (LINK). Abstract, edited.]

Development of a Specific CHIKV-E2 Monoclonal Antibody for Chikungunya Diagnosis

Authors: Jaemoo Kim, Jihyun Yang, Young Bong Kim, Hee-Jung Lee, Sehyun Kim, Haryoung Poo

Research Article / First Online: 18 June 2019



Chikungunya fever is a vector-borne viral disease transmitted to humans by chikungunya virus (CHIKV)-infected mosquitoes. There have been many outbreaks of CHIKV infection worldwide, and the virus poses ongoing risks to global health. To prevent and control CHIKV infection, it is important to improve the current CHIKV diagnostic approaches to allow for the detection of low CHIKV concentrations and to correctly distinguish CHIKV infections from those due to other mosquito-transmitted viruses, including dengue virus (DENV), Japanese encephalitis virus (JEV), and Zika virus (ZIKV). Here, we produced monoclonal antibodies (mAbs) against the CHIKV envelope 2 protein (CHIKV-E2) and compared their sensitivity and specificity with commercially available mAbs using enzyme-linked immunosorbent assays (ELISA). Two anti-CHIKV-E2 mAbs, 19-1 and 21-1, showed higher binding affinities to CHIKV-E2 protein than the commercial mAbs did. In particular, the 19-1 mAb had the strongest binding affinity to inactivated CHIKV. Moreover, the 19-1 mAb had very little cross-reactivity with other mosquito-borne viruses, such as ZIKV, JEV, and DENV. These results suggest that the newly produced anti-CHIKV-E2 mAb, 19-1, could be used for CHIKV diagnostic approaches.

Keywords: Chikungunya virus – (CHIKV) Envelope 2 – Monoclonal antibody – Diagnosis – Sensitivity – Specificity




This work was supported by Grants from the R&D Convergence Program of National Research Council of Science & Technology (No. CAP-16-02-KIST) and the National Research Foundation of Korea (No. NRF-2016M3A9B6918584).

Author Contributions

HP designed the experiments; JK performed the experiments; SK, HL, and YK contributed to analyze cross-reactivities of anti-CHIKV-E2 antibodies to arboviruses; JK and JY analyzed the experiments and drafted the manuscript; HP supervised the experiments, analyzed results, and wrote the manuscript. All authors approved the final manuscript.


Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no competing interests.

Animal and Human Rights Statement

Animal experiments were approved by the Institutional Animal Care and Use Committee (IACUC) of the Korea Research Institute of Bioscience and Biotechnology (KRIBB) and performed according to the Guidelines for Animal Experiments of the KRIBB.

Keywords: Chikungunya fever; Monoclonal antibodies; Diagnostic tests.


#Detection of #oseltamivir #resistant #zoonotic and #animal #influenza A viruses using the rapid influenza antiviral resistance test (Influenza Other Respir Viruses, abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Influenza Other Respir Viruses. 2019 Jun 11. doi: 10.1111/irv.12661. [Epub ahead of print]

Detection of oseltamivir-resistant zoonotic and animal influenza A viruses using the rapid influenza antiviral resistance test.

Hodges EN1,2, Mishin VP1, De la Cruz J1,3, Guo Z1, Nguyen HT1,3, Fallows E4, Stevens J1, Wentworth DE1, Davis CT1, Gubareva LV1.

Author information: 1 Influenza Division, Centers for Disease Control and Prevention (CDC), Atlanta, Georgia. 2 CNI Advantage, Atlanta, Georgia. 3 Battelle Memorial Institute, Atlanta, Georgia. 4 Becton, Dickinson and Company, Research Triangle Park, North Carolina.



Mutations in the influenza virus neuraminidase (NA) that cause reduced susceptibility to the NA inhibitor (NAI) oseltamivir may occur naturally or following antiviral treatment. Currently, detection uses either a traditional NA inhibition assay or gene sequencing to identify known markers associated with reduced inhibition by oseltamivir. Both methods are laborious and require trained personnel. The influenza antiviral resistance test (iART), a prototype system developed by Becton, Dickinson and Company for research use only, offers a rapid and simple method to identify such viruses. This study investigated application of iART to influenza A viruses isolated from non-human hosts with a variety of NA subtypes (N1-N9).

© 2019 The Authors. Influenza and Other Respiratory Viruses Published by John Wiley & Sons Ltd.

KEYWORDS: antiviral drugs; avian; influenza A virus; resistance; zoonotic

PMID: 31187572 DOI: 10.1111/irv.12661

Keywords: Influenza A; Avian Influenza; Antivirals; Drugs Resistance; Diagnostic tests.


What is the role of rapid #molecular #testing for seniors and other at-risk #adults with #RSV #infections? (J Clin Virol., abstract)

[Source: Journal of Clinical Virology, full page: (LINK). Abstract, edited.]

Journal of Clinical Virology / Available online 25 May 2019 / In Press, Accepted Manuscript

What is the role of rapid molecular testing for seniors and other at-risk adults with respiratory syncytial virus infections?

Steven J. Drews a, Angela R. Branche b, Ann R. Falsey c, Nelson Lee d

{a} 2B1.03 WMC University of Alberta Hospital, 8440 112th St NW, Edmonton, Alberta, T6J 1L9, Canada; {b} University of Rochester, 601 Elmwood Avenue, Box 689, Rochester, NY 14642, USA; {c} 1425 Portland Avenue, Rochester General Hospital, Rochester, NY 14621, USA; {d} Division of Infectious Diseases, Department of Medicine, University of Alberta, Clinical Sciences Building (CSB), 1-124, 11350-83 Avenue NW, Edmonton, Alberta, T6G 2G3, Canada

Received 13 February 2019, Revised 21 May 2019, Accepted 22 May 2019, Available online 25 May 2019. DOI: https://doi.org/10.1016/j.jcv.2019.05.010



  • RSV has substantial burden of morbidity and mortality in older and at-risk adults
  • Currently, little incentive exists for clinicians to order routine RSV tests in adults
  • In future, it will be essential to have established standardized testing protocols
  • Outcome data to underpin recommendations and give confidence to order RSV molecular tests are needed



Lower respiratory tract infections are a leading cause of hospitalization and viruses are important causal pathogens, especially in the elderly, immunocompromised patients and those with respiratory or cardiovascular comorbidities. Respiratory syncytial virus (RSV) is recognized as comprising a substantial burden of morbidity and mortality in older and at-risk adults, and the emergence of rapid point-of-care molecular testing has made it possible to confirm an RSV diagnosis accurately, in a clinically actionable timeframe. RSV patients have significantly higher healthcare resource use (including hospital stays and emergency room/urgent care visits) than non-RSV matched controls, especially if aged ≥65 years, a longer length of hospitalization than those with influenza, and associated costs nearly three times higher. We found no direct clinical outcome data specific to rapid molecular testing for RSV in adults and very little in children. There is very limited evidence that prompt diagnosis may reduce hospital length of stay but this and other outcome parameters need confirmation in larger, prospective clinical trials. Regarding reducing inappropriate antibiotic prescribing, the picture is mixed and testing alone is unlikely to change entrenched habits. There is little incentive for clinicians to order routine RSV tests in adults given the absence of a specific antiviral therapy. However, with numerous vaccine and antiviral candidates in clinical development, we believe it is good practice to plan and start establishing standardized testing protocols – perhaps as part of outcome studies. For especially vulnerable patients, e.g., immunocompromised and transplant patients, prompt accurate RSV diagnosis may prevent disease spread and save lives.

Keywords: respiratory syncytial virus – point-of-care testing – polymerase chain reaction – adult – immunocompromised patient – health – resource

© 2019 Published by Elsevier B.V.

Keywords: RSV; Diagnostic tests.


#Oral #fluid: non-invasive alternative for #parvovirus B19 #diagnosis? (J Clin Virol., abstract)

[Source: Journal of Clinical Virology, full page: (LINK). Abstract, edited.]

Journal of Clinical Virology / Available online 19 May 2019 / In Press, Accepted Manuscript

Oral fluid: non-invasive alternative for parvovirus B19 diagnosis?

Rogier Bodewes a, Jeroen Kerkhof a, Jeroen Cremer a, Daphne B. Gijselaar a, Bettie C.G. Voordouw a,b, Irene K. Veldhuijzen c, Maarten Schipper d, Robvan Binnendijk e

{a} Center for Infectious Disease Research, Diagnostics andlaboratorySurveillance (IDS), National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands; {b} Department of Viroscience, ErasmusMC, Rotterdam, The Netherlands; {c} Centre for Infectious Diseases, Epidemiology and Surveillance, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands; {d}
Department of Statistics, Computer Science and Modelling – SIM, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands; {e} Centre for Immunology of Infectious Diseases and Vaccines, National Institute for Public Health and the Environment (RIVM), Bilthoven, The Netherlands

Received 3 September 2018, Revised 18 April 2019, Accepted 17 May 2019, Available online 19 May 2019. DOI: https://doi.org/10.1016/j.jcv.2019.05.008

Under a Creative Commons license



  • Parvovirus B19 (B19 V) causes exanthema in children, similar to rubella and measles virus
  • A B19V-qPCR on oral fluid was evaluated as a non-invasive alternative for serum IgM
  • On basis of 116 children with exanthema, sensitivity of the qPCR on oral fluid reached 70%
  • B19V detection in oral fluid is a suitable public health alternative in the diagnosis of fifth disease




Infections with parvovirus B19 (B19 V) have been associated with a wide range of disease manifestations of which erythema infectiosum (fifth disease) in children is most common. Clinical signs following infection of children with B19 V can be similar to measles and rubella. Laboratory detection of B19 V infections is based on detection of B19V-specific IgM antibodies by enzyme immunoassay (IgM-EIA) and/or B19 V DNA by quantitative PCR (qPCR) on blood samples. The need for invasive sampling can be a barrier for public health diagnostics.


To evaluate the use of a dual target B19V-qPCR directed against the NS1 and VP2 of B19 V on oral fluid samples as a non-invasive alternative for laboratory diagnosis of B19 V infections in children below 12 years of age with exanthema.

Study design

Oral fluid and serum samples were collected from 116 children with exanthema. All serum samples were tested by IgM-EIA/IgG-EIA, while all oral fluid and 56 serum samples were tested by B19V-qPCR.


B19V-specific IgM antibodies were detected in 25 of 116 children in the study. B19V DNA was detected in oral fluid in 17 of the 25 children who were IgM positive, as well as two children who were IgM-equivocal or negative. The child with the equivocal IgM had a high quantity of B19 V DNA in oral fluid (7 log IU/ml), compatible with an acute B19V infection. The IgM-negative child was IgG-positive and 4 log IU/ml B19 V DNA was detected in the oral fluid sample, suggesting an acute infection and a falsely negative IgM. Sample size calculations indicated that oral fluid samples for qPCR should be collected from 2-3 children during outbreaks of exanthema to achieve similar sensitivity as IgM-EIA for one child (≥0.9) to confirm or exclude B19 V.


Results indicate that oral fluid samples are a suitable public health alternative for detection of B19 V infections, potentially lowering the barriers for sampling.

Keywords: parvovirus B19 – PCR – serology – exanthema – children – fifth disease

Keywords: Parvovirus B19; Fifth Disease; Diagnostic tests; Serology.


Generation and Selection of a #Panel of Pan- #Filovirus Single-Chain #Antibodies using Cell-Free Ribosome Display (Am J Trop Med Hyg., abstract)

[Source: US National Library of Medicine, full page: (LINK). Abstract, edited.]

Am J Trop Med Hyg. 2019 May 6. doi: 10.4269/ajtmh.18-0658. [Epub ahead of print]

Generation and Selection of a Panel of Pan-Filovirus Single-Chain Antibodies using Cell-Free Ribosome Display.

Kunamneni A1,2, Clarke EC2, Ye C2, Bradfute SB2, Durvasula R2,1.

Author information: 1 Department of Medicine, Loyola University Medical Center, Chicago, Illinois. 2 Department of Internal Medicine, Center for Global Health, University of New Mexico, Albuquerque, New Mexico.



Filoviruses, which include ebolaviruses and marburgvirus, can cause outbreaks of highly lethal hemorrhagic fever. This disease causes significant morbidity and mortality in humans and non-human primates, with human fatality rates reaching 90% during some outbreaks. Currently, there are a lack of licensed vaccines or antivirals for these viruses. Since early symptoms of filovirus infection mimic more common diseases, there is a strong unmet public health and biodefense need for broad-spectrum filovirus rapid diagnostics. We have generated a panel of mouse single-chain Fv-antibodies (scFvs) to filovirus glycoproteins (GPs) using cell-free ribosome display and determined their cross-reactivity profiles to all known filovirus species. Two scFvs (4-2 and 22-1) were able to detect all known Ebolavirus and Marburgvirusspecies. This is the first report on ribosome display scFvs that can detect a broad set of filovirus GPs, which demonstrates the potential for use in diagnostics.

PMID: 31074409 DOI: 10.4269/ajtmh.18-0658

Keywords: Filovirus; Ebola; Marburg; Diagnostic tests.