SUMMARY The source of the severe acute respiratory syndrome (SARS) epidemic was traced to wildlife market civets and ultimately to bats. Subsequent hunting for novel coronaviruses (CoVs) led to the discovery of two additional human and over 40 animal CoVs, including the prototype lineage C betacoronaviruses, Tylonycteris bat CoV HKU4 and Pipistrellus bat CoV HKU5; these are phylogenetically closely related to the Middle East respiratory syndrome (MERS) CoV, which has affected more than 1,000 patients with over 35% fatality since its emergence in 2012. All primary cases of MERS are epidemiologically linked to the Middle East. Some of these patients had contacted camels which shed virus and/or had positive serology. Most secondary cases are related to health care-associated clusters. The disease is especially severe in elderly men with comorbidities. Clinical severity may be related to MERS-CoV9s ability to infect a broad range of cells with DPP4 expression, evade the host innate immune response, and induce cytokine dysregulation. Reverse transcription-PCR on respiratory and/or extrapulmonary specimens rapidly establishes diagnosis. Supportive treatment with extracorporeal membrane oxygenation and dialysis is often required in patients with organ failure. Antivirals with potent in vitro activities include neutralizing monoclonal antibodies, antiviral peptides, interferons, mycophenolic acid, and lopinavir. They should be evaluated in suitable animal models before clinical trials. Developing an effective camel MERS-CoV vaccine and implementing appropriate infection control measures may control the continuing epidemic.

/pdf/middle-east-respiratory-syndrome-coronavirus-another-3rpyownr3e.pdf

Middle East Respiratory Syndrome Coronavirus: Another Zoonotic Betacoronavirus Causing SARS-Like Disease

https://www.thelancet.com/pdfs/journals/lancet/PIIS0140-6736(11)60281-X.pdf

Chikungunya: a re-emerging virus

Summary: Molecular technology has changed the way that clinical laboratories diagnose and manage many infectious diseases. Excellent sensitivity, specificity, and speed have made molecular assays an attractive alternative to culture or enzyme immunoassay methods. Many molecular assays are commercially available and FDA approved. Others, especially those that test for less common analytes, are often laboratory developed. Laboratories also often modify FDA-approved assays to include different extraction systems or additional specimen types. The Clinical Laboratory Improvement Amendments (CLIA) federal regulatory standards require clinical laboratories to establish and document their own performance specifications for laboratory-developed tests to ensure accurate and precise results prior to implementation of the test. The performance characteristics that must be established include accuracy, precision, reportable range, reference interval, analytical sensitivity, and analytical specificity. Clinical laboratories are challenged to understand the requirements and determine the types of experiments and analyses necessary to meet the requirements. A variety of protocols and guidelines are available in various texts and documents. Many of the guidelines are general and more appropriate for assays in chemistry sections of the laboratory but are applied in principle to molecular assays. This review presents information that laboratories may consider in their efforts to meet regulatory requirements.

Validation of laboratory-developed molecular assays for infectious diseases

The first known cases of Middle East respiratory syndrome (MERS), associated with infection by a novel coronavirus (CoV), occurred in 2012 in Jordan but were reported retrospectively. The case first to be publicly reported was from Jeddah, in the Kingdom of Saudi Arabia (KSA). Since then, MERS-CoV sequences have been found in a bat and in many dromedary camels (DC). MERS-CoV is enzootic in DC across the Arabian Peninsula and in parts of Africa, causing mild upper respiratory tract illness in its camel reservoir and sporadic, but relatively rare human infections. Precisely how virus transmits to humans remains unknown but close and lengthy exposure appears to be a requirement. The KSA is the focal point of MERS, with the majority of human cases. In humans, MERS is mostly known as a lower respiratory tract (LRT) disease involving fever, cough, breathing difficulties and pneumonia that may progress to acute respiratory distress syndrome, multiorgan failure and death in 20 % to 40 % of those infected. However, MERS-CoV has also been detected in mild and influenza-like illnesses and in those with no signs or symptoms. Older males most obviously suffer severe disease and MERS patients often have comorbidities. Compared to severe acute respiratory syndrome (SARS), another sometimes- fatal zoonotic coronavirus disease that has since disappeared, MERS progresses more rapidly to respiratory failure and acute kidney injury (it also has an affinity for growth in kidney cells under laboratory conditions), is more frequently reported in patients with underlying disease and is more often fatal. Most human cases of MERS have been linked to lapses in infection prevention and control (IPC) in healthcare settings, with approximately 20 % of all virus detections reported among healthcare workers (HCWs) and higher exposures in those with occupations that bring them into close contact with camels. Sero-surveys have found widespread evidence of past infection in adult camels and limited past exposure among humans. Sensitive, validated reverse transcriptase real-time polymerase chain reaction (RT-rtPCR)-based diagnostics have been available almost from the start of the emergence of MERS. While the basic virology of MERS-CoV has advanced over the past three years, understanding of the interplay between camel, environment, and human remains limited.

/pdf/mers-coronavirus-diagnostics-epidemiology-and-transmission-138610lrnv.pdf

MERS coronavirus: diagnostics, epidemiology and transmission

Chikungunya virus (CHIKV) is an alphavirus transmitted by mosquitoes, mostly Aedes aegypti and Aedes albopictus. After half a century of focal outbreaks of acute febrile polyarthralgia in Africa and Asia, the disease unexpectedly spread in the past decade with large outbreaks in Africa and around the Indian Ocean and rare autochthonous transmission in temperate areas. This emergence brought new insights on its pathogenesis, notably the role of the A226V mutation that improved CHIKV fitness in Ae. albopictus and the possible CHIKV persistence in deep tissue sanctuaries for months after infection. Massive outbreaks also revealed new aspects of the acute stage: the high number of symptomatic cases, unexpected complications, mother-to-child transmission, and low lethality in debilitated patients. The follow-up of patients in epidemic areas has identified frequent, long-lasting, rheumatic disorders, including rare inflammatory joint destruction, and common chronic mood changes associated with quality-of-life impairment. Thus, the globalization of CHIKV exposes countries with Aedes mosquitoes both to brutal outbreaks of acute incapacitating episodes and endemic long-lasting disorders.

/pdf/chikungunya-virus-infection-5d8k4vu9f5.pdf

Chikungunya Virus Infection

Performance and clinical validation of the RealStar® MERS-CoV Kit for detection of Middle East respiratory syndrome coronavirus RNA

Molecular detection of Bordetella pertussis DNA is a sensitive and specific method for the rapid diagnosis of pertussis. In this study, a new molecular assay for the detection and differentiation of Bordetella spp. based on automated DNA extraction and real-time PCR was evaluated. The analytical sensitivity of the new assay was determined by Probit analysis of serial dilutions of both cloned PCR products IS481 and IS1001 and cell suspensions of B. pertussis, B. parapertussis, and B. bronchiseptica. The specificity was analyzed by testing a number of pathogens producing respiratory infections. Moreover, a total of 92 clinical samples were investigated. The results were compared to those obtained by an in-house assay based on manual DNA extraction, followed by real-time PCR and detection of IS481. The analytical sensitivity of the new assay for the detection of IS481 and IS1001 was determined to be 2.2 and 1.2 genome equivalents/μl, respectively. The analytical sensitivity for the detection of B. pertussis, B. parapertussis, and B. bronchiseptica was determined to be 1.6, 1.0, and 2.7 genome equivalents/μl, respectively. When clinical specimens were tested with the new assay, 46 of 92 were found to be positive for Bordetella DNA. With the in-house assay, 45 samples tested positive. The new molecular assay proved to be suitable for the rapid diagnosis of pertussis in the routine diagnostic laboratory.

Detection and Differentiation of Bordetella spp. by Real-Time PCR

A novel commercial Chikungunya virus real-time reverse transcription-PCR (RT-PCR) kit was evaluated on a comprehensive panel of original patient samples. The assay was 100% sensitive and specific in comparison to a published real-time RT-PCR. Viral loads from both assays were highly correlated. The kit proved to be suitable for routine use in patient care.

Performance of the RealStar Chikungunya Virus Real-Time Reverse Transcription-PCR Kit

Whole blood has been found to be a reliable matrix for the detection and quantitation of cytomegalovirus (CMV) DNA. In this study, the performance of the artus CMV LightCycler (LC) PCR kit in conjunction with automated sample preparation on a BioRobot EZ1 workstation was evaluated. The accuracy, linearity, analytical sensitivity, and inter- and intra-assay variations were determined. A total of 102 clinical EDTA whole-blood samples were investigated, and results were compared with those obtained with the in vitro diagnostics (IVD)/Conformite Europeene (CE)-labeled CMV HHV6,7,8 R-gene quantification kit. When the accuracy of the new kit was tested, seven of eight results were found to be within +/-0.5 log(10) unit of the expected panel results. Determination of linearity resulted in a quasilinear curve over more than 5 log units. The lower limit of detection of the assay was determined to be 139 copies/ml in EDTA whole blood. The interassay variation ranged from 15 to 58%, and the intra-assay variation ranged from 7 to 35%. When clinical samples were tested and the results were compared with those of the routinely used IVD/CE-labeled assay, 53 samples tested positive and 13 samples tested negative by both of the assays. One sample was found to be positive with the artus CMV LC PCR kit only, and 35 samples tested positive with the routinely used assay only. The majority of discrepant results were found with low-titer samples. In conclusion, use of the artus CMV LC PCR kit in conjunction with automated sample preparation on the BioRobot EZ1 workstation may be suitable for the detection and quantitation of CMV DNA in EDTA whole blood in the routine low-throughput laboratory; however, low-positive results may be missed by this assay.

https://jcm.asm.org/content/jcm/46/4/1241.full.pdf

Detection of Cytomegalovirus (CMV) DNA in EDTA Whole-Blood Samples: Evaluation of the Quantitative artus CMV LightCycler PCR Kit in Conjunction with Automated Sample Preparation

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Performance and clinical validation of the RealStar® MERS-CoV Kit for detection of Middle East respiratory syndrome coronavirus RNA

Detection and Differentiation of Bordetella spp. by Real-Time PCR

Performance of the RealStar Chikungunya Virus Real-Time Reverse Transcription-PCR Kit

Detection of Cytomegalovirus (CMV) DNA in EDTA Whole-Blood Samples: Evaluation of the Quantitative artus CMV LightCycler PCR Kit in Conjunction with Automated Sample Preparation