Real-time polymerase chain reaction (PCR) is a frequently used technique in molecular diagnostics. To date, practical guidelines for the complete process of optimization and validation of commercial and in-house developed molecular diagnostic methods are scare. Therefore, we propose a practical guiding principle for the optimization and validation of real-time PCR assays. Based on literature, existing guidelines, and personal experience, we created a checklist that can be used in different steps of the development and validation process of commercial and in-house developed real-time PCR assays. Furthermore, determination of target values and reproducibility of internal quality controls are included, which allows a statistical follow-up of the performance of the assay. Recently, we used this checklist for the development of various qualitative and quantitative assays for microbiological and hematological applications, for which accreditation according to ISO 15189:2007 was obtained. In our experience, the use of the proposed guidelines leads to a more efficient and standardized optimization and validation. Ultimately, this results in reliable and robust molecular diagnostics. The proposed checklist is independent of environment, equipment, and specific applications and can be used in other laboratories. A worldwide consensus on this kind of checklist should be aimed at.

Viral infections are common complications of pregnancy, with a wide range of obstetric and neonatal sequelae. Currently, there are limited data on whether SARS-CoV-2 is vertically transmitted in pregnant women tested positive for the virus. Here we describe a case of a known SARS-CoV-2-positive woman giving preterm birth to two fetuses with SARS-CoV-2 positive testing in placental tissue and amniotic fluid. The placental histological examinations showed chronic intervillositis and extensive intervillous fibrin depositions with ischemic necrosis of the surrounding villi.

Rothia dentocariosa is a rare cause of endocarditis. It occurs most frequently in patients with prior heart conditions. Although the clinical course is typically subacute, it has a high rate of complications. In particular, the reported incidence of mycotic aneurysms is as high as 25%. Penicillin is the treatment of choice, but additional complications may necessitate prompt surgical intervention. As far as we know, this paper reports the first case of repeated subarachnoid hemorrhages due to R. dentocariosa endocarditis.

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Swab transport systems should preserve the viability and stability of micro-organisms in clinical specimens throughout transport and storage. eSwab, a nylon-tipped swab in liquid medium, designed for better specimen collection and less micro-organism entrapment, was evaluated for the maintenance of viability and quantitative survival of Escherichia coli, Streptococcus agalactiae and Candida albicans. The quantitative elution method was used to evaluate eSwab in vitro. In vitro, the recovery of the three micro-organisms was higher in eSwab (97-100%) as compared to the Copan Venturi Transystem (CVT) (86-96%) at room temperature (RT) for time point 0 h and remained similar after 6 h. E. coli and C. albicans proliferated in both transport systems when preserved beyond 6 h. At 4 degrees C, the recovery of eSwab was higher (>94%) compared to CVT (77-94%) for the micro-organisms tested. eSwab did not only meet the Clinical Laboratory and Standards Institute (CLSI) criteria for microbiological transport devices, but as its recovery rate in vitro was higher than that of CVT, it might also enhance the sensitivity of bacterial culture in the future.

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The results of our study seem to indicate a better sensitivity for the Respifinder. Analysis of patient samples is necessary to evaluate the clinical performance.