Optimization of the STARlet workflow for semi-automatic SARS-CoV-2 screening of swabs and deep respiratory materials using the RealAccurate Quadruplex SARS-CoV-2 PCR kit and Allplex SARS-CoV-2 PCR kit.

The severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) pandemic triggered the implementation of large-scale screenings in the health care and in the general population. Consequently, medical laboratories have to apply lean laboratory management to design workflows that are able to process large batches within short turnaround times while maintaining flexibility to use different SARS-CoV-2 reverse transcription polymerase chain reactions (RT-PCRs) and to be able to process a variety of clinical samples. We validated two SARS-CoV-2 PCR assays on the STARlet workflow: Allplex SARS-CoV-2 PCR kit and RealAccurate Quadruplex SARS-CoV-2 PCR kit. Furthermore, we optimized and validated the STARlet workflow for semi-automatic screening for SARS-CoV-2 in upper respiratory swabs and deep respiratory materials (sputa, bronchoalveolar lavage, and aspirate). Strikingly, guanidine-containing lysis buffers allow for easy processing and can enhance sensitivity of SARS-COV-2 screening since sampling in these buffers may preserve viral transcripts as evident by the higher copy numbers of the SARS-CoV-2 N gene. Moreover, using the principles of lean laboratory management, several bottlenecks that are typical for medical laboratories were addressed. We show that lean laboratory management resulted in significant reduction of the turnaround times of the SARS-CoV-2 PCR in our laboratory. This report thus describes a useful framework for laboratories to implement similar semi-automated workflows.IMPORTANCEThe SARS-CoV-2 pandemic triggered the implementation of large-scale screenings in the health care and in the general population. Consequently, medical laboratories had to adapt and evolve workflows that are able to process large batches within short turnaround times while maintaining flexibility to use different assays and to be able to process a variety of clinical samples. We describe how the need for increased outputs and greater flexibility was addressed with respect to clinical samples and assays (Allplex SARS-CoV-2 PCR and RealAccurate Quadruplex SARS-CoV-2 PCR). Strikingly, we found that upper respiratory swabs collected in guanidine-containing lysis buffers both improved the ease of processing as well as enhanced the sensitivity of the SARS-CoV-2 screening. This report thus describes a useful framework for laboratories to implement and optimize similar semi-automated workflows.

Short Communication: Prolonged COVID-19 Infection in a Patient with Newly Diagnosed HIV/AIDS.

A 38-year-old male patient presented to the emergency department with fever and dyspnea. Hospitalization was warranted and soon coronavirus disease 2019 (COVID-19) was diagnosed based on a positive SARS-CoV-2-PCR. Over the following weeks his condition gradually worsened, leading to admission at the intensive care unit. Because of unexplained weight loss before admission, a HIV screening was performed. HIV was confirmed and additional tests showed an undetectable CD4+ T cell count, alongside a number of co-infections. Convalescent plasma therapy, which has been shown to be effective in severe humoral immunodeficiency was tried, but was not effective. One week after the HIV diagnosis, antiretroviral therapy was started and finally, 3 months after the initial positive test and after partial recovery of cellular immunity, the COVID-19 virus was cleared. In the end, the patient made a full recovery. Our case demonstrates a prolonged COVID-19 infection in a patient with undiagnosed HIV with severely impaired cellular immunity.

C-reactive protein to rule out complicated pneumococcal disease manifestations: a retrospective cohort study in adults with pneumococcal bacteraemia.

OBJECTIVES: To explore the negative predictive value (NPV) of C-reactive protein (CRP) at admission to exclude complicated disease manifestations of pneumococcal disease. METHODS: A Dutch multicentre retrospective cohort study was conducted between 01-01-2012 and 30-06-2020. Adults with positive blood cultures for Streptococcus pneumoniae, whose CRP was measured at admission and whose infection focus was known, were included. Electronic medical and microbiological records were reviewed. RESULTS: Of the 832 bacteraemic patients enrolled, 30% had complicated manifestations of pneumococcal disease; most frequent were pleural effusion (8.9%), pleural empyema (5.4%) and meningitis (7.5%). Compared to solitary pneumonia, patients with pleural effusion and empyema presented with higher CRP levels. Although low CRP levels did not exclude complicated disease in general, a CRP level < 114 mg/L at admission could reliably exclude empyema among adult pneumonia patients with an NPV of 93% and a specificity of 26%. However, in cases where pleural fluid was present, CRP levels were mostly > 114 mg/L, such that suspicion of empyema could only be ruled out in a minority of cases (10%). CONCLUSIONS: Complicated manifestations are prevalent in adult pneumococcal bacteraemia. Low blood CRP levels can reliably exclude the development of pulmonary empyema. Practical value may be largest in settings without thoracic imaging at hand.

The drop in reported invasive pneumococcal disease among adults during the first COVID-19 wave in the Netherlands explained.

OBJECTIVES: Streptococcus pneumoniae is the leading bacterial pathogen causing respiratory infections. Since the COVID-19 pandemic emerged, less invasive pneumococcal disease (IPD) was identified by surveillance systems worldwide. Measures to prevent transmission of SARS-CoV-2 also reduce transmission of pneumococci, but this would gradually lead to lower disease rates. DESIGN: Here, we explore additional factors contributing to the instant drop in pneumococcal disease cases captured in surveillance. RESULTS: Our observations on referral practices and other impediments to diagnostic testing indicate that residual IPD has likely occurred but remained undetected by conventional hospital-based surveillance. CONCLUSIONS: Depending on the setting, we discuss alternative monitoring strategies that could improve understanding of pneumococcal disease dynamics.

Autochthonous Human Case of Seoul Virus Infection, the Netherlands.

Orthohantaviruses are a group of rodentborne viruses with a worldwide distribution. The orthohantavirus Seoul virus (SEOV) can cause hemorrhagic fever with renal syndrome in humans and is distributed worldwide, like its reservoir host, the rat. Cases of SEOV in wild and pet rats have been described in several countries, and human cases have been reported in the United Kingdom, France, Canada, and the United States. In the Netherlands, SEOV has previously been found in wild brown rats. We describe an autochthonous human case of SEOV infection in the Netherlands. This patient had nonspecific clinical symptoms of an orthohantavirus infection (gastrointestinal symptoms and distinct elevation of liver enzymes). Subsequent source investigation revealed 2 potential sources, the patient's feeder rats and a feeder rat farm. At both sources, a high prevalence of SEOV was found in the rats. The virus closely resembled the Cherwell and Turckheim SEOV strains that were previously found in Europe.

Increased overall survival after introduction of structured bedside consultation in Staphylococcus aureus bacteraemia.

Staphylococcus aureus bacteraemia (SAB) is a common and severe disease. In 2012, a structured bedside consultation (SBC) was introduced at Rijnstate Hospital. We analysed the effect of this SBC on the overall survival of patients with SAB and the effect on the diagnostic workup. We performed a retrospective cohort study, including all patients over 18 years with SAB from 2009 until 2017. The cases preceding versus those after implementation of SBC in 2012 were compared. In total, 613 episodes of SAB were analysed: 234 cases before and 379 cases since SBC. In 484 patients at risk for a complicated course, there was no significant difference in the 30-day survival (77 versus 82%, p = 0.18); however, an increase in 365-day survival was seen (56 versus 64%, p = 0.05). Overall, more patients received adequate therapy, both in the first 2 weeks (67.8 versus 86.7%, p < 0.001), as in complicated SAB (70.5 versus 93.2%, p < 0.001). In 21% of patients with transoesophageal echocardiogram (TEE) following a negative or inconclusive TTE, endocarditis was diagnosed. In patients at risk for complicated SAB, the PET scan revealed a metastatic infection which was not clinically suspected in 65% of positive PET scans. Structured bedside consultation is associated with a better 365-day survival in patients at risk for complicated SAB. Moreover, the additional value of TEE and the PET scan was shown. We strongly advise compliance to SBC in all patients at risk for complicated SAB and the use of both TEE and PET scans in these patients. Even in uncomplicated SAB, TEE or PET scan can reveal metastatic infections.

Zidovudine continuous infusion in an HIV-infected pregnant woman with a phobia of swallowing pills.

The authors report the difficulties of preventing mother-to-child transmission in a pregnant HIV-infected woman with a phobia of swallowing pills. After multiple attempts and just as many failures, the authors ended up with cART consisting of small tablets of nevirapine, lamivudine and a continuous intravenous infusion of zidovudine given via an elastomeric pump at home. This case demonstrates the difficulties that HIV physicians can encounter in pregnant women who have difficulties in swallowing tablets. In exceptional circumstances, continuous infusion of zidovudine may be an option, even in an outpatient setting.

Urinary Tract Infection Caused by a Capnophilic Proteus mirabilis Strain.

From a urine sample from a patient with a urinary tract infection, a carbon dioxide-dependent Proteus mirabilis strain was isolated. It is important to perform urine cultures in 5% carbon dioxide and an anaerobic atmosphere if bacteria prominent in Gram stains do not grow on routine media in ambient air.

September through October 2010 multi-centre study in the Netherlands examining laboratory ability to detect enterovirus 68, an emerging respiratory pathogen.

During September and October 2010, the Dutch Public Health Institute detected an enterovirus (EV) 68 (EV68) epidemic in the Netherlands through general practitioner-based surveillance of acute respiratory infections. EV68 shares phenotypic and genotypic properties with human rhinovirus (HRV). Despite increased EV and HRV detections, Dutch clinical laboratories did not identify EV68. To assess the capability of Dutch clinical laboratories to detect EV68, ten laboratories with more than eight detected EV and HRV cases in September and October 2010 provided information about their detection algorithms and testing results for a 2010 Dutch EV68 strain. For EV detection mostly stool specimens (median 49%), respiratory specimens (median 27%) and cerebrospinal fluid (median 22%) were used. For HRV detection only respiratory specimens were used. Except for the Seeplex® RV15ACE EV-specific assay, all EV and 73% of HRV assays, including those of the Public Health Institute, were able to detect EV68. Two-step EV RT-PCR protocols were the most sensitive. Thus, laboratories might have misidentified EV68 as HRV. In addition, EV68 cases might have also been missed because patients with respiratory diseases are usually not tested for EV infection. Therefore, clinical laboratories should include EV detection in the differential diagnosis of patients presenting with respiratory symptoms.

Rapid enterovirus molecular testing in cerebrospinal fluid reduces length of hospitalization and duration of antibiotic therapy in children with aseptic meningitis.

We studied the potential benefits of introducing a rapid enterovirus molecular test in children with enterovirus meningitis. The 2 groups of pediatric patients were comparable with respect to clinical and laboratory data, but differed in availability of enterovirus test results. In the control group, the results were available within 3 to 7 days, whereas in the study group, rapid enterovirus molecular test results were available within 3 to 24 hours. The median duration of hospitalization and the duration of antibiotics were significantly reduced to, respectively, 2 days and 1 day in the study group when compared with the control group (P < 0.001). Mean costs per patient calculation showed an average reduction of more than US $1450 (P < 0.001).

Prevalence of xenotropic murine leukaemia virus-related virus in patients with chronic fatigue syndrome in the Netherlands: retrospective analysis of samples from an established cohort.

OBJECTIVE: The presence of the retrovirus xenotropic murine leukaemia virus-related virus (XMRV) has been reported in peripheral blood mononuclear cells of patients with chronic fatigue syndrome. Considering the potentially great medical and social relevance of such a discovery, we investigated whether this finding could be confirmed in an independent European cohort of patients with chronic fatigue syndrome. DESIGN: Analysis of a well defined cohort of patients and matched neighbourhood controls by polymerase chain reaction. SETTING: Certified (ISO 15189) laboratory of clinical virology in a university hospital in the Netherlands. Population Between December 1991 and April 1992, peripheral blood mononuclear cells were isolated from 76 patients and 69 matched neighbourhood controls. In this study we tested cells from 32 patients and 43 controls from whom original cryopreserved phials were still available. MAIN OUTCOME MEASURES: Detection of XMRV in peripheral blood mononuclear cells by real time polymerase chain reaction assay targeting the XMRV integrase gene and/or a nested polymerase chain reaction assay targeting the XMRV gag gene. RESULTS: We detected no XMRV sequences in any of the patients or controls in either of the assays, in which relevant positive and negative isolation controls and polymerase chain reaction controls were included. Spiking experiments showed that we were able to detect at least 10 copies of XMRV sequences per 10(5) peripheral blood mononuclear cells by real time as well as by nested polymerase chain reaction, demonstrating high sensitivity of both assays. CONCLUSIONS: This study failed to show the presence of XMRV in peripheral blood mononuclear cells of patients with chronic fatigue syndrome from a Dutch cohort. These data cast doubt on the claim that XMRV is associated with chronic fatigue syndrome in the majority of patients.

Prevalence of human parechovirus in the Netherlands in 2000 to 2007.

Infection with human parechovirus 3 (HPeV3) was described for the first time in Japan in 2004 and reportedly is more often associated with severe disease than infection with HPeV1 or HPeV2. In 2004, infections with HPeV3 were observed for the first time in The Netherlands. Genetic analysis showed several different lineages, suggesting endemic circulation. We analyzed 163 cell culture isolates from the same number of patients tested in routine virological laboratories as part of the national enterovirus surveillance program. Isolates were collected between 2000 and 2007 and could not be characterized by routine methods. In total, 155 isolates (95%) were found positive for HPeV by a reverse transcription-PCR assay targeting the 5' untranslated region, explaining the majority of the diagnostic deficit in enterovirus surveillance for these years. Typing of the isolates by use of partial genome sequencing of the VP1/2A region revealed the presence of 55 HPeV1, 2 HPeV2, 89 HPeV3, 1 HPeV4, and 8 HPeV5 isolates. We compared isolation dates, age groups affected, and clinical pictures, which were reported as part of the routine surveillance. Clear differences in epidemiology were observed, with HPeV3 occurring at intervals of 2 years and in the spring-summer season, whereas HPeV1 was observed in small numbers throughout each year, with a low in the summer months. HPeV3 infection affected younger children than HPeV1 infection and was significantly more often associated with fever, meningitis, and viremia.