Andrea Zbinden

IVDR regulation represents a major challenge for diagnostic microbiology laboratories. IVDR complicates a broad range of aspects and poses a risk given the high diversity of pathogens (including rare but highly virulent microbes) and the large variety of samples submitted for analysis. The regular emergence of new pathogens (including Echovirus E-11, Adenovirus 41, Monkeypox virus, Alongshan virus, and Enterovirus D68, as recent examples in Europe in the post SARS-CoV-2 era) is another factor that makes IVDR regulation risky, because its detrimental effect on production of in-house tests will negatively impact knowledge and expertise in the development of new diagnostic tests. Moreover, such regulations negatively impact the availability of diagnostic tests, especially for neglected pathogens, and has a detrimental effect on the overall costs of the tests. The increased regulatory burden of IVDR may thereby pose an important risk for public health. Taken together, it will have a negative impact on the financial balance of diagnostic microbiology laboratories (especially small ones). The already-high standards of quality management of all ISO-accredited and Swissmedic-authorized laboratories render IVDR law of little value, at least in Switzerland, while tremendously increasing the regulatory burden and associated costs. Eventually, patients will need to pay for diagnostic assays outside of the framework of their insurance in order to obtain a proper diagnostic assessment, which may result in social inequity. Thus, based on the risk assessment outlined above, the coordinated commission for clinical microbiology proposes adjusting the IvDO ordinance by (i) introducing an obligation to be ISO 15189 accredited and (ii) not implementing the IvDO 2028 milestone.

Zusammenfassung. Für die Diagnostik einer Lyme-Borreliose, welche durch Borrelia burgdorferi verursacht wird, ist nebst der Klinik die Borrelien-Serologie zentral. Die Borrelien-Serologie besteht aus zwei Stufen: einem sensitiven Suchtest wie einem Enzym-Immuno-Assay und einem spezifischen Bestätigungstest wie zum Beispiel einem Immunoblot. Bei einem Suchtest können Borrelien-spezifische IgM- und IgG-Antikörper, aber auch unspezifische Antikörper nachgewiesen werden, beispielsweise kreuzreagierende Antikörper gegen Treponema pallidum (Erreger der Lues). Deshalb müssen reaktive Suchtestresultate mit einem spezifischen Test wie einem Immunoblot bestätigt werden. Dabei werden die Antikörper gegen die verschiedenen Borrelien-Antigene aufgeschlüsselt, was eine Beurteilung über eine mögliche Kreuzreaktion und gegebenenfalls eine Einteilung in ein Früh- oder Spätstadium der Lyme-Borreliose erlaubt. Für die Diagnostik der Neuroborreliose wird die Borrelien-spezifische, intrathekale Antikörperbildung bestimmt, wobei zwingend eine Serum- und eine Liquorprobe vom gleichen Entnahmezeitpunkt analysiert werden müssen. Die Diagnose der Lyme-Borreliose erfordert eine Gesamtschau der serologischen Befunde zusammen mit der Klinik.

Introduction: Screening of hepatitis B surface antigen (HBsAg) and individual-donation nucleic acid amplification testing (ID-NAT) of blood donors have become standard to detect hepatitis B virus (HBV) infection. However, there is still a residual risk of HBV transmission by blood components of donors suffering from occult HBV infection (OBI). Therefore, many countries implemented universal testing of anti-HBV core antigen (anti-HBc) antibodies in order to increase blood safety. In Switzerland, anti-HBc testing is not part of the routine blood donor-screening repertoire. Therefore, we sought to assess prevalence of donors with OBI in a Swiss blood donor collective. Methods: Blood donations were prospectively investigated for the presence of anti-HBc antibodies during two time periods (I: all donors, March 2017; II: first-time donors only, April 2017 until February 2018). Anti-HBc-positive findings were confirmed by an anti-HBc neutralization test. Discarded plasma samples of anti-HBc-confirmed positive donors were ultracentrifuged and subsequently retested by regular HBV-ID-NAT to search for traces of HBV. Results: During time period I, 78 (1.6%) individuals out of 4,923 donors were confirmed anti-HBc-positive. Sixty-nine (88%) anti-HBc-positive samples were available and processed by ultracentrifugation followed by repeat HBV-ID-NAT. Four samples (5.8%) were found positive for HBV DNA. Sixty-five (94.2%) samples remained HBV NAT-negative upon ultracentrifugation. During time period II, 56 (0.9%) donor samples out of 6,509 exhibited anti-HBc-confirmed positive. Fifty-five (98%) samples could be reassessed by HBV-ID-NAT upon ultracentrifugation. Three (5.5%) samples contained HBV DNA and 52 (94.5%) samples remained HBV NAT-negative. Conclusion: Overall, we detected 7 viremic OBI carriers among 11,432 blood donors, which tested negative for HBV by standard HBV-ID-NAT and HBsAg screening. In contrast, OBI carriers showed positive anti-HBc findings which could be confirmed in 83.8% of the cases. Thus, OBI might be missed by the current HBV screening process of Swiss blood donors. We suggest to review current HBV screening algorithm. Extended donor screening by anti-HBc testing may unmask OBI carriers and contribute to blood safety for the recipient of blood products.

Background: Aeromonas hydrophila is a gram-negative facultative anaerobic coccobacillus, which is an environmental opportunistic pathogen. A. hydrophila are involved in several infectious diseases such as gastroenteritis, septicemia and wound infections. However, gastroenteritis caused by Aeromonas spp. are rare and the clinical relevance of Aeromonas species in stool specimens is still under debate. Case presentation: Our case concerns a 32-year-old woman who presented at hospital with a worsening watery diarrhea and fever requiring intensive care. A cholera-like illness was diagnosed. The patient had a past history of an anti-Hu syndrome with a myenteric ganglionitis. A molecular multiplex RT-PCR (QIAstat-Dx Gastrointestinal Panel, QIAGEN) covering a broad spectrum of diverse gastrointestinal pathogens performed directly from the stool was negative but the stool culture revealed growth of A. hydrophila. Further investigations of the A. hydrophila strain in cell cultures revealed the presence of a cytotoxic enterotoxin. Conclusions: Although A. hydrophila rarely causes gastroenteritis, Aeromonas spp. should be considered as a causative agent of severe gastroenteritis with a cholera-like presentation. This case highlights the need to perform culture methods from stool samples when PCR-based methods are negative and gastrointestinal infection is suspected.

Background: Accurate identification of fastidious Gram-negative rods (GNR) by conventional phenotypic characteristics is a challenge for diagnostic microbiology. The aim of this study was to evaluate the use of molecular methods, e.g., 16S rRNA gene sequence analysis for identification of fastidious GNR in the clinical microbiology laboratory.Results: A total of 158 clinical isolates covering 20 genera and 50 species isolated from 1993 to 2010 were analyzed by comparing biochemical and 16S rRNA gene sequence analysis based identification. 16S rRNA gene homology analysis identified 148/158 (94%) of the isolates to species level, 9/158 (5%) to genus and 1/158 (1%) to family level. Compared to 16S rRNA gene sequencing as reference method, phenotypic identification correctly identified 64/158 (40%) isolates to species level, mainlyAggregatibacter aphrophilus,Cardiobacterium hominis,Eikenella corrodens, Pasteurella multocida, and 21/158 (13%) isolates correctly to genus level, notablyCapnocytophagasp.; 73/158 (47%) of the isolates were not identified or misidentified.Conclusions: We herein propose an efficient strategy for accurate identification of fastidious GNR in the clinical microbiology laboratory by integrating both conventional phenotypic methods and 16S rRNA gene sequence analysis. We conclude that 16S rRNA gene sequencing is an effective means for identification of fastidious GNR, which are not readily identified by conventional phenotypic methods.

Four Gram-stain-positive, catalase-negative, coccus-shaped bacterial strains were isolated from multiple blood cultures of patients with endocarditis, meningitis and spondylodiscitis. The isolates were tentatively identified as viridans streptococci on the basis of phenotypic characteristics. Comparative 16S rRNA gene sequencing studies showed that the organisms were members of the Streptococcus mitis group but did not correspond to any recognized species. The nearest phylogenetic relative was S. mitis ATCC 49456^T, with 98.6 % sequence similarity. The representative strain AZ_3a^Tshowed less than 96.8, 97.6, 94.5 and 95.5 % similarity to the phylogenetically most closely related species byrecA,rpoB,sodAandgroELgene sequence analysis, respectively. DNA–DNA hybridization analyses showed a low reassociation value of 32.2 % between strain AZ_3a^Tand S. mitis DSM 12643^T. Reassociation values with members of other S. mitis group species ranged from 27.3 to 49.7 %. The G+C content of the DNA was 40.0 mol%. Based on our biochemical and molecular analyses, the isolates represent a novel species, for which the nameStreptococcustigurinussp. nov. is proposed. The type strain is AZ_3a^T( = CCOS 600^T = DSM 24864^T).

Opportunistic invasive fungal infections are a major cause of mortality in immunocompromised patients. Early diagnosis of invasive aspergillosis and proper identification of the causative agent is crucial for guidance of therapy. Accurate differentiation of Aspergillus lentulus, a filamentous fungus often misidentified as atypical Aspergillus fumigatus, is of concern as multiple antifungal drugs show a reduced susceptibility. This is the first report, to our knowledge, of a proven pulmonary invasive fungal infection caused by A. lentulus after heart transplantation.

We recently described the novel speciesStreptococcus tigurinussp. nov. belonging to theStreptococcus mitisgroup. The type strain AZ_3a^TofS. tigurinuswas originally isolated from a patient with infective endocarditis. According to its phenotypic and molecular characteristics,S. tigurinusis most closely related toStreptococcus mitis,Streptococcus pneumoniae,Streptococcus pseudopneumoniae,Streptococcus oralis, andStreptococcus infantis. Accurate identification ofS. tigurinusis facilitated by 16S rRNA gene analysis. We retrospectively analyzed our 16S rRNA gene molecular database, which contains sequences of all clinical samples obtained in our institute since 2003. We detected 17 16S rRNA gene sequences which were assigned toS. tigurinus, including sequences from the 3S. tigurinusstrains described previously.S. tigurinusoriginated from normally sterile body sites, such as blood, cerebrospinal fluid, or heart valves, of 14 patients and was initially detected by culture or broad-range 16S rRNA gene PCR, followed by sequencing. The 14 patients had serious invasive infections, i.e., infective endocarditis (n= 6), spondylodiscitis (n= 3), bacteremia (n= 2), meningitis (n= 1), prosthetic joint infection (n= 1), and thoracic empyema (n= 1). To evaluate the presence ofStreptococcus tigurinusin the endogenous oral microbial flora, we screened saliva specimens of 31 volunteers. After selective growth, alpha-hemolytic growing colonies were analyzed by matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) and subsequent molecular methods.S. tigurinuswas not identified among 608 strains analyzed. These data indicate thatS. tigurinusis not widely distributed in the oral cavity. In conclusion,S. tigurinusis a novel agent of invasive infections, particularly infective endocarditis.

Proper identification of Streptococcus pneumoniae by conventional methods remains problematic. The discriminatory power of the 16S rRNA gene, which can be considered the “gold standard” for molecular identification, is too low to differentiate S. pneumoniae from closely related species such as Streptococcus pseudopneumoniae , Streptococcus mitis , and Streptococcus oralis in the routine clinical laboratory. A 313-bp part of recA was selected on the basis of variability within the S. mitis group, showing recA fragment. We show that recA analysis is a useful tool for proper identification to species level within the S. mitis group, in particular, for pneumococci.