Mentor-mentee relationship in clinical microbiology.

Clinical microbiology is a field in constant evolution, with increasing technological opportunities and a growing emphasis on human and social issues. Maintaining knowledge and skills and anticipating future changes is challenging both for laboratory managers and for all the co-workers. Training and succession preparation represents a unique opportunity to adapt/prepare future generations according to the evolutions of the field. The aim of this review is to provide to clinical microbiologists a reflection on ongoing technological and social changes in their field and a deepening of the central role of preparing future generations to these changes through a fruitful mentor-mentee relationship. This narrative review relies on selected publications addressing mentor-mentee interactions in various academic fields, on interview with our colleagues and pairs, as well as on our personal experience. From the qualities and aspects that emerged as necessary for a productive mentor-mentee interaction, we selected and discuss five of them for the mentor: the role and responsibility, the positioning, the vision, the scientific credibility, and the moral credibility, as well as five for the mentee: creativity, flexibility, energy, responsibility, and self evaluation. This review emphasizes the importance of both the scientific and the ethical credibility of the mentor and the mentee as well as the importance of human and social values such as solidarity, equality, equity, respectfulness, and empathy, and might support mentor and mentee in the field of clinical microbiology and also in the field of infectious disease in their intent for a fruitful interaction

Methods for Real-Time PCR-Based Diagnosis of Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia abortus Infections in an Opened Molecular Diagnostic Platform.

The advances in molecular biology of the last decades have dramatically improved the field of diagnostic bacteriology. In particular, PCR-based technologies have impacted the diagnosis of infections caused by obligate intracellular bacteria such as pathogens from the Chlamydiacae family. Here, we describe a real-time PCR-based method using the Taqman technology for the diagnosis of Chlamydia pneumoniae, Chlamydia psittaci, and Chlamydia abortus infection. The method presented here can be applied to various clinical samples and can be adapted on opened molecular diagnostic platforms

Assessment of SARS-CoV-2 tests costs and reimbursement tariffs readjustments during the COVID-19 pandemic.

While laboratories have been facing limited supplies of reagents for diagnostic tests throughout the course of the COVID-19 pandemic, national and international health plans, as well as billing costs, have been constantly adjusted in order to optimize the use of resources. We aimed to assess the impact of SARS-CoV-2 test costs and reimbursement tariff adjustments on diagnostic strategies in Switzerland to determine the advantages and disadvantages of different costs and resource saving plans. We specifically assessed the cost of diagnostic SARS-COV-2 RT-PCR using five different approaches: i) in-house platform, ii) cobas 6800® (Roche, Basel, Switzerland), iii) GeneXpert® SARS-CoV-2 test (Cepheid, Sunnyvale, CA, USA), iv) VIASURE SARS-CoV-2 (N1 + N2) Real-Time PCR Detection Kit for BD MAX™ (Becton Dickinson, Franklin Lake, NJ, USA), v) cobas® Liat® SARS-CoV-2 & Influenza A/B (Roche, Basel, Switzerland). We compared these costs to the evolution of the reimbursement tariffs. The cost of a single RT-PCR test varied greatly (as did the volume of tests performed), ranging from as high as 180 CHF per test at the beginning of the pandemic (February to April 2020) to as low as 82 CHF per test at the end of 2020. Depending on the time period within the pandemic, higher costs did not necessarily mean greater benefits for the laboratories. The costs of molecular reagents for rapid tests were higher than of those for classic RT-PCR platforms, but the rapid tests had reduced turnaround times (TATs), thus improving patient care and enabling more efficient implementation of isolation measures, as well as reducing the burden of possible nosocomial infections. At the same time, there were periods when the production or distribution of these reagents was insufficient, and only the use of several different molecular platforms allowed us to sustain the high number of tests requested. Cost-saving plans need to be thoroughly assessed and constantly adjusted according to the epidemiological situation, the clinical context and the national resources in order to always guarantee that the highest performing diagnostic solutions are available. Not all cost-saving strategies guarantee good analytical performance

Viral load of SARS-CoV-2 across patients and compared to other respiratory viruses.

RT-PCRs to detect SARS-CoV-2 RNA is key to manage the COVID-19 pandemic. We analyzed SARS-CoV-2 viral loads from 22'323 RT-PCR results according to samples types, gender, age, and health units. Viral load did not show any difference across age and appears to be a poor predictor of disease outcome. SARS-CoV-2 viral load showed similar high viral loads than the one observed for RSV and influenza B. The importance of viral load to predict contagiousness and to assess disease progression is discussed

Impact of different SARS-CoV-2 assays on laboratory turnaround time.

Introduction. Clinical microbiology laboratories have had to cope with an increase in the volume of tests due to the emergence of the SARS-CoV-2 virus. Short turnaround times (TATs) are important for case tracing and to help clinicians in patient management. In such a context, high-throughput systems are essential to process the bulk of the tests. Rapid tests are also required to ensure shorter TATs for urgent situations. In our laboratory, SARS-CoV-2 assays were initially implemented on our custom platform using a previously published method. The commercial cobas 6800 (Roche diagnostics) assay and the GeneXpert Xpress (Cepheid) SARS-CoV-2 assay were implemented on 24 March and 8 April 2020, respectively, as soon as available.Hypothesis/Gap Statement. Despite the abundant literature on SARS-CoV-2 assays, the articles focus mainly on the diagnostic performances. This is to our knowledge the first article that specifically studies the TAT of different assays.Aim. We aimed to describe the impact of various SARS-CoV-2 assays on the TAT at the beginning of the outbreak.Methodology. In this study, we retrospectively analysed the TAT of all SARS-CoV-2 assays performed in our centre between 24 February and 9 June, 2020.Results. We retrieved 33 900 analyses, with a median TAT of 6.25 h. TATs were highest (6.9 h) when only our custom platform was used (24 February to 24 March, 2020). They were reduced to 6.1 h when the cobas system was introduced (24 March to 8 April, 2020). The implementation of the GeneXpert further reduced the median TAT to 4.8 h (8 April to 9 June, 2020). The GeneXpert system had the shortest median TAT (1.9 h), followed by the cobas (5.5 h) and by our custom platform (6.9 h).Conclusion. This work shows that the combination of high-throughput systems and rapid tests allows the efficient processing of a large number of tests with a short TAT. In addition, the use of a custom platform allowed the quick implementation of an in-house test when commercial assays were not yet available

Genome of the carbapenemase-producing clinical isolate Elizabethkingia miricola EM_CHUV and comparative genomics with Elizabethkingia meningoseptica and Elizabethkingia anophelis: evidence for intrinsic multidrug resistance trait of emerging pathogens.

Elizabethkingia miricola is a Gram-negative non-fermenting rod emerging as a life-threatening human pathogen. The multidrug-resistant (MDR) carbapenemase-producing clinical isolate E. miricola EM_CHUV was recovered in the setting of severe nosocomial pneumonia. In this study, the genome of E. miricola EM_CHUV was sequenced and a functional analysis was performed, including a comparative genomic study with Elizabethkingia meningoseptica and Elizabethkingia anophelis. The resistome of EM_CHUV revealed the presence of a high number of resistance genes, including the presence of the blaGOB-13 and blaB-9 carbapenemase-encoding genes. Twelve mobility genes, with only two of them located in the proximity of resistance genes, and four potential genomic islands were identified in the genome of EM_CHUV, but no prophages or CRISPR sequences. Ten restriction-modification system (RMS) genes were also identified. In addition, we report the presence of a putative conjugative plasmid (pEM_CHUV) that does not encode any antibiotic resistance genes. Altogether, these findings point towards a limited number of DNA exchanges with other bacteria and suggest that multidrug resistance is an intrinsic trait of E. miricola owing to the presence of a high number of resistance genes within the bacterial core genome

Detection of respiratory bacterial pathogens causing atypical pneumonia by multiplex Lightmix^® RT-PCR.

Pneumonia is a severe infectious disease. In addition to common viruses and bacterial pathogens (e.g. Streptococcus pneumoniae), fastidious respiratory pathogens like Chlamydia pneumoniae, Mycoplasma pneumoniae and Legionella spp. can cause severe atypical pneumonia. They do not respond to penicillin derivatives, which may cause failure of antibiotic empirical therapy. The same applies for infections with B. pertussis and B. parapertussis, the cause of pertussis disease, that may present atypically and need to be treated with macrolides. Moreover, these fastidious bacteria are difficult to identify by culture or serology, and therefore often remain undetected. Thus, rapid and accurate identification of bacterial pathogens causing atypical pneumonia is crucial. We performed a retrospective method evaluation study to evaluate the diagnostic performance of the new, commercially available Lightmix <sup>®</sup> multiplex RT-PCR assay that detects these fastidious bacterial pathogens causing atypical pneumonia. In this retrospective study, 368 clinical respiratory specimens, obtained from patients suffering from atypical pneumonia that have been tested negative for the presence of common agents of pneumonia by culture and viral PCR, were investigated. These clinical specimens have been previously characterized by singleplex RT-PCR assays in our diagnostic laboratory and were used to evaluate the diagnostic performance of the respiratory multiplex Lightmix <sup>®</sup> RT-PCR. The multiplex RT-PCR displayed a limit of detection between 5 and 10 DNA copies for different in-panel organisms and showed identical performance characteristics with respect to specificity and sensitivity as in-house singleplex RT-PCRs for pathogen detection. The Lightmix <sup>®</sup> multiplex RT-PCR assay represents a low-cost, time-saving and accurate diagnostic tool with high throughput potential. The time-to-result using an automated DNA extraction device for respiratory specimens followed by multiplex RT-PCR detection was below 4 h, which is expected to significantly improve diagnostics for atypical pneumonia-associated bacterial pathogens

Genomics of the new species Kingella negevensis: diagnostic issues and identification of a locus encoding a RTX toxin.

Kingella kingae, producing the cytotoxic RTX protein, is a causative agent of serious infections in humans such as bacteremia, endocarditis and osteoarticular infection, especially in young children. Recently, Kingella negevensis, a related species, has been isolated from the oral cavity of healthy children. In this study, we report the isolation of K. negevensis strain eburonensis, initially misidentified as K. kingae with MALDI-TOF MS, from a vaginal specimen of a patient suffering of vaginosis. The genome sequencing and analysis of this strain together with comparative genomics of the Kingella genus revealed that K. negevensis possesses a full homolog of the rtx operon of K. kingae involved in the synthesis of the RTX toxin. We report that a K. kingae specific diagnostic PCR, based on the rtxA gene, was positive when tested on K. negevensis strain eburonensis DNA. This cross-amplification, and risk of misidentification, was confirmed by in silico analysis of the target gene sequence. To overcome this major diagnostic issue we developed a duplex real-time PCR to detect and distinguish K. kingae and K. negevensis. In addition to this, the identification of K. negevensis raises a clinical issue in term of pathogenic potential given the production of a RTX hemolysin

Diagnostic strategies for SARS-CoV-2 infection and interpretation of microbiological results.

To face the current COVID-19 pandemic, diagnostic tools are essential. It is recommended to use real-time RT-PCR for RNA viruses in order (a) to perform a rapid and accurate diagnostic, (b) to guide patient care and management and (c) to guide epidemiological strategies. Further studies are warranted to define the role of serological diagnosis and a possible correlation between serological response and prognosis. The aim was to guide clinical microbiologists in the use of these diagnostic tests and clinicians in the interpretation of their results. A search of literature was performed through PubMed and Google Scholar using the keywords SARS-CoV-2, SARS-CoV-2 molecular diagnosis, SARS-CoV-2 immune response, SARS-CoV-2 serology/antibody testing, coronavirus diagnosis. The present review discusses performances, limitations and use of current and future diagnostic tests for SARS-CoV-2. Real-time RT-PCR remains the reference method for diagnosis of SARS-CoV-2 infection. On the other hand, notwithstanding its varying sensitivity according to the time of infection, serology represents a valid asset (a) to try to solve possible discrepancies between a highly suggestive clinical and radiological presentation and negative RT-PCR, (b) to solve discrepancies between different PCR assays and (c) for epidemiological purposes

Improving the molecular diagnosis of Chlamydia psittaci and Chlamydia abortus infection with a species-specific duplex real-time PCR.

Chlamydia psittaci and Chlamydia abortus are closely related intracellular bacteria exhibiting different tissue tropism that may cause severe but distinct infection in humans. C. psittaci causes psittacosis, a respiratory zoonotic infection transmitted by birds. C. abortus is an abortigenic agent in small ruminants, which can also colonize the human placenta and lead to foetal death and miscarriage. Infections caused by C. psittaci and C. abortus are underestimated mainly due to diagnosis difficulties resulting from their strict intracellular growth. We developed a duplex real-time PCR to detect and distinguish these two bacteria in clinical samples. The first PCR (PCR1) targeted a sequence of the 16S-23S rRNA operon allowing the detection of both C. psittaci and C. abortus. The second PCR (PCR2) targeted the coding DNA sequence CPSIT_0607 unique to C. psittaci. The two PCRs showed 100 % detection for ≥ 10 DNA copies per reaction (1000 copies ml- 1). Using a set of 120 samples, including bacterial reference strains, clinical specimens and infected cell culture material, we monitored 100 % sensitivity and 100 % specificity for the detection of C. psittaci and C. abortus for PCR1. When PCR1 was positive, PCR2 could discriminate C. psittaci from C. abortus with a positive predictive value of 100 % and a negative predictive value of 88 %. In conclusion, this new duplex PCR represents a low-cost and time-saving method with high-throughput potential, expected to improve the routine diagnosis of psittacosis and pregnancy complication in large-scale screening programs and also during outbreaks