Visceral leishmaniasis in a lung transplant recipient: usefulness of highly sensitive real-time polymerase chain reaction for preemptive diagnosis.

We report the case of a lung transplant recipient in whom the diagnosis of visceral leishmaniasis (VL) was made by detection of parasites in a peripheral blood smear when the parasite load already reached 8.9 × 103 parasites/mL. We demonstrated that the VL diagnosis could have been done months before the development of symptoms by the use of Leishmania-specific real-time polymerase chain reaction (PCR), suggesting the role of preemptive PCR-based diagnosis in transplant recipients at risk for VL.

A battery of tandem mass spectrometry assays with stable isotope-dilution for the quantification of 15 anti-tuberculosis drugs and two metabolites in patients with susceptible-, multidrug-resistant- and extensively drug-resistant tuberculosis.

OBJECTIVE: Anti-tuberculosis (antiTB) drugs are characterized by an important inter-interindividual pharmacokinetic variability poorly predictable from individual patients' characteristics. Therapeutic drug monitoring (TDM) may therefore be beneficial for patients with Mycobacterium tuberculosis infection, especially for the management of multidrug/extensively drug resistant- (MDR/XDR)-TB. Our objective was to develop robust HPLC-MS/MS methods for plasma quantification of 15 antiTB drugs and 2 metabolites, namely rifampicin, isoniazid plus N-acetyl-isoniazid, pyrazinamide, ethambutol (the conventional quadritherapy for susceptible TB) as well as combination of agents against MDR/XDR-TB: bedaquiline, clofazimine, delamanid and its metabolite M1, levofloxacin, linezolid, moxifloxacin, pretomanid, rifabutin, rifapentine, sutezolid, and cycloserine. METHODS: Plasma protein precipitation was used for all analytes except cycloserine, which was analyzed separately after derivatization with benzoyl chloride. AntiTB quadritherapy drugs (Pool1) were separated by Hydrophilic Interaction Liquid Chromatography (column Xbridge BEH Amide, 2.1 × 150 mm, 2.5 µm, Waters®) while MDR/XDR-TB agents (Pool 2) and cycloserine (as benzoyl derivative) were analyzed by reverse phase chromatography on a column XSelect HSS T3, 2.1 × 75 mm, 3.5 µm (Waters®). All runs last <7 min. Quantification was performed by selected reaction monitoring electrospray tandem mass spectrometry, using stable isotopically labelled internal standards. RESULTS: The method covers the clinically relevant plasma levels and was extensively validated based on FDA recommendations, with intra- and inter-assay precision (CV) < 15% over the validated ranges. Application of the method is illustrated by examples of TDM for two patients treated for drug-susceptible- and MDR-TB. CONCLUSION: Such convenient extraction methods and the use of stable isotope-labelled drugs as internal standards provide an accurate and precise quantification of plasma concentrations of all major clinically-used antiTB drugs regimens and is optimally suited for clinically efficient TDM against tuberculosis.

The dark side of SARS-CoV-2 rapid antigen testing: screening asymptomatic patients.

Several reports showed SARS-CoV-2 rapid antigen tests (RATs) performances among COVID-19 symptomatic subjects in outpatient settings during periods of highest incidence of infections and high rates of hospital admissions, but few data are present for asymptomatic patients. We investigated the role of RATs in an emergency department, as a novel screening tool before admission for COVID-19 asymptomatic patients. A total of 116 patients were screened on admission in a 250-bed community hospital in Morges, Switzerland. RAT detected 2/7 RT-PCR-positive patients and delivered two false-positive results. These data suggest the non-fiability of RATs screening in this clinical scenario.

Universal admission screening strategy for COVID-19 highlighted the clinical importance of reporting SARS-CoV-2 viral loads.

Previously limited to symptomatic patients, our hospital introduced a universal admission screening strategy for coronavirus disease 2019 on 25 April 2020. All patients were tested by RT-PCR. We observed decreased viral loads linked to increased screening of asymptomatic patients highlighting the fact that viral load values could guide infection control decisions.

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

BACKGROUND: 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. OBJECTIVES: The aim was to guide clinical microbiologists in the use of these diagnostic tests and clinicians in the interpretation of their results. SOURCES: 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. CONTENT: The present review discusses performances, limitations and use of current and future diagnostic tests for SARS-CoV-2. IMPLICATIONS: 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.

The rapid molecular test Xpert MTB/RIF ultra: towards improved tuberculosis diagnosis and rifampicin resistance detection.

BACKGROUND: Tuberculosis diagnosis has dramatically improved since the introduction of the rapid molecular test Xpert MTB/RIF (Xpert) detecting M. tuberculosis and rifampicin resistance directly from clinical specimens, therefore shortening the turnaround time, reducing patient's isolation period and decreasing the time to start anti-TB drugs. The new version, Xpert MTB/RIF Ultra (Ultra), displays a higher sensitivity and an improved rifampicin resistance detection. Both tests have been endorsed by the World Health Organisation. AIMS: Xpert and Ultra rapidly became widespread and paved the way for new approaches and new paradigms as well as for the development of molecular point-of-care tests (POCTs). In this narrative review, we aimed to address their performance in the diagnosis of tuberculosis and to discuss the expectations of these tests as well as their limits and the unmet needs. SOURCES: Peer-reviewed publications addressing the diagnostic performance of Ultra and Xpert. CONTENT: We focused on publications that evaluated the performance of Ultra and Xpert on the same group of patients or the same set of specimens in different tuberculosis-burden settings. IMPLICATIONS: The studies published so far reported an increased sensitivity of Ultra when compared to Xpert, which represents a benefit for tuberculosis diagnosis. The fact that such a sensitive assay cannot distinguish between alive and dead bacilli emphasizes that caution should be exercised regarding indications and interpretation of results. Additional studies are needed to determine the true performance for the diagnosis of extrapulmonary tuberculosis because of the great diversity of the specimens.

Whole-genome sequencing for rapid, reliable and routine investigation of Mycobacterium tuberculosis transmission in local communities.

Contact investigations following the diagnosis of active tuberculosis (TB) are paramount for the control of the disease. Epidemiological data are very powerful for contact tracing but might be delayed and/or difficult to integrate, especially in the setting of multiple contact-tracing investigations. The aim of this study was to address the added-value of whole-genome sequencing (WGS) to routine local TB surveillance systems. From November 2016 to July 2017, the local TB surveillance system identified three clusters that could constitute a unique larger outbreak. Epidemiological and clinical information were integrated with WGS genotyping data of Mycobacterium tuberculosis strains obtained using a simple DNA extraction method coupled with sequencing using an Illumina MiSeq platform and an in-house bioinformatics pipeline for single nucleotide polymorphism (SNP) analysis. Epidemiological investigations identified three putative TB clusters potentially interrelated including eight patients with active TB. Seven M. tuberculosis isolates were available and analysed by WGS. Using a 5-SNP threshold to define recent transmission, WGS-based genotyping supported the occurrence of the three clusters as well as a link between clusters 1 and 2 (SNP ≤1), constituting a larger outbreak. This outbreak was clearly delineated by refuting a potential link with the third cluster (SNP >500). Genotyping data did not support the belonging of patient 7 to any studied cluster. This study illustrates the usefulness of WGS genotyping for routine TB surveillance systems in local communities to rapidly confirm or disprove epidemiological hypotheses and delineate TB clusters, especially in the context of multiple contact-tracing investigations.

How to: identify non-tuberculous Mycobacterium species using MALDI-TOF mass spectrometry.

BACKGROUND: The implementation of MALDI-TOF MS for microorganism identification has changed the routine of the microbiology laboratories as we knew it. Most microorganisms can now be reliably identified within minutes using this inexpensive, user-friendly methodology. However, its application in the identification of mycobacteria isolates has been hampered by the structure of their cell wall. Improvements in the sample processing method and in the available database have proved key factors for the rapid and reliable identification of non-tuberculous mycobacteria isolates using MALDI-TOF MS. AIMS: The main objective is to provide information about the proceedings for the identification of non-tuberculous isolates using MALDI-TOF MS and to review different sample processing methods, available databases, and the interpretation of the results. SOURCES: Results from relevant studies on the use of the available MALDI-TOF MS instruments, the implementation of innovative sample processing methods, or the implementation of improved databases are discussed. CONTENT: Insight about the methodology required for reliable identification of non-tuberculous mycobacteria and its implementation in the microbiology laboratory routine is provided. IMPLICATIONS: Microbiology laboratories where MALDI-TOF MS is available can benefit from its capacity to identify most clinically interesting non-tuberculous mycobacteria in a rapid, reliable, and inexpensive manner.

Mentor-mentee relationship in clinical microbiology.

BACKGROUND: 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. AIMS: 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. SOURCES: 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. CONTENT: 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. IMPLICATIONS: 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.

Nanomechanical sensor applied to blood culture pellets: a fast approach to determine the antibiotic susceptibility against agents of bloodstream infections.

OBJECTIVES: The management of bloodstream infection, a life-threatening disease, largely relies on early detection of infecting microorganisms and accurate determination of their antibiotic susceptibility to reduce both mortality and morbidity. Recently we developed a new technique based on atomic force microscopy capable of detecting movements of biologic samples at the nanoscale. Such sensor is able to monitor the response of bacteria to antibiotic's pressure, allowing a fast and versatile susceptibility test. Furthermore, rapid preparation of a bacterial pellet from a positive blood culture can improve downstream characterization of the recovered pathogen as a result of the increased bacterial concentration obtained. METHODS: Using artificially inoculated blood cultures, we combined these two innovative procedures and validated them in double-blind experiments to determine the susceptibility and resistance of Escherichia coli strains (ATCC 25933 as susceptible and a characterized clinical isolate as resistant strain) towards a selection of antibiotics commonly used in clinical settings. RESULTS: On the basis of the variance of the sensor movements, we were able to positively discriminate the resistant from the susceptible E. coli strains in 16 of 17 blindly investigated cases. Furthermore, we defined a variance change threshold of 60% that discriminates susceptible from resistant strains. CONCLUSIONS: By combining the nanomotion sensor with the rapid preparation method of blood culture pellets, we obtained an innovative, rapid and relatively accurate method for antibiotic susceptibility test directly from positive blood culture bottles, without the need for bacterial subculture.

Added value of molecular assay Xpert MTB/RIF compared to sputum smear microscopy to assess the risk of tuberculosis transmission in a low-prevalence country.

Airborne precautions are required at hospital admission for patients with suspected pulmonary tuberculosis. The isolation is maintained until 3 serially collected sputum smears are acid-fast bacilli negative, a time- and labor-intensive method with limited sensitivity and specificity, which has a great impact on patient flow management. We evaluated the possibility of replacing the result of microscopy by the semiquantitative result of the molecular point-of-care test Xpert MTB/RIF to assess patients' transmission risk to quickly guide airborne isolation decisions in low-endemic countries. The performance of the Xpert MTB/RIF, used as a first-line test, was compared to the results of microscopy for specimens (n=242) collected from May 2010 to December 2014 in Lausanne, Switzerland. The sensitivity and specificity of Xpert MTB/RIF were 91.5% (65/71) and 99.6% (170/171), respectively, vs. 64.8% (46/71) and 94.2% (161/171) for microscopy. Samples with negative Xpert MTB/RIF were all smear negative for Mycobacterium tuberculosis (negative predictive value, 100%). The semiquantitative results of Xpert MTB/RIF-high, medium, low or very low-were found to correlate with acid-fast bacilli detection: positive predictive value of 100% (6/6), 96.5% (27/28), 52.2% (12/23) and 11.1% (1/9) respectively. Finally, when including clinical criteria, we identified 11 smear-negative but Xpert MTB/RIF-positive patients with a significant transmission potential. In conclusion, our data support the introduction of an Xpert MTB/RIF-based strategy as a replacement of smear microscopy for a faster and more accurate management of tuberculosis patients' transmission risk in a low-prevalence country.

Microbial diagnosis of bloodstream infection: towards molecular diagnosis directly from blood.

When a bloodstream infection (BSI) is suspected, most of the laboratory results-biochemical and haematologic-are available within the first hours after hospital admission of the patient. This is not the case for diagnostic microbiology, which generally takes a longer time because blood culture, which is to date the reference standard for the documentation of the BSI microbial agents, relies on bacterial or fungal growth. The microbial diagnosis of BSI directly from blood has been proposed to speed the determination of the etiological agent but was limited by the very low number of circulating microbes during these paucibacterial infections. Thanks to recent advances in molecular biology, including the improvement of nucleic acid extraction and amplification, several PCR-based methods for the diagnosis of BSI directly from whole blood have emerged. In the present review, we discuss the advantages and limitations of these new molecular approaches, which at best complement the culture-based diagnosis of BSI.

Blood culture-based diagnosis of bacteraemia: state of the art.

Blood culture remains the best approach to identify the incriminating microorganisms when a bloodstream infection is suspected, and to guarantee that the antimicrobial treatment is adequate. Major improvements have been made in the last years to increase the sensitivity and specificity and to reduce the time to identification of microorganisms recovered from blood cultures. Among other factors, the introduction in clinical microbiology laboratories of the matrix-assisted laser desorption ionization time-of-flight mass spectrometry technology revolutionized the identification of microorganisms whereas the introduction of nucleic-acid-based methods, such as DNA hybridization or rapid PCR-based test, significantly reduce the time to results. Together with traditional antimicrobial susceptibility testing, new rapid methods for the detection of resistance mechanisms respond to major epidemiological concerns such as methicillin-resistant Staphylococcus aureus, extended-spectrum ß-lactamase or carbapenemases. This review presents and discusses the recent developments in microbial diagnosis of bloodstream infections based on blood cultures.

Cryptococcus neoformans meningitis with negative cryptococcal antigen: Evaluation of a new immunochromatographic detection assay.

Detection of cryptococcal antigen in serum or cerebrospinal fluid allows cryptococcal meningitis diagnosis within few hours with >90% sensitivity. In an HIV-positive patient with Cryptococcus neoformans meningitis, initial antigen detection by immunoagglutination was negative. We thus evaluated a new immunochromatographic detection assay that exhibited a higher sensitivity.