No abstract

Baseline characteristics and outcomes of 1591 patients infected with SARS-CoV-2 admitted to ICUs of the Lombardy Region, Italy. JAMA.

There have been reports of increasing numbers of cases of malaria among migrants and travelers. Although microscopic examination of blood smears remains the "gold standard" in diagnosis, this method suffers from insufficient sensitivity and requires considerable expertise. To improve diagnosis, a multiplex real-time PCR was developed. One set of generic primers targeting a highly conserved region of the 18S rRNA gene of the genus Plasmodium was designed; the primer set was polymorphic enough internally to design four speciesspecific probes for P. falciparum, P. vivax, P. malarie, and P. ovale. Real-time PCR with species-specific probes detected one plasmid copy of P. falciparum, P. vivax, P. malariae, and P. ovale specifically. The same sensitivity was achieved for all species with real-time PCR with the 18S screening probe. Ninety-seven blood samples were investigated. For 66 of them (60 patients), microscopy and real-time PCR results were compared and had a crude agreement of 86% for the detection of plasmodia. Discordant results were reevaluated with clinical, molecular, and sequencing data to resolve them. All nine discordances between 18S screening PCR and microscopy were resolved in favor of the molecular method, as were eight of nine discordances at the species level for the species-specific PCR among the 31 samples positive by both methods. The other 31 blood samples were tested to monitor the antimalaria treatment in seven patients. The number of parasites measured by real-time PCR fell rapidly for six out of seven patients in parallel to parasitemia determined microscopically. This suggests a role of quantitative PCR for the monitoring of patients receiving antimalaria therapy.

Rapid diagnosis of active Mycobacterium tuberculosis (Mtb) infection remains a clinical and laboratory challenge. We have analyzed the cytokine profile (interferon-γ (IFN-γ), tumor necrosis factor-α (TNF-α) and interleukin-2 (IL-2)) of Mtb-specific T cells by polychromatic flow cytometry. We studied Mtb-specific CD4+ T cell responses in subjects with latent Mtb infection and active tuberculosis disease. The results showed substantial increase in the proportion of single-positive TNF-α Mtb-specific CD4+ T cells in subjects with active disease, and this parameter was the strongest predictor of diagnosis of active disease versus latent infection. We validated the use of this parameter in a cohort of 101 subjects with tuberculosis diagnosis unknown to the investigator. The sensitivity and specificity of the flow cytometry-based assay were 67% and 92%, respectively, the positive predictive value was 80% and the negative predictive value was 92.4%. Therefore, the proportion of single-positive TNF-α Mtb-specific CD4+ T cells is a new tool for the rapid diagnosis of active tuberculosis disease.

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.

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.

Conventional methods are sometimes insufficient to identify human bacterial pathogens, and alternative techniques, often molecular, are required. Matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) identified with a valid score 45.9% of 410 clinical isolates from 207 different difficult-to-identify species having required 16S rRNA gene sequencing. MALDI-TOF MS might represent an alternative to 16S rRNA gene sequencing.The technique matrix-assisted laser desorption ionizationtime-of-flight mass spectrometry (MALDI-TOF MS) has recently been introduced in diagnostic microbiology laboratories and has been shown to be a promising method to replace conventional phenotypic identification for the majority of bacterial pathogens routinely found in clinical samples (1,3,8,15,17).For a subset of bacterial isolates, conventional techniques which mainly rely on commercial biochemical kit tests fail to provide a definitive identification of these infrequent isolates. In those cases, alternative techniques are required. That could be an expanded battery of biochemical tests, high-performance liquid chromatography (HPLC), or sequencing of housekeeping bacterial genes such as 16S rRNA genes and comparing the obtained sequences to those in databases (4). Since 16S rRNA gene sequencing is both time-consuming and costly, we wanted to assess the potential of MALDI-TOF MS, which is based on the study of proteic profiles, to replace it.We investigated a collection of 1,405 isolates on which 16S rRNA gene sequencing was performed in the last 8 years in our laboratory due to a lack of satisfactory identification by conventional methods such as Vitek2 or the API system (bioMérieux, Marcy l'Etoile, France). We selected for each species a maximum of 5 isolates. This yielded a total of 433 bacterial isolates representing 207 different species from 84 different genera. For 23 isolates, no growth could be obtained from frozen stock. A total of 410 isolates were consequently further studied. Acquisition of protein mass spectra of strains by MALDI-TOF MS was done on a Microflex LT instrument (Bruker Daltonics, Leipzig, Germany) using the FlexControl 3.0 software (Bruker Daltonics) after a formic acid-acetonitrile extraction step was performed as previously described (1). Automated data analysis of raw spectra was performed by the BioTyper automation 2.0 software (Bruker Daltonics), using a library of 3,290 spectra (database update of 2 September 2008) with default settings. According to the criteria proposed by the manufacturer, a result was considered valid at the species level whenever the score value attributed by BioTyper was x Ն 2.0, valid at the genus level when the score was 1.7 Յ x Ͻ 2, and no reliable identification when the score was x Ͻ 1.7. 16S rRNA gene sequencing and identification were performed by standard methods (5).For routine isolates, MALDI-TOF MS has shown overall correct identification levels often greater than 85% (1,3,8,15,17). In the present study, MALDI-TOF MS yielded a score of...

Originally composed of the single family Chlamydiaceae, the Chlamydiales order has extended considerably over the last several decades. Chlamydia-related bacteria were added and classified into six different families and family-level lineages: the Criblamydiaceae, Parachlamydiaceae, Piscichlamydiaceae, Rhabdochlamydiaceae, Simkaniaceae, and Waddliaceae. While several members of the Chlamydiaceae family are known pathogens, recent studies showed diverse associations of Chlamydia-related bacteria with human and animal infections. Some of these latter bacteria might be of medical importance since, given their ability to replicate in free-living amoebae, they may also replicate efficiently in other phagocytic cells, including cells of the innate immune system. Thus, a new Chlamydiales-specific real-time PCR targeting the conserved 16S rRNA gene was developed. This new molecular tool can detect at least five DNA copies and show very high specificity without cross-amplification from other bacterial clade DNA. The new PCR was validated with 128 clinical samples positive or negative for Chlamydia trachomatis or C. pneumoniae. Of 65 positive samples, 61 (93.8%) were found to be positive with the new PCR. The four discordant samples, retested with the original test, were determined to be negative or below detection limits. Then, the new PCR was applied to 422 nasopharyngeal swabs taken from children with or without pneumonia; a total of 48 (11.4%) samples were determined to be positive, and 45 of these were successfully sequenced. The majority of the sequences corresponded to Chlamydia-related bacteria and especially to members of the Parachlamydiaceae family.The Chlamydiales order contains obligate intracellular bacteria separated in 7 different families and family-level lineages, the Chlamydiaceae, the Criblamydiaceae, the Parachlamydiaceae, the Piscichlamydiaceae, the Rhabdochlamydiaceae, the Simkaniaceae, and the Waddliaceae (16,(23)(24)(25). Some of these bacteria are established pathogens and, for instance, Chlamydia trachomatis, C. psittaci, and C. pneumoniae from the Chlamydiaceae family can cause significant human infections. The other families constitute a group called Chlamydia-related bacteria (also referred to as Chlamydia-like organisms), which has been yet poorly investigated. Like the Chlamydiaceae, these Chlamydia-related bacteria are obligate intracellular bacteria that also exhibit a biphasic developmental cycle. Serological and molecular studies have implicated some species in various human and animal infections. Parachlamydia acanthamoebae is associated with human pneumonia (6,12,26,27) and might cause bovine abortions (5, 38, 39), and Simkania negevensis might be responsible for respiratory infections, especially in children (18,20,22,28,32,33,35), whereas Waddlia chondrophila has been reported to cause abortion in bovines (14,40) and is strongly suspected to cause miscarriage in humans (3, 4). Some of these newly discovered Chlamydia-related bacteria that resist digestion by several environmental am...