Abstract
Staphylococcus aureus
is the most frequent aetiology of bone and joint infections (BJI) and can cause relapsing and chronic infections. One of the main factors involved in the chronicization ...of staphylococcal BJIs is the internalization of
S. aureus
into osteoblasts, the bone-forming cells. Previous studies have shown that
S. aureus
triggers an impairment of osteoblasts function that could contribute to bone loss. However, these studies focused mainly on the extracellular effects of
S. aureus
. Our study aimed at understanding the intracellular effects of
S. aureus
on the early osteoblast differentiation process. In our in vitro model of osteoblast lineage infection, we first observed that internalized
S. aureus
8325-4 (a reference lab strain) significantly impacted
RUNX2
and
COL1A1
expression compared to its non-internalized counterpart 8325-4
∆
fnbAB
(with deletion of
fnbA
and
fnbB
). Then, in a murine model of osteomyelitis, we reported no significant effect for
S. aureus
8325-4 and 8325-4
∆
fnbAB
on bone parameters at 7 days post-infection whereas
S. aureus
8325-4 significantly decreased trabecular bone thickness at 14 days post-infection compared to 8325-4
∆
fnbAB
. When challenged with two clinical isogenic strains isolated from initial and relapse phase of the same BJI, significant impairments of bone parameters were observed for both initial and relapse strain, without differences between the two strains. Finally, in our in vitro osteoblast infection model, both clinical strains impacted alkaline phosphatase activity whereas the expression of bone differentiation genes was significantly decreased only after infection with the relapse strain. Globally, we highlighted that
S. aureus
internalization into osteoblasts is responsible for an impairment of the early differentiation in vitro and that
S. aureus
impaired bone parameters in vivo in a strain-dependent manner.
In recent years, metagenomic Next-Generation Sequencing (mNGS) has increasingly been used for an accurate assumption-free virological diagnosis. However, the systematic workflow evaluation on ...clinical respiratory samples and implementation of quality controls (QCs) is still lacking.
A total of 3 QCs were implemented and processed through the whole mNGS workflow: a no-template-control to evaluate contamination issues during the process; an internal and an external QC to check the integrity of the reagents, equipment, the presence of inhibitors, and to allow the validation of results for each sample. The workflow was then evaluated on 37 clinical respiratory samples from patients with acute respiratory infections previously tested for a broad panel of viruses using semi-quantitative real-time PCR assays (28 positive samples including 6 multiple viral infections; 9 negative samples). Selected specimens included nasopharyngeal swabs (n = 20), aspirates (n = 10), or sputums (n = 7).
The optimal spiking level of the internal QC was first determined in order to be sufficiently detected without overconsumption of sequencing reads. According to QC validation criteria, mNGS results were validated for 34/37 selected samples. For valid samples, viral genotypes were accurately determined for 36/36 viruses detected with PCR (viral genome coverage ranged from 0.6 to 100%, median = 67.7%). This mNGS workflow allowed the detection of DNA and RNA viruses up to a semi-quantitative PCR Ct value of 36. The six multiple viral infections involving 2 to 4 viruses were also fully characterized. A strong correlation between results of mNGS and real-time PCR was obtained for each type of viral genome (R
ranged from 0.72 for linear single-stranded (ss) RNA viruses to 0.98 for linear ssDNA viruses).
Although the potential of mNGS technology is very promising, further evaluation studies are urgently needed for its routine clinical use within a reasonable timeframe. The approach described herein is crucial to bring standardization and to ensure the quality of the generated sequences in clinical setting. We provide an easy-to-use single protocol successfully evaluated for the characterization of a broad and representative panel of DNA and RNA respiratory viruses in various types of clinical samples.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Background
Protracted viral shedding is common in hospitalized patients with COVID-19 pneumonia, and up to 40% display signs of pulmonary fibrosis on computed tomography (CT) after hospital ...discharge. We hypothesized that COVID-19 patients with acute respiratory failure (ARF) who die in intensive care units (ICU) have a lower viral clearance in the respiratory tract than ICU patients discharged alive, and that protracted viral shedding in respiratory samples is associated with patterns of fibroproliferation on lung CT. We, therefore, conducted a retrospective observational study, in 2 ICU of Lyon university hospital.
Results
129 patients were included in the study, of whom 44 (34%) died in ICU. 432 RT-PCR for SARS-CoV-2 were performed and 137 CT scans were analyzed. Viral load was significantly higher in patients deceased as compared to patients alive at ICU discharge (
p
< 0.001), after adjustment for the site of viral sampling and RT-PCR technique. The median time to SARS-CoV-2 negativation on RT-PCR was 19 days CI
95 %
:15–21 in patients alive at ICU discharge and 26 days CI
95 %
:17-infinity in non-survivors at ICU discharge. Competitive risk regression identified patients who died in ICU and age as independent risk factors for longer time to SARS-CoV-2 negativation on RT-PCR, while antiviral treatment was independently associated with shorter time. None of the CT scores exploring fibroproliferation (i.e., bronchiectasis and reticulation scores) were significantly associated with time to SARS-CoV-2 negativation.
Conclusions
Viral load in respiratory samples is significantly lower and viral shedding significantly shorter in ICU survivors of COVID-19 associated acute respiratory failure. Protracted viral shedding is unrelated to occurrence of fibrosis on lung CT.
Biofilm formation, intra-osteoblastic persistence, small-colony variants (SCVs) and the dysregulation of agr, the major virulence regulon, are possibly involved in staphylococcal bone and joint ...infection (BJI) pathogenesis. We aimed to investigate the contributions of these mechanisms among a collection of 95 Staphylococcus aureus clinical isolates from 64 acute (67.4%) and 31 chronic (32.6%) first episodes of BJI. The included isolates were compared for internalization rate, cell damage and SCV intracellular emergence using an ex vivo model of human osteoblast infection. Biofilm formation was assessed in a microbead immobilization assay (BioFilm Ring test). Virulence gene profiles were assessed by DNA microarray. Seventeen different clonal complexes were identified among the screened collection. The staphylococcal internalization rate in osteoblasts was significantly higher for chronic than acute BJI isolates, regardless of the genetic background. Conversely, no differences regarding cytotoxicity, SCV emergence, biofilm formation and virulence gene distribution were observed. Additionally, agr dysfunction, detected by the lack of delta-toxin production using whole-cell matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) analysis (n = 15; 15.8%), was significantly associated with BJI chronicity, osteoblast invasion and biofilm formation. These findings provide new insights into MSSA BJI pathogenesis, suggesting the correlation between chronicity and staphylococcal osteoblast invasion. This adaptive mechanism, along with biofilm formation, is associated with agr dysfunction, which can be routinely assessed by delta-toxin detection using MALDI-TOF spectrum analysis, possibly providing clinicians with a diagnostic marker of BJI chronicity at the time of diagnosis.
Vancomycin-intermediate Staphylococcus aureus (VISA) is associated with genetic changes that may also impact upon pathogenicity. In the current study, we compared the virulence of clinical VISA ...strains with their isogenic vancomycin-susceptible progenitors (VSSA).
Production of the critical virulence protein, α toxin, was assessed using Western blot analysis and was correlated to agr activity using a bioluminescent agr-reporter. Cytotoxicity and intracellular persistence were compared ex vivo for VSSA and VISA within non-professional phagocytes (NPP). Virulence and host immune responses were further explored in vivo using a murine model of bacteraemia.
VISA isolates produced up to 20-fold less α toxin compared with VSSA, and this was corroborated by either loss of agr activity due to agr mutation, or altered agr activity in the absence of mutation. VISA were less cytotoxic towards NPP and were associated with enhanced intracellular persistence, suggesting that NPP may act as a reservoir for VISA. Infection with VSSA strains produced higher mortality in a murine bacteraemia model (≥90% 7-day mortality) compared with infection with VISA isolates (20% to 50%, p <0.001). Mice infected with VISA produced a dampened immune response (4.6-fold reduction in interleukin-6, p <0.001) and persistent organ bacterial growth was observed for VISA strains out to 7 days.
These findings highlight the remarkable adaptability of S. aureus, whereby, in addition to having reduced antibiotic susceptibility, VISA alter the expression of pathogenic factors to circumvent the host immune response to favour persistent infection over acute virulence.
The holistic approach of the human immune system is based on the study of its components collectively driving a functional response to an immunogenic stimulus. To appreciate a specific immune ...dysfunction, a condition is mimicked ex vivo and the immune response induced is assessed. The application field of such assays are broad and expanding, from the diagnosis of primary and secondary immunodeficiencies, immunotherapy for cancer to the management of patients at-risk for infections and vaccination. These assays are immune monitoring tools that may contribute to a personalised and precision medicine. The purpose of this review is to describe immune functional assays available in the setting of non-HIV acquired immune deficiency. First, we will address the use of theses assays in the diagnosis of opportunistic infections such as viral reactivation. Secondly, we will report the usefulness of these assays to assess vaccine efficacy and to manage immunosuppressive therapies.
Recent research on
Staphylococcus aureus
vaccine development has focused on active immunization against Panton–Valentine leukocidin (PVL), a potent leukotoxin associated with both superficial and ...severe deep-seated infections. PVL prevalence is highly variable worldwide, but it is unknown to what extent immunity to PVL varies between patients from geographic areas with different PVL-positive
S. aureus
prevalences. We conducted a retrospective multicentric study of anti-PVL and anti-alpha-toxin (Hla) antibody levels in uninfected adult patients from France (low PVL prevalence;
n
= 200), Algeria (moderate prevalence;
n
= 143), and Senegal (high prevalence;
n
= 228). The antibody levels were quantified by an enzyme-linked immunosorbent assay (ELISA) procedure. Because Hla is present in virtually all
S. aureus
strains, its corresponding antibody levels were considered to reflect population exposure to
S. aureus
. Compared with French participants, the average anti-PVL antibody levels were 2.5-fold and 8.2-fold higher in Algerian and Senegalese participants, respectively (
p
< 0.001). Conversely, anti-Hla antibody levels did not differ between participants from the three countries, suggesting that the observed differences in anti-PVL antibody levels were not biased by variations in population exposure to
S. aureus
. Hence, anti-PVL antibody levels in the general populations of France, Algeria, and Senegal vary widely and match variations in PVL-positive
S. aureus
strain prevalence, with an increasing north-to-south gradient. To conclude, immunity to PVL in a given population correlates with local PVL prevalence. This finding can help to inform PVL vaccine strategies.
Patients with autoimmune polyendocrinopathy syndrome type 1 (APS-1) caused by autosomal recessive AIRE deficiency produce autoantibodies that neutralize type I interferons (IFNs)
, conferring a ...predisposition to life-threatening COVID-19 pneumonia
. Here we report that patients with autosomal recessive NIK or RELB deficiency, or a specific type of autosomal-dominant NF-κB2 deficiency, also have neutralizing autoantibodies against type I IFNs and are at higher risk of getting life-threatening COVID-19 pneumonia. In patients with autosomal-dominant NF-κB2 deficiency, these autoantibodies are found only in individuals who are heterozygous for variants associated with both transcription (p52 activity) loss of function (LOF) due to impaired p100 processing to generate p52, and regulatory (IκBδ activity) gain of function (GOF) due to the accumulation of unprocessed p100, therefore increasing the inhibitory activity of IκBδ (hereafter, p52
/IκBδ
). By contrast, neutralizing autoantibodies against type I IFNs are not found in individuals who are heterozygous for NFKB2 variants causing haploinsufficiency of p100 and p52 (hereafter, p52
/IκBδ
) or gain-of-function of p52 (hereafter, p52
/IκBδ
). In contrast to patients with APS-1, patients with disorders of NIK, RELB or NF-κB2 have very few tissue-specific autoantibodies. However, their thymuses have an abnormal structure, with few AIRE-expressing medullary thymic epithelial cells. Human inborn errors of the alternative NF-κB pathway impair the development of AIRE-expressing medullary thymic epithelial cells, thereby underlying the production of autoantibodies against type I IFNs and predisposition to viral diseases.
Over recent years, there has been increasing interest in the use of the anelloviruses, the major component of the human virome, for the prediction of post-transplant complications such as severe ...infections. Due to an important diversity, the comprehensive characterization of this viral family over time has been poorly studied. To overcome this challenge, we used a metagenomic next-generation sequencing (mNGS) approach with the aim of determining the individual anellovirus profile of autologous stem cell transplant (ASCT) patients. We conducted a prospective pilot study on a homogeneous patient cohort regarding the chemotherapy regimens that included 10 ASCT recipients. A validated viral mNGS workflow was used on 108 plasma samples collected at 11 time points from diagnosis to 90 days post-transplantation. A complex interindividual variability in terms of abundance and composition was noticed. In particular, a strong sex effect was found and confirmed using quantitative PCR targeting torque teno virus, the most abundant anellovirus. Interestingly, an important turnover in the anellovirus composition was observed during the course of the disease revealing a strong intra-individual variability. Although more studies are needed to better understand anellovirus dynamics, these findings are of prime importance for their future use as biomarkers of immune competence.
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