Unexpected associations between respiratory viruses and bacteria with Pulmonary Function Testing in children suffering from Cystic Fibrosis (MUCOVIB study).

BACKGROUND: Various bacterial and viral assemblages composing Cystic Fibrosis (CF) lung microbiota contribute to long-term lung function decline over time. Yet, the impact of individual microorganisms on pulmonary functions remains uncertain in children with CF. METHODS: As part of the 'Mucoviscidosis, respiratory VIruses, intracellular Bacteria and fastidious organisms'' project, children with CF were longitudinally followed in a Swiss multicentric study. Respiratory samples included mainly throat swabs and sputa samples for bacterial culture and 16S rRNA metagenomics and nasopharyngeal swabs for respiratory virus detection by molecular assays. Percentage of predicted Forced Expiratory Volume in one second (FEV1%) and Lung Clearance Index (LCI) were recorded. RESULTS: Sixty-one children, of whom 20 (32.8%) presented with at least one pulmonary exacerbation, were included. Almost half of the 363 nasopharyngeal swabs tested by RT-PCR were positive for a respiratory virus, mainly rhinovirus (26.5%). From linear mixed-effects regression models, P. aeruginosa (-11.35, 95%CI [-17.90; -4.80], p = 0.001) was significantly associated with a decreased FEV1%, whereas rhinovirus was associated with a significantly higher FEV1% (+4.24 95%CI [1.67; 6.81], p = 0.001). Compared to conventional culture, 16S rRNA metagenomics showed a sensitivity and specificity of 80.0% and 85.4%, respectively for detection of typical CF pathogens. However, metagenomics detected a bacteria almost twice more often than culture. CONCLUSIONS: As expected, P. aeruginosa impacted negatively on FEV1% while rhinovirus was surprisingly associated with better FEV1%. Culture-free assays identifie significantly more pathogens than standard culture, with disputable clinical correlation.

Case Report: Stepwise Anti-Inflammatory and Anti-SARS-CoV-2 Effects Following Convalescent Plasma Therapy With Full Clinical Recovery.

In these times of COVID-19 pandemic, concern has been raised about the potential effects of SARS-CoV-2 infection on immunocompromised patients, particularly on those receiving B-cell depleting agents and having therefore a severely depressed humoral response. Convalescent plasma can be a therapeutic option for these patients. Understanding the underlying mechanisms of convalescent plasma is crucial to optimize such therapeutic approach. Here, we describe a COVID-19 patient who was deeply immunosuppressed following rituximab (anti-CD20 monoclonal antibody) and concomitant chemotherapy for chronic lymphoid leukemia. His long-term severe T and B cell lymphopenia allowed to evaluate the treatment effects of convalescent plasma. Therapeutic outcome was monitored at the clinical, biological and radiological level. Moreover, anti-SARS-CoV-2 antibody titers (IgM, IgG and IgA) and neutralizing activity were assessed over time before and after plasma transfusions, alongside to SARS-CoV-2 RNA quantification and virus isolation from the upper respiratory tract. Already after the first cycle of plasma transfusion, the patient experienced rapid improvement of pneumonia, inflammation and blood cell counts, which may be related to the immunomodulatory properties of plasma. Subsequently, the cumulative increase in anti-SARS-CoV-2 neutralizing antibodies due to the three additional plasma transfusions was associated with progressive and finally complete viral clearance, resulting in full clinical recovery. In this case-report, administration of convalescent plasma revealed a stepwise effect with an initial and rapid anti-inflammatory activity followed by the progressive SARS-CoV-2 clearance. These data have potential implications for a more extended use of convalescent plasma and future monoclonal antibodies in the treatment of immunosuppressed COVID-19 patients.

Chlamydia pneumoniae and Mycoplasma pneumoniae in children with cystic fibrosis: impact on bacterial respiratory microbiota diversity.

OBJECTIVES: The contribution of intracellular and fastidious bacteria in Cystic fibrosis (CF) pulmonary exacerbations, and progressive lung function decline remains unknown. This project aimed to explore their impact on bacterial microbiota diversity over time in CF children. METHODS: Sixty-one children enrolled in the MUCOVIB multicentre prospective cohort provided 746 samples, mostly nasopharyngeal swabs, throat swabs and sputa which were analysed using culture, specific real-time qPCRs and 16S rRNA amplicon metagenomics. RESULTS: Chlamydia pneumoniae (n = 3) and Mycoplasma pneumoniae (n = 1) were prospectively documented in 6.6% of CF children. Microbiota alpha-diversity in children with a documented C. pneumoniae was highly variable, similarly to children infected with Staphylococcus aureus or Pseudomonas aeruginosa. The transition from routine follow-up visits to pulmonary exacerbation (n = 17) yielded variable changes in diversity indexes with some extreme loss of diversity. CONCLUSIONS: The high rate of C. pneumoniae detection supports the need for regular screenings in CF patients. A minor impact of C. pneumoniae on the microbial community structure was documented. Although detected in a single patient, M. pneumoniae should also be considered as a possible aetiology of lung infection in CF subjects.

CAPNOCYTOPHAGA CANIMORSUS ENDOPHTHALMITIS AFTER CATARACT SURGERY LINKED TO SALIVARY DOG-TO-HUMAN TRANSMISSION.

PURPOSE: To describe a case of acute postoperative bacterial endophthalmitis because of Capnocytophaga canimorsus after cataract surgery, with probable contamination through salivary droplets of dog two days after the procedure. METHODS: An 83-year-old woman who underwent uncomplicated cataract extraction with intraocular lens implantation, presented 12 days later with acute pain, redness, and vision loss in her left eye. Visual acuity was hand motion and clinical findings suggested the diagnosis of acute postoperative endophthalmitis. The patient underwent diagnostic vitrectomy, intravitreal ceftazidime/vancomycin injection and received oral moxifloxacin (400 mg/day). Two days later, she underwent complete pars-plana vitrectomy because of the absence of clinical improvement. Vitreous samples showed gram-negative bacterium on direct examination but cultures remained sterile, which prompted the realization of a broad-range bacterial polymerase chain reaction analysis. RESULTS: Polymerase chain reaction on the vitreous sample detected C. canimorsus, a fastidious gram-negative bacterium of the oral canine flora. When asked for recent contact with dogs, the patient reported having proceeded to an intensive tooth care session for her dog at postoperative Day 2. Intravenous ceftriaxone (2 g/day) was added to the treatment. Anterior and posterior segment inflammation slowly resolved, and final visual acuity was 20/160. CONCLUSION: Although very rare, this complication suggests that patients undergoing ocular surgery should avoid contact with salivary secretions of pets during the early postoperative period. Diagnostic broad-range bacterial polymerase chain reaction is useful to detect unconventional or slow-growing agents in vitreous samples.

Phylogenomics reveal that Mycobacterium kansasii subtypes are species-level lineages. Description of Mycobacterium pseudokansasii sp. nov., Mycobacterium innocens sp. nov. and Mycobacterium attenuatum sp. nov.

Among the species Mycobacterium kansasii, seven subtypes have been previously reported based on the PCR and the restriction fragment length polymorphism of the gene hsp65. Here, we used whole-genome sequencing to refine M. kansasii taxonomy and correct multiple inconsistencies. Average nucleotide identity (ANI) values between M. kansasii subtypes ranged from 88.4 to 94.2 %, lower than the accepted 95-96 % cut-off for species delineation. In addition, Mycobacterium gastri was closer to the M. kansasii subtypes 1, 2, 3, 4 and 5 than M. kansasii subtype 6. The recently described species Mycobacterium persicum shared 99.77 % ANI with M. kansasii subtype 2. Consistent with the ANI results, the digital DNA-DNA hybridization value was below the 70 % threshold for species delineation between subtypes and above it within subtypes as well as between subtype 2 and M. persicum. Furthermore, core-genome phylogeny confirmed the current M. kansasii species to be polyphyletic. Hence, we propose (i) Mycobacterium pseudokansasii sp. nov., replacing subtype 3, with the type strain MK142T(=CCUG 72128T=DSM 107152T), (ii) Mycobacterium innocens sp. nov., replacing subtype 5, with the type strain MK13T (=CCUG 72126T=DSM 107161T), and (iii) Mycobacterium attenuatum sp. nov., replacing subtype 6, with the type strain MK41T(=CCUG 72127T=DSM 107153T). Subtype 4 represents a new species-level lineage based on the genomic data but no strain was available. No genome sequence or strain was available for subtype 7. The proposed nomenclature will facilitate the identification of the most pathogenic subtype 1 as M. kansasii by clinicians while the new species names suggest the attenuated pathogenicity of the other subtypes.

Role of bi-weekly serum galactomannan screening for the diagnosis of invasive aspergillosis in haematological cancer patients.

Invasive aspergillosis (IA) is a life-threatening infection affecting haematological cancer patients with chemotherapy-induced neutropenia. The diagnosis of IA often relies on the detection of galactomannan (GM) in serum or bronchoalveolar lavage fluid (BAL). Bi-weekly serum GM screening has been proposed for a pre-emptive therapeutic approach of IA in patients not receiving mold-active prophylaxis. We have analysed all IA cases among patients with haematological malignancies and prolonged chemotherapy-induced neutropenia (>14 days) in our institution over a 10-year period (2007-2017). Serum GM was measured twice weekly and mold-active prophylaxis was not routinely administered. Thirty IA cases were observed and a positive serum GM was the first indicator of IA in 10 (33%) of them, which represents a need of approximately 500 GM tests for the detection of a single IA case. In the other 20 (67%) cases, suggestive chest CT lesion was the first sign of IA and bronchoscopy was required in 15 (50%) cases with negative serum GM for establishing the diagnosis of probable/proven IA. A positive serum GM was associated with a worse prognosis (57% 12-week survival vs 100% among serum GM-negative patients, P = .006), irrespective of the timing of GM positivity compared to CT. We concluded that bi-weekly serum GM screening demonstrated limited benefit in this population.

Determining the analytical specificity of PCR-based assays for the diagnosis of IA: What is Aspergillus?

A wide array of PCR tests has been developed to aid the diagnosis of invasive aspergillosis (IA), providing technical diversity but limiting standardisation and acceptance. Methodological recommendations for testing blood samples using PCR exist, based on achieving optimal assay sensitivity to help exclude IA. Conversely, when testing more invasive samples (BAL, biopsy, CSF) emphasis is placed on confirming disease, so analytical specificity is paramount. This multicenter study examined the analytical specificity of PCR methods for detecting IA by blind testing a panel of DNA extracted from a various fungal species to explore the range of Aspergillus species that could be detected, but also potential cross reactivity with other fungal species. Positivity rates were calculated and regression analysis was performed to determine any associations between technical specifications and performance. The accuracy of Aspergillus genus specific assays was 71.8%, significantly greater (P < .0001) than assays specific for individual Aspergillus species (47.2%). For genus specific assays the most often missed species were A. lentulus (25.0%), A. versicolor (24.1%), A. terreus (16.1%), A. flavus (15.2%), A. niger (13.4%), and A. fumigatus (6.2%). There was a significant positive association between accuracy and using an Aspergillus genus PCR assay targeting the rRNA genes (P = .0011). Conversely, there was a significant association between rRNA PCR targets and false positivity (P = .0032). To conclude current Aspergillus PCR assays are better suited for detecting A. fumigatus, with inferior detection of most other Aspergillus species. The use of an Aspergillus genus specific PCR assay targeting the rRNA genes is preferential.

Microbiota present in cystic fibrosis lungs as revealed by whole genome sequencing.

Determination of the precise composition and variation of microbiota in cystic fibrosis lungs is crucial since chronic inflammation due to microorganisms leads to lung damage and ultimately, death. However, this constitutes a major technical challenge. Culturing of microorganisms does not provide a complete representation of a microbiota, even when using culturomics (high-throughput culture). So far, only PCR-based metagenomics have been investigated. However, these methods are biased towards certain microbial groups, and suffer from uncertain quantification of the different microbial domains. We have explored whole genome sequencing (WGS) using the Illumina high-throughput technology applied directly to DNA extracted from sputa obtained from two cystic fibrosis patients. To detect all microorganism groups, we used four procedures for DNA extraction, each with a different lysis protocol. We avoided biases due to whole DNA amplification thanks to the high efficiency of current Illumina technology. Phylogenomic classification of the reads by three different methods produced similar results. Our results suggest that WGS provides, in a single analysis, a better qualitative and quantitative assessment of microbiota compositions than cultures and PCRs. WGS identified a high quantity of Haemophilus spp. (patient 1) or Staphylococcus spp. plus Streptococcus spp. (patient 2) together with low amounts of anaerobic (Veillonella, Prevotella, Fusobacterium) and aerobic bacteria (Gemella, Moraxella, Granulicatella). WGS suggested that fungal members represented very low proportions of the microbiota, which were detected by cultures and PCRs because of their selectivity. The future increase of reads' sizes and decrease in cost should ensure the usefulness of WGS for the characterisation of microbiota.

Species-specific recognition of Aspergillus fumigatus by Toll-like receptor 1 and Toll-like receptor 6.

BACKGROUND: Aspergillus fumigatus causes invasive aspergillosis, a potentially fatal infection in oncohematological patients. Innate immune detection of A. fumigatus involves Toll-like receptor (TLR) 4 and TLR2, which forms a heterodimer with either TLR1 or TLR6. The role of those coreceptors in Aspergillus sensing is unknown. METHODS: Cytokine production was measured in bone marrow-derived macrophages (BMDMs) from wild-type (WT) and TLR-deficient mice after incubation with a WT and an immunogenic RodA-deficient (ΔrodA-47) strain of A. fumigatus and in lungs from these mice after intranasal mold inoculation. Aspergillus fumigatus-mediated NF-κB activation was measured in HEK293T cells transfected with plasmids expressing mouse or human TLRs. RESULTS: Bone marrow-derived macrophages from TLR1- and TLR6-deficient mice produced lower amounts of interleukin 12p40, CXCL2, interleukin 6, and tumor necrosis factor α than BMDMs from WT mice after stimulation with A. fumigatus. Lungs from TLR1- and TLR6-deficient mice had diminished CXCL1 and CXCL2 production and increased fungal burden after intranasal inoculation of ΔrodA A. fumigatus compared with lungs from WT mice. ΔrodA strain-mediated NF-κB activation was observed in HEK293T cells expressing mouse TLR2/1, mouse TLR2/6, and human TLR2/1 but not human TLR2/6. CONCLUSIONS: Innate immune detection of A. fumigatus is mediated by TLR4 and TLR2 together with TLR1 or TLR6 in mice and TLR1 but not TLR6 in humans.

Development of a duplex real-time PCR for the detection of Rickettsia spp. and typhus group rickettsia in clinical samples.

Molecular diagnosis using real-time polymerase chain reaction (PCR) may allow earlier diagnosis of rickettsiosis. We developed a duplex real-time PCR that amplifies (1) DNA of any rickettsial species and (2) DNA of both typhus group rickettsia, that is, Rickettsia prowazekii and Rickettsia typhi. Primers and probes were selected to amplify a segment of the 16S rRNA gene of Rickettsia spp. for the pan-rickettsial PCR and the citrate synthase gene (gltA) for the typhus group rickettsia PCR. Analytical sensitivity was 10 copies of control plasmid DNA per reaction. No cross-amplification was observed when testing human DNA and 22 pathogens or skin commensals. Real-time PCR was applied to 16 clinical samples. Rickettsial DNA was detected in the skin biopsies of three patients. In one patient with severe murine typhus, the typhus group PCR was positive in a skin biopsy from a petechial lesion and seroconversion was later documented. The two other patients with negative typhus group PCR suffered from Mediterranean and African spotted fever, respectively; in both cases, skin biopsy was performed on the eschar. Our duplex real-time PCR showed a good analytical sensitivity and specificity, allowing early diagnosis of rickettsiosis among three patients, and recognition of typhus in one of them.

Dominant TNF-α+ Mycobacterium tuberculosis-specific CD4+ T cell responses discriminate between latent infection and active disease.

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.

Outbreak of Mycobacterium haemophilum infections after permanent makeup of the eyebrows.

We report a Mycobacterium haemophilum outbreak after permanent make-up of the eyebrows performed by the same freelance artist. Twelve patients presented an eyebrow lesion and cervical lymphadenitis. All were treated with antibiotics. Surgery was required in 10 cases. M. haemophilum DNA was identified in the make-up ink.

Mycobacterium tuberculosis aortic graft infection with recurrent hemoptysis: a case report.

INTRODUCTION: Mycobacterium tuberculosis may cause a large variety of clinical presentations due to its ability to disseminate by contiguity or hematogenously. Tuberculosis may remain undiagnosed for years due to the chronic course of the disease, with potentially life-threatening long-term complications. CASE PRESENTATION: In this case report, we describe a tuberculous aortic graft infection in a 72-year-old man documented by polymerase chain reaction and cultures. The patient presented with three episodes of hemoptysis following a remote history of miliary tuberculosis. The infection was treated by graft replacement and prolonged antimycobacterial therapy. CONCLUSION: Tuberculous infection of a vascular graft is an uncommon complication, but should be considered in patients with an intravascular device and a history of previous tuberculosis, especially when hematogenous spread may have occurred a few months after surgery, or when an active mycobacterial infection is present in close proximity to the graft.

Failure of voriconazole to cure disseminated zygomycosis in an immunocompromised child.

UNLABELLED: Voriconazole is increasingly used as a first-line agent for empirical antifungal therapy of prolonged febrile neutropenia in paediatric cancer patients. We describe the case of a 9-year-old patient with stage IV Burkitt lymphoma, who developed pulmonary and splenic zygomycosis while receiving voriconazole for persistent febrile neutropenia. The causative agent, Absidia corymbifera, was identified by broad-range fungal PCR in a lung biopsy sample. The patient was successfully treated with a combination of partial resection of the left upper lobe and antifungal therapy with high-dose liposomal amphotericin B followed by oral itraconazole as demonstrated by resolving pulmonary infiltrates on serial high resolution CT scans. CONCLUSION: This case emphasises that the lack of in vitro activity of voriconazole against zygomycetes is clinically relevant. Failure of voriconazole in suspected fungal infection should be investigated for the possibility of zygomycosis. Broad-range polymerase chain reaction may be able to identify the causative organism when cultures remain sterile.