From Investigating a Case of Cellulitis to Exploring Nosocomial Infection Control of ST1 Legionella pneumophila Using Genomic Approaches.

Legionella pneumophila can cause a large panel of symptoms besides the classic pneumonia presentation. Here we present a case of fatal nosocomial cellulitis in an immunocompromised patient followed, a year later, by a second case of Legionnaires' disease in the same ward. While the first case was easily assumed as nosocomial based on the date of symptom onset, the second case required clear typing results to be assigned either as nosocomial and related to the same environmental source as the first case, or community acquired. To untangle this specific question, we applied core-genome multilocus typing (MLST), whole-genome single nucleotide polymorphism and whole-genome MLST methods to a collection of 36 Belgian and 41 international sequence-type 1 (ST1) isolates using both thresholds recommended in the literature and tailored threshold based on local epidemiological data. Based on the thresholds applied to cluster isolates together, the three methods gave different results and no firm conclusion about the nosocomial setting of the second case could been drawn. Our data highlight that despite promising results in the study of outbreaks and for large-scale epidemiological investigations, next-generation sequencing typing methods applied to ST1 outbreak investigation still need standardization regarding both wet-lab protocols and bioinformatics. A deeper evaluation of the L. pneumophila evolutionary clock is also required to increase our understanding of genomic differences between isolates sampled during a clinical infection and in the environment.

Successful Treatment of Spondylodiscitis and Infectious Sacroiliitis due to Listeria monocytogenes using Meropenem as Salvage Therapy.

Infectious sacroiliitis is a rare disease that can occur in conjunction with other osteoarticular infections. Furthermore, it is very unusual for Listeria monocytogenes to be identified in an osteoarticular infection, with spondylodiscitis being the most frequent such infection. We report a case of spondylodiscitis with infectious sacroiliitis due to infection with L. monocytogenes treated successfully with meropenem as salvage therapy. LEARNING POINTS: It is very unusual for Listeria monocytogenes to be identified in an osteoarticular infection.L. monocytogenes can cause infectious sacroiliitis, which is a rare disease.Meropenem has been proposed as salvage therapy and was successful in our case.

Mortality Rate and Predictors Among Patients with COVID-19 Related Acute Respiratory Failure Requiring Mechanical Ventilation: a Retrospective Single Centre Study.

AIM: The objective of the study was to assess mortality rates in COVID-19 patients suffering from acute respiratory distress syndrome (ARDS) who also requiring mechanical ventilation. The predictors of mortality in this cohort were analysed, and the clinical characteristics recorded. MATERIAL AND METHOD: A single centre retrospective study was conducted on all COVID-19 patients admitted to the intensive care unit of the Epicura Hospital Center, Province of Hainaut, Belgium, between March 1st and April 30th 2020. RESULTS: Forty-nine patients were included in the study of which thirty-four were male, and fifteen were female. The mean (SD) age was 68.8 (10.6) and 69.5 (12.6) for males and females, respectively. The median time to death after the onset of symptoms was eighteen days. The median time to death, after hospital admission was nine days. By the end of the thirty days follow-up, twenty-seven patients (55%) had died, and twenty-two (45%) had survived. Non-survivors, as compared to those who survived, were similar in gender, prescribed medications, COVID-19 symptoms, with similar laboratory test results. They were significantly older (p = 0.007), with a higher co-morbidity burden (p = 0.026) and underwent significantly less tra-cheostomy (p < 0.001). In multivariable logistic regression analysis, no parameter significantly predicted mortality. CONCLUSIONS: This study reported a mortality rate of 55% in critically ill COVID-19 patients with ARDS who also required mechanical ventilation. The results corroborate previous findings that older and more comorbid patients represent the population at most risk of a poor outcome in this setting.

Exposure to SARS-CoV-2 in Hospital Environment: Working in a COVID-19 Ward Is a Risk Factor for Infection.

Aims. Health care workers (HCWs) are at risk of acquiring the Severe Acute Respiratory Syndrome Coronavirus 2 Infection (SARS-CoV-2). The aim of the study is to determine the SARS-CoV-2 positivity rates during the first epidemiologic peak among HCWs of a south Belgian hospital and to identify risks factors for infection. Methods. All hospital staff who worked during the first epidemiological peak were asked to answer a questionnaire regarding demographical data, function, type of working unit, type of contact with patients, eventual symptomatology, and the positivity of reverse transcription-polymerase chain reaction (RT-PCR) testing or immunoassay. Results. A total of 235 questionnaires were collected; 90 (38%) HCWs tested positive for SARS-CoV-2 from either RT-PCR or immunoassay testing. The positivity rate of HCWs between wards was statistically different (p = 0.004) and was higher in COVID-19 wards than Intensive Care Unit (ICU) and Emergency Department (ED). A total of 114 (49%) HCWs presented SARS-CoV-2-compatible symptomatology; 79 (88%) were positive on either RT-PCR or immunoassay testing; 74 (37%) HCWs were unable to work during the studied period; 5 were hospitalized. No deaths were reported. Multivariate logistic regression modeling showed that having symptoms was highly associated with test positivity (OR 23.3, CI 11.1, 53.1, p-value < 0.001). Working in a COVID-19 ward against working in ICU or ED was also predictive of positivity among HCWs (OR 3.25, CI 1.50, 7.28, p-value = 0.003). Discussion and Conclusions. This study shows a higher positivity rate compared to already reported positivity rates among HCWs. Reported differences in positivity rates depend on many factors, such as local crisis intensity, screening strategy, training in use of self-protective equipment, and study selection bias. HCWs working in COVID-19 wards, in comparison to ED and ICU, seemed at greater risk of being infected in this study. This could be explained by the disparity of HCWs' experience in handling self-protective equipment and knowledge in infection prevention. Hence, care should be taken in proper training for less-experienced HCWs during hospital epidemics. The latter could increase HCWs' protection and consequently decrease work absenteeism, ensuring enhanced continuity of patient care during hospital crisis. Rapid quarantine of symptomatic HCWs could reduce contamination rates, as having symptoms was highly associated with test positivity in this study.

Predictive Accuracy of COVID-19 World Health Organization (WHO) Severity Classification and Comparison with a Bayesian-Method-Based Severity Score (EPI-SCORE).

Assess the predictive accuracy of the WHO COVID-19 severity classification on COVID-19 hospitalized patients. The secondary aim was to compare its predictive power with a new prediction model, named COVID-19 EPI-SCORE, based on a Bayesian network analysis. Methods: We retrospectively analyzed a population of 295 COVID-19 RT-PCR positive patients hospitalized at Epicura Hospital Center, Belgium, admitted between March 1st and April 30th, 2020. Results: Our cohort's median age was 73 (62-83) years, and the female proportion was 43%. All patients were classified following WHO severity classification at admission. In total, 125 (42.4%) were classified as Moderate, 69 (23.4%) as Severe, and 101 (34.2%) as Critical. Death proportions through these three classes were 11.2%, 33.3%, and 67.3%, respectively, and the proportions of critically ill patients (dead or needed Invasive Mechanical Ventilation) were 11.2%, 34.8%, and 83.2%, respectively. A Bayesian network analysis was used to create a model to analyze predictive accuracy of the WHO severity classification and to create the EPI-SCORE. The six variables that have been automatically selected by our machine learning algorithm were the WHO severity classification, acute kidney injury, age, Lactate Dehydrogenase Levels (LDH), lymphocytes and activated prothrombin time (aPTT). Receiver Operation Characteristic (ROC) curve indexes hereby obtained were 83.8% and 91% for the models based on WHO classification only and our EPI-SCORE, respectively. Conclusions: Our study shows that the WHO severity classification is reliable in predicting a severe outcome among COVID-19 patients. The addition to this classification of a few clinical and laboratory variables as per our COVID-19 EPI-SCORE has demonstrated to significantly increase its accuracy.

Hydroxychloroquine in a G6PD-Deficient Patient with COVID-19 Complicated by Haemolytic Anaemia: Culprit or Innocent Bystander?

Hydroxychloroquine has been used worldwide as a first-line treatment for patients hospitalized with COVID-19. Little is known about COVID-19 and its effects on patients with congenital red blood cell disorders. We report a case of haemolytic anaemia in a 32-year-old patient and a fortuitous highlighting of G6PD deficiency. We reviewed the literature to assess the risk of hydroxychloroquine use in this context. LEARNING POINTS: A rapid drop in haemoglobin in COVID-19 patients should alert physicians to test for haemolytic anaemia and enzymopathies.Our review of the literature shows that use of hydroxychloroquine is safe in G6PD-deficient patients.

Sudden Development of Subcutaneous Nodules Shortly after Radioiodine Treatment for Thyroid Cancer Caused by Self-Limiting Sarcoidosis.

BACKGROUND: Sarcoidosis is a multisystemic disease producing non-caseating granulomas. The aetiology and pathogenesis are unknown. We herewith report an atypical case of cutaneous sarcoidosis. CASE PRESENTATION: A 50-year-old female presented with an onset of multiple subcutaneous nodules on her 4 limbs. These nodules appeared concomitantly with the initiation of radioactive iodine therapy for papillary thyroid cancer. These nodules were not obvious on inspection of the skin, but easily felt on palpation.The biopsy of the subcutaneous nodules revealed hypodermic non-caseating granulomas consistent with sarcoidosis. The patient underwent an 18F-fluorodeoxyglucose positron emission tomography (PET) scan study that revealed, besides the subcutaneous nodules, multiple hypermetabolic mediastinal lymphadenopathies and cervical adenopathies. Biopsy of the mediastinal lymphadenopathy showed neither granulomas nor neoplastic cells. Cervical biopsy revealed neoplastic cells of thyroid origin. Laboratory tests were normal. Bronchoalveolar lavage showed a normal CD4/CD8 T-cell ratio.A diagnosis of cutaneous sarcoidosis was established, as well as a recurrence of the cancerous disease. The subcutaneous nodules regressed spontaneously in the absence of any treatment. DISCUSSION AND CONCLUSION: Sarcoidosis is a multisystemic disease of unknown origin. This case illustrates an uncommon occurrence of sarcoidosis, triggered by radioactive iodine therapy. Radioiodine may lead to immunological changes, especially affecting the Th1/Th2 ratio, which may promote the emergence of sarcoidosis in genetically predisposed patients. There is still much to discover to fully understand the pathogenesis of sarcoidosis. LEARNING POINTS: The immunopathogenesis of sarcoidosis is poorly understood, as well as the environmental factors involved.Radioactive iodine therapy for thyroid cancer treatment may be an environmental trigger.The immunological changes induced by radioiodine, especially with respect to the Th1/Th2 ratio, may promote occurrence of sarcoidosis in genetically predisposed patients.

Objective Olfactory Findings in Hospitalized Severe COVID-19 Patients.

OBJECTIVE: We investigate the prevalence of the self-reported and objective sudden loss of smell (SLS) in patients with severe coronavirus disease 2019 (COVID-19). METHODS: Severe COVID-19 patients with self-reported SLS were recruited at hospitalization discharge. Epidemiological and clinical data were collected. The Sino-nasal Outcome Test-22 (SNOT-22) was used to evaluate rhinological complaints. Subjective olfactory and gustatory functions were assessed with the National Health and Nutrition Examination Survey (NHNES). Objective SLS was evaluated using psychophysical tests. Potential associations between olfactory evaluation and the clinical outcomes (duration of hospitalization; admission biology; one month serology (IgG), and chest computed tomography findings) were studied. RESULTS: Forty-seven patients completed the study (25 females). Subjectively, eighteen (38.3%) individuals self-reported subjective partial or total SLS. Among them, only three and four were anosmic and hyposmic, respectively (38.9%). Considering the objective evaluation in the entire cohort, the prevalence of SLS was 21.3%. Elderly patients and those with diabetes had lower objective olfactory evaluation results than young and non-diabetic individuals. CONCLUSIONS: The prevalence of SLS in severe COVID-19 patients appears to be lower than previously estimated in mild-to-moderate COVID-19 forms. Future comparative studies are needed to explore the predictive value of SLS for COVID-19 severity.

Clinical and epidemiological characteristics of 1420 European patients with mild-to-moderate coronavirus disease 2019.

BACKGROUND: The clinical presentation of European patients with mild-to-moderate COVID-19 infection is still unknown. OBJECTIVE: To study the clinical presentation of COVID-19 in Europe. METHODS: Patients with positive diagnosis of COVID-19 were recruited from 18 European hospitals. Epidemiological and clinical data were obtained through a standardized questionnaire. Bayesian analysis was used for analysing the relationship between outcomes. RESULTS: A total of 1,420 patients completed the study (962 females, 30.7% of healthcare workers). The mean age of patients was 39.17 ± 12.09 years. The most common symptoms were headache (70.3%), loss of smell (70.2%), nasal obstruction (67.8%), cough (63.2%), asthenia (63.3%), myalgia (62.5%), rhinorrhea (60.1%), gustatory dysfunction (54.2%) and sore throat (52.9%). Fever was reported by 45.4%. The mean duration of COVID-19 symptoms of mild-to-moderate cured patients was 11.5 ± 5.7 days. The prevalence of symptoms significantly varied according to age and sex. Young patients more frequently had ear, nose and throat complaints, whereas elderly individuals often presented fever, fatigue and loss of appetite. Loss of smell, headache, nasal obstruction and fatigue were more prevalent in female patients. The loss of smell was a key symptom of mild-to-moderate COVID-19 patients and was not associated with nasal obstruction and rhinorrhea. Loss of smell persisted at least 7 days after the disease in 37.5% of cured patients. CONCLUSION: The clinical presentation of mild-to-moderate COVID-19 substantially varies according to the age and the sex characteristics of patients. Olfactory dysfunction seems to be an important underestimated symptom of mild-to-moderate COVID-19 that needs to be recognized as such by the WHO.

An Atypical Case of Vasculitis: When 'Occult' 18FDG-PET Scan Findings Create a Classification Dilemma.

We describe a 66-year old patient with a recurrent ulcer on her right ankle. Biopsy revealed medium-vessel vasculitis consistent with cutaneous polyarteritis nodosa. There were no signs or symptoms suggestive of systemic vasculitis, but a 18FDG-PET scan showed areas of increased uptake around the large arteries and the pelvic and shoulder girdles. These findings suggested polymyalgia rheumatica in the setting of large-vessel vasculitis. This case thus supports the statement from the Chapel-Hill consensus conference that classification of systemic vasculitis by vessel size is based on the vessels predominantly involved, but vessels of other sizes may also be affected. LEARNING POINTS: The classification of vasculitis needs further refinement as some patients may not easily fit into just one category.Conditions that include aspects of different vasculitis also exist and cannot be classified using the Chapel Hill nomenclature.The role of PET scan in the assessment of PAN needs further evaluation.

Heparin-binding, hemagglutinin-specific IFN-gamma synthesis at the site of infection during active tuberculosis in humans.

RATIONALE: Tuberculosis (TB) remains a major cause of mortality. A better understanding of the immune responses to mycobacterial antigens may be helpful to develop improved vaccines and diagnostics. OBJECTIVES: The mycobacterial antigen heparin-binding hemagglutinin (HBHA) induces strong IFN-γ responses by circulating lymphocytes from subjects latently infected with Mycobacterium tuberculosis, and low responses associated with CD4(+) regulatory T (Treg) cells in patients with TB. Here, we investigated HBHA-specific IFN-γ responses at the site of the TB disease. METHODS: Bronchoalveolar lavages, pleural fluids, and blood were prospectively collected from 61 patients with a possible diagnosis of pulmonary or pleural TB. HBHA-specific IFN-γ production was analyzed by flow cytometry and ELISA. The suppressive effect of pleural Treg cells was investigated by depletion experiments. MEASUREMENTS AND MAIN RESULTS: The percentages of HBHA-induced IFN-γ(+) alveolar and pleural lymphocytes were higher for pulmonary (P < 0.0001) and for pleural (P < 0.01) TB than for non-TB controls. Local CD4(+) and CD8(+) T cells produced the HBHA-specific IFN-γ. This local secretion was not suppressed by Treg lymphocytes, contrasting with previously reported data on circulating lymphocytes. CONCLUSIONS: Patients with TB display differential effector and regulatory T-cell responses to HBHA in local and circulating lymphocytes with a predominant effector CD4(+) and CD8(+) response locally, compared with a predominant Treg response among circulating lymphocytes. These findings may be helpful for the design of new vaccines against TB, and the detection of HBHA-specific T cells at the site of the infection may be a promising tool for the rapid diagnosis of active TB.

Heparin-binding-hemagglutinin-induced IFN-gamma release as a diagnostic tool for latent tuberculosis.

BACKGROUND: The detection of latent tuberculosis infection (LTBI) is a major component of tuberculosis (TB) control strategies. In addition to the tuberculosis skin test (TST), novel blood tests, based on in vitro release of IFN-gamma in response to Mycobacterium tuberculosis-specific antigens ESAT-6 and CFP-10 (IGRAs), are used for TB diagnosis. However, neither IGRAs nor the TST can separate acute TB from LTBI, and there is concern that responses in IGRAs may decline with time after infection. We have therefore evaluated the potential of the novel antigen heparin-binding hemagglutinin (HBHA) for in vitro detection of LTBI. METHODOLOGY AND PRINCIPAL FINDINGS: HBHA was compared to purified protein derivative (PPD) and ESAT-6 in IGRAs on lymphocytes drawn from 205 individuals living in Belgium, a country with low TB prevalence, where BCG vaccination is not routinely used. Among these subjects, 89 had active TB, 65 had LTBI, based on well-standardized TST reactions and 51 were negative controls. HBHA was significantly more sensitive than ESAT-6 and more specific than PPD for the detection of LTBI. PPD-based tests yielded 90.00% sensitivity and 70.00% specificity for the detection of LTBI, whereas the sensitivity and specificity for the ESAT-6-based tests were 40.74% and 90.91%, and those for the HBHA-based tests were 92.06% and 93.88%, respectively. The QuantiFERON-TB Gold In-Tube (QFT-IT) test applied on 20 LTBI subjects yielded 50% sensitivity. The HBHA IGRA was not influenced by prior BCG vaccination, and, in contrast to the QFT-IT test, remote (>2 years) infections were detected as well as recent (<2 years) infections by the HBHA-specific test. CONCLUSIONS: The use of ESAT-6- and CFP-10-based IGRAs may underestimate the incidence of LTBI, whereas the use of HBHA may combine the operational advantages of IGRAs with high sensitivity and specificity for latent infection.

Regulatory T cells depress immune responses to protective antigens in active tuberculosis.

RATIONALE: Tuberculosis (TB) remains a leading cause of death, and the role of T-cell responses to control Mycobacterium tuberculosis infections is well recognized. Patients with latent TB infection develop strong IFN-gamma responses to the protective antigen heparin-binding hemagglutinin (HBHA), whereas patients with active TB do not. OBJECTIVES: We investigated the mechanism of this difference and evaluated the possible involvement of regulatory T (Treg) cells and/or cytokines in the low HBHA T-cell responses of patients with active TB. METHODS: The impact of anti-transforming growth factor (TGF)-beta and anti-IL-10 antibodies and of Treg cell depletion on the HBHA-induced IFN-gamma secretion was analyzed, and the Treg cell phenotype was characterized by flow cytometry. MEASUREMENTS AND MAIN RESULTS: Although the addition of anti-TGF-beta or anti-IL-10 antibodies had no effect on the HBHA-induced IFN-gamma secretion in patients with active TB, depletion of CD4(+)CD25(high)FOXP3(+) T lymphocytes resulted in the induction by HBHA of IFN-gamma concentrations that reached levels similar to those obtained for latent TB infection. No effect was noted on the early-secreted antigen target-6 or candidin T-cell responses. CONCLUSIONS: Specific CD4(+)CD25(high)FOXP3(+) T cells depress the T-cell-mediated immune responses to the protective mycobacterial antigen HBHA during active TB in humans.

Heparin-binding hemagglutinin, from an extrapulmonary dissemination factor to a powerful diagnostic and protective antigen against tuberculosis.

Interactions of Mycobacterium tuberculosis with macrophages have long been recognized to be crucial to the pathogenesis of tuberculosis. The role of non-phagocytic cells is less well known. We have discovered a M. tuberculosis surface protein that interacts specifically with non-phagocytic cells, expresses hemagglutination activity and binds to sulfated glycoconjugates. It is therefore called heparin-binding hemagglutinin (HBHA). HBHA-deficient M. tuberculosis mutant strains are significantly impaired in their ability to disseminate from the lungs to other tissues, suggesting that the interaction with non-phagocytic cells, such as pulmonary epithelial cells, may play an important role in the extrapulmonary dissemination of the tubercle bacillus, one of the key steps that may lead to latency. Latently infected human individuals mount a strong T cell response to HBHA, whereas patients with active disease do not, suggesting that HBHA is a good marker for the immunodiagnosis of latent tuberculosis, and that HBHA-specific Th1 responses may contribute to protective immunity against active tuberculosis. Strong HBHA-mediated immuno-protection was shown in mouse challenge models. HBHA is a methylated protein and its antigenicity in latently infected subjects, as well as its protective immunogenicity strongly depends on the methylation pattern of HBHA. In both mice and man, the HBHA-specific IFN-gamma was produced by both the CD4(+) and the CD8(+) T cells. Furthermore, the HBHA-specific CD8(+) T cells expressed bactericidal and cytotoxic activities to mycobacteria-infected macrophages. This latter activity is most likely perforin mediated. Together, these observations strongly support the potential of methylated HBHA as an important component in future, acellular vaccines against tuberculosis.

Effector functions of heparin-binding hemagglutinin-specific CD8+ T lymphocytes in latent human tuberculosis.

BACKGROUND: Most individuals infected with Mycobacterium tuberculosis do not develop tuberculosis (TB) and can be regarded as being protected by an appropriate immune response to the infection. The characterization of the immune responses of individuals with latent TB may thus be helpful in the definition of correlates of protection and the development of new vaccine strategies. The highly protective antigen heparin-binding hemagglutinin (HBHA) induces strong interferon (IFN)- gamma responses during latent, but not active, TB. Because of the recently recognized importance of CD8(+) T lymphocytes in anti-TB immunity, we characterized the CD8(+) T lymphocyte responses to HBHA in subjects with latent TB. RESULTS: HBHA-specific CD8(+) T lymphocytes expressed memory cell markers and synthesized HBHA-specific IFN- gamma . They also restricted mycobacterial growth and expressed cytotoxicity by a granule-dependent mechanism. This activity was associated with the intracellular expression of HBHA-induced perforin. Surprisingly, the perforin-producing CD8(+) T lymphocytes were distinct from the IFN- gamma -producing CD8(+) T lymphocytes. CONCLUSION: During latent TB, the HBHA-specific CD8(+) T lymphocyte population expresses all 3 effector functions associated with CD8(+) T lymphocyte-mediated protective immune mechanisms, which supports the notion that HBHA may be protective in humans and suggests that markers of HBHA-specific CD8(+) T lymphocyte responses may be useful in the monitoring of protection.

Optimal kinetics for quantification of antigen-induced cytokines in human peripheral blood mononuclear cells by real-time PCR and by ELISA.

Real-time polymerase chain reaction (PCR) has recently been described as a new tool to measure and accurately quantify mRNA levels. In this study, we have applied this technique to evaluate cytokine mRNA synthesis induced by antigenic stimulation with purified protein derivative (PPD) or heparin-binding haemagglutinin (HBHA) in human peripheral blood mononuclear cells (PBMC) from Mycobacterium tuberculosis-infected individuals. Whereas PPD and HBHA optimally induced IL-2 mRNA after respectively 8 and 16 to 24 h of in vitro stimulation, longer in vitro stimulation times were necessary for optimal induction of interferon-gamma (IFN-gamma) mRNA, respectively 16 to 24 h for PPD and 24 to 96 h for HBHA. IL-13 mRNA was optimally induced by in vitro stimulation after 16-48 h for PPD and after 48 to 96 h for HBHA. Comparison of antigen-induced Th1 and Th2 cytokines appears, therefore, valuable only if both cytokine types are analysed at their optimal time point of production, which, for a given cytokine, may differ for each antigen tested. Results obtained by real-time PCR for IFN-gamma and IL-13 mRNA correlated well with those obtained by measuring the cytokine concentrations in cell culture supernatants, provided they were high enough to be detected. We conclude that real-time PCR can be successfully applied to the quantification of antigen-induced cytokine mRNA and to the evaluation of the Th1/Th2 balance, only if the kinetics of cytokine mRNA appearance are taken into account and evaluated for each cytokine measured and each antigen analysed.