Invasive fungal infections in immunocompromised hosts : epidemiology and diagnostics

This doctoral project aimed to attain a more in-depth knowledge of the epidemiology and diagnosis of invasive fungal infections. In Study I, we evaluated the diagnostic performance of PCR/ESI-MS. This novel broad- spectrum molecular test combines multiplex PCR with electrospray ionisation for diagnosing mould lung infections in haematological patients. We analysed bronchoalveolar lavage samples from 27 patients, and PCR/ESI-MS was able to correctly identify all cases of proven and probable invasive fungal infection. It also detected additional cases missed by the standard diagnostic tests, including four cases of Mucorales. PCR/ESI-MS had a clinical impact on the antifungal therapy in 44% of the cases. In Study II, we prospectively investigated the incidence of influenza-associated pulmonary aspergillosis (IAPA) in 12 Swedish intensive care units. Patients were screened for IAPA at inclusion using serum Galactomannan, β-d-Glucan, and a respiratory sample for fungal culture. Bronchoalveolar lavage was performed when feasible. The study included 55 patients, of which five (9%) were classified as probable IAPA according to a recently published case definition. An important finding was that four of these patients lacked traditional risk factors. Patients diagnosed with IAPA had a statistically higher ICU mortality, 60%, compared to 8% in patients without IAPA. Our study is the first prospective study to confirm the association between influenza and IAPA and the first to report data from the Nordic region. Based on our findings, we recommend implementing screening for IAPA for all influenza patients receiving mechanical ventilation in Sweden. In Study III, we present the development of a novel porcine model of invasive candidiasis using monitoring and interventions relevant to intensive care. The final model utilised corticosteroids, repeated Candida inoculation, and continuous endotoxin to simulate the clinical situation where invasive candidiasis occurs. The model consistently demonstrated quantifiable growth of Candida in blood and organs. The proposed model could be a valuable tool for studying pathophysiology and testing new therapies for invasive candidiasis. In Study IV, we prospectively evaluated the efficacy of the commercial T2Candida test for diagnosing intraabdominal candidiasis in surgical patients admitted to intensive care. T2Candida is a new direct-on-blood test that combines PCR with magnetic resonance targeting five clinically relevant Candida species and delivers a result within a few hours. In total, 134 patients were tested with T2Candida and 1,3-β-D-glucan parallel to blood cultures. T2Candida had lower sensitivity for non-candidemia intraabdominal candidiasis compared to 1,3-β-D-glucan, but comparable performance for intraabdominal candidiasis with concurrent candidemia. Still, T2Candida could serve as a valuable complement to 1,3-β-D-glucan in situations where a rapid result can help guide treatment or surgical intervention. In Study V, we retrospectively evaluated the efficacy of reduced-dose trimethoprim- sulfamethoxazole (7.5-15 mg trimethoprim /kg/day) compared to the standard dose (>15-20 mg trimethoprim/kg/day) for treating Pneumocystis jirovecii pneumonia in haematological patients. The study included 113 patients from six Swedish University hospitals, with 80 patients receiving reduced and 33 receiving the standard dose. The groups had no clinically relevant or statistically significant difference in respiratory function or mortality. In patients with mild to moderate pneumonia, defined as PaO2/FiO2 >200 mmHg, all 44 patients receiving the reduced dose were alive at 30 days. The results indicate that the reduced dose is effective and safe for treating mild to moderate Pneumocystis jirovecii pneumonia

No findings of SARS-CoV-2 in conjunctival swabs from patients at an emergency outpatient ophthalmological healthcare facility in a Swedish county hospital : a cross-sectional study

Background COVID-19 is caused by SARS-CoV-2. Virus has been found in conjunctiva of hospitalised patients with COVID-19. Conjunctivitis has also been reported as a presenting symptom of disease. Objective The aims of the study were to investigate the prevalence of SARS-CoV-2 in the conjunctiva and throat among patients presenting at the emergency outpatient ophthalmological healthcare facility at a county hospital along with investigating the seroprevalence of SARS-CoV-2 among staff at the department. Methods and Analysis Swabs from conjunctiva and throat of patients were analysed with real-time reverse transcriptase PCR (RT-PCR) for SARS-CoV-2. Blood samples for serological analysis were obtained from staff. A questionnaire was used to investigate symptoms associated with COVID-19 during the last 3 months as well as symptoms for which the patients were seeking ophthalmological healthcare. Results In total, 68 patients and 70 individuals from the staff were included in the study. Conjunctivitis was observed in 7% of patients. One patient, presenting with reduced visual acuity due to preretinal haemorrhage in the macula, was positive for SARS-CoV-2 in throat swab. Contact tracing was negative. All other RT-PCR tests were negative. Seropositivity for SARS-CoV-2 was found in 4% of staff. Conclusions Our study demonstrated low prevalence of SARS-CoV-2 among patients as well as low seroprevalence of SARS-CoV-2 IgG-antibodies among staff at the ophthalmological ward. The risk for contracting COVID-19 at the department was small. Follow-up investigation is planned

An experimental porcine model of invasive candidiasis

Abstract Background Invasive candidiasis (IC) is a severe and often fatal fungal infection that affects critically ill patients. The development of animal models that mimic human disease is essential for advancing our understanding of IC pathophysiology and testing experimental or novel treatments. We aimed to develop a large animal model of IC that could provide a much-needed addition to the widely used murine models. Results A total of 25 pigs (including one control), aged between 9 and 12 weeks, with a median weight of 25.1 kg (IQR 24.1–26.2), were used to develop the porcine IC model. We present the setup, the results of the experiments, and the justification for the changes made to the model. The experiments were conducted in an intensive care setting, using clinically relevant anaesthesia, monitoring and interventions. The final model used corticosteroids, repeated Candida inoculation, and continuous endotoxin. The model consistently demonstrated quantifiable growth of Candida in blood and organs. The registered physiological data supported the development of the sepsis-induced circulatory distress observed in IC patients in the ICU. Conclusions Our proposed porcine model of IC offers a potential new tool in the research of IC

The kinetics of SARS-CoV-2 viremia in COVID-19 patients receiving remdesivir

Detection of SARS-CoV-2 RNA in serum, viremia, has been linked to disease severity and outcome. The kinetics of viremia in patients receiving remdesivir has not been thoroughly studied and could help predict treatment response and outcome. We investigated the kinetics of SARS-CoV-2 viremia and factors associated with baseline viremia, viral clearance and 30-day mortality in patients receiving remdesivir. An observational study including 378 hospitalised patients (median age 67 years, 67% male) sampled with serum SARS-CoV-2 RT-PCR within +/- 24 h of initiation of remdesivir treatment. Baseline viremia was present in 206 (54%) patients with a median Ct value of 35.3 (IQR = 33.3-37.1). In patients with baseline viremia, the estimated probability of viral clearance was 72% by day 5. Ct values decreased significantly during remdesivir treatment for viremic patients, indicating an increase in viral load. In total, 44 patients (12%) died within 30 days, and mortality was significantly associated with viremia at baseline (OR = 2.45, p = 0.01) and lack of viral clearance by day 5 (OR = 4.8, p = < 0.01). Viral clearance was not associated with any individual risk factor. Viremia appears to be a prognostic marker before and during remedesivir treatment. The resolution of viremia was similar to patients not receiving remdesivir in other studies, and the decrease in Ct values during treatment questions the antiviral capacity of remdesivir in vivo. Prospective studies are warranted to confirm our findings

Low rate of COVID-19 seroconversion in health-care workers at a Department of Infectious Diseases in Sweden during the later phase of the first wave; a prospective longitudinal seroepidemiological study

Background: Health-care workers are at risk of contracting and transmitting SARS-CoV-2. The aim of this study was to investigate the prevalence of SARS-CoV-2 IgG antibodies and the rate of seroconversion in an environment with high exposure to SARS-CoV-2. Methods: 131 health-care workers at the Department of Infectious Diseases in Vasteras, Sweden, were included in the study. Abbott's SARS-COV-2 IgG immunoassay was used with a signal cut-off ratio of >= 1.4. Every third week from the beginning of May, blood samples were drawn, and the participants completed a questionnaire regarding symptoms consistent with COVID-19 and the result of any SARS-CoV-2 PCR performed since the last sampling occasion. Participants with IgG antibodies against SARS-CoV-2 were re-sampled only on the sixth and last occasion. Results: At the start of the study, 18 (15%) participants had SARS-CoV-2 IgG antibodies. At the end, 25 (19%) of 131 participants were seropositive. One case of asymptomatic infection was detected, and two cases with PCR-confirmed COVID-19 did not develop IgG antibodies. Conclusion: The low rate of seroconversion during the study suggests that it is possible to prevent transmission of SARS-COV-2 in a high-exposure environment. Compliance with adequate infection control guidelines is the likely explanation of our findings

Pre-exposure to mechanical ventilation and endotoxemia increases Pseudomonas aeruginosa growth in lung tissue during experimental porcine pneumonia

Background: Immune system suppression during critical care contributes to the risk of acquired bacterial infections with Pseudomonas (P.) aeruginosa. Repeated exposure to endotoxin can attenuate systemic inflammatory cytokine responses. Mechanical ventilation affects the systemic inflammatory response to various stimuli. Aim: To study the effect of pre-exposure to mechanical ventilation with and without endotoxin-induced systemic inflammation on P. aeruginosa growth and wet-to-dry weight measurements on lung tissue and plasma and bronchoalveolar lavage levels of tumor necrosis factor alpha, interleukins 6 and 10. Methods: Two groups of pigs were exposed to mechanical ventilation for 24 hours before bacterial inoculation and six h of experimental pneumonia (total experimental time 30 h): A(30h+Etx) (n = 6, endotoxin 0.063 mu g x kg(-1) x h(-1)) and B-30h (n = 6, saline). A third group, C-6h (n = 8), started the experiment at the bacterial inoculation unexposed to endotoxin or mechanical ventilation (total experimental time 6 h). Bacterial inoculation was performed by tracheal instillation of 1x10(11) colony-forming units of P. aeruginosa. Bacterial cultures and wet-to-dry weight ratio analyses were done on lung tissue samples postmortem. Separate group comparisons were done between A(30h+Etx) vs.B-30h (Inflammation) and B-30h vs. C-6h (Ventilation Time) during the bacterial phase of 6 h. Results: P. aeruginosa growth was highest in A(30h+Etx), and lowest in C-6h (Inflammation and Ventilation Time both p<0.05). Lung wet-to-dry weight ratios were highest in A(30h+Etx) and lowest in B-30h (Inflammation p<0.01, Ventilation Time p<0.05). C-6h had the highest TNF-alpha levels in plasma (Ventilation Time p<0.01). No differences in bronchoalveolar lavage variables between the groups were observed. Conclusions: Mechanical ventilation and systemic inflammation before the onset of pneumonia increase the growth of P. aeruginosa in lung tissue. The attenuated growth of P. aeruginosa in the non-pre-exposed animals (C-6h) was associated with a higher systemic TNF-alpha production elicited from the bacterial challenge

The clinical impact of implementing GenMark ePlex blood culture panels for around-the-clock blood culture identification; a prospective observational study

Background:Implementing rapid molecular blood culture diagnostics in the clinical management of sepsis is essential for early pathogen identification and resistance gene testing. The GenMark ePlex blood culture panels offer a broad microbial spectrum with minimal hands-on time and approximately 1.5 h to result. Therefore, ePlex can be utilized at times when the clinical microbiology laboratory is unavailable. Methods:From 23 October 2019 to 30 December 2019, consecutive non-duplicate positive blood cultures signalling microbial growth at the 24 h/7 days-a-week available clinical chemistry laboratory between 9 pm and 7 am were analysed with ePlex. All blood cultures were transported to the microbiology laboratory the following day for conventional identification and antibiotic susceptibility testing. Results:We used ePlex to test 91 blood cultures, of which 86 had confirmed microbial growth. Eighty-one were positive for ePlex target pathogens. The ePlex results were in complete agreement with conventional methods in 72/81 (88.9%) of cases and available within a median of 10.9 h earlier. Resistance gene targets (11mecA and 1 CTX-M) were concordant with phenotypic susceptibility in all cases. In 18/86 (20.9%) of the patient cases, there was an opportunity to optimize antimicrobial therapy based on the ePlex result. The ePlex result affected clinical decision-making in 4/86 (4.7%) of the cases and reduced the average time to effective antimicrobial therapy by 8.9 h. Conclusions:Our implementation of ePlex is a feasible option to attain around-the-clock blood culture identification in many hospitals. It can significantly reduce time-to-pathogen identification and have an impact on clinical decision-making

PCR with electrospray ionization-mass spectrometry on bronchoalveolar lavage for detection of invasive mold infections in hematological patients.

Invasive mold infections are life-threatening complications in patients with hematological malignancies. Conventional microbiological methods for diagnosing invasive pulmonary mold infections have low sensitivity, and molecular methods are being developed. Detection of molds using PCR with a narrow spectrum has been reported, but data with broad-spectrum PCR are lacking. In this study, the diagnostic performance and utility of a broad-spectrum PCR (broad-spectrum PCR with subsequent electrospray ionization-mass spectrometry, PCR/ESI-MS) for detection of molds in bronchoalveolar lavage (BAL) in 27 hematological patients with a new pulmonary infiltrate was analyzed. Using the revised EORTC/MSG criteria, PCR/ESI-MS was the only positive microbiological test in patients with proven invasive mold infection (n = 2) and correctly identified all cases of probable invasive pulmonary aspergillosis (n = 5). In patients with a possible invasive mold infection (n = 5), PCR/ESI-MS was positive in three patients. Mucorales was identified with PCR/ESI-MS in four patients that were all culture negative. The PCR/ESI-MS results had a clinical impact on antifungal therapy in 12 (44%) of the patients: modification of treatment in 6 (22%) patients and discontinuation in 6 (22%) patients. This study provides proof of concept that routine use of a broad-spectrum PCR for molds in bronchoalveolar lavage in immunocompromised patients is sensitive, fast, and has an impact on clinical decision-making

Long-lasting T-cell response to SARS-CoV-2 antigens after vaccination-a prospective cohort study of HCWs working with COVID-19 patients

Background: Vaccination against SARS-CoV-2 reduces the risk of hospitalisation and death, but vaccine-induced IgG antibodies against the spike protein (IgG S) decline over time. Less is known about the nature of the vaccine-induced T-cell response to SARS-CoV-2 antigens. Methods: IgG antibodies against nucleocapsid protein (IgG N), IgG S, and T-cell response towards SARS-CoV-2 antigens were determined in samples taken between November 2020 and November 2021 from a cohort of healthcare workers at an Infectious Diseases Department. RT-PCR screening for SARS-CoV-2 was encouraged once every four weeks in addition to testing when symptomatic or identified through contact tracing. Vaccination data were collected at the end of the study. Results: At inclusion, T-cell response to SARS-CoV-2 antigens was found in 10/15 (66.7%) of participants with a previous/current COVID-19 infection and in 9/54 (16.7%) of participants with no prior/current history of COVID-19 infection. All participants with complete follow-up (n = 59) received two doses of a SARS-CoV-2 vaccine during the study. All participants demonstrated detectable IgG (S) antibodies at the end of the study, in median 278 days (IQR 112) after the second vaccine dose. All but four participants displayed T-cell responses towards SARS-CoV-2 antigens. IgG S antibody levels correlated with time since the second vaccine dose. In addition, previous COVID-19 infection and the strength of the S1 T-cell response correlated with IgG S antibody levels. However, no correlation was demonstrated between the strength of the T-cell response and time since the second vaccine dose. Conclusion: COVID-19 vaccination induces robust T-cell responses that remain for at least nine months