Risk factors for ventilator-associated lower respiratory tract infection in COVID-19, a retrospective multicenter cohort study in Sweden.

BACKGROUND: Ventilator-associated lower respiratory tract infections (VA-LRTI) increase morbidity and mortality in intensive care unit (ICU) patients. Higher incidences of VA-LRTI have been reported among COVID-19 patients requiring invasive mechanical ventilation (IMV). The primary objectives of this study were to describe clinical characteristics, incidence, and risk factors comparing patients who developed VA-LRTI to patients who did not, in a cohort of Swedish ICU patients with acute hypoxemic respiratory failure due to COVID-19. Secondary objectives were to decipher changes over the three initial pandemic waves, common microbiology and the effect of VA-LTRI on morbidity and mortality. METHODS: We conducted a multicenter, retrospective cohort study of all patients admitted to 10 ICUs in southeast Sweden between March 1, 2020 and May 31, 2021 because of acute hypoxemic respiratory failure due to COVID-19 and were mechanically ventilated for at least 48 h. The primary outcome was culture verified VA-LRTI. Patient characteristics, ICU management, clinical course, treatments, microbiological findings, and mortality were registered. Logistic regression analysis was conducted to determine risk factors for first VA-LRTI. RESULTS: Of a total of 536 included patients, 153 (28.5%) developed VA-LRTI. Incidence rate of first VA-LRTI was 20.8 per 1000 days of IMV. Comparing patients with VA-LRTI to those without, no differences in mortality, age, sex, or number of comorbidities were found. Patients with VA-LRTI had fewer ventilator-free days, longer ICU stay, were more frequently ventilated in prone position, received corticosteroids more often and were more frequently on antibiotics at intubation. Regression analysis revealed increased adjusted odds-ratio (aOR) for first VA-LRTI in patients treated with corticosteroids (aOR 2.64 [95% confidence interval [CI]] [1.31-5.74]), antibiotics at intubation (aOR 2.01 95% CI [1.14-3.66]), and days of IMV (aOR 1.05 per day of IMV, 95% CI [1.03-1.07]). Few multidrug-resistant pathogens were identified. Incidence of VA-LRTI increased from 14.5 per 1000 days of IMV during the first wave to 24.8 per 1000 days of IMV during the subsequent waves. CONCLUSION: We report a high incidence of culture-verified VA-LRTI in a cohort of critically ill COVID-19 patients from the first three pandemic waves. VA-LRTI was associated with increased morbidity but not 30-, 60-, or 90-day mortality. Corticosteroid treatment, antibiotics at intubation and time on IMV were associated with increased aOR of first VA-LRTI.

Reduced susceptibility of clinical strains of Mycobacterium tuberculosis to reactive nitrogen species promotes survival in activated macrophages.

BACKGROUND: Drugs such as isoniazid (INH) and pretomanid (PRT), used against Mycobacterium tuberculosis are active partly through generation of reactive nitrogen species (RNS). The aim of this study was to explore variability in intracellular susceptibility to nitric oxide (NO) in clinical strains of M. tuberculosis. METHOD: Luciferase-expressing clinical M. tuberculosis strains with or without INH resistance were exposed to RNS donors (DETA/NO and SIN-1) in broth cultures and bacterial survival was analysed by luminometry. NO-dependent intracellular killing in a selection of strains was assessed in interferon gamma/lipopolysaccharide-activated murine macrophages using the NO inhibitor L-NMMA. RESULTS: When M. tuberculosis H37Rv was compared to six clinical isolates and CDC1551, three isolates with inhA mediated INH resistance showed significantly reduced NO-susceptibility in broth culture. All strains showed a variable but dose-dependent susceptibility to RNS donors. Two clinical isolates with increased susceptibility to NO exposure in broth compared to H37Rv were significantly inhibited by activated macrophages whereas there was no effect on growth inhibition when activated macrophages were infected by clinical strains with higher survival to NO exposure in broth. Furthermore, the most NO-tolerant clinical isolate showed increased resistance to PRT both in broth culture and the macrophage model compared to H37Rv in the absence of mutational resistance in genes associated to reduced susceptibility against PRT or NO. CONCLUSION: In a limited number of clinical M. tuberculosis isolates we found a significant difference in susceptibility to NO between clinical isolates, both in broth cultures and in macrophages. Our results indicate that mycobacterial susceptibility to cellular host defence mechanisms such as NO need to be taken into consideration when designing new therapeutic strategies.

Asymptomatic Helminth Infection in Active Tuberculosis Is Associated with Increased Regulatory and Th-2 Responses and a Lower Sputum Smear Positivity.

BACKGROUND: The impact of intestinal helminth infection on the clinical presentation and immune response during active tuberculosis (TB) infection is not well characterized. Our aim was to investigate whether asymptomatic intestinal helminth infection alters the clinical signs and symptoms as well as the cell mediated immune responses in patients with active TB. METHODOLOGY: Consecutive, newly diagnosed TB patients and healthy community controls (CCs) were recruited in North-west Ethiopia. TB-score, body mass index and stool samples were analyzed. Cells from HIV-negative TB patients (HIV-/TB) and from CCs were analyzed for regulatory T-cells (Tregs) and cytokine responses using flow cytometry and ELISPOT, respectively. RESULTS: A significantly higher ratio of helminth co-infection was observed in TB patients without HIV (Helm+/HIV-/TB) compared to HIV negative CCs, (40% (121/306) versus 28% (85/306), p = 0.003). Helm+/HIV-/TB patients showed significantly increased IL-5 secreting cells compared to Helm-/HIV-/TB (37 SFU (IQR:13-103) versus 2 SFU (1-50); p = 0.02, n = 30). Likewise, levels of absolute Tregs (9.4 (3.2-16.7) cells/µl versus 2.4 (1.1-4.0) cells/µl; p = 0.041) and IL-10 secreting cells (65 SFU (7-196) versus 1 SFU (0-31); p = 0.014) were significantly higher in Helm+/HIV-/TB patients compared to Helm-/HIV-/TB patients. In a multivariate analysis, a lower rate of sputum smear positivity for acid fast bacilli, lower body temperature, and eosinophilia were independently associated with helminth infection in TB patients. CONCLUSIONS: Asymptomatic helminth infection is associated with increased regulatory T-cell and Th2-type responses and a lower rate of sputum smear positivity. Further studies are warranted to investigate the clinical and immunological impact of helminth infection in TB patients.

Vitamin D enhances IL-1ß secretion and restricts growth of Mycobacterium tuberculosis in macrophages from TB patients.

The emergence of multidrug-resistant strains of Mycobacterium tuberculosis (MTB), the bacterium responsible for tuberculosis (TB), has rekindled the interest in the role of nutritional supplementation of micronutrients, such as vitamin D, as adjuvant treatment. Here, the growth of virulent MTB in macrophages obtained from the peripheral blood of patients with and without TB was studied. The H37Rv strain genetically modified to express Vibrio harveyi luciferase was used to determine the growth of MTB by luminometry in the human monocyte-derived macrophages (hMDMs) from study subjects. Determination of cytokine levels in culture supernatants was performed using a flow cytometry-based bead array technique. No differences in intracellular growth of MTB were observed between the different study groups. However, stimulation with 100nM 1,25-dihydroxyvitamin D significantly enhanced the capacity of hMDMs isolated from TB patients to control the infection. This effect was not observed in hMDMs from the other groups. The interleukin (IL)-1ß and IL-10 release by hMDMs was clearly increased upon stimulation with 1,25-dihydroxyvitamin D. Furthermore, the 1,25-dihydroxyvitamin D stimulation also led to elevated levels of TNF-α (tumor necrosis factor-alpha) and IL-12p40. It was concluded that vitamin D triggers an inflammatory response in human macrophages with enhanced secretion of cytokines, as well as enhancing the capacity of hMDMs from patients with active TB to restrict mycobacterial growth.

The impact of asymptomatic helminth co-infection in patients with newly diagnosed tuberculosis in north-west Ethiopia.

BACKGROUND: Areas endemic of helminth infection, tuberculosis (TB) and HIV are to a large extent overlapping. The aim of this study was to assess the impact of asymptomatic helminth infection on the immunological response among TB patients with and without HIV, their house hold contacts and community controls. METHODOLOGY: Consecutive smear positive TB patients (n = 112), their household contacts (n = 71) and community controls (n = 112) were recruited in Gondar town, Ethiopia. Stool microscopy, HIV serology, serum IgE level, eosinophil and CD4 counts were performed and tuberculosis patients were followed up for 3 months after initiation of anti-TB treatment. RESULTS: Helminth co-infection rate was 29% in TB patients and 21% in both community control and household contacts (p = 0.3) where Ascaris lumbricoides was the most prevalent parasite. In TB patients the seroprevalence of HIV was 47% (53/112). Eosinophilia and elevated IgE level were significantly associated with asymptomatic helminth infection. During TB treatment, the worm infection rate of HIV+/TB patients declined from 31% (10/32) at week 0 to 9% (3/32) at week 2 of TB treatment, whereas HIV-/TB patients showed no change from baseline to week 2, 29% (13/45) vs. 22.2% (10/45). This trend was stable at week 8 and 12 as well. CONCLUSION: One third of smear positive TB patients were infected with helminths. Eosinophilia and elevated IgE level correlated with asymptomatic worm infection, indicating an effect on host immunity. The rate of worm infection declined during TB treatment in HIV+/TB co-infected patients whereas no decline was seen in HIV-/TB group.

Early treatment response evaluated by a clinical scoring system correlates with the prognosis of pulmonary tuberculosis patients in Ethiopia: a prospective follow-up study.

BACKGROUND: In resource-limited settings the monitoring of tuberculosis (TB) patients is challenging, and early identification of TB patients with a high mortality risk is important. The aim of this study was to investigate prospectively whether early changes in a clinical scoring system (TB score) can predict treatment outcome in Ethiopian patients with pulmonary tuberculosis. METHOD: TB patients (n = 250) and blood donors (n = 82) were recruited prospectively at Gondar University Hospital, Ethiopia. Clinical scoring was performed using an interview-based questionnaire and clinical examination. RESULTS: Among TB patients (53.6% of whom were HIV co-infected) the median TB score declined from week 0 to week 2 (8 (interquartile range (IQR) 6-9) vs 4 (IQR 2-6)) and dropped to a low level at week 8, which was still significantly higher than that found in blood donors (2 (IQR 1-4) vs 0 (IQR 0-1), p < 0.0001). Patients who died had a significantly higher TB score at week 0, week 2, and week 8 than survivors. Mortality was associated with a failure to achieve a decrease greater than 25% in the TB score at 2 weeks. Baseline CD4 + cell counts (< 200 cells/mm³) were associated with mortality but not with initial TB score results. CONCLUSIONS: The TB score was increased during the first 2 months of treatment among patients who died. Failure to achieve a greater than 25% decrease in TB score after 2 weeks of treatment was associated with increased mortality. Repeated clinical scoring during the intensive phase of TB treatment could be useful to identify high-risk patients.

Resistance to first-line anti-TB drugs is associated with reduced nitric oxide susceptibility in Mycobacterium tuberculosis.

BACKGROUND AND OBJECTIVE: The relative contribution of nitric oxide (NO) to the killing of Mycobacterium tuberculosis in human tuberculosis (TB) is controversial, although this has been firmly established in rodents. Studies have demonstrated that clinical strains of M. tuberculosis differ in susceptibility to NO, but how this correlates to drug susceptibility and clinical outcome is not known. METHODS: In this study, 50 sputum smear- and culture-positive patients with pulmonary TB in Gondar, Ethiopia were included. Clinical parameters were recorded and drug susceptibility profile and spoligotyping patterns were investigated. NO susceptibility was studied by exposing the strains to the NO donor DETA/NO. RESULTS: Clinical isolates of M. tuberculosis showed a dose- and time-dependent response when exposed to NO. The most frequent spoligotypes found were CAS1-Delhi and T3_ETH in a total of nine known spoligotypes and four orphan patterns. There was a significant association between reduced susceptibility to NO (>10% survival after exposure to 1 mM DETA/NO) and resistance against first-line anti-TB drugs, in particular isoniazid (INH). Patients infected with strains of M. tuberculosis with reduced susceptibility to NO showed no difference in cure rate or other clinical parameters but a tendency towards lower rate of weight gain after two months of treatment, independent of antibiotic resistance. CONCLUSION: There is a correlation between resistance to first-line anti-TB drugs and reduced NO susceptibility in clinical strains of M. tuberculosis. Further studies including the mechanisms of reduced NO susceptibility are warranted and could identify targets for new therapeutic interventions.

Identification of drug susceptibility pattern and mycobacterial species in sputum smear positive pulmonary tuberculosis patients with and without HIV co-infection in north west Ethiopia.

BACKGROUND: Ethiopia is among the high-burden countries of tuberculosis (TB) in the world Since mycobacterial culture and susceptibility testing are not routinely performed in Ethiopia, recent data on susceptibility patterns and the mycobacterial species cultured from sputum smear positive patients are limited. OBJECTIVES: The aim was to determine first line anti-TB drug susceptibility of Mycobacterium tuberculosis isolates obtained from consecutive newly diagnosed smear positive pulmonary TB patients in north west Ethiopia. METHODOLOGY: A retrospective cross sectional study was conducted using previously collected sputum samples (n=180) kept at the referral hospital of the University of Gondar at -20 degrees C. Sputum samples were cultured on Lowenstein Jensen (LJ) medium. Conventional Polymerase Chain Reaction (PCR) using RD4 primers to identify the M. tuberculosis complex was performed on cultured isolates. Ninety eight (84.4%) of the 116 isolates identified as M. tuberculosis were tested for their drug susceptibility pattern using the proportion method Clinical baseline data including body mass index, body temperature, clinical symptoms and erythrocyte sedimentation rate were obtained. RESULTS: The culture retrieval rate of previously frozen sputum samples was 64.4% (116/180). All the isolated mycobacterial species (n=116) were confirmed as belonging to the M. tuberculosis complex by PCR. Of 98 isolates for which the drug susceptibility test was done, 15.3% (15/98) were found to be resistant to one or more antimycobacterial drugs, and resistance to isoniazid and streptomycin was most common with 8.2% (8/98) and 6.1% (6/98) respectively. TB patients co infected with HIV had increased erythrocyte sedimentation rate, higher age and lower sputum smear grade than HIV negative TB patients. CONCLUSIONS: No mycobacteria other than M. tuberculosis were detected in sputum smear positive TB-patients. Although no multi drug resistant strain was observed, relatively high rates of INH resistance were found in this region. Culture facilities are urgently needed in regional centers to increase diagnostic sensitivity and monitor developing trends of drug resistance in Ethiopia.

Kinetics of the QuantiFERON-TB Gold In-Tube test during treatment of patients with sputum smear-positive tuberculosis in relation to initial TST result and severity of disease.

The QuantiFERON-TB Gold In-Tube test (QFN) measures interferon-gamma production in response to Mycobacterium tuberculosis antigens. Our aim was to assess the kinetics of the QFN and initial tuberculin skin test (TST) result in relation to severity of disease in a tuberculosis (TB) endemic area. Smear-positive TB patients (n = 71) were recruited at Gondar University Hospital, Ethiopia. The TST, QFN, CD4+ cell count and clinical symptoms (TB score) were assessed and followed up during treatment. From baseline to 7 months after treatment, there was a significant decrease in QFN reactivity (93.8% to 62.5% in HIV-negative/TB; 70.3% to 33.3% in HIV-positive/TB patients) down to a level comparable to a control group of blood donors (51.2%). The agreement between TST and QFN was poor in TB patients compared to healthy controls. A negative TST correlated to more advanced TB in contrast to a negative QFN test. We conclude that the QFN reactivity is significantly reduced at the end of treatment against active TB to the background level of healthy blood donors, and that the agreement between TST and QFN is poor including correlation to the severity of disease.

Nitric oxide production in the exhaled air of patients with pulmonary tuberculosis in relation to HIV co-infection.

BACKGROUND: Nitric oxide (NO) is essential for host defense in rodents, but the role of NO during tuberculosis (TB) in man remains controversial. However, earlier observations that arginine supplementation facilitates anti-TB treatment, supports the hypothesis that NO is important in the host defense against TB. Local production of NO measured in fractional exhaled air (FeNO) in TB patients with and without HIV co-infection has not been reported previously. Thus, our aim was to investigate levels of FeNO in relation to clinical symptoms and urinary NO metabolites (uNO). METHODS: In a cross sectional study, FeNO and uNO were measured and clinical symptoms, chest x-ray, together with serum levels of arginine, tumor necrosis factor alpha (TNF-alpha) and interleukin 12 (IL-12) were evaluated in sputum smear positive TB patients (HIV+/TB, n = 36, HIV-/TB, n = 59), their household contacts (n = 17) and blood donors (n = 46) from Gondar University Hospital, Ethiopia. RESULTS: The proportion of HIV-/TB patients with an increased FeNO level (> 25 ppb) was significantly higher as compared to HIV+/TB patients, but HIV+/TB patients had significantly higher uNO than HIV-/TB patients. HIV+ and HIV-/TB patients both had lower levels of FeNO compared to blood donors and household contacts. The highest levels of both uNO and FeNO were found in household contacts. Less advanced findings on chest x-ray, as well as higher sedimentation rate were observed in HIV+/TB patients as compared to HIV-/TB patients. However, no significant correlation was found between FeNO and uNO, chest x-ray grading, clinical symptoms, TNF-alpha, IL-12, arginine levels or sedimentation rate. CONCLUSION: In both HIV negative and HIV co infected TB patients, low levels of exhaled NO compared to blood donors and household were observed. Future studies are needed to confirm whether low levels of exhaled NO could be a risk factor in acquiring TB and the relative importance of NO in human TB.