Laboratory Findings and Biomarkers in Long COVID: What Do We Know So Far? Insights into Epidemiology, Pathogenesis, Therapeutic Perspectives and Challenges.

Long COVID (LC) encompasses a constellation of long-term symptoms experienced by at least 10% of people after the initial SARS-CoV-2 infection, and so far it has affected about 65 million people. The etiology of LC remains unclear; however, many pathophysiological pathways may be involved, including viral persistence; a chronic, low-grade inflammatory response; immune dysregulation and a defective immune response; the reactivation of latent viruses; autoimmunity; persistent endothelial dysfunction and coagulopathy; gut dysbiosis; hormonal and metabolic dysregulation; mitochondrial dysfunction; and autonomic nervous system dysfunction. There are no specific tests for the diagnosis of LC, and clinical features including laboratory findings and biomarkers may not specifically relate to LC. Therefore, it is of paramount importance to develop and validate biomarkers that can be employed for the prediction, diagnosis and prognosis of LC and its therapeutic response, although this effort may be hampered by challenges pertaining to the non-specific nature of the majority of clinical manifestations in the LC spectrum, small sample sizes of relevant studies and other methodological issues. Promising candidate biomarkers that are found in some patients are markers of systemic inflammation, including acute phase proteins, cytokines and chemokines; biomarkers reflecting SARS-CoV-2 persistence, the reactivation of herpesviruses and immune dysregulation; biomarkers of endotheliopathy, coagulation and fibrinolysis; microbiota alterations; diverse proteins and metabolites; hormonal and metabolic biomarkers; and cerebrospinal fluid biomarkers. At present, there are only two reviews summarizing relevant biomarkers; however, they do not cover the entire umbrella of current biomarkers, their link to etiopathogenetic mechanisms or the diagnostic work-up in a comprehensive manner. Herein, we aim to appraise and synopsize the available evidence on the typical laboratory manifestations and candidate biomarkers of LC, their classification based on pathogenetic mechanisms and the main LC symptomatology in the frame of the epidemiological and clinical aspects of the syndrome and furthermore assess limitations and challenges as well as potential implications in candidate therapeutic interventions.

Socioeconomic inequality and stillbirth rate disparities among native and foreign mothers: evidence from Greece.

We study, for the first time, stillbirth differentials among native and migrant populations in Greece using national vital registration microdata for the period of 2010-2014. We employ conventional demographic measures and propose a standardization procedure to delineate the effect of selected distributions of livebirths on the observed stillbirth rates. The stillbirth rate among immigrant mothers is 40% higher than among natives, an excess which persists throughout gestational intervals and age groups. Our findings also show a clear gradient of stillbirth rates by maternal education, favoring more educated women, and this finding applies to both native and immigrant mothers. Our standardization methodology shows that the distribution of births by maternal educational level and age play a role; this finding implies that elevated immigrant stillbirth rates may be attributed to a certain extent to the specific characteristics of this group, since immigrant women have, on average, a younger age structure and lower educational attainment. Supplementary Information: The online version contains supplementary material available at 10.1007/s43545-022-00410-y.

Economic crisis and stillbirth ratios: Evidence from Southern Europe.

In this paper we assess the impact of the recent European recession on stillbirth indices over the course of the 2000s and 2010s; the analysis focuses on four Southern European countries (Greece, Italy, Spain, Portugal), which were seriously affected by the sovereign debt crisis from around 2008 to 2017. We use national vital statistics and established economic indicators for the period 2000-2017; stillbirth ratios (stillbirths per 1000 livebirths) are the chosen response variable. For the purpose of the study, we employ correlation analysis and fit regression models. The overall impact of economic indicators on the stillbirth indices is sizeable and statistically robust. We find that a healthy economy is associated with low and declining levels of stillbirth measures. In contrast, economic recession appears to have an adverse effect (Greece, Italy and Spain), or an unclear impact (Portugal), on the stillbirth outcome. This study provides evidence of the adverse effect of the European sovereign debt crisis and ensuing period of austerity on a scarcely explored aspect of health.

Activity of Sulbactam-Durlobactam and Comparators Against a National Collection of Carbapenem-Resistant Acinetobacter baumannii Isolates From Greece.

BACKGROUND: Acinetobacter baumannii is a leading cause of healthcare-associated infections worldwide, due to both its persistence in the hospital setting and ability to acquire high levels of antibiotic resistance. Carbapenem-resistant A. baumannii isolates (CRAB) limit the activity of current antimicrobial regimens and new alternatives or adjuncts to traditional antibiotics are urgently needed. Durlobactam is a novel broad-spectrum inhibitor of serine-type ß-lactamases that restores sulbactam (SUL) activity against A. baumannii. The sulbactam-durlobactam (SD) combination has recently completed Phase 3 testing in the global ATTACK trial. OBJECTIVES: The aim of this study is to evaluate the in vitro activity of SD versus comparators against a representative nationwide collection of CRAB isolates. METHODS: One hundred ninety CRAB isolates were collected from clinical samples of patients hospitalized in 11 hospitals throughout Greece during 2015. In vitro activities of SD and comparators (SUL alone, amikacin, minocycline, imipenem, meropenem, colistin, SD and imipenem combined with SD) were determined by broth microdilution. RESULTS: Durlobactam restored sulbactam activity against the majority of the strains tested, with SD exhibiting the lowest MIC90 (8 µg/ml) relative to the other single comparators tested; 87.9% of the isolates had SD MICs ≤4/4 µg/ml. The most active comparator was colistin (MIC90 = 16 µg/ml). The addition of imipenem further lowered the MIC90 of SD by one two-fold dilution. CONCLUSIONS: This study demonstrated the potential utility of SD for the treatment of infections caused by A. baumannii. If its clinical efficacy is confirmed, SD may be an important therapeutic option for CRAB infections.

Characteristics of meropenem heteroresistance in Klebsiella pneumoniae carbapenemase (KPC)-producing clinical isolates of K. pneumoniae.

Meropenem heteroresistance was investigated in six apparently meropenem-susceptible, Klebsiella pneumoniae carbapenemase (KPC)-producing K. pneumoniae (KPC-KP) clinical isolates, compared with that in carbapenemase-negative, meropenem-susceptible controls. In population analyses, the KPC-KP isolates grew at meropenem concentrations of 64 to 256 microg/ml. Heteroresistant colonies had significantly elevated expression of the bla(KPC) gene compared with the native populations but did not retain heteroresistance when subcultured in drug-free media. Time-kill assays indicated that meropenem alone was not bactericidal against KPC-KP but efficiently killed the control strains.

In vivo acquisition of a plasmid-mediated bla(KPC-2) gene among clonal isolates of Serratia marcescens.

Three patients admitted to a Greek hospital were infected with Serratia marcescens isolates that exhibited reduced susceptibility to carbapenems and harbored Klebsiella pneumoniae carbapenemase (KPC) enzymes. In two of these cases, the patients were initially infected by carbapenem-susceptible S. marcescens isolates. Molecular typing and plasmid analysis suggested that all three patients had clonally indistinguishable isolates of S. marcescens that acquired a plasmid-mediated bla(KPC-2) gene during the hospitalization.

A simple phenotypic method for the differentiation of metallo-beta-lactamases and class A KPC carbapenemases in Enterobacteriaceae clinical isolates.

BACKGROUND: The increasing frequency of class A KPC enzymes and class B metallo-beta-lactamases (MBLs) among Enterobacteriaceae as well as their possible co-production makes their early differentiation urgent. METHODS: A simple phenotypic algorithm employing three combined-disc tests consisting of meropenem alone and with phenylboronic acid (PBA), EDTA, or both PBA and EDTA was designed for the differentiation of KPC and MBL enzymes. Augmentation of the zone of inhibition by >or=5 mm was considered a positive combined-disc test result. A total of 141 genotypically confirmed carbapenemase-positive Enterobacteriaceae clinical isolates (63 KPC producers, 47 MBL producers, and 31 KPC and MBL producers) with various carbapenem MICs were examined. For comparison, 84 genotypically confirmed carbapenemase-negative Enterobacteriaceae clinical isolates [39 extended-spectrum beta-lactamase (ESBL) producers, 22 AmpC producers, and 23 ESBL and AmpC producers] were also tested. RESULTS: The phenotypic algorithm was able to differentiate MBL from KPC producers as well as to detect the possible co-production of both carbapenemases (positive result only with the combined-disc test using meropenem alone and with both PBA and EDTA). The method detected all KPC or MBL producers (sensitivity 100%) as well as 30 of the KPC and MBL producers (sensitivity 96.8%). All three combined-disc tests were negative for non-carbapenemase-producing isolates, except two ESBL and AmpC producers that gave positive combined-disc tests using meropenem alone and with PBA and both PBA and EDTA (specificity for KPC detection 98.8%). CONCLUSIONS: This phenotypic method is very helpful to detect carbapenemase production and provides a simple algorithm for the differentiation of KPC and MBL enzymes, especially in regions where KPC- and MBL-possessing Enterobacteriaceae are highly prevalent.

Evaluation of boronic acid disk tests for differentiating KPC-possessing Klebsiella pneumoniae isolates in the clinical laboratory.

The worldwide increase in the occurrence and dissemination of KPC beta-lactamases among gram-negative pathogens makes critical the early detection of these enzymes. Boronic acid disk tests using different antibiotic substrates were evaluated for detection of KPC-possessing Klebsiella pneumoniae isolates. A total of 57 genotypically confirmed KPC-possessing K. pneumoniae isolates with varying carbapenem MICs were examined. To measure the specificity of the tests, 106 non-KPC-possessing isolates (89 K. pneumoniae and 17 Escherichia coli isolates) were randomly selected among those exhibiting reduced susceptibility to cefoxitin, expanded-spectrum cephalosporins, or carbapenems. As many as 56, 53, and 40 of the non-KPC-possessing isolates harbored extended-spectrum beta-lactamases, metallo-beta-lactamases, and plasmid-mediated AmpC beta-lactamases, respectively. By use of CLSI methodology and disks containing imipenem, meropenem, or cefepime, either alone or in combination with 400 microg of boronic acid, all 57 KPC producers gave positive results (sensitivity, 100%) whereas all 106 non-KPC producers were negative (specificity, 100%). The meropenem duplicate disk with or without boronic acid demonstrated the largest differences in inhibition zone diameters between KPC producers and non-KPC producers. By use of disks containing ertapenem, all isolates were correctly differentiated except for five AmpC producers that gave false-positive results (sensitivity, 100%; specificity, 95.3%). These practical and simple boronic acid disk tests promise to be very helpful for the accurate differentiation of KPC-possessing K. pneumoniae isolates, even in regions where different broad-spectrum beta-lactamases are widespread.

Use of boronic acid disk tests to detect extended- spectrum beta-lactamases in clinical isolates of KPC carbapenemase-possessing enterobacteriaceae.

We evaluated boronic acid (BA)-based methods for their ability to detect extended-spectrum beta-lactamases (ESBLs) among clinical isolates of KPC-producing members of the Enterobacteriaceae family. A total of 155 isolates of Klebsiella pneumoniae (n = 141), Escherichia coli (n = 6), Enterobacter aerogenes (n = 6), and Klebsiella oxytoca (n = 2) genotypically confirmed to be KPC producers were analyzed. As many as 118 isolates harbored ESBLs (103 harbored SHV-type ESBLs, 13 harbored CTX-M-type ESBLs, and 2 harbored both SHV- and CTX-M-type ESBLs); the remaining 37 isolates were genotypically negative for ESBL production. The CLSI ESBL confirmatory test was positive for 79 of the 118 ESBL producers (sensitivity, 66.9%), while all 37 non-ESBL producers were negative (specificity, 100%). When a > or =5-mm increase in the zone diameter of either the cefotaxime (CTX)-clavulanate (CA) or the ceftazidime (CAZ)-CA disks containing BA compared with the zone diameter of the CTX or CAZ disks containing BA was considered to be a positive result for ESBL production, the method detected all 118 ESBL producers (sensitivity, 100%) and showed no false-positive results for non-ESBL producers (specificity, 100%). Double-disk synergy tests, in which disks of CTX, CAZ, aztreonam, or cefepime in combination with BA were placed at distances of 20, 25, and 30 mm (center to center) from a disk containing amoxicillin (amoxicilline)-clavulanate-BA, were able to detect 116 (98.3%), 101 (85.6%), and 28 (23.7%) of the ESBL-positive isolates, respectively; no false-positive results for non-ESBL-producing isolates were detected. Our results demonstrate that the modified CLSI ESBL confirmatory test with antibiotic disks containing BA is the most accurate phenotypic method for the detection of ESBLs in Enterobacteriaceae producing KPC carbapenemases.

Evaluation of imipenem/imipenem+EDTA disk method for detection of metallo-beta-lactamase-producing Klebsiella pneumoniae isolated from blood cultures.

The objective of this study was to evaluate the imipenem (IMP) and IMP+EDTA (IMP/IMP+EDTA) disk method for the detection of metallo-beta-lactamases (MBLs) in clinical isolates of Klebsiella pneumoniae with various MIC levels to IMP. Forty-one blood isolates of K. pneumoniae with MIC to IMP ranging from < or =0.5 to > or =16 microg/ml were examined. The MICs were determined by VITEK-2 (bioMerieux Vitek two, France). Disks of 10 microg IMP with and without the addition of 0.5 M EDTA were used for the IMP/IMP+EDTA disk method. The E-test (AB Biodisk, Solna, Sweden) for MBL detection was also used. All isolates were examined for the bla (VIM-1) gene by PCR and for clonality of VIM-1-producing isolates by pulsed-field gel electrophoresis (PFGE). All isolates with MIC values of IMP < or =0.5 microg/ml exhibited no differences in inhibition zone diameters (IZD) produced by IMP and IMP+EDTA disks, whereas the isolates with MICs > or =1 microg/ml showed an increase in IZD, ranging from 8 to 26 mm. All isolates with MIC values of > or =1 microg/ml were found positive for the bla (VIM-1) gene by PCR and for MBL production by the E-test, whereas none of isolates with MICs <0.5 microg/ml was found positive by any of the tests. DNA restriction fragments generated by PFGE of VIM-1-producing isolates were classified in four main types. The IMP/IMP+EDTA disk method is simple to perform, sensitive, and specific for detection of MBL-producing K. pneumoniae clinical isolates. K. pneumoniae isolates with MICs of IMP > or =1 microg/ml and/or IZD produced by IMP disk <19 mm should be tested for MBL production.

Two cases of severe sepsis caused by Bacillus pumilus in neonatal infants.

Bacillus pumilus is an environmental contaminant that has been rarely associated with clinical infections. Here, two cases of severe sepsis caused by B. pumilus are described in two full-term neonates; one in a female infant with no factors predisposing her to infection and the other in a male infant requiring mechanical ventilation and an intravenous catheter. In both cases, the micro-organism was recovered from repeated blood cultures and was identified using biochemical assays and 16S rRNA gene sequencing. Both infants were successfully treated with vancomycin. This report reveals the potential role of B. pumilus as a bloodstream pathogen during infancy.

Prevalence of asymptomatic bacteriuria in type 2 diabetic subjects with and without microalbuminuria.

BACKGROUND: Diabetic subjects, especially women, show high prevalence of asymptomatic bacteriuria (ASB). The aim of the present study was to evaluate the prevalence of ASB in subjects with type 2 diabetes mellitus (T2D) with and without microalbuminuria (MA). FINDINGS: A hundred diabetic subjects with MA (53 males/47 females, mean age +/- standard deviation: 65.5 +/- 11.1 years) and 100 diabetic subjects without MA (52 males/48 females, mean age +/- standard deviation: 65.4 +/- 11.3 years), consecutively attending the outpatient diabetes clinic of our hospital were recruited in the study. Subjects with overt diabetic nephropathy or nephropathy from other causes were excluded. In addition, subjects with symptoms of urinary track infection or use of antimicrobial drugs in the last 14 days were excluded by the study.Diabetic subjects with MA showed increased prevalence of ASB compared to diabetic subjects without MA (21% versus 8%, P < 0.001, respectively). Escherichia coli was the most prevalent pathogen isolated in diabetic subjects with and without MA (12% versus 3.0%, P = 0.01, respectively) followed by Proteus mirabilis (6% versus 5%, P = 0.75, respectively) and Klebsiella spp (5% versus 1%, P = 0.09, respectively). Univariate logistic analysis showed that ASB was associated with the presence of coronary artery disease [odds ratio (OR): 0.29, 95% Confidence Intervals (95% CI): 0.09-0.95, P = 0.04] and gender (OR: 0.09, 95% CI: 0.02-0.35, P < 0.001) in the diabetic study group with MA. CONCLUSIONS: ASB is more prevalent among T2D subjects with MA. Screening for ASB is warranted in diabetic patients especially if pyuria is detected in urine analysis since ASB has been found to be a risk factor for developing symptomatic urinary tract infection.

Persistence of a clone of glycopeptide-resistant Enterococcus faecalis among patients in an intensive care unit of a Greek hospital.

OBJECTIVES: To investigate an outbreak of glycopeptide-resistant Enterococcus faecalis (GREF) in the intensive care unit (ICU) of 'G. Gennimatas' General Hospital, Athens, Greece. MATERIALS AND METHODS: Between August 2000 and November 2001, 20 highly GREF isolates were recovered from severe infections of separate patients in the ICU. The isolates were tested by PCR, PFGE, mating experiments and plasmid analysis. RESULTS: All isolates carried the vanA gene. Nineteen isolates fitted to one clone by macrorestriction analysis with four subclones being consecutively detected. Each subclone seemed to predominate for a specific time period. Additionally, four GREF isolates related to the ICU clone were recovered from other wards of the hospital. CONCLUSIONS: Our findings indicate that a monoclonal GREF outbreak persisted for more than 1 year in a large Greek hospital. The rate of GREF isolation declined after the application of infection control measures.

Multiple liver abscesses associated with bacteremia due to Leuconostoc lactis.

Leuconostoc species, which are members of the family Streptococcacae, have only recently been recognized as potential pathogens. We describe a patient with type II diabetes mellitus who had multiple liver abscesses associated with bacteremia due to Leuconostoc lactis. To our knowledge, this is the first case of this association to be reported in the literature.