Comparison of the measured values of quantitative SARS-CoV-2 spike antibody assays.

BACKGROUND: The concentration of antibodies against the SARS-CoV-2 spike protein is frequently being measured for clinical and epidemiological purposes. The aim of this study was to examine whether the results of different quantitative SARS-CoV-2 spike antibody assays are comparable. MATERIAL AND METHODS: The Siemens SARS-CoV-2 IgG, Abbott SARS-CoV-2 IgG II Quant, Roche ElecsysT Anti-SARS-CoV-2 S, and Euroimmun Anti-SARS-CoV-2-QuantiVac assay were compared with 110 sera from patients 6-9 months after SARS-CoV-2 infection and the WHO First International SARS-CoV-2 antibody standard 20/136. The antibody values were converted into WHO binding antibody units (BAU)/ml. The diagnostic sensitivity of the assays was determined and the antibody values were compared. RESULTS: The diagnostic sensitivity ranged from 57.3% (Euroimmun) to 100% (Roche). The antibody concentration values of different assays correlated with Pearson coefficients of correlation between 0.729 and 0.953. The geometric mean antibody values of the Abbott, Siemens and Euroimmun assay varied by a factor of 1.1-1.2. The geometric mean antibody values of the Roche assay were 2.4-2.8 times higher than those from the other assays. The assays yielded varying results with the WHO International antibody standard. CONCLUSIONS: The quantitative SARS-CoV-2 antibody assays from Abbott, Siemens, Roche and Euroimmun correlate strongly but differ in the antibody concentrations. Therefore, the same assay should be used when testing patients repeatedly. In addition, the name of the assay used and the manufacturer should be indicated along with the test results.

Sensitivity of SARS-CoV-2 antibody tests with late convalescent sera.

SARS-CoV-2-specific IgM antibodies wane during the first three months after infection and IgG antibody levels decline. This may limit the ability of antibody tests to identify previous SARS-CoV-2 infection at later time points. To examine if the diagnostic sensitivity of antibody tests falls off, we compared the sensitivity of two nucleoprotein-based antibody tests, the Roche Elecsis II Anti-SARS-CoV-2 and the Abbott SARS-CoV-2 IgG assay and three glycoprotein-based tests, the Abbott SARS-CoV-2 IgG II Quant, Siemens Atellica IM COV2T and Euroimmun SARS-CoV-2 assay with 53 sera obtained 6 months after SARS-CoV-2 infection. The sensitivity of the Roche, Abbott SARS-CoV-2 IgG II Quant and Siemens antibody assays was 94.3% (95% confidence interval (CI) 84.3-98.8%), 98.1 % (95% CI: 89.9-100%) and 100 % (95% CI: 93.3-100%). The sensitivity of the N-based Abbott SARS-CoV-2 IgG and the glycoprotein-based Euroimmun ELISA was 45.3 % (95% CI: 31.6-59.6%) and 83.3% (95% CI: 70.2-91.9%). The nucleoprotein-based Roche and the glycoprotein-based Abbott receptor binding domain (RBD) and Siemens tests were more sensitive than the N-based Abbott and the Euroimmun antibody tests (p = 0.0001 to p = 0.039). The N-based Abbott antibody test was less sensitive 6 months than 4-10 weeks after SARS-CoV-2 infection (p = 0.0001). The findings show that most SARS-CoV-2 antibody assays correctly identified previous infection 6 months after infection. The sensitivity of pan-Ig antibody tests was not reduced at 6 months when IgM antibodies have usually disappeared. However, one of the nucleoprotein-based antibody tests significantly lost diagnostic sensitivity over time.

The Activation of Mucosal-Associated Invariant T (MAIT) Cells Is Affected by Microbial Diversity and Riboflavin Utilization in vitro.

Recent research has demonstrated that MAIT cells are activated by individual bacterial or yeasts species that possess the riboflavin biosynthesis pathway. However, little is known about the MAIT cell activating potential of microbial communities and the contribution of individual community members. Here, we analyze the MAIT cell activating potential of a human intestinal model community (SIHUMIx) as well as intestinal microbiota after bioreactor cultivation. We determined the contribution of individual SIHUMIx community members to the MAIT cell activating potential and investigated whether microbial stress can influence their MAIT cell activating potential. The MAIT cell activating potential of SIHUMIx was directly related to the relative species abundances in the community. We therefore suggest an additive relationship between the species abundances and their MAIT cell activating potential. In diverse microbial communities, we found that a low MAIT cell activating potential was associated with high microbial diversity and a high level of riboflavin demand and vice versa. We suggest that microbial diversity might affect MAIT cell activation via riboflavin utilization within the community. Microbial acid stress significantly reduced the MAIT cell activating potential of SIHUMIx by impairing riboflavin availability through increasing the riboflavin demand. We show that MAIT cells can perceive microbial stress due to changes in riboflavin utilization and that riboflavin availability might also play a central role for the MAIT cell activating potential of diverse microbiota.

Mucosal-associated invariant T-Cell (MAIT) activation is altered by chlorpyrifos- and glyphosate-treated commensal gut bacteria.

Mucosal-associated invariant T-cells (MAIT) can react to metabolites of the vitamins riboflavin and folate which are produced by the human gut microbiota. Since several studies showed that the pesticide chlorpyrifos (CPF) and glyphosate (GLP) can impair the gut microbiota, the present study was undertaken to investigate the impact of CPF and GLP treatment on the metabolism of gut microbiota and the resulting bacteria-mediated modulation of MAIT cell activity. Here, Bifidobacterium adolescentis (B. adolescentis), Lactobacillus reuteri (L. reuteri), and Escherichia coli (E. coli) were treated with CPF (50-200 µM) or GLP (75-300 mg/L) and then used in MAIT cell stimulation assays as well as in vitamin and proteome analyses. All three bacteria were nonpathogenic and chosen as representatives of a healthy human gut microflora. The results showed that E. coli activated MAIT cells whereas B. adolescentis and L. reuteri inhibited MAIT cell activation. CPF treatment significantly increased E. coli-mediated MAIT cell activation. Treatment of B. adolescentis and L. reuteri with CPF and GLP weakened the inhibition of MAIT cell activation. Riboflavin and folate production by the test bacteria was influenced by CPF treatment, whereas GLP had only minor effects. Proteomic analysis of CPF-treated E. coli revealed changes in the riboflavin and folate biosynthesis pathways. The findings here suggest that the metabolism of the analyzed bacteria could be altered by exposure to CPF and GLP, leading to an increased pro-inflammatory immune response.

Effect of sub-MIC concentrations of metronidazole, vancomycin, clindamycin and linezolid on toxin gene transcription and production in Clostridium difficile.

Clostridium difficile is the major cause of hospital-acquired infectious diarrhoea. Several antimicrobials are known to induce and promote C. difficile-associated diarrhoea (CDAD). The impact of metronidazole (MTR), vancomycin (VAN), clindamycin (CLI) and linezolid (LZD) on growth, toxin gene transcription and toxin production in C. difficile was investigated. Four C. difficile strains were grown with and without sub-MIC concentrations of MTR, VAN, CLI and LZD (0.5x MIC) and growth was measured by colony counts. Toxin production was detected using ELISA (for toxin A) and a cytotoxicity assay (for toxin B) in culture supernatants and also in sonicated cells. Real-time PCR was used to measure transcription of the toxin A and B genes. The aim of this work was to combine analysis of toxin A and B production by ELISA or cell culture assay with transcriptomic analysis. The four strains showed similar growth and different levels of toxin production in the absence of antibiotics. An antibiotic-free control showed toxin production at a late stage when the plateau phase of bacterial growth was reached, whereas antibiotic-exposed strains showed earlier toxin production. All of the antibiotics used except CLI increased the transcription rate of toxin genes. The findings of this study show that sub-MIC concentrations of antibiotics can cause changes in gene transcription of the major virulence factors of C. difficile. This study describes a new method for transcriptomic analysis of toxin genes in C. difficile.

Antimicrobial phenotypes and molecular basis in clinical strains of Clostridium difficile.

Clostridium difficile remains the leading cause of nosocomial-acquired diarrhea. This study investigated antimicrobial susceptibility patterns of C. difficile over a 3-year period. Three hundred seventeen C. difficile isolates recovered between 2002 and 2004 were analyzed for their susceptibility to erythromycin (ERY), clindamycin (CLI), moxifloxacin (MXF), doxycycline (DOX), vancomycin (VAN), and metronidazole (MTR) by Etest. The molecular basis for resistance was investigated using polymerase chain reaction (PCR) and DNA sequencing. PCR ribotyping was used to differentiate strains. All strains were susceptible to VAN and MTR. Resistance rates to ERY/CLI, MXF, and DOX increased during the study period. Eighty-four (26.5%) strains exhibited resistance against ERY/CLI, MXF, and DOX. Prevalence of resistance genes was as follows: ermB, 83; ermQ, 0; ermFS, 1; tetM, 84; tetP, 0; tetO, 2; and gyrA mutation, 76. These results indicate an increasing trend in the prevalence of combined resistance against macrolide-lincosamide-streptogramin B antibiotics, fluoroquinolones, and tetracycline in C. difficile. The lack of understanding of antibiotic resistance mechanisms in C. difficile and the increased resistant strains warrants further investigations.

Prevalence of the ermB gene in Clostridium difficile strains isolated at a university teaching hospital from 1987 through 1998.

We analyzed 226 strains of Clostridium difficile for the presence of erythromycin ribosomal methylase B (ermB) genes. Forty-four strains (19.4%) carried ermB genes and were resistant to erythromycin. Toxin A and toxin B gene sequences were identified in 81.9% of these 44 strains. Strains of C. difficile that carry ermB genes are common etiologic agents of C. difficile-associated diarrhea.

Prevalence and characteristics of bacteria and host factors in an outbreak situation of antibiotic-associated diarrhoea.

Antibiotic-associated diarrhoea (AAD) represents a clinical entity leading to prolonged hospital stays and diagnostic and therapeutic procedures, and results in additional costs. The aim of the present study was to assess the prevalence and characteristics of different bacteria in stools of patients with AAD. The reliability of diagnostic procedures under routine conditions was evaluated. Host factors were also analysed. From June 2002 to April 2003 89 cases of diarrhoea were reported at a hospital unit for internal medicine. Clostridium difficile and Clostridium perfringens toxin enzyme-immunoassays (EIAs), and culture for C. difficile, C. perfringens and Staphylococcus aureus were performed on stool samples from all patients. Toxin production was determined in isolated S. aureus strains. In vitro susceptibility of S. aureus for oxacillin and of C. difficile for vancomycin, metronidazole, linezolid, fusidic acid and tetracycline was tested. Host factors, such as age, comorbidities, antibiotic exposure and contact with other patients, were evaluated. Twenty-six stools were positive for C. difficile toxins by an EIA technique, while C. difficile was cultured from 39. C. difficile was isolated from 21 stools that were EIA negative. Additionally, from 28 stools S. aureus and/or C. perfringens could be isolated. Nine samples contained only S. aureus and/or C. perfringens. Thirty-one stools were negative in all tests. All C. difficile isolates were susceptible to vancomycin and metronidazole. Age >60 years, and diseases of the vascular system, the heart, the kidneys and the lungs were identified as risk factors for acquiring C. difficile in this setting (P values < 0.05). Stool culture for C. difficile was shown to be more sensitive than toxin EIA in this study. Risk factors for the acquisition of C. difficile in outbreak situations seem to differ from risk factors in the normal hospital setting. The role of toxin-producing S. aureus in cases of AAD needs further investigation.

High rates of sustained virological response in hepatitis C virus-infected injection drug users receiving directly observed therapy with peginterferon alpha-2a (40KD) (PEGASYS) and once-daily ribavirin.

This retrospective study evaluated the efficacy and tolerability of directly observed therapy with peginterferon alfa-2a and once-daily ribavirin (RBV) for chronic hepatitis C in 49 opioid-addicted injection drug users (IDUs) participating in a drug treatment program at a specialized outpatient center. Patients also received prophylactic citalopram to minimize the risk of interferon-induced depression. Patients had daily access to and support from specialist physicians, nurses and counseling services at the center, and a 24-hour helpline. Sustained virological response was achieved by 48 of 49 patients (98%) overall, including 20 of 21 (95%) hepatitis C virus (HCV) Genotype 1/4-infected patients and 28 of 28 (100%) Genotype 2/3-infected patients. Treatment was well tolerated, and no unexpected side effects of peginterferon treatment were seen. The safety profile of once-daily RBV was not different from twice-daily dosing. Decline in hemoglobin levels was similar to those reported in clinical trials including once-daily RBV and did not lead to dose reduction or treatment withdrawal. Our data demonstrate that HCV-infected IDUs on stable L-polamidone (methadone) or buprenorphine maintenance can be successfully and safely treated with peginterferon alfa-2a and RBV in an optimal substitution setting.

OPT-80, a macrocyclic antimicrobial agent for the treatment of Clostridium difficile infections: a review.

BACKGROUND: Clostridium difficile-associated diarrhoea has become a major problem over the last years. Increasing incidence and more severe clinical cases initiated the search for new treatment options. OBJECTIVE: OPT-80, also known as tiacumicin B, lipiarmycin or PAR-101, is a macrocyclic antimicrobial with little or no systemic absorption after oral administration and narrow activity spectrum against Gram-positive aerobic and anaerobic bacteria. METHODS: Data on OPT-80 available from published studies, presentations at conferences and the manufacturer were collected and reviewed. RESULTS/CONCLUSION: The in vitro studies of OPT-80 and clinical C. difficile strains showed high activity at low concentrations. Safety and efficacy of the drug were found to be favourable. More Phase II and III clinical trials are to be completed.

Drugs of the 21st century: telithromycin (HMR 3647)--the first ketolide.

Telithromycin (HMR 3647) is the first ketolide introduced into clinical practice. Ketolides are semisynthetic derivates of erythromycin A that carry novel biological properties on the erythronolide A ring. This new class of antimicrobials was designed to overcome current resistance mechanisms against erythromycin A within Gram-positive cocci. Ketolides do not induce macrolide-lincosamide-streptogramin B (MLS(B)) resistance and are active against erythromycin resistance methylase gene (erm)-carrying Gram-positive cocci. This review summarizes published data on telithromycin and intends to define the challenge that a new antimicrobial brings to medical practice.

In vitro activity of OPT-80 against Clostridium difficile.

Clostridium difficile remains the major cause of nosocomial diarrhea. Reports on impaired susceptibility of C. difficile to metronidazole and vancomycin and frequent relapses of patients after therapy necessitate the search for new substances. With this study, the activity of OPT-80, a new macrocycle, against 207 C. difficile strains and against other obligately anaerobic bacteria was tested. OPT-80 showed high in vitro activity against all C. difficile strains tested.

Prevalence and association of macrolide-lincosamide-streptogramin B (MLS(B)) resistance with resistance to moxifloxacin in Clostridium difficile.

Clostridium difficile remains the leading cause of nosocomially acquired diarrhoea. C. difficile usually exhibits resistance against beta-lactam antibiotics, whereas susceptibility to other drugs may vary. This study investigated the antimicrobial susceptibility of C. difficile to different antibiotics over a period of time and characterizes molecular mechanisms for resistance. One hundred and seventy-three toxigenic and 19 non-toxigenic C. difficile strains, recovered from patients in two university hospitals in Germany between 1986 and 2001, were investigated for their susceptibility to erythromycin, clindamycin, moxifloxacin, vancomycin and metronidazole employing the Etest. The genetic background for resistance was analysed using PCR and DNA sequencing. All strains were susceptible to vancomycin and metronidazole. Resistance to erythromycin, clindamycin and moxifloxacin was found in 27%, 36% and 12% of the tested strains, respectively. High-level resistance (MIC > 128 mg/L) against erythromycin and clindamycin was detected in 25% of the strains tested. Thirty-four of the macrolide-lincosamide-streptogramin B (MLS(B))-resistant strains carried the erythromycin resistance methylase gene. The results indicate an increase in the prevalence of resistance to MLS(B) and fluoroquinolone antibiotics in C. difficile. Fluoroquinolone resistance is associated with resistance to MLS(B) antimicrobials.

Effect of moxifloxacin versus imipenem/cilastatin treatment on the mortality of mice infected intravenously with different strains of Bacteroides fragilis and Escherichia coli.

OBJECTIVES: To study the effect of moxifloxacin versus imipenem/cilastatin (hereafter referred to as imipenem) treatment on the mortality of mice infected intravenously with different strains of Bacteroides fragilis and Escherichia coli. METHODS: Groups of 20 mice each were infected intravenously with different strains of B. fragilis [moxifloxacin and imipenem susceptible or resistant, and enterotoxin (ET) positive or negative] and E. coli (moxifloxacin and imipenem susceptible). Twenty-four hours post-infection, intravenous therapy with either moxifloxacin (2.0 mg twice a day) or imipenem (2.4 mg three times a day) was started and continued for 3 days. Control groups were left untreated. Survival rates were recorded at day 7 post-infection. At that time, surviving mice were killed and numbers of bacteria in the liver and kidneys were determined. RESULTS: If compared with untreated animals, mice treated with either moxifloxacin or imipenem showed significantly improved survival (P < 0.001). There was no significant difference (P = 0.97) in the survival rates comparing the two treatment regimens irrespective of the ET positivity or the susceptibility to moxifloxacin or imipenem of the infective B. fragilis strain. However, there was a tendency that B. fragilis was recovered more often from the liver and kidneys of mice infected with ET positive strains. CONCLUSIONS: The data show that moxifloxacin was as efficacious as imipenem in reducing the mortality rate of mice suffering from a severe systemic aerobic/anaerobic infection.

Therapeutic efficacy of moxifloxacin in a murine model of severe systemic mixed infection with Escherichia coli and Bacteroides fragilis.

The therapeutic efficacy of moxifloxacin was studied in an experimental murine model of a systemic aerobic/anaerobic mixed infection and compared to therapies with either imipenem or ciprofloxacin plus metronidazole. Groups of 20 mice each were intravenously (iv) infected with approximately 2.5 x 10(6) colony forming units (CFU) of Escherichia coli and 2 x 10(7)CFU of Bacteroides fragilis. Iv therapy was started 24 h post-infection (pi) with either moxifloxacin, imipenem, or ciprofloxacin plus metronidazole, for 3 days. A control group of 20 mice was left untreated. Survival rate at day seven pi was recorded, mice were then sacrificed and bacterial organ contents of livers and kidneys were determined. All mice treated survived at day seven, while six animals of the untreated group died. B. fragilis was not detected in any of the treated mice. E. coli was found in two of the moxifloxacin-treated mice and in two and five of the ciprofloxacin plus metronidazole and imipenem-treated animals, respectively. The results indicate that a therapy of severe mixed aerobic/anaerobic infections with moxifloxacin might be feasible and possibly be as efficacious as current therapy regimens with ciprofloxacin plus metronidazole or imipenem.