An overview of the data obtained during the validation of an optimized MALDI-TOF MS Biotyper database for the identification of anaerobic bacteria.

This data in brief article presents the data obtained during the validation of the optimized Biotyper Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS) database. The validation was performed by the different expertise laboratories, collaborating within the European Network for the Rapid Identification of Anaerobes (ENRIA) project, using 6309 human clinical anaerobic bacterial strains. Different databases were compared with each other; the db 5989 database (V5 database); the V5 database complimented with Main Spectral Profiles (MSPs) of ENRIA strains added to the next update of the database; and the V5 database complimented with the MSPs of all anaerobic clinical isolates collected within the ENRIA project. For a comprehensive discussion of the full dataset, please see the research article that accompanies this data article (Veloo et al., 2018) [1].

Validation of MALDI-TOF MS Biotyper database optimized for anaerobic bacteria: The ENRIA project.

Within the ENRIA project, several 'expertise laboratories' collaborated in order to optimize the identification of clinical anaerobic isolates by using a widely available platform, the Biotyper Matrix Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS). Main Spectral Profiles (MSPs) of well characterized anaerobic strains were added to one of the latest updates of the Biotyper database db6903; (V6 database) for common use. MSPs of anaerobic strains nominated for addition to the Biotyper database are included in this validation. In this study, we validated the optimized database (db5989 [V5 database] + ENRIA MSPs) using 6309 anaerobic isolates. Using the V5 database 71.1% of the isolates could be identified with high confidence, 16.9% with low confidence and 12.0% could not be identified. Including the MSPs added to the V6 database and all MSPs created within the ENRIA project, the amount of strains identified with high confidence increased to 74.8% and 79.2%, respectively. Strains that could not be identified using MALDI-TOF MS decreased to 10.4% and 7.3%, respectively. The observed increase in high confidence identifications differed per genus. For Bilophila wadsworthia, Prevotella spp., gram-positive anaerobic cocci and other less commonly encountered species more strains were identified with higher confidence. A subset of the non-identified strains (42.1%) were identified using 16S rDNA gene sequencing. The obtained identities demonstrated that strains could not be identified either due to the generation of spectra of insufficient quality or due to the fact that no MSP of the encountered species was present in the database. Undoubtedly, the ENRIA project has successfully increased the number of anaerobic isolates that can be identified with high confidence. We therefore recommend further expansion of the database to include less frequently isolated species as this would also allow us to gain valuable insight into the clinical relevance of these less common anaerobic bacteria.

How to isolate, identify and determine antimicrobial susceptibility of anaerobic bacteria in routine laboratories.

BACKGROUND: There has been increased interest in the study of anaerobic bacteria that cause human infection during the past decade. Many new genera and species have been described using 16S rRNA gene sequencing of clinical isolates obtained from different infection sites with commercially available special culture media to support the growth of anaerobes. Several systems, such as anaerobic pouches, boxes, jars and chambers provide suitable anaerobic culture conditions to isolate even strict anaerobic bacteria successfully from clinical specimens. Beside the classical, time-consuming identification methods and automated biochemical tests, the use of matrix-assisted laser desorption/ionization time-of-flight mass spectrometry has revolutionized identification of even unusual and slow-growing anaerobes directly from culture plates, providing the possibility of providing timely information about anaerobic infections. AIMS: The aim of this review article is to present methods for routine laboratories, which carry out anaerobic diagnostics on different levels. SOURCES: Relevant data from the literature mostly published during the last 7 years are encompassed and discussed. CONTENT: The review involves topics on the anaerobes that are members of the commensal microbiota and their role causing infection, the key requirements for collection and transport of specimens, processing of specimens in the laboratory, incubation techniques, identification and antimicrobial susceptibility testing of anaerobic bacteria. Advantages, drawbacks and specific benefits of the methods are highlighted. IMPLICATIONS: The present review aims to update and improve anaerobic microbiology in laboratories with optimal conditions as well as encourage its routine implementation in laboratories with restricted resources.

A multi-center ring trial for the identification of anaerobic bacteria using MALDI-TOF MS.

Inter-laboratory reproducibility of Matrix Assisted Laser Desorption Time-of-Flight Mass Spectrometry (MALDI-TOF MS) of anaerobic bacteria has not been shown before. Therefore, ten anonymized anaerobic strains were sent to seven participating laboratories, an initiative of the European Network for the Rapid Identification of Anaerobes (ENRIA). On arrival the strains were cultured and identified using MALDI-TOF MS. The spectra derived were compared with two different Biotyper MALDI-TOF MS databases, the db5627 and the db6903. The results obtained using the db5627 shows a reasonable variation between the different laboratories. However, when a more optimized database is used, the variation is less pronounced. In this study we show that an optimized database not only results in a higher number of strains which can be identified using MALDI-TOF MS, but also corrects for differences in performance between laboratories.

Incidence of Propionibacterium acnes in initially culture-negative thioglycollate broths-a prospective cohort study at a Danish University Hospital.

The aim of this study was to prospectively investigate the incidence of Propionibacterium acnes in thioglycollate broths reported as culture-negative at the Department of Clinical Microbiology, Rigshospitalet, to evaluate whether 5 days of incubation was enough to find all relevant cases. Five hundred thioglycollate broths reported as culture-negative after 5 days were consecutively collected and incubated for at least a further 9 days (at least 14 days of incubation in total). Only tissue samples from sterile sites of the body (n = 298), bone tissue (n = 197) and foreign material (n = 5) were included in this study. Samples were divided into two groups: infected group and control group. This made it possible to compare findings between groups, thereby making it possible to estimate the level of true-positive findings and contamination. Samples from 296 participants were included in this study. After exclusion criteria were met, P. acnes was cultured from ten out of 151 patients (6.6%) in the infected group and from one out of 138 participants (0.7%) in the control group. This resulted in more findings of P. acnes in the infected group on day 14 than on day 5 (p 0.002). Furthermore, P. acnes was cultured more often from bone tissue and tissue surrounding foreign materials on day 14 than on day 5 (p 0.04). Clinical microbiology laboratories should consider incubating thioglycollate broths for at least 14 days to find all relevant cases of P. acnes, especially when it comes to bone tissue and tissue surrounding foreign materials.

The optimization and validation of the Biotyper MALDI-TOF MS database for the identification of Gram-positive anaerobic cocci.

Gram-positive anaerobic cocci (GPAC) account for 24%-31% of the anaerobic bacteria isolated from human clinical specimens. At present, GPAC are under-represented in the Biotyper MALDI-TOF MS database. Profiles of new species have yet to be added. We present the optimization of the matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) database for the identification of GPAC. Main spectral profiles (MSPs) were created for 108 clinical GPAC isolates. Identity was confirmed using 16S rRNA gene sequencing. Species identification was considered to be reliable if the sequence similarity with its closest relative was ≥98.7%. The optimized database was validated using 140 clinical isolates. The 16S rRNA sequencing identity was compared with the MALDI-TOF MS result. MSPs were added from 17 species that were not yet represented in the MALDI-TOF MS database or were under-represented (fewer than five MSPs). This resulted in an increase from 53.6% (75/140) to 82.1% (115/140) of GPAC isolates that could be identified at the species level using MALDI-TOF MS. An improved log score was obtained for 51.4% (72/140) of the strains. For strains with a sequence similarity <98.7% with their closest relative (n = 5) or with an inconclusive sequence identity (n = 4), no identification was obtained by MALDI-TOF MS or in the latter case an identity with one of its relatives. For some species the MSP of the type strain was not part of the confined cluster of the corresponding clinical isolates. Also, not all species formed a homogeneous cluster. It emphasizes the necessity of adding sufficient MSPs of human clinical isolates.

Whole genome sequencing as a tool for phylogenetic analysis of clinical strains of Mitis group streptococci.

Identification of Mitis group streptococci (MGS) to the species level is challenging for routine microbiology laboratories. Correct identification is crucial for the diagnosis of infective endocarditis, identification of treatment failure, and/or infection relapse. Eighty MGS from Danish patients with infective endocarditis were whole genome sequenced. We compared the phylogenetic analyses based on single genes (recA, sodA, gdh), multigene (MLSA), SNPs, and core-genome sequences. The six phylogenetic analyses generally showed a similar pattern of six monophyletic clusters, though a few differences were observed in single gene analyses. Species identification based on single gene analysis showed their limitations when more strains were included. In contrast, analyses incorporating more sequence data, like MLSA, SNPs and core-genome analyses, provided more distinct clustering. The core-genome tree showed the most distinct clustering.

Routine disc diffusion antimicrobial susceptibility testing of Clostridium difficile and association with PCR ribotype 027.

Reduced susceptibility to metronidazole and vancomycin in Clostridium difficile has been reported, which emphasises the need for simple antimicrobial susceptibility testing methods. The aim of this study was to apply a published disc diffusion method and zone diameter breakpoint correlates to the European Committee on Antimicrobial Susceptibility Testing (EUCAST) epidemiological minimum inhibitory concentration (MIC) cut-off values in a routine setting. Metronidazole and vancomycin zone diameters from 2702 isolates were recorded. Fifteen isolates had a metronidazole zone diameter below the published breakpoint (<23 mm) and five isolates had a vancomycin zone diameter below the published breakpoint (<19 mm), most of which were polymerase chain reaction (PCR) ribotype 027. The total number of PCR ribotype 027 was 29 (1.1 %). Overall, C. difficile PCR ribotype 027 isolates had smaller zone diameters than non-027 isolates. The disc diffusion method is very simple and inexpensive, and the published zone diameter breakpoints will detect C. difficile isolates with reduced susceptibility to metronidazole and vancomycin.

Comparison of Rosco Neo-Sensitabs with Oxoid paper disks in EUCAST disk diffusion antimicrobial susceptibility testing on Mueller-Hinton agar.

This study compared Neo-Sensitabs with Oxoid paper disks using the European Committee on Antimicrobial Susceptibility Testing (EUCAST) disk diffusion antimicrobial susceptibility test on Mueller-Hinton agar. The EUCAST-recommended quality control strains (Escherichia coli ATCC 25922, Pseudomonas aeruginosa ATCC 27853, Staphylococcus aureus ATCC 29213 and Enterococcus faecalis ATCC 29212) (Part I) and clinical isolates (Part II) were investigated. In Part I of the study, 27 combinations of antimicrobial agents were tested on four quality control strains repeatedly up to 60 times and zone diameters of tablets and disks were compared. In Part II of the study, 351 clinical isolates were included to cover a broad range of species, as well as resistance mechanisms. In Part I, four major deviations (>1 mm outside quality control ranges) were observed with Neo-Sensitabs. In one case with P. aeruginosa ATCC 27853 (meropenem), there was a corresponding major deviation (2 mm) with the Oxoid disk. The three remaining major deviations with Neo-Sensitabs were observed with meropenem (2 mm) in E. coli ATCC 25922 and with ciprofloxacin (2 mm) and gentamicin (3 mm) in P. aeruginosa ATCC 27853. For Oxoid disks, there were only minor deviations (=1 mm outside quality control ranges) in these three cases. In Part II, there were six discrepancies, susceptible versus resistant, in 3,533 comparisons between the two methods with the clinical isolates. The Rosco Neo-Sensitabs appear to be a possible alternative to Oxoid paper disks for EUCAST disk diffusion antimicrobial susceptibility testing on Mueller-Hinton agar.

Multidrug-resistant Bacteroides fragilis group on the rise in Europe?

We report a case of multidrug-resistance (MDR) in a strain of Bacteroides fragilis from a blood culture and abdominal fluid in a Danish patient. The patient had not been travelling for several years and had not received antibiotics prior to the present case. We also summarize the cases that have been reported to date of MDR B. fragilis group in Europe. As far as we know, a case like this with MDR B. fragilis has not been described in Scandinavia before.

Antimicrobial susceptibility testing of Clostridium difficile using EUCAST epidemiological cut-off values and disk diffusion correlates.

With the emergence of reduced susceptibility of Clostridium difficile to metronidazole and vancomycin the value of antimicrobial susceptibility testing has increased. The aim of our study was to evaluate disk diffusion for susceptibility testing of C. difficile by comparing disk diffusion results with MICs from gradient tests and to propose zone diameter breakpoint correlates for the EUCAST epidemiological cut-off values (ECOFFs) recently published. We tested 211 clinical isolates of C. difficile, from patients with diarrhoea hospitalized at Aarhus and Odense University Hospitals, Denmark. Furthermore, ten clinical isolates of C. difficile from the Anaerobe Reference Laboratory, University Hospital of Wales, with known reduced susceptibility to either metronidazole or vancomycin, were included. Isolates were tested with Etest gradient strips and disk diffusion towards metronidazole, vancomycin and moxifloxacin on Brucella Blood Agar supplemented with hemin and vitamin K. We found an excellent agreement between inhibition zone diameter and MICs. For each MIC value, the inhibition zones varied from 0 to 8 mm, with 93% of values within 6 mm for metronidazole, 95% of values within 4 mm for vancomycin, and 98% of values within 4 mm for moxifloxacin. With proposed zone diameter breakpoints for metronidazole, vancomycin and moxifloxacin of WT ≥ 23 mm, WT ≥ 19 and WT ≥ 20 mm, respectively, we found no very major errors and only major errors below 2%. In conclusion, we suggest that disk diffusion is an option for antimicrobial susceptibility testing of C. difficile.

The long-term pharmacokinetics and safety of adding low-dose ritonavir to a nelfinavir 1,250 mg twice-daily regimen in HIV-infected patients.

OBJECTIVES: To evaluate the long-term pharmacokinetics and safety of adding ritonavir 100 mg twice-daily to a nelfinavir 1250 mg twice-daily regimen in HIV-infected patients. METHODS: This was a prospective, randomized, open-label, controlled 24-week study. Sixteen patients receiving a nelfinavir 1250 mg twice-daily regimen with plasma viral load <1000 HIV-1 RNA copies/mL were randomized to continue treatment or to have ritonavir 100 mg twice-daily added. Safety, including fasting lipid levels, was evaluated at weeks 4, 12 and 24. Patients who were randomized to have ritonavir added (n=9) participated in three 12-h pharmacokinetic evaluations at baseline, week 4 and week 24. RESULTS: Increases in median nelfinavir steady-state plasma concentrations at 12 h (C(12)) from 512 to 773 ng/mL [median difference 450 ng/mL; 95% confidence interval (CI) 116--1510 ng/mL] and in median active nelfinavir metabolite M 8 C(12) from 107 to 603 ng/mL (median difference 545 ng/mL; 95% CI 370--891) were seen after the addition of low-dose ritonavir (baseline to week 24). There were no differences between the nelfinavir or M 8 pharmacokinetic parameters at weeks 4 and 24. No significant changes or differences in the concentration of fasting total cholesterol, low-density lipoprotein (LDL) cholesterol or total triglycerides or in the occurrence of adverse events were observed within or between the two groups. CONCLUSIONS: Nelfinavir and especially M 8 concentrations are increased when low-dose ritonavir is added to a nelfinavir-containing regimen. The combination seems to be safe and the nelfinavir/ritonavir regimen could be an option in patients with low nelfinavir+M 8 concentrations.

Pharmacokinetic interaction between rifampin and the combination of indinavir and low-dose ritonavir in HIV-infected patients.

Rifampin is an important drug in the treatment of tuberculosis, but administration of rifampin in combination with protease inhibitors is complicated because of drug-drug interactions. A prospective, controlled, multiple-dose study involving 6 HIV-infected patients receiving a combination of indinavir (800 mg) and ritonavir (100 mg) twice a day was performed to evaluate whether the inducing effect of rifampin on the drug-metabolizing enzyme cytochrome P450 (CYP) 3A4 could be overcome by the inhibitory effect of ritonavir. Pharmacokinetic evaluations of steady-state concentrations of indinavir and ritonavir were performed before and after administration of rifampin (300 mg every day for 4 days). An 87% reduction (from 837 to 112 ng/mL) in median indinavir and a 94% reduction (from 431 to 27 ng/mL) in median ritonavir concentrations were seen 12 h after the last dose of rifampin was administered (P=.031). These results strongly indicate that the administration of rifampin with a combination of indinavir (800 mg) and ritonavir (100 mg) could lead to subtherapeutic concentrations of indinavir.

Low-dose indinavir in combination with low-dose ritonavir: steady-state pharmacokinetics and long-term clinical outcome follow-up.

OBJECTIVES: To evaluate the long-term efficacy and pharmacokinetics of indinavir (IDV)/ritonavir (RTV) 400/100 mg twice a day in combination with two nucleoside reverse transcriptase inhibitors. METHODS: The study was retrospective with a prospective pharmacokinetic study at a single centre. All HIV-1-infected patients who started the regimen in the period from January 1999 to February 2001 were included in the study. Plasma HIV RNA and CD4 cell counts were recorded from baseline to week 120. Results were evaluated as intention-to-treat and on-treatment analyses with separate analyses for protease inhibitor naive and experienced patients. Patients who were still on the regimen by August 2001 were asked to participate in a pharmacokinetic evaluation. RESULTS: Twenty-one patients started treatment with the regimen (median follow-up: 116 weeks). The percentage of patients with below 20 HIV-1 RNA copies/mL was 70.0% at week 120 and the median CD4 cell count increased from 320 to 607 cells/microL (P=0.062). The median IDV morning and evening Cmin were 434 ng/mL and 220 ng/mL, respectively. CONCLUSIONS: Treatment with the IDV/RTV 400/100 mg regimen appears to be efficacious for up to 2 years. However, rather low IDV Cmin suggests that the regimen should be evaluated further before its widespread use and that the regimen probably should be guided by pharmacokinetic evaluation.

The use of calcium carbonate in nelfinavir-associated diarrhoea in HIV-1-infected patients.

OBJECTIVES: To evaluate the efficacy of oral calcium supplements in HIV-infected patients with nelfinavir (NFV)-associated diarrhoea, and to investigate the influence on the pharmacokinetics of nelfinavir and the active metabolite M8. METHODS: An open-label prospective trial with enrolment of 15 patients with NFV-associated diarrhoea. Study subjects received either calcium carbonate or calcium gluconate/calcium carbonate in addition to highly active antiretroviral therapy (HAART), and were randomized to (i) calcium supplements for 14 days followed by 14 without calcium supplements, or (ii) 14 days without calcium supplements followed by calcium supplements for 14 days. Clinical endpoint was the severity of diarrhoea, graded and summarized for the specific 14 day-period. In the pharmacokinetic evaluation concentrations of NFV and M8 were measured before morning dosing, and 3 h after dosing. RESULTS: Nine patients were treated with calcium carbonate, and six with calcium gluconate/calcium carbonate. In the paired analysis, neither of the groups had a significant improvement in diarrhoea score when treated with calcium supplements (P = 0.34 and 0.46, respectively). We found no significant differences in the concentrations of NFV and M8 between the calcium and control periods. CONCLUSIONS: Oral calcium supplements did not significantly improve nelfinavir-associated diarrhoea. In the pharmacokinetic analysis calcium supplements did not induce major alterations in plasma concentrations of NFV and M8.

Simultaneous quantitative determination of the HIV protease inhibitors indinavir, amprenavir, ritonavir, lopinavir, saquinavir, nelfinavir and the nelfinavir active metabolite M8 in plasma by liquid chromatography.

A simple HPLC method that quantitates all six currently available protease inhibitors and the nelfinavir active metabolite M8 in one assay is presented. A 500-microliter plasma sample was treated by liquid-liquid extraction with a mixture of heptane and ethyl acetate. After evaporation, the residue was redissolved in sodium dihydrogenphosphate and acetonitrile and washed twice with heptane. Chromatography was performed with an analytical C(18) column. Ultraviolet detection at 210 and 239 nm was used. The present method is associated with high accuracy and low imprecision in the concentration range of 25-5000 ng/ml of all six protease inhibitors and M8. This makes it suitable for monitoring purposes.

[Lactic acidosis type B. A life-threatening adverse effect of antiretroviral therapy]. / Lactatacidose type B. En livstruende bivirkning til antiretroviral behandling.

A thirty-year old HIV-positive woman, who had been receiving antiretroviral therapy (protease inhibitor, lamivudine and stavudine) for seven months, was diagnosed with severe lactic acidosis type B, most likely induced by nucleoside reverse transcriptase inhibitor treatment. The antiretroviral therapy was ceased, and she was treated with isotonic sodium bicarbonate intravenously. She made a full recovery apart from a perceptive hearing deficit.

[HIV protease inhibitors and interactions]. / Hiv-proteasehaemmere og interaktioner.

The protease inhibitors are a new class of drugs for the treatment of HIV infection. Results of treatment have proved beneficial to HIV positive patients, resulting in slower disease progression to aids and death. However the potential of drug interactions is high because of the drug-transporting P-glycoprotein and cytochrome P450 3A4-mediated metabolism. Administration of protease inhibitors may result in increased or decreased concentrations of co-administered drugs, and the plasma concentration of protease inhibitors may be affected by other drugs. It is possible to take advantage of the interactions by combining two protease inhibitors. Attention is drawn to the protease inhibitors and their possible interactions because of the advantages and disadvantages this implies. It is possible to monitor interactions by measuring plasma concentrations of the protease inhibitors.

[Experiences with antiretroviral combination therapy]. / Erfaringer med antiretroviral kombinationsbehandling.

The purpose of the study was to evaluate the efficacy of treating of HIV infected patients with two nucleoside analogues and one protease inhibitor in clinical practice. Sixty-one patients were included and followed with respect to plasma HIV-RNA, CD4 cell count and side effects up to one year. Median plasma HIV-RNA was reduced from 20,000 to < 200 copies/ml, and the percentage of patients with plasma HIV-RNA < 200 copies per ml increased from 7.5% to 65.2%. The CD4 cell count increased from 180 to 300 x 10(6)/l. Among 49 patients who remained on therapy, the percentage of patients with plasma HIV-RNA < 200 copies/ml increased from 9.8% to 73.7%. It is concluded that triple drug antiretroviral treatment shows significant effects on plasma HIV-RNA and CD4 cell count, and that the results obtained in clinical practice are comparable to results reported from controlled clinical trials.