Total knee arthroplasty infection due to Abiotrophia defectiva

The first documented case of knee alloarthroplasty infection due to Abiotrophia defectiva, formerly known as nutritionally variant streptococci (NVS) and Streptococcus defectivus, is presented. The microbiology of this bacterium is discussed and clinical features of previously reported cases of infections by NVS are reviewed briefly.     

Best practice in primary care pathology: review 9

This ninth best-practice review examines two series of common primary care questions in laboratory medicine: (i) potassium abnormalities and (ii) venous leg ulcer microbiology. The review is presented in question-and-answer format, referenced for each question series. The recommendations represent a précis of guidance found using a standardised literature search of national and international guidance notes, consensus statements, health policy documents and evidence-based medicine reviews, supplemented by MEDLINE EMBASE searches to identify relevant primary research documents. They are not standards but form a guide to be set in the clinical context. Most are consensus rather than evidence-based. They will be updated periodically to take account of new information.     

Compliance of clinical microbiology laboratories in the United States with current recommendations for processing respiratory tract specimens from patients with cystic fibrosis

Respiratory tract specimens from patients with cystic fibrosis (CF) require unique processing by clinical microbiology laboratories to ensure detection of all potential pathogens. The present study sought to determine the compliance of microbiology laboratories in the United States with recently published recommendations for CF respiratory specimens. Microbiology laboratory protocols from 150 of 190 (79%) CF care sites were reviewed. Most described the use of selective media for Burkholderia cepacia complex (99%), Staphylococcus aureus (82%), and Haemophilus influenzae (89%) and identified the species of all gram-negative bacilli (87%). Only 52% delineated the use of agar diffusion assays for susceptibility testing of Pseudomonas aeruginosa. Standardizing laboratory practices will improve treatment, infection control, and our understanding of the changing epidemiology of CF microbiology.     

Accuracy of methods used for susceptibility testing of Haemophilus influenzae in United Kingdom laboratories

Antibiotic susceptibility test reports on 1841 strains of Haemophilus influenzae from 25 microbiology laboratories were compared with results obtained with the same strains at The London Hospital Medical College. Of strains found to be sensitive to the antibiotics tested, 0.5% were reported as tetracycline-resistant, 1.6% as ampicillin-resistant, and 6.2% as trimethoprim-resistant. Of strains found to be resistant to these antibiotics, 37% were reported as tetracycline-sensitive, 27% as ampicillin-sensitive, and 66.7% as trimethoprim-sensitive. Factors found to be of significance in improving accuracy of sensitivity reporting included use of chromogenic cephalosporin and low-content antibiotic discs for detection of ampicillin resistance, and use of lysed blood agar rather than chocolated blood agar to detect trimethoprim sensitivity.     

What should a laboratory physician expect from a microbiology laboratory?

Remarkable changes are affecting the discipline of Clinical Pathology/Laboratory Medicine in Japan. Laboratories are changing from revenue centers to cost centers that have many serious problems(ex. closure of the clinical laboratories in the hospitals and outsourcing of laboratory tests due to restructuring in response to economic aspect, limited numbers of certified laboratory physicians, and other factors). And many clinicians in university hospitals do not know what they should expect correctly from the microbiology laboratory. Therefore, we, laboratory physicians and medical technologists must modify our behavior effectively and establish a good collaborative partnership with physicians, nurses and other health care professionals. The microbiology laboratory should provide information that will affect clinical management guidelines for obtaining specimens, microbial identification, antimicrobial susceptibilities, reporting of data and educational updating. Leadership and management skills must be increasingly critical to the success of laboratory physicians in and outside of academic centers.     

Diagnostic Stewardship: the Central Role of Clinical Microbiology Laboratories

Diagnostic stewardship aims to improve diagnostic test utilization through evidence-based practices to improve care, quality, safety, and costs. Diagnostic stewardship is a collaborative effort that brings together multidisciplinary groups that have a common interest in promoting and ensuring best testing practices. For infectious disease testing, clinical microbiology laboratories are perhaps best positioned within their health care systems to lead these efforts, as they are not only diagnostic experts, but also directly oversee many of the tools and choices available to improve test performance and utilization. While some interventions may not fall under the direct purview of clinical microbiology laboratories, their expertise is nevertheless essential to inform these efforts, as well. Multiple stewardship strategies have been evaluated, providing laboratories with several opportunities to implement evidence-based practice changes to improve quality and outcomes. Further research is needed to continue advancing practice for well-established and emerging tests alike.     

Bacterial genome sequencing in clinical microbiology: a pathogen-oriented review

In recent years, whole-genome sequencing (WGS) has been perceived as a technology with the potential to revolutionise clinical microbiology. Herein, we reviewed the literature on the use of WGS for the most commonly encountered pathogens in clinical microbiology laboratories: Escherichia coli and other Enterobacteriaceae, Staphylococcus aureus and coagulase-negative staphylococci, streptococci and enterococci, mycobacteria and Chlamydia trachomatis. For each pathogen group, we focused on five different aspects: the genome characteristics, the most common genomic approaches and the clinical uses of WGS for (i) typing and outbreak analysis, (ii) virulence investigation and (iii) in silico antimicrobial susceptibility testing. Of all the clinical usages, the most frequent and straightforward usage was to type bacteria and to trace outbreaks back. A next step toward standardisation was made thanks to the development of several new genome-wide multi-locus sequence typing systems based on WGS data. Although virulence characterisation could help in various particular clinical settings, it was done mainly to describe outbreak strains. An increasing number of studies compared genotypic to phenotypic antibiotic susceptibility testing, with mostly promising results. However, routine implementation will preferentially be done in the workflow of particular pathogens, such as mycobacteria, rather than as a broadly applicable generic tool. Overall, concrete uses of WGS in routine clinical microbiology or infection control laboratories were done, but the next big challenges will be the standardisation and validation of the procedures and bioinformatics pipelines in order to reach clinical standards.     

Congenital bacterial sepsis in very preterm infants.

The results of body fluid and surface cultures from 148 preterm infants less than 33 weeks gestational age obtained routinely on admission to a neonatal intensive care unit were reviewed. The aim was to determine the occurrence of congenital bacterial sepsis in this population and to examine whether surface cultures yielded information helpful in management. Gastric aspirate and umbilical, nasal and ear swabs were cultured and the results were compared to those of blood cultures. Nine infants (5.4%) had congenital bacterial sepsis diagnosed by positive blood cultures. Only the results of microscopy of gastric aspirate were available within hours of birth and before the results of blood culture. Microscopy of gastric aspirate, demonstrating pus cells, alone had a sensitivity of 0.86 in predicting congenital sepsis but a specificity of 0.49; the specificity, however, rose to 0.80 if both organisms and pus cells were observed on microscopy. Thus, only this combination was a useful pre-indicator of congenital sepsis. In infants who did not develop septicaemia, treatment was modified only if Streptococcus agalactiae was cultured from surface sites; in all such cases, the organism was grown from the ear swab. Our results demonstrate that congenital bacterial sepsis is common amongst very preterm infants admitted for neonatal intensive care but routine screening of surface cultures should be restricted to an ear swab only.     

Effects of restructuring on the performance of microbiology laboratories in Alberta

OBJECTIVE: To evaluate the error rates of organism identification and antibiotic susceptibility proficiency testing challenges before, during, and after microbiology laboratory restructuring in Alberta. METHODS: Alberta Health substantially reduced and redistributed laboratory funds to the regional health authorities in 1995, forcing a dramatic restructure of services. Many rural hospitals expanded their microbiology test menus, and urban centers consolidated microbiology testing into a centralized high-volume laboratory. The Laboratory Proficiency Testing Program of the College of Physicians and Surgeons of Alberta mailed regular test profile surveys to microbiology laboratories during the restructure period to determine the type and extent of changes in services. Based on the types of tests and the extent of analysis being done, most rural B-level and some C-level laboratories were reclassified to the A level. The Laboratory Proficiency Testing Program reviewed the error rates of proficiency challenges based on the performance of different levels of laboratories before and after the period of restructure. RESULTS: Overall performance has improved according to the number of errors documented on identification and susceptibility challenges for laboratories that remained at the same classification (ie, A or C). The number of major identification errors for laboratories that were reclassified increased, but the rate of major susceptibility errors decreased. More reclassified laboratories do not have dedicated registered technologist(s) who perform microbiology testing and are not supervised by an on-site pathologist and/or medical microbiologist compared with laboratories that remained at the same classification. CONCLUSIONS: Microbiology laboratory restructuring will have adverse effects on the quality of complex testing if experienced technologists are not retained and services are not medically supervised.     

An interprovincial external quality assessment of the ability of Canadian laboratories to detect the vancomycin and penicillin resistance of Enterococcus faecium D366

Objectives: To evaluate the ability of Canadian laboratories to identify enterococci and detect low-level resistance to penicillin, ampicillin and vancomycin in five provinces and two territories by two external quality assessment schemes.
Methods: Enterococcus faecium , strain D366, with minimum inhibitory concentrations for vancomycin and penicillin of 32 and 16 mg/L respectively, was distributed during a routine proficiency survey. Laboratories were required to culture and identify the isolate and to test antimicrobial susceptibility. Participants were assessed against consensus reference values.
Results: Three hundred and sixty-four hospital, commercial and public-health laboratories participated, using their established procedures for patient samples. The isolate was identified to the species level by 222 (61%) laboratories and to the genus level by a further 98 participants. Forty-four failed to meet the expected standard. Vancomycin resistance was detected by 94%. Those reporting a falsely susceptible result used disk diffusion testing. Penicillin resistance was noted by 250 of 258 laboratories reporting on this agent. An incorrect ampicillin-susceptible finding was reported by 62 of 147 laboratories using automated microdilution or agar dilution methods.
Conclusions: Most laboratories identified the isolate to an appropriate level. Detection of low-level vancomycin and penicillin resistance was achieved by the majority. Ampicillin resistance was less readily detected.     

Delineation of Streptococcus dysgalactiae, its subspecies, and its clinical and phylogenetic relationship to Streptococcus pyogenes

The taxonomic status and structure of Streptococcus dysgalactiae have been the object of much confusion. Bacteria belonging to this species are usually referred to as Lancefield group C or group G streptococci in clinical settings in spite of the fact that these terms lack precision and prevent recognition of the exact clinical relevance of these bacteria. The purpose of this study was to develop an improved basis for delineation and identification of the individual species of the pyogenic group of streptococci in the clinical microbiology laboratory, with a special focus on S. dysgalactiae. We critically reexamined the genetic relationships of the species S. dysgalactiae, Streptococcus pyogenes, Streptococcus canis, and Streptococcus equi, which may share Lancefield group antigens, by phylogenetic reconstruction based on multilocus sequence analysis (MLSA) and 16S rRNA gene sequences and by emm typing combined with phenotypic characterization. Analysis of concatenated sequences of seven genes previously used for examination of viridans streptococci distinguished robust and coherent clusters. S. dysgalactiae consists of two separate clusters consistent with the two recognized subspecies dysgalactiae and equisimilis. Both taxa share alleles with S. pyogenes in several housekeeping genes, which invalidates identification based on single-locus sequencing. S. dysgalactiae, S. canis, and S. pyogenes constitute a closely related branch within the genus Streptococcus indicative of recent descent from a common ancestor, while S. equi is highly divergent from other species of the pyogenic group streptococci. The results provide an improved basis for identification of clinically important pyogenic group streptococci and explain the overlapping spectrum of infections caused by the species associated with humans.     

Surveillance of linezolid resistance in Germany, 2001–2002

A surveillance study was performed throughout Germany from November 2001 to June 2002 to assess the prevalence of linezolid-resistant isolates among Gram-positive bacteria from routine susceptibility data and to compare the in-vitro activity of linezolid to that of other antibacterial agents. Each of 86 laboratories provided routine susceptibility data for 100 consecutive isolates. Most laboratories (c. 60%) used the disk diffusion test. Laboratories were also requested to send a representative sample of their isolates, as well as all isolates reported as intermediate or resistant to linezolid, to a reference laboratory for MIC determination. Susceptibility data for 8594 isolates were evaluated. Sites of infection were skin and soft tissue (29.9%), upper and lower respiratory tract (19.1%), foreign body or catheter (10.5%), or urinary tract (9.8%). Routine linezolid susceptibility data were reported for 6433 isolates. The prevalence of linezolid resistance, as reported to the clinician, was 0.4% in Staphylococcus aureus, 0.3% in Staphylococcus epidermidis, 2.9% in Enterococcus faecalis, 2.3% in Enterococcus faecium, 1.4% in Streptococcus pyogenes and 2.9% in Streptococcus agalactiae. Linezolid resistance was not detected in Streptococcus pneumoniae or in viridans group streptococci. Sixty-nine of 115 isolates reported as intermediate or resistant to linezolid were retested, but none was resistant to linezolid. Linezolid exhibited excellent in-vitro activity against representative isolates of the six most frequently encountered species (MIC90, 1-2 mg/L). The prevalence of resistance to linezolid was very low in Germany. Organisms reported as linezolid-resistant should be retested, either in the same laboratory with an alternative method or in a reference laboratory.     

Association of viridans group streptococci from pregnant women with bacterial vaginosis and upper genital tract infection

The prevalence and role of viridans group streptococci in the female genital tract have not been well described. In this study of 482 pregnant women, 147 (30%) were culture positive for viridans group streptococci. Of 392 women with predominant Lactobacillus morphotypes by Gram stain (normal), 110 (28%) were colonized with viridans group streptococci, compared with 37 (41%) of 90 women with bacterial vaginosis (BV) (P = 0.02). To determine whether any species were associated with BV, 177 consecutively isolated viridans group streptococci from the vagina were identified to the species level by using the Facklam scheme. The most frequently isolated species from the vagina was Streptococcus intermedius (13%), followed by Streptococcus acidominimus (6%), Streptococcus constellatus (5%), Streptococcus sanguis II (4%), Streptococcus mitis (2%), Streptococcus salivarius (2%), Streptococcus morbillorum (2%), Streptococcus sanguis I (1%), Streptococcus mutans (0.2%), and Streptococcus uberis (0.2%) with an average of 1.2 species per woman. The distribution of the species among women with BV compared with normal women was not significantly different, with the exception of two species which were associated with BV: S. acidominimus (18% versus 3%, P less than 0.001) and S. morbillorum (6% versus 0.7%, P = 0.005). Amniotic fluid and placenta cultures yielded 54 isolates: S. sanguis II (13 isolates), S. acidominimus (9 isolates), S. intermedius (10 isolates), S. constellatus (3 isolates), S. mitis (4 isolates), S. sanguis I (4 isolates), S. morbillorum (5 isolates), S. mutans (2 isolates), S. uberis (1 isolate), mannitol-positive S. intermedius (1 isolate), and 2 isolates which were not classified. The distribution of species isolated from the upper genital tract was not a reflection of the distribution in the lower genital tract. Dextran-producing species of viridans group streptococci may have a greater pathogenic potential in the placenta than the non-dextran-producing species.     

Antibiotic susceptibilities of streptococci from the mouth and blood of patients treated with penicillin or lincomycin and clindamycin.

Patients undergoing dental extractions were non-randomly allocated to three groups, one of which received no antibiotic, one benzylpenicillin followed by oral penicillin for 5 days, and the third intramuscular lincomycin followed by oral clindamycin. Dental extraction was performed at the beginning of the course of chemotherapy. Streptococci were isolated from the extracted teeth, from blood cultures collected before and immediately after dental extraction, and from sutures removed from the gums 5-7 days after the operation. The species of these organisms was determined, and their susceptibilities to penicillin, clindamycin, cephaloridine, erythromycin and tetracycline were assessed. The majority of streptococci isolated from teeth belonged to the species Streptococcus sanguis, S. mitior, S. mutans and S. milleri. Occasional isolates of each of these organisms collected before the antibiotic could take effect were resistant to penicillin. Three of these species, but not S. mutans, were the commonest streptococci to be isolated from the blood after dental extraction. Penicillin completely suppressed dental bacteriaemia under the conditions of our investigation, and lincomycin reduced the incidence by about 60 per cent. The commonest streptococci from sutures were also S. sanguis, S. mitior, S. mutans and S. milleri. S. faecalis was also isolated, but only in patients who had received antibiotics. Among the non-faecalis organisms, penicillin resistance was significantly more frequent among isolates from patients given penicillin than from patients not given this antibiotic, and clindamycin resistance was significantly more frequent among isolates from patients given lincomycin and clindamycin than from patients not given these antibiotics.     

Measurement of microbial alpha-amylases with p-nitrophenyl glycosides as the substrate complex.

The detection of alpha-amylase is commonly used in clinical microbiology laboratories to aid in differentiating Streptococcus bovis from other streptococci. It is also useful in identifying Eikenella corrodens and the gravis subspecies of Corynebacterium diphtheriae and in separating species of the genera Bacteroides, Clostridium, Actinomyces, and Bacillus. Currently, the most frequently used procedure utilizes starch as the substrate and iodine as the indicator. Starch is incorporated into a agar medium, the isolate is inoculated on the surface, and the medium is incubated for 24 to 48 h. A 15-min test containing p-nitrophenyl polyglycosides as the substrate complex was developed to yield results comparable with the agar-based starch test. The reagent was made in liquid form, 0.20 ml per tube, and could be incubated either in ambient air or at 35 degrees C. When dried, the p-nitrophenyl polyglycoside reagent could be stored at 0 degrees C for 4 weeks.     

Quality assessment of Microbe Base antimicrobial susceptibility data.

AIMS--To assess the quality of centres contributing antimicrobial susceptibility data to a centralised database. METHODS--Twelve organisms were distributed to 31 regional microbiology laboratories contributing data to a centralised susceptibility database. Participants were asked to determine susceptibilities to certain antibiotics by their routine method and return the data to the Department of Microbiology, Royal Hallamshire Hospital, Sheffield, for analysis. RESULTS--Results for the overwhelming majority of organism/antibiotic combinations were in agreement with expected results. Reasons for discrepancies included the non-bimodal distribution of susceptibilities, the use of different content discs, and, more importantly, minimum inhibitory concentrations falling close to breakpoint values. CONCLUSIONS--It is inevitable that any large multicentre database will contain a degree of inaccurate data. This study has highlighted several areas where discrepant results have occurred and has enabled Glaxo Laboratories to approach individual laboratories to address this problem. This study emphasises the value and consistency of Microbe Base as the largest database, of its kind, nationally.     

Antibiotic resistance patterns of group B streptococci in pregnant women.

This study examined the antibiotic resistance patterns of group B streptococci (GBS) isolated from gravid women. A total of 156 vaginal and cervical isolates of GBS were examined for resistance to penicillin, ampicillin, clindamycin, cefoxitin, gentamicin, and erythromicin. No resistance to penicillin or ampicillin was found, nor was penicillinase production demonstrated. A high level of resistance to gentamicin was noted (91%). Of the isolates examined, 9, 9.5, and 15.3% exhibited either resistance or intermediate susceptibility to erythromycin, clindamycin, and cefoxitin, respectively. Thirty strains (19%) exhibited a multiple antibiotic resistance pattern. Given the high penicillin and ampicillin treatment failure rates when attempting to eradicate vaginal GBS colonization and our findings of higher and multiple drug resistance patterns of GBS, the selection of an alternative antibiotic regimen is of considerable clinical importance. We recommend that routine reporting of GBS susceptibilities by clinical laboratories be adopted.     

Distribution and incidence of viridans streptococcal species in routine clinical specimens

Five hundred consecutive isolates of viridans streptococci were identified to the species level in an effort to determine their distribution and incidence in routine clinical specimens. Viridans streptococci accounted for significant percentages of streptococcal isolates from urine, wounds, body fluids, and blood. The most commonly isolated strains belonged to the Streptococcus milleri, Streptococcus mitis, Streptococcus sanguis I, and Streptococcus sanguis II species. Patient charts were reviewed in order to investigate the possible role as a urinary pathogen of strains belonging to a subgroup of S. milleri. Although these strains frequently are isolated from urine, they appear to play no pathogenic role in urinary tract infections.     

Use of an oligonucleotide array for laboratory diagnosis of bacteria responsible for acute upper respiratory infections

We developed a diagnostic array of oligonucleotide probes targeting species-specific variable regions of the genes encoding topoisomerases GyrB and ParE of respiratory bacterial pathogens. Suitable broad-range primer sequences were designed based on alignment of gyrB/parE sequences from nine different bacterial species. These species included Corynebacterium diphtheriae, Fusobacterium necrophorum, Haemophilus influenzae, Legionella pneumophila, Moraxella catarrhalis, Mycoplasma pneumoniae, Staphylococcus aureus, Streptococcus pneumoniae, and Streptococcus pyogenes. Specific probe sequences were selected by comparative analysis against the European Bioinformatics Database, as well as gyrB/parE sequences generated for this study. To verify specificity, at least six initial oligonucleotide probe sequences per bacterial species were tested by hybridization on a solid glass support using culture collection strains as templates. Finally, three oligonucleotide probes per bacterial species were utilized to examine 65 middle ear fluid and 29 throat swab samples. The sensitivities of the developed assay compared to classic culture from middle ear fluid samples for H. influenzae, M. catarrhalis, and S. pneumoniae were 96 (93 for culture), 73 (93 for culture), and 100% (78% for culture), respectively. No cross-reactivity with bacterial species belonging to the normal oral flora was observed when the 29 throat swab samples were studied. The sensitivity of the assay to detect S. pyogenes from these samples was 93% (80% for culture). These results provide a proof of concept for the diagnostic use of microarray technology based on broad-range topoisomerase gene amplification, followed by hybridization and specific detection of bacterial species.