Post-mortem microbiology in sudden death: sampling protocols proposed in different clinical settings

BackgroundAutopsies, including minimally invasive autopsies, are a powerful tool for determination of the cause of death. When a patient dies from an infection, microbiology is crucial to identify the causative organism. Post-mortem microbiology (PMM) aims to detect unexpected infections causing sudden deaths; confirm clinically suspected but unproven infection; evaluate the efficacy of antimicrobial therapy; identify emergent pathogens; and recognize medical errors. Additionally, the analysis of the thanatomicrobiome may help to estimate the post-mortem interval.AimsThe aim was to provide advice in the collection of PMM samples and to propose sampling guidelines for microbiologists advising autopsy pathologists facing different sudden death scenarios.SourcesA multidisciplinary team with experts in various fields of microbiology and autopsies on behalf of the ESGFOR (ESCMID – European Society of Clinical Microbiology and Infectious Diseases – study group of forensic and post-mortem microbiology and in collaboration with the European Society of Pathology) developed this narrative review based on a literature search using MedLine and Scopus electronic databases supplemented with their own expertise.ContentThese guidelines address measures to prevent sample contamination in autopsy microbiology; general PMM sampling technique; protocols for PMM sampling in different scenarios and using minimally invasive autopsy; and potential use of the evolving post-mortem microbiome to estimate the post-mortem interval.ImplicationsAdequate sampling is paramount to identify the causative organism. Meaningful interpretation of PMM results requires careful evaluation in the context of clinical history, macroscopic and histological findings. Networking and closer collaboration among microbiologists and autopsy pathologists is vital to maximize the yield of PMM.     

Development of microbiological field methodology for water and food-chain hygiene analysis of Campylobacter spp. and Yersinia spp. in Burkina Faso,West Africa

Field-adaptable research methods for identifying Campylobacter sp., Yersinia sp. and other pathogenic and indicator bacteria were designed in Finland and tested in Burkina Faso. Several bacterial groups were also validated from artificially contaminated samples. Campylobacter strains were cultivated using an innovative gas generation system: The ‘Portable Microbe Enrichment Unit’ (PMEU) which provides microaerobic gas flow into the enrichment broth. This enhanced cultivation system produced rapid growth of several isolates of campylobacteria from water and chicken samples. The latter were obtained from local marketplace samples. No yersinias were found in the field studies, whereas they were readily recovered from the spiked samples, as well as Salmonella sp. and Escherichia coli strains. The PMEU method turned out to be reliable for monitoring of water and food hygiene in remote locations.     

Normal flora and bacterial vaginosis in pregnancy: an overview

Abstract

The female genital tract is an intricate, yet balanced ecosystem that hosts a variety of different residential microflora. The physiological changes that occur during pregnancy may disrupt this balanced ecosystem and predispose women to a potentially pathogenic microbiota. Bacteria that are associated with bacterial vaginosis (BV) are opportunistic pathogens that frequently form part of this microbiota. The overgrowth of and infections with these bacteria are linked to poor obstetric outcomes and increased transmission of other reproductive tract infections (RTIs). These infections increase women's susceptibility of acquiring HIV, the rates of HIV shedding and the development of acquired immune deficiency syndrome (AIDS) in HIV-infected patients. It is unknown how the plethora of bacterial species associated with BV contributes to the dynamics of this condition. The use of high-throughput methods have led to the in-depth investigation of different BV-related bacterial species and the functional capabilities of these species. However, the pathogenesis of BV is still poorly defined and the role of individual BV-related bacterial species in specific pregnancy complications is unclear and controversial. The majority of BV infections are asymptomatic and successful diagnosis is complicated by the lack of reliable and standardized diagnostic tests.     

Etiology,clinical features,management, and outcomes of skin and soft tissue infections in hospitalized children: A 10-year review

PurposeThis study aimed to describe the etiology, clinical features, hospital course, and outcomes of hospitalized children with skin and soft tissue infections (SSTIs) and to test if clinical and laboratory variables at admission could differentiate between community-acquired methicillin-resistant Staphylococcus aureus (CA-MRSA) and community-acquired methicillin-sensitive S. aureus (CA-MSSA).MethodsWe reviewed the clinical, laboratory, treatment, and outcome data for children hospitalized with SSTIs, aged 0–18 years at MacKay Children's Hospital between 2010 and 2019. Multivariable logistic regression was used to identify independent predictors of CA-MRSA and CA-MSSA SSTIs.ResultsA total of 1631 patients were enrolled. Erysipelas/cellulitis (73.8%) was the most common pediatric SSTI type, followed by acute lymphadenitis (13.6%) and abscess/furuncle/carbuncle (8.6%). Among the 639 culture-positive isolates (purulent SSTIs), 142 (22.2%) were CA-MSSA and 363 (56.8%) were CA-MRSA. The age group 0–1 month (OR, 6.52; 95% CI 1.09–38.92; P = 0.04) and local lymph node reaction (OR, 2.47; 95% CI 1.004–6.08; P = 0.049) were independent factors for differentiating children with CA-MSSA from those with CA-MRSA SSTIs. MRSA isolates in our cohort were highly susceptible to glycopeptides (100%), linezolid (100%), daptomycin (100%), and sulfamethoxazole/trimethoprim (98.6%) but were significantly less susceptible to clindamycin compared with MSSA (34.2% vs. 78.2%, P < 0.001).ConclusionS. aureus is the leading pathogen of culture-proven SSTIs in hospitalized children with MRSA accounting for more than half. Determining the optimal empirical antibiotics in CA-SSTIs may rely on the patient's age, disease severity, and local epidemiologic data.     

Assessment of the usefulness of performing bacterial identification and antimicrobial susceptibility testing 24 h a day in a clinical microbiology laboratory

The impact of inoculating agar media with positive blood cultures and of performing bacterial identification and antimicrobial susceptibility testing (AST) for positive urine cultures, blood cultures and certain fluid cultures after day hours (night service (NS)) was evaluated in a clinical microbiology laboratory. The impact of the NS was assessed in terms of decreases in the delays from the time of sampling to the time at which results became available and of the consequences for patient management and antimicrobial treatment. Two major benefits were obtained: initiation of earlier appropriate treatment, and change to a reduced-spectrum but still efficient regimen. The hours of laboratory testing and the availability and transmission of results to the clinical staff were recorded. Concurrently, these hours were estimated as though laboratory tests had been performed in the absence of NS. Reductions in delay were defined as the differences between the hours actually spent and the estimated hours. Economic concerns were also considered. Overall, 430 samples for which an identification and/or AST were performed during the NS were included in the study. The NS led to the implementation of earlier appropriate therapy in 97 cases (22.6%), and to the change to reduced-spectrum but still efficient regimens in 23 additional cases (5.3%). In conclusion, there appeared to be benefits from a system providing bacterial identification and AST overnight, but a study of the cost-effectiveness of the NS would be useful to back up this observation.     

Haematology and clinical chemistry in rats: Comparison of different blood collection sites

Blood samples from male and female rats were collected from four different sampling sites by the same technicians and analysed by the same procedures. The sampling sites were the abdominal aorta, orbital venous plexus, dorsal anastomotic orbital vein and sublingual vein. Values obtained in blood samples collected from peripheral sites were compared to those from the abdominal aorta, a sampling site which is normally unaffected by the sampling technique.There were significant differences in haematological parameters, particularly in leucocyte counts which were higher in samples collected from the peripheral sites than in those withdrawn from the central one. No significant changes were observed in coagulation parameters. A significant increase in clinical chemistry parameters related to soft tissue damage, namely creatinine kinase, lactate dehydrogenase, hydroxybutyrate dehydrogenase and aspartate aminotransferase, was seen in samples collected from both orbital sites.From this study it can be concluded that haematological and biochemical values obtained from rats in toxicological studies using different sampling sites are reliable both in males and females, provided that they are compared to values obtained from the same site in untreated controls. Sampling from the orbital plexus proved to be the least invasive method.     

Complement in clinical medicine: Clinical trials,case reports and therapy monitoring

Research during past decades made it evident that complement is involved in more tasks than fighting infections, but has important roles in other immune surveillance and housekeeping functions. If the balance between complement activation and regulation is out of tune, however, complement can quickly turn against the host and contribute to adverse processes that result in various clinical conditions. Whereas clinical awareness was initially focused on complement deficiencies, excessive activation and insufficient regulation are frequently the dominant factors in complement-related disorders. The individual complement profile of a patient often determines the course and severity of the disease, and the pathophysiological involvement of complement may be highly diverse. As a consequence, complement assays have evolved as essential tools not only in initial diagnosis but also for following disease progression and for monitoring complement-targeted therapies, which become increasingly available in routine clinical use. We herein review the current state of complement-directed drug candidates in clinical evaluation and provide an overview of extended indications considered for the FDA-approved inhibitor eculizumab. Furthermore we review the literature describing cases reports and case series where eculizumab has been used “off-label”. Finally, we give a summary of the currently available tests to measure complement profiles and discuss their suitability in diagnostics and treatment monitoring. With complement finally entering the clinical arena, there are intriguing opportunities for treating complement-mediated diseases. However, this progress also requires a new awareness about complement pathophysiology, adequate diagnostic tools and suitable treatment options among clinicians treating patients with such disorders.     

Antilogic,a new supervised machine learning software for the automatic interpretation of antibiotic susceptibility testing in clinical microbiology: proof-of-concept on three frequently isolated bacterial species

ObjectiveAntibiotic susceptibility testing (AST) is necessary in order to adjust empirical antibiotic treatment, but the interpretation of results requires experience and knowledge. We have developed a machine learning software that is capable of reading AST images without any human intervention and that automatically interprets the AST, based on a database of antibiograms that have been clinically validated with European Committee on Antimicrobial Susceptibility Testing rules.MethodsWe built a database of antibiograms that were labelled by senior microbiologists for three species: Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. We then developed Antilogic, a Python software based on an original image segmentation module and supervised learning models that we trained against the database. Finally, we blind tested Antilogic against a validation set of 5100 photos of antibiograms.ResultsWe trained Antilogic against a database of 18072 pictures of antibiograms. Overall agreement against the validation set reached 97% (16 855/17 281) regarding phenotypes. The severity rate of errors was also evaluated: 1.66% (287/17 281) were major errors and 0.80% (136/17 281) were very major errors. After implementation of uncertainty quantifications, the rate of errors decreased to 0.80% (114/13 451) and 0.42% (51/13 451) for major and very major errors respectively.DiscussionAntilogic is the first machine learning software that has been developed for AST interpretation. It is based on a novel approach that differs from the typical diameter measurement and expert system approach. Antilogic is a proof of concept that artificial intelligence can contribute to faster and easier diagnostic methods in the field of clinical microbiology.     

Use of broth enrichment and real-time PCR to exclude the presence of methicillin-resistant Staphylococcus aureus in clinical samples: a sensitive screening approach

A rapid and sensitive method for excluding the presence of methicillin-resistant Staphylococcus aureus (MRSA) in clinical samples was developed and evaluated. The method utilised an MRSA-selective enrichment broth for 16 h, followed by PCR quantification of the nuc gene. Samples below a quantitative PCR threshold were reported as MRSA-negative. Broths from PCR-positive samples were subcultured for MRSA isolation. Clinical samples (n = 334) in a constructed high prevalence population were analysed in parallel with a selective plating method. The new broth-PCR assay increased the number of positive samples by 35% (49 vs. 66), and 94% of negative samples were reported within 24 h. To reduce costs and workload, 665 clinical samples were grown separately in enrichment broth and then pooled in the PCR step. The broth-PCR assay increased the number of MRSA positive samples from 11 to 15 compared with selective plating. Most (89%) of the culture-negative samples were also PCR-negative and could be reported within 24 h. The growth of 25 European EMRSA strains was tested in the selective enrichment broth. On average, the MRSA strains showed a 300 000-fold increase in CFU, compared with 30-fold for the eight methicillin-sensitive Staphylococcus aureus strains tested.     

The trend of environmental and clinical methicillin-resistant Staphylococcus aureus in a hospital in Taiwan: Impact of USA300

BackgroundThe environment may facilitate transmission of health care-associated methicillin-resistant Staphylococcus aureus (MRSA) and the pathogen is frequently shed by patients. However, the molecular characteristics and genetic relatedness between clinical and environmental MRSA isolates remain largely unclear in the clinical setting.MethodsA total of 100 hospitalized patients with MRSA infection and 25 hospitalized patients without MRSA infection were enrolled in a medical center, Taiwan in 2019. Environmental and clinical MRSA isolates were characterized by antibiotic susceptibility testing and molecular methods.ResultsIn the study, we detected 17 MRSA isolates in the environment that surrounded 15 MRSA-infected patients and one environmental MRSA isolate from one patient without MRSA infection. The molecular analysis revealed a high genetic diversity within either environmental or clinical MRSA isolates, while the USA300 clone (pulsotype AI, SCCmec IV, ST8, PVL-positive) accounts for 39% (7/18) of environmental and 33% (7/21) of clinical MRSA isolates. Moreover, 13 of the 15 MRSA-infected patients had identical paired clinical-environmental MRSA isolates, which exhibited indistinguishable genetic relatedness and highly similar antibiotic susceptibility phenotype, suggesting a possible transmission cycle of MRSA in the hospital.ConclusionsThe environmental MRSA was closely linked to MRSA isolated from patients, suggesting that the environment may act as a reservoir of MRSA. Besides, the USA300 MRSA has become a major clone in the hospital setting. An effective and rigorous approach to environmental cleaning and decontamination is suggested to eradicate MRSA in the hospital.     

Detection of Panton–Valentine leukocidin gene in Staphylococcus aureus by LightCycler PCR: clinical and epidemiological aspects

The prevalence of the Panton-Valentine leukocidin (PVL) gene in Staphylococcus aureus was investigated with a simple, reproducible and rapid real-time LightCycler SYBR Green I PCR assay. The PVL gene was detected in one isolate from 65 patients with S. aureus bacteraemia, in four isolates from 55 patients with respiratory tract infections, and in two isolates from 91 patients with cutaneous infections. In contrast, 15 of 25 cutaneous isolates of methicillin-resistant S. aureus (MRSA) were positive. All PVL-positive cutaneous MRSA isolates were community-acquired and comprised three different clones as determined by pulsed-field gel electrophoresis. The PVL gene was detected in isolates from patients with recurrent primary skin infections and S. aureus bacteraemia, but PVL did not seem to be an important virulence factor in the pathogenesis of staphylococcal bacteraemia.     

Nightly asthma caused by allergens in silk-filled bed quilts: clinical and immunologic studies

Bed quilts filled with silk waste were found to cause nightly attacks of asthma. The first symptoms appeared on an average after 7 mo of exposure at a mean patient age of 30 yr. About 50% of the patients could be classified as atopic. Extract of the filling material revealed positive scratch and RAST tests in most patients. The bed quilts were advertised as being filled with pure wild silk (from silk moths of the genus Antheraea feeding on oak leaves), but it was found that in most cases the filling also included waste of cultivated silk (Bombyx mori). The origin of the allergen was sought. Since textile products of silk are considered to be practically nonallergenic, some type of contaminant appeared the most likely candidate. No allergen was found in cocoons, chrysalis (pupa), or moths of Antheraea. However, some cocoons of Bombyx mori, usually in small amounts, and excretions of the silkworm did contain allergen. High concentrations of allergen were found in an extract of living insects of the genus Anthrenus that was present in one batch of Bombyx mori cocoons. Thus, the silk waste appeared to be contaminated with material from the silkworms and also with infesting insects, which together create a very potent allergen repertoire that after a short incubation time, in a considerable frequency, elicits a rise in asthmatic symptoms and high serum IgE antibody levels.     

Sublingual therapeutic immunization with a polyvalent bacterial preparation in patients with recurrent respiratory infections: immunomodulatory effect on antigen-specific memory CD4+ T cells and impact on clinical outcome

Recurrent respiratory tract infections (RRTIs) are common clinical conditions in individuals with alterations of the immune function. A prospective open pilot study in a cohort of patients with RRTIs has been performed to assess whether sublingual immunization with a polyvalent bacterial vaccine could exert an immunomodulatory effect on the antigen‐specific immunological responses and have an impact on the clinical outcome. Seventeen patients with RRTIs were recruited. An oral polyvalent bacterial preparation (Bactek ® ) was administered to all patients daily for 6 months. Immunological assessment was performed at baseline and at the end of immunization. Immunological measurements included: T cell‐specific proliferations of CD3⁺CD4⁺ and CD3⁺CD8⁺ to Bactek® antigens, total immunoglobulin levels, antibodies to pneumococcal polysaccharide and tetanus toxoid and B, T and natural killer (NK) cell subsets. There was a significant increase in the proliferative capacity of CD3⁺CD4⁺ T cells specific to Bactek® antigens at month 6 in comparison to baseline (P < 0·0001). A significant increase in total CD3⁺ T cells was also observed (P < 0·05). No significant differences were observed between baseline and month 6 in levels of total immunoglobulins, specific antibodies and B, T or NK cell subsets. A significant reduction in the patient's rate of RRTIs was observed compared with 1 year prior to initiation of therapy (P < 0·0001). The results demonstrate that long‐term administration of a sublingual polyvalent bacterial preparation in patients with RRTIs exerts an immune stimulating effect on CD4⁺ T helper cell responses to bacterial antigens which could be associated with clinical benefit.     

‘Information is information’: a public perspective on incidental findings in clinical and research genome‐based testing

The potential for genomic incidental findings is increasing with the use of genome‐based testing. At the same time approaches to clinical decision making are shifting to shared decision‐making models involving both the healthcare community and the public. The public's voice has been nearly absent in discussions on managing incidental findings. We conducted nine focus groups and nine interviews (n = 63) with a broad cross‐section of lay public groups to elucidate public viewpoints on incidental findings that could occur as a result of genome‐based testing in clinical and research situations. Data were analyzed using qualitative content analysis. Participants wanted incidental findings disclosed to them whether or not these were clinical or research findings. Participants used different terms to define and describe incidental findings; they wanted to know that incidental findings are possible and be given a choice to learn about them. Personal utility was an important reason for disclosure, and participants believed that managing information is a shared responsibility between professionals and themselves. Broad public input is needed in order to understand and incorporate the public's perspective on management of incidental findings as disclosure guidelines, and policies are developed in clinical and research settings.     

A study of indefinite for dysplasia in Barrett’s oesophagus: reproducibility of diagnosis,clinical outcomes and predicting progression with AMACR (α‐methylacyl‐CoA‐racemase)

Sonwalkar S A, Rotimi O, Scott N, Verghese E, Dixon M, Axon A T R A & Everett S M
(2010) Histopathology 56, 900–907
A study of indefinite for dysplasia in Barrett’s oesophagus: reproducibility of diagnosis, clinical outcomes and predicting progression with AMACR (α‐methylacyl‐CoA‐racemase) Aims: To assess interobserver variation in the diagnosis of dysplasia in Barrett’s oesophagus, especially indefinite dysplasia (IND) using the revised Vienna classification. A secondary aim was to study clinical outcome of IND cases and to evaluate expression of alpha‐methyl‐CoA racemase (AMACR) as a marker predictive of progression. Methods and results: Cases of Barrett’s oesophagus and dysplasia over a 20 year period were assessed. Three experienced histopathologists reviewed 101 cases on set criteria in a blinded fashion. Slides were immunostained for AMACR and evaluated for the presence, extent and location of AMACR expression. Clinical and progression data were collected. Overall agreement for the diagnosis of dysplasia was fair (k = 0.35) but that for IND was poor (k = 0.18). 6 IND cases progressed after a median follow‐up of 31.4 months to a higher grade. The sensitivity of AMACR for the detection of abnormality was 22% for IND and specificity 100%. The positive predictive value of AMACR for progression was 0.44 and the negative predictive value was 0.92. Conclusion: Fair agreement was achieved for the diagnosis of dysplasia but poor agreement for IND. A proportion of IND cases progress. Re‐diagnosis or consensus diagnosis did not predict progression. AMACR shows promise as a marker to indicate IND patients in need of more intensive surveillance.     

Advancing neurotrophic factors as treatments for age-related neurodegenerative diseases: developing and demonstrating “clinical proof-of-concept” for AAV-neurturin (CERE-120) in Parkinson's disease

Neurotrophic factors have long shown promise as potential therapies for age-related neurodegenerative diseases. However, 20 years of largely disappointing clinical results have underscored the difficulties involved with safely and effectively delivering these proteins to targeted sites within the central nervous system. Recent progress establishes that gene transfer can now likely overcome the delivery issues plaguing the translation of neurotrophic factors. This may be best exemplified by adeno-associated virus serotype-2-neurturin (CERE-120), a viral-vector construct designed to deliver the neurotrophic factor, neurturin to degenerating nigrostriatal neurons in Parkinson's disease. Eighty Parkinson's subjects have been dosed with CERE-120 (some 7+ years ago), with long-term, targeted neurturin expression confirmed and no serious safety issues identified. A double-blind, controlled Phase 2a trial established clinical “proof-of-concept” via 19 of the 24 prescribed efficacy end points favoring CERE-120 at the 12-month protocol-prescribed time point and all but one favoring CERE-120 at the 18-month secondary time point (p = 0.007 and 0.001, respectively). Moreover, clinically meaningful benefit was seen with CERE-120 on several specific protocol-prescribed, pairwise, blinded, motor, and quality-of-life end points at 12 months, and an even greater number of end points at 18 months. Because the trial failed to meet the primary end point (Unified Parkinson's Disease Rating Scale motor-off, measured at 12 months), a revised multicenter Phase 1/2b protocol was designed to enhance the neurotrophic effects of CERE-120, using insight gained from the Phase 2a trial. This review summarizes the development of CERE-120 from its inception through establishing “clinical proof-of-concept” and beyond. The translational obstacles and issues confronted, and the strategies applied, are reviewed. This information should be informative to investigators interested in translational research and development for age-related and other neurodegenerative diseases.