Análisis microbiológico post mórtem

Post-mortem microbiology is useful in both clinical and forensic autopsies, and allows a suspected infection to be confirmed. Indeed, it is routinely applied to donor studies in the clinical setting, as well as in sudden and unexpected death in the forensic field. Implementation of specific sampling techniques in autopsy can minimize the possibility of contamination, making interpretation of the results easier. Specific interpretation criteria for post-mortem cultures, the use of molecular diagnosis, and its fusion with molecular biology and histopathology have led to post-mortem microbiology playing a major role in autopsy. Multidisciplinary work involving microbiologists, pathologists, and forensic physicians will help to improve the achievements of post-mortem microbiology, prevent infectious diseases, and contribute to a healthier population.     

Identification of bacteria through 16S rRNA sequencing: principles, methods and applications in clinical microbiology

Phylogenetic relationships among prokaryotes can be inferred from comparisons of their 16S rRNA (or 16S rDNA) sequences. This has had an enormous repercussion on bacterial taxonomy, leading to the currently applied system of classification, and allowing a rapid and precise identification of bacteria. In clinical microbiology, molecular identification based on 16S rDNA sequencing is applied fundamentally to bacteria whose identification by means of other types of techniques turns out impossible, difficult, or requires a lot of time. Amplification of the gene to be sequenced uses preferably DNA extracted from a bacterial pure culture, but can be achieved also directly from a clinical sample. The latter has led to the discovery of new pathogens. Bearing in mind its potential, as the technical resources improve and the prize becomes more competitive, the identification based on 16S rDNA sequencing will certainly find a wider application in the clinical microbiology laboratory.     

Real-time PCR

PCR based assays are currently used routinely in most microbiology laboratories. But, with few exceptions, they are restricted to the field of virology, especially to a limited number of viral targets with important economical interest for which commercially well standardized assays are available. For several reasons, it has had a poor implementation of the PCR assays into routine diagnostics for other infectious diseases despite they are advantageous. Combined with automated sample isolation of nucleic acids, real-time PCR gives an ideal platform for the development of molecular assays for a wide range of infectious agents with clinical interest. Because of its advantages, as simplicity, rapidity and minor risk of contamination, real-time PCR will go replace conventional PCR assays and its use will extend to a wide range of applications in clinical microbiology.     

Movies as a teaching resource for infectious diseases and clinical microbiology

Since its inception, the cinema has constantly provided a reflection of infectious diseases because of their omnipresence in life and their importance to individuals and society. Few infectious diseases escape its eye, to the extent that the cinema constitutes an authentic treatise on these phenomena. The cinema is a very valuable educational resource, able to supplement classical teaching methods and to encourage critical thinking among students. The enormous flow of information, images, sounds, consequences, situations, and points of view that it provides should not be wasted and can be of great use, both in the spread of ideas and in training in infectious diseases and clinical microbiology.     

Community-acquired bacterial pneumonias

Community-acquired bacterial pneumonias are among the most common of infectious diseases. The diagnosis of the etiological agent is becoming more challenging and more critical as new organisms are recognized as pathogens, and as well established agents become increasingly resistant to antimicrobial agents. The value of clinical microbiology laboratory data in the management of pneumonia is controversial. A well recognized, published guideline suggests that no laboratory studies be performed. Yet common practice and a more recent guideline advocate routine collection of sputum for Gram's stain and culture, along with traditional blood work. Given the increasing need to distinguish among a long list of possible pathogens and the need to recognize antibiotic resistance, it seems most prudent to include microbiological studies.This information can be used to guide initial therapy, and perhaps limit the overutilization of broad-spectrum antimicrobials. However, a prerequisite for the use of all cunently available test methods is their deployment in patients for whom there is clinical and radiographic evidence of pneumonia because recovery of a microorganism, especially from sputum, will occur with or without this clinical condition.     

Electronic tools for infectious diseases and microbiology

Electronic tools for infectious diseases and medical microbiology have the ability to change the way the diagnosis and treatment of infectious diseases are approached. Medical information today has the ability to be dynamic, keeping up with the latest research or clinical issues, instead of being static and years behind, as many textbooks are. The ability to rapidly disseminate information around the world opens up the possibility of communicating with people thousands of miles away to quickly and efficiently learn about emerging infections. Electronic tools have expanded beyond the desktop computer and the Internet, and now include personal digital assistants and other portable devices such as cellular phones. These pocket-sized devices have the ability to provide access to clinical information at the point of care. New electronic tools include e-mail listservs, electronic drug databases and search engines that allow focused clinical questions. The goal of the present article is to provide an overview of how electronic tools can impact infectious diseases and microbiology, while providing links and resources to allow users to maximize their efficiency in accessing this information. Links to the mentioned Web sites and programs are provided along with other useful electronic tools.     

Role of clinical microbiology laboratories in the management and control of infectious diseases and the delivery of health care.

Modern medicine has led to dramatic changes in infectious diseases practice. Vaccination and antibiotic therapy have benefited millions of persons. However, constrained resources now threaten our ability to adequately manage threats of infectious diseases by placing clinical microbiology services and expertise distant from the patient and their infectious diseases physician. Continuing in such a direction threatens quality of laboratory results, timeliness of diagnosis, appropriateness of treatment, effective communication, reduction of health care-associated infections, advances in infectious diseases practice, and training of future practitioners. Microbiology laboratories are the first lines of defense for detection of new antibiotic resistance, outbreaks of foodborne infection, and a possible bioterrorism event. Maintaining high-quality clinical microbiology laboratories on the site of the institution that they serve is the current best approach for managing today's problems of emerging infectious diseases and antimicrobial agent resistance by providing good patient care outcomes that actually save money.     

Métodos de diagnóstico rápido en microbiología clínica: necesidades clínicas

The diagnostic methods of infectious diseases should be fast, accurate, simple and affordable. The speed of diagnosis can play a crucial role in healing the patient, allowing the administration of appropriate antibiotic treatment. One aspect that increasingly determines the need for rapid diagnostic techniques is the increased rates of serious infections caused by multidrug resistant bacteria, which cause a high probability of error in the empirical treatment. Some of the conventional methods such as Gram staining or antigen detection can generate results in less than 1 hour but lack sensitivity.Today we are witnessing a major change in clinical microbiology laboratories with the technological advances such as molecular diagnostics, digital microbiology and mass spectrometry. There are several studies showing that these changes in the microbiological diagnosis reduce the generation time of the test results, which has an obvious clinical impact.However, if we look into the future, other new technologies which will cover the needs required for a rapid microbiological diagnosis are on the horizon. This review provides an in depth analysis of the clinical impact that the implementation of rapid diagnostic techniques will have on unmet clinical needs.     

A clinical evaluation of the new laboratory method that diagnoses bacterial infection, using silkworm larvae plasma

Based upon the phenomenon that the peptidoglycan, a common component of Gram positive and negative bacteria, reacts specifically with silkworm larvae plasma (SLP), a new laboratory method named "SLP test" was developed to measure the reaction products in plasma quantitatively as SLP. This SLP test seems to be able to diagnose both Gram positive and negative bacterial infection. So we evaluated its usefulness in diagnosing clinical infectious diseases. This study included 14 patients with result to positive bacterial blood culture, 22 patients with bacterial local infection, 7 patients without any evidence of bacterial infection, and 19 healthy volunteers. It seemed that the cut-off value of this SLP test should be set at 0.6 ng/ml. The sensitivity and specificity of this SLP test were 57.1%, 100%, respectively. A significant difference was not detected statistically between SLP values of patients with Gram positive and Gram negative bacterial infectious diseases. So the SLP test did not appeared specific to either Gram positive or Gram negative bacteria. This test may become a new method diagnosing bacterial infectious disease.     

Perspective on the clone library method for infectious diseases

Recently, culture-independent molecular methods, such as DNA sequencing techniques targeting the 16S-ribosomal RNA (rRNA) gene and/or other housekeeping genes with Sanger method-based technologies, next generation sequencing (NGS), and metagenomic analysis, have been developed for detecting microorganisms in the human body; these can provide information on microbiomes of samples from individuals with or without infectious diseases. Determining the bacterial species is crucial in identifying causative bacteria of upper and lower respiratory tract infections, especially for Streptococcus species, but NGS analysis is often not precise enough to identify bacteria at the species level. This review briefly introduces previous observations of the microbiome of samples from various respiratory and other infections assessed using the clone library method with Sanger sequencing of the 16S-rRNA gene. On analysis of 16S-rRNA gene-sequence data of bronchoalveolar lavage fluid obtained from pneumonia lesions in patients with bacterial pneumonia and lung abscess, anaerobes are often detected in non-elderly patients with pneumonia, and the detection rate of Staphylococcus aureus in patients with hospital-acquired pneumonia is lower than that previously reported. Analysis of pleural effusion samples from patients with pleurisy indicated a more important role of anaerobes than previous believed. The other topics reviewed include microbiomes of nontuberculous mycobacteriosis and lower respiratory tract infections in children with permanent tracheostomy due to neuromuscular disorders, in nasal discharge, in bacterial vaginosis, in the intracystic fluid of postoperative maxillary cyst, and in bacterial conjunctivitis; urine microbiota in urethritis; fecal microbiota; and newly detected infectious organisms in the human respiratory tract.     

Clinical microbiology and the outpatient

The boundaries between "hospital" and "extrahospital" microbiology do not exist as such and it would be more appropriate to speak of the microbiological care of inpatients or outpatients. Therefore, today, the development of clinical microbiology obligatorily the outpatient. The strengthening of this interventional setting should entail both individual diagnosis of outpatients as well as epidemiological surveillance of infectious diseases in the non-hospital setting, detection of levels of resistance among the most common pathogens and, moreover, the control of this resistance in the healthy population.     

The application of the polymerase chain reaction of cerebrospinal fluid in the clinical management of AIDS-related CNS disorders

In AIDS patients central nervous system (CNS) illness may be caused by HIV disease itself or by opportunistic agents, resulting in serious morbidity such as behavioral and motor disturbances, meningitis or encephalitis, among other disorders. Early diagnosis can allow specific treatment (e.g., antimicrobial treatment) that may prevent, ameliorate, or slow the catastrophic sequelae of infection, as well as reduce the need for expensive diagnostic procedures. Conventional microbiology techniques have proven inadequate for the diagnosis of most AIDS-related CNS diseases. However, the development in the past decade of the application of polymerase chain reaction (PCR) to clinical specimens has facilitated the early diagnosis of a number of infectious diseases in these patients. The technique permits the amplification of target nucleic acids such that common laboratory methods may then be used for diagnosis. The application of PCR to cerebrospinal fluid for early diagnosis of AIDS-related neurologic complications has been an impressive example of the application of PCR and may form the basis of new algorithms for diagnosis and possibly the evaluation of treatment protocols.     

Antiinfective applications of toll-like receptor 9 agonists

The innate immune system detects pathogens by the presence of highly conserved pathogen-expressed molecules, which trigger host immune defenses. Toll-like receptor (TLR) 9 detects unmethylated CpG dinucleotides in bacterial or viral DNA, and can be stimulated for therapeutic applications with synthetic oligodeoxynucleotides containing immune stimulatory "CpG motifs." TLR9 activation induces both innate and adaptive immunity. The TLR9-induced innate immune activation can be applied in the prevention or treatment of infectious diseases, and the adaptive immune-enhancing effects can be harnessed for improving vaccines. This article highlights the current understanding of the mechanism of action of CpG oligodeoxynucleotides, and provides an overview of the preclinical data and early human clinical trial results, applying these TLR9 agonists in the field of infectious diseases.     

新世纪的临床微生物学

临床微生物学在过去20年取得了全面发展,但依然面临许多新问题,包括分子生物学诊断的权利受限,缺乏专业人力资源,费用调配,获得实验室资助,如何及时正确鉴定和控制耐药菌株传播及新发和再发的传染病的及时诊断等。分子生物学技术的迅速发展为微生物的检测创造了新的机遇。本文根据美国微生物学院2008年院士学术报告会报道,就新世纪临床微生物学的现状、难题和机遇、急需解决的问题及未来趋势做一评述,并对学科发展提一些建议。     

Bacterial genome sequencing and its use in infectious diseases

The availability of genome sequences is revolutionising the fields of bacteriology and infectious diseases. By mid-2007, 479 bacterial genomes from 352 distinct species have been sequenced, including representatives of all notable human pathogens. Additionally, the genomes of several strains from each of 55 species have been sequenced. This tremendous amount of genomic data has led to unprecedented advances in pathogen diagnosis, genotyping, detection of virulence, and detection of resistance to antibiotics. We review current achievements in these fields and potential developments in the future for the clinical microbiology laboratory.     

Fact and fiction in tuberculosis vaccine research: 10 years later

Tuberculosis is one of the most deadly infectious diseases. The situation is worsening because of co-infection with HIV and increased occurrence of drug resistance. Although the BCG vaccine has been in use for 90 years, protection is insufficient; new vaccine candidates are therefore needed. 12 potential vaccines have gone into clinical trials. Ten are aimed at prevention of tuberculosis and, of these, seven are subunit vaccines either as adjuvanted or viral-vectored antigens. These vaccines would be boosters of BCG-prime vaccination. Three vaccines are recombinant BCG constructs-possible replacements for BCG. Additional vaccine candidates will enter clinical trials in the near future, including postexposure vaccines for individuals with latent infection. In the long term, vaccines that prevent or eradicate infection with Mycobacterium tuberculosis would be the best possible option. Improved knowledge of immunology, molecular microbiology, cell biology, biomics, and biotechnology has paved the way towards an effective and safe vaccine against tuberculosis. The pipeline of new vaccine candidates from preclinical to clinical testing could be accelerated by development of biomarkers that can predict the clinical outcome of tuberculosis.     

Evaluation of a multiplex PCR kit for detection of 17 respiratory pathogens in hospitalized patients

BackgroundRapid pathogen identification is critical for optimizing diagnosis and treatment of infectious diseases. Multiplex polymerase chain reaction (PCR) is a sensitive, broad-spectrum molecular detection technique that is simple and rapid to perform. It is capable of simultaneously screening for multiple pathogens within a short time range. Here, we designed and evaluated a multiplex PCR kit for the identification of 17 common respiratory pathogens in clinical samples from hospitalized patients.MethodsA total of 452 samples from hospitalized patients, including 242 respiratory and 210 non-respiratory samples, were analyzed for 13 bacteria and 4 fungi by a multiplex fluorescent PCR kit. The diagnostic performance of the kit was assessed by considering routine microbiology as the reference standard.ResultsThe overall positivity rate of the multiplex PCR kit was 86.9%, much higher than that noted on routine microbiology (56.9%). Furthermore, the co-infection detection rate was also significantly higher than that noted on routine microbiology (69.5% vs. 15.0%). Compared with routine microbiology, kit sensitivity was >90% for detection of most target bacteria, with a negative predictive value (NPV) of >99%, especially for detection of Pseudomonas aeruginosa, Klebsiella pneumoniae, Acinetobacter baumannii, Stenotrophomonas maltophilia, Enterococcus faecium, Enterococcus faecalis and Escherichia coli. The kit was noted to be particularly superior in identifying Stenotrophomonas maltophilia and Streptococcus pneumoniae as compared to routine microbiology. The multiplex PCR kit was noted to be less sensitive (33.3–59.6%) and more specific (93.9–100.0%) for detection of mycobacteria and fungi.ConclusionsOur multiplex PCR kit offers a rapid and sensitive diagnosis of common bacterial pneumonia, although sensitivity for mycobacteria and fungi warrants enhancement. Further optimization includes minimizing false positivity and increasing relevance to clinical application.     

Applications of molecular genetics to gastrointestinal and liver diseases. I. Technical approaches

Recent developments in recombinant DNA techniques have allowed an understanding of the molecular genetics of many diseases, some affecting the gastrointestinal tract and liver. DNA probes which detect sequences within or near disease genes can be selected by direct approaches, if the gene product or primary gene function is known, or by indirect methods when the chromosomal location is known. Such probes have resulted in extensive family studies which can now define risks to family members of developing a genetic disease. The development of the polymerase chain reaction will also be of considerable use in clinical genetics and in the diagnosis of some infectious diseases. The techniques are summarized and examples of their use are given. A glossary of terms is also provided.     

Manifestations of Epstein-Barr virus-associated disorders in liver

Abstract: Epstein-Barr virus is a ubiquitous virus associated with a variety of different diseases and disorders. The manifestations of Epstein-Barr virus-associated diseases or disorders within the liver, which involve a broad spectrum of histologic and clinical features, ranging from hepatitis through lymphoproliferative disorders to lymphoma, are presented. An important aspect of Epstein-Barr virus expression and infection is the biology of the Epstein-Barr virus. Documentation of infection can be performed using serology to detect the interaction of Epstein-Barr virus with the immune system, and the detection of EBV proteins and use of molecular biologic techniques to identify the presence of EBV RNA, and DNA sequences. Of particular utility are in situ hybridization, Southern blot analysis, and polymerase chain reaction as diagnostic methods to identify specific RNA or DNA sequences. Epstein-Barr virus-associated diseases and disorders including infectious mononucleosis, sporadic fatal infectious mononucleosis, X-linked proliferative disorder (Duncan's disease), post-transplant lymphoproliferative disorders, lymphoma, and AIDS are discussed. The histopathologic findings present in liver associated with each disease are presented with illustrative examples. Handling the tissue and interaction with clinical services are also discussed as a method for appropriate diagnosis of Epstein-Barr virus-driven processes affecting the liver.     

Intravenous Immunoglobulin (IVIg) for Refractory and Difficult-to-treat Infections

Traditionally, intravenous immunoglobulin (IVIg) has been used as replacement therapy for patients with primary or secondary immunoglobulin deficiencies. Increasingly, IVIg is being used (in doses higher than for replacement therapy) in certain bacterial or viral infectious diseases. A variety of modes of action have been attributed to the beneficial effects of IVIg, including its interaction with T-cell function, antigen-presenting cell maturation/presentation, combined with a general "tune down" effect on inflammatory reactions. More often, IVIg is being evaluated in clinical trials for the treatment of refractory and difficult-to-treat chronic infections. The evidence, molecular mechanisms, and rationale for the use of adjunct IVIg therapy in infectious diseases are reviewed, and its potential use in the adjunct treatment of difficult-to-treat drug-resistant tuberculosis discussed.