Clinical Microbiology

In clinical microbiology, the combination of MALDI-TOF MS plays a key role in bacterial identification, susceptibility testing, and nucleic acid targeting and facilitated a better workflow for the timely management of positive blood cultures and other samples.

From: Recent Developments in Applied Microbiology and Biochemistry , 2019

Challenges in Clinical Microbiology Testing

Laura Chandler , in Accurate Results in the Clinical Laboratory, 2013

Introduction

Clinical microbiology is a discipline that encompasses a broad range of testing methodologies, and it is complex in terms of organisms and methods used to isolate and identify them. Although significant improvements in testing methodologies have been made, clinical microbiology remains heavily reliant on culture-based methods and phenotypic methods for identification of culture organisms. The wide variety of pathogens and testing methods that are available makes microbiological testing challenging, and thus error detection and correction are important components of quality microbiology laboratory testing. Errors may occur at all stages of testing (pre-analytical, analytical, and post-analytical), and an error in one stage of testing is likely to overlap with or lead to errors in other stages (e.g., incorrect specimen collection can lead to culture, identification, and reporting of organisms that are not involved in the disease process and to incorrect or unnecessary antimicrobial therapy as a result). In the clinical microbiology laboratory, as in every other discipline, the frequency of analytical errors has been reduced considerably with the implementation of quality control and quality assurance programs. Despite the improvements in microbiological testing, microorganisms remain a constant challenge, and errors do occasionally occur. This chapter discusses some of the common interferences in the clinical microbiology laboratory.

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Microbiologic Diagnosis of Lung Infection

Niaz Banaei MD , ... Benjamin A. Pinsky MD, PhD , in Murray and Nadel's Textbook of Respiratory Medicine (Sixth Edition), 2016

Introduction

The clinical microbiology laboratory plays a critical role in diagnosis and management of patients with lower respiratory tract infections. By providing pathogen detection and identification and susceptibility testing the laboratory provides the basis of optimal empirical antimicrobial therapy and individually tailored regimens. 1 The microbiology laboratory also provides epidemiologic data that assist the hospital epidemiologist in the prevention, detection, investigation, and termination of nosocomial outbreaks. 2 When correctly and promptly used, the information provided by the clinical microbiology laboratory improves clinical outcomes, reduces unnecessary utilization of antibiotics, and prevents nosocomial transmissions. 3,4

The primary aim of this chapter is to assist clinicians in efficient and effective utilization of the resources of the clinical microbiology laboratory in diagnosis of the causes of infections of the lower respiratory tract. This chapter assumes that clinical laboratories are using validated methods and reporting quality-assured results and does not delve into technical or operational aspects of the clinical microbiology laboratory. For additional information on laboratory operation, the reader is referred to the latest edition of the Manual of Clinical Microbiology (American Society for Microbiology). 5

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First Principles of Clinical Microbiology: Collection, Handling, and Diagnostics

Michael A. Bachman , William D. LeBar , in Encyclopedia of Microbiology (Fourth Edition), 2019

Introduction to Clinical Microbiology

Clinical microbiology focuses on the isolation and characterization of infectious organisms so they can be managed and treated in patients. Infections can be caused by bacteria, fungi, viruses, and parasites. To diagnose an infection, a sample must be collected from a patient at a body site where the detection of a pathogen or its associated biomarkers is likely to signify disease. The specimen must be transported to the laboratory in a manner that preserves the specimen for the intended testing. Then the specimen must be tested in a way that is sensitive and specific for the suspected organism causing the disease. Finally, these results must be communicated back to a clinician in a way that he or she can interpret and act on appropriately.

Clinical microbiology is arguably the first discipline of personalized medicine. As an example, a patient has the signs and symptoms of a urinary tract infection, including increased urgency, frequency, and pain with urination. A urine sample is collected and cultured quantitatively. Within 24   h, the clinical microbiology laboratory reports the species and quantity of bacteria found in the urine. 1–2   days later, the laboratory reports the susceptibility of that patient's bacterial isolate to a panel of antibiotics approved to treat urinary tract infections. The patient's clinician can then choose an antibiotic that is predicted to be effective against that patient's infection.

A pioneering microbiologist, Robert Koch, established the paradigm of clinical microbiology that is still in practice today. In proving that the bacterium Bacillus anthracis caused the disease anthrax, he developed what have become known as Koch's postulates which are: (1) the microorganism must be observed in all cases of the disease, (2) the microorganism must be isolated and grown in pure culture, (3) microorganisms from the pure culture, when inoculated into a susceptible animal, must reproduce the disease, (4) the microorganism must be observed in and recovered from the experimentally diseased animal. Many pathogens have been linked to disease by fulfilling these postulates. For these pathogens, clinical microbiology labs can diagnose infections in patients with compatible signs and symptoms by isolating the microorganism in pure culture, or detecting it by an alternative method. When Koch's postulates have not been met, interpretation of detection of the microorganism in a patient can be challenging or impossible.

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Advances in Diagnostic Microbiology

GrÉgory Dubourg , Pierre-Edouard Fournier , in Infectious Diseases (Fourth Edition), 2017

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Clinical microbiology laboratories play a central role in optimizing the management of infectious diseases.

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Syndrome-based sampling and molecular testing, as well as extended automation, are major improvements in the clinical microbiology workflow.

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Mass spectrometry enables a rapid and cost-effective identification of bacterial and fungal pathogens, cultivated on agar or within blood culture vials.

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Real-time genomics has reached a stage when it may impact infectious disease outbreaks.

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Point-of-care assays and laboratories reduce the time to diagnosis and allow better patient management.

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Biomarker Characterization by MALDI–TOF/MS

Yi-Tzu Cho , ... Jentaie Shiea , in Advances in Clinical Chemistry, 2015

3.1 Identifying pathogens and microorganisms

Clinical microbiology laboratory plays an important role in patient care by providing the cause of infection and antimicrobial susceptibility data to physicians. Rapid diagnosis of pathogens is important for initiating effective antibiotic administration and improving the outcomes of treatment. Conventional diagnosis of microorganisms uses phenotypic identification and gene sequencing, which is tedious and time-consuming. In contrast, MALDI–TOF/MS is a simple, rapid, reproducible, and low-cost technique that has been successfully applied to identify pathogens [135–143]. Based on characteristic peptide and protein profiles obtained from intact cells, MALDI–TOF/MS allows a highly discriminatory identification of bacteria, yeasts, and filamentous fungi even after 10   min of culture. With the use of a database to identify microorganisms, the reliability and accuracy of this approach have been demonstrated, and systems (including instrument and software) are already commercially available [135–143]. The applications of MALDI–TOF/MS in research on pathogens and microorganisms include identification of pathogens from positive blood cultures and urine, real-time diagnosis of blood stream infections, and detection of antibiotic resistance bacteria [135–143].

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Scientific Advances in the Diagnosis of Emerging and Reemerging Viral Human Pathogens

Rahma Ait Hammou , ... Moulay Mustapha Ennaji , in Emerging and Reemerging Viral Pathogens, 2020

Conclusion

Clinical microbiology laboratories at the local level have an increasing responsibility to provide rapid and accurate diagnostic services for emerging (new) and reemerging infectious diseases, especially those diseases for which significant mortality or morbidity may occur as the result of a delay in diagnosis. Rapid, accurate diagnosis of emerging and reemerging infectious diseases may also be critical at the local level to ensure optimal infection control. Detection of these pathogens has often required esoteric procedures such as conventional PCR, which could be performed only at referral laboratories or, recently, at public health laboratories.

Recent technical advances in molecular diagnostics have resulted in the development of user-friendly automated testing platforms, such as real-time PCR. These novel-testing methods can be used to detect emerging and reemerging pathogens as well as common pathogens and have the potential for broadscale use in smaller laboratories in close proximity to the delivery of care.

While writing this review, a large outbreak of influenza virus type A (H3N2) was peaking in the United States, and new influenza virus type A strains (H5N1, H9N2) have been associated with both avian and human influenza in regions of the Far East. The apparent significant morbidity and mortality associated with these new influenza virus strains emphasize the need for rapid, accurate laboratory diagnostic capabilities at the local level. As is the case for SARS, agents of bioterrorism, and the other pathogens, rapid diagnostic methods, such as real-time PCR, and microarray will likely play a major role in the early and sensitive detection of emerging and reemerging infectious diseases encountered in the future.

Otherwise, a class of small RNAs implicated in the diagnosis of these diseases is miRNAs and is considered an essential mediator of host response to pathogens. Since several microbes have evolved to exploit their pleiotropic characteristics, identification of key genes and pathways in terms of activation, enhancement, repression, or silent, which are essential to facilitate the immune response, is based on the elucidation of the roles of miRNAs in host response to infectious disease. The complex regulatory network within which miRNAs are embedded makes unpicking the roles of miRNAs tough but not impossible. Integrating large miRNA and mRNA datasets using advanced statistical techniques (in a "systems biology" approach) will facilitate the unpicking of these complex networks.

Overall, miRNAs have multiple targets, and therefore any vaccines or treatments that harness miRNAs may produce off-target effects compromising safety; however, there are challenges that must be overcome. With the objective to improve the cross-study reproducibility of the findings, especially in the context of ex-miRNA biomarkers identification, universal endogenous controls are needed, and a more standardized approach to biomarker studies may also help. Initiatives devoted to harnessing the diagnostic and therapeutic potential of extracellular RNAs such as The National Institute for Health Extracellular Communication Consortium can facilitate this.

As the literature and experimental studies on miRNAs are developing, the potential for new miRNA therapeutics, diagnostics/prognostics, and vaccines becomes tangibly closer. Translating the insights of miRNA studies into improving the lives of patients is the critical next step.

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Use of the microbiology laboratory

T. Wallace MacFarlane BDS, DDS, FRCPath, FDSRCPS (Glasgow) , Lakshman P. Samaranayake BDS, DDS, MIBiol, MRCPath , in Clinical Oral Microbiology, 1989

The use of laboratory investigations in the management of antimicrobial therapy

The clinical microbiologist can on occasions give a presumptive diagnosis of a disease and suggest suitable treatment by examining a Gram film of material prepared from the lesion. In other circumstances, once certain organisms have been isolated and identified, antimicrobial sensitivity can sometimes be predicted, e.g. strict anaerobes are usually sensitive to metronidazole and Candida albicans is almost always sensitive to both nystatin and amphotericin B. However, it is essential to base rational therapy on the results of laboratory antibiotic tests.

Susceptibility of organisms to antimicrobial agents

In clinical microbiology a microbe is susceptible to an antimicrobial agent if it is inhibited by a concentration of the drug normally obtained in human tissues after a standard therapeutic dose. The reverse is true for a resistant organism. Organisms are considered intermediate in susceptibility if the inhibiting concentration of the antimicrobial agent is slightly higher than that obtained with a therapeutic dose.

Laboratory testing for antimicrobial sensitivity

The action of an antimicrobial drug against an organism can be measured qualitatively (disc diffusion tests), semi-quantitatively (break-point tests), or quantitatively (MIC or MBC tests, see below). These in vitro tests indicate whether the expected therapeutic concentration of the drug given in standard dosage inhibits the growth of a given organism in vivo.

Laboratory results can only give an indication of the activity of the drug in vitro, and its effect in vivo depends on factors such as the ability of the drug to reach the site of infection and the immune status of the host. A strong host defence response may give the impression of 'successful' drug therapy, even although the infecting organism was 'resistant' to a specific drug when laboratory tests were used.

Disc diffusion tests

The most commonly used method of testing the sensitivity of a microorganism to an antimicrobial agent is the disc diffusion test. In this technique, both test and control organisms are inoculated on to the same sensitivity agar plate, as shown in Figure 13.5. The 'Oxford staphylococcus' is used as a control in most tests as it is sensitive to most antimicrobial agents at therapeutic drug concentrations. The drug-impregnated filter paper discs are placed between the control and test zone. After 18 hours incubation at 37 °C, a zone of growth inhibition is observed surrounding the disc, depending on the sensitivity of a particular organism to a given agent. The zones of growth inhibition can now be compared with the controls and the organism reported as sensitive, resistant, or moderately sensitive to the antimicrobial agent, as shown in Figure 13.5.

Figure 13.5. Disc diffusion (Stoke's) method of antibiotic sensitivity testing. Sensitive control (Oxford staphylococcus) is spread over top and bottom thirds of plate. The specimen (or test organism) is spread over middle third. This particular test organism is sensitive to ampicillin (A) and cephaloridine (C), moderately sensitive to erythromycin (E) and resistant to penicillin (PG).

Antimicrobial sensitivity tests of this type can be divided into primary (direct) and secondary (indirect). A primary test is carried out by inoculating the clinical sample, say pus, directly on to the test zone of the plate. The advantage of this is that the overall sensitivity results for the organisms present in pus will be available after 24–48 hours incubation. This is particularly useful when treating debilitated patients with acute infections, e.g. dentoalveolar abscesses. Secondary sensitivity tests are performed on a pure culture of the isolates originally present in the specimen but the results are not available for at least 2–4 days after sampling (see Figure 13.2).

Assessment of minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC)

These tests give a quantitative assessment of the potency of an antibiotic. A range of two-fold dilutions of an antimicrobial agent can be incorporated into a suitable broth in a series of tubes (tube dilution technique). The broth is inoculated with a standardized suspension of the test organism and incubated for 18 hours. The minimum concentration of the drug which inhibits the growth of the test organism in the tube is recorded as the MIC. Subsequently, a standard inoculum from each of the tubes in which no growth occurred may be subcultured on to blood agar to determine the minimum concentration of the drug required to kill the organism (MBC). The MBC is defined as the minimum concentration of drug which kills 99.9% of the test microorganisms in the original inoculum. A variation of the MIC test, called the 'breakpoint' or 'critical' concentration test is a semi-quantitative version of the standard MIC test. In this method, which may be carried out by incorporating antimicrobial agents into either broth or agar, a limited number of drug concentrations are used rather than an entire series of doubling dilutions. These tests are not routinely performed but are useful in patients with serious infections where optimal antimicrobial therapy is essential. Examples include streptococci isolated from blood cultures from patients with infective endocarditis and some bacterial strains causing septicaemia in immunosuppressed patients.

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The Mycoses

Joseph J. Nania MD , Peter F. Wright MD , in Kendig & Chernick's Disorders of the Respiratory Tract in Children (Eighth Edition), 2012

Diagnosis

Most clinical microbiology labs will not routinely identify Cryptococcus isolates to the species level. Although cultures may take up to a week to become positive, Cryptococcus spp. grow on most clinical media, including standard radiometric blood culturing systems. However, in the diagnosis of pulmonary disease, both the sensitivity and specificity of cultures of respiratory secretions are questionable. 63 The majority of cryptococcal isolates from sputum are thought to represent colonization. 69 Conversely, isolation of the organism from blood, cerebrospinal fluid (CSF), or other sterile body sites is reasonably sensitive and would be putative evidence of pulmonary cryptococcosis when clinical or radiographic signs are suggestive. It is important to note, however, that in immunocompromised hosts, other pulmonary (and nonpulmonary) opportunistic infections may be present simultaneously. Mycobacterium tuberculosis, nontuberculous mycobacteria, cytomegalovirus, Nocardia, and P. jiroveci (formerly P. carinii) have all been reported as co-pathogens, highlighting the need for appropriate studies to rule out such diagnoses in severely compromised patients. 69 Direct histopathologic identification of C. neoformans from biopsy specimens is both sensitive and specific for the diagnosis of pulmonary infection. Several different stains can be used to identify the yeast in tissue, including the nonspecific Grocott-Gomori methenamine–silver nitrate and others such as mucicarmine, which stains the polysaccharide capsule red. 70 Additionally, a monoclonal antibody against the main capsular polysaccharide antigen (GXM) has been used for specific identification of the yeast in tissue. 70 In examination of CSF, India ink has long been used to identify C. neoformans. The sensitivity is about 80% for AIDS patients and 50% for non-AIDS patients with cryptococcal meningitis, and it has been used on other clinical specimens as well. 70

Detection of the capsular polysaccharide of C. neoformans in blood or CSF by latex agglutination or enzyme immunoassay is a reliable method of diagnosing disseminated infection. For the diagnosis of cryptococcal meningitis, CSF antigen detection assays are at least 90% sensitive and specific. However, these techniques are thought to be less sensitive in the patient with isolated pulmonary cryptococcosis. 71

Antibodies to C. neoformans can develop in response to either colonization or infection and are not useful in diagnosis. 63

As previously described, imaging of the chest by plain radiograph and CT is not diagnostic. Isolated or multiple nodules, pulmonary masses, lobar consolidation, cavitary lesions, pleural effusion, diffuse interstitial infiltrates, and adult respiratory distress–like appearance have all been described with pulmonary cryptococcosis. 64 If signs of increased intracranial pressure or other signs suggestive of CNS infection are present, magnetic resonance imaging or CT of the head to rule out hydrocephalus and cryptococcomas is indicated.

Given the limitations of noninvasive methods, invasive procedures are often necessary to confirm the diagnosis of cryptococcosis, especially when involvement is limited to the lungs. Either fine-needle aspiration or biopsy may be required to confirm the diagnosis. When pleural effusions are present, cultures of thoracentesis fluid are positive in about 40% of AIDS patients. 69 Some authors have questioned the need for examination of spinal fluid in nonimmunocompromised patients with pulmonary cryptococcosis, no overt signs of CNS infection, and a negative serum antigen. 70 However, given the frequency of concomitant meningitis, lumbar puncture with measurement of opening pressure, CSF India ink preparation, a cryptococcal antigen assay, and routine studies are generally recommended. 70

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Current and Emerging Technologies for the Diagnosis of Microbial Infections

Susan M. Novak-Weekley , Elizabeth M. Marlowe , in Methods in Microbiology, 2015

Abstract

In today's clinical microbiology laboratory, automation is being introduced that will change the nature of how clinical specimens are processed and analysed. Over the last several years, many microbiology laboratories have implemented automation to process liquid specimens which have historically been inoculated to media manually. In some institutions, this automation has been able to free up staff to concentrate on other tasks and has resulted in increased efficiency in the laboratory setting. In addition to efficiency, there are ergonomic gains in the workplace due to the pre-analytical plating instruments since tasks that are manual such as de-capping and re-capping specimens are now performed by the automated processor. This functionality reduces the ergonomic impact of the manual task and improves the work place environment for the employee. This pre-analytical plating instrumentation is now being integrated within a suite of instruments referred to as Total Laboratory Automation, or TLA, which includes digital plate reading (DPR) and middleware technology applied to culture analysis. DPR and associated middleware allow the laboratory to analyse cultures in a new and innovative way. The inoculated media is imaged using a camera in the "smart incubator", and the image presented to the technologist via the computer screen at the bench. The analysis of the culture occurs using the digital image. Further workup, such as picking a colony for mass spectrometry or automated identification, is being automated as well. This chapter will discuss the new and innovative automation solutions available for the clinical microbiology laboratory today.

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Automation

Amanda J. Fife , Derrick W.M. Crook , in Methods in Microbiology, 1999

1 Processes Requiring Visual Analysis or Manual Dexterity

In a clinical microbiology laboratory, two areas depend on visual analysis or manual dexterity. First, the examination and recognition of specific characteristics of bacterial colonies growing on agar. This is a skill which requires pattern recognition and takes months, if not years, for a person to learn. Second, purifying organisms from a mixed growth by isolating individual bacterial colonies (picking colonies) requires high degrees of manual skill and hand-eye co-ordination. These skills, which are unique to clinical microbiology, take prolonged practice to perfect and depend on memorising a large body of information. A major part of the laboratory activity in bacteriology continues to depend on these processes. Third, microscopy is used for examination of a wide range of samples and tests. These include examination of: Gram stains of fresh clinical material or organisms isolated from specimens; stools for parasites; tissue culture cells for evidence of a cytopathic effect and performing cell counts on samples such as cerebrospinal fluid. Much of medical mycology is dependent on visual recognition. Electron microscopy is also available in some laboratories to aid viral diagnosis. These activities share much in common with other specialties of pathology such as histopathology, cytology and haematology which also utilise microscopy extensively. The results from these processes are largely dependent on producing a descriptive written report which, again, increases the complexity over those processes which can produce a numerical result. Therefore, full laboratory automation for performing these analyses and producing a test result will depend on highly sophisticated image analysis, advanced artificial intelligence and robotics.

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