Laboratory and clinical diagnosis of infections.pptx

Laboratory and clinical diagnosis of infections in infection prevention and control

1. CLINICAL AND LABORATORY DIAGNOSIS OF INFECTIONS

2. INTRODUCTION Some infectious diseases are distinctive enough to be identified clinically. Most pathogens, however, can cause a wide spectrum of clinical syndromes in humans. Conversely, a single clinical syndrome may result from infection with any one of many pathogens. Influenza virus infection, for example, causes a wide variety of respiratory syndromes that cannot be distinguished clinically from those caused by streptococci, mycoplasmas, or more than 100 other viruses. Most often, therefore, it is necessary to use microbiologic laboratory methods to identify a specific etiologic agent. Diagnostic medical microbiology is the discipline that identifies etiologic agents of disease.

3. The staff of a clinical microbiology laboratory should be qualified to advise the physician as well as process specimens. The physician should supply salient information about the patient, such as age and sex, tentative diagnosis or details of the clinical syndrome, date of onset, significant exposures, prior antibiotic therapy, immunologic status, and underlying conditions. The clinical microbiologist participates in decisions regarding the microbiologic diagnostic studies to be performed, the type and timing of specimens to be collected, and the conditions for their transportation and storage. Above all, the clinical microbiology laboratory, whenever appropriate, should provide an interpretation of laboratory results.

4. DIAGNOSTIC PROCESS It includes three phases Preanalytic phase Analytical phase Post analytical phase

5. Manifestation of disease The manifestations of an infection depend on many factors, including the site of acquisition or entry of the microorganism; organ or system tropisms of the microorganism; microbial virulence; the age, sex, and immunologic status of the patient; underlying diseases or conditions; and the presence of implanted prosthetic devices or materials . The signs and symptoms of infection may be localized, or they may be systemic, with fever, chills, and hypotension. In some instances the manifestations of an infection are sufficiently characteristic to suggest the diagnosis; however, they are often nonspecific.

6. Laboratory diagnosis of infectious diseases The general approaches to laboratory diagnosis vary with different microorganisms and infectious diseases. T he types of methods used are a combination of direct microscopic examinations, culture, antigen detection, and antibody detection (serology). Nucleic acid amplification (NAA) assays that allow direct detection of genomic components of pathogens are now being used in many clinical microbiology laboratories. M ultiplexed polymerase chain reaction (PCR) platforms that enable rapid detection of multiple potential pathogens in a single test reaction are also available.

7. Specimen Specimens intended for cultivation of microorganisms can be divided into two types: • those from sites that are normally sterile • those from sites that usually have a commensal flora Body sites that are normally sterile: Blood and bone marrow, Cerebrospinal fluid, Serous fluids, Tissues, Lower respiratory tract and Bladder Body sites that have a normal commensal flora: Mouth, nose and upper respiratory tract, Skin, Gastrointestinal tract, Female genital tract and Urethra

8. Specimen collection Take the appropriate specimen, e.g. blood and cerebrospinal fluid in suspected meningitis. Collect the specimen at the appropriate time, during the acute phase of the disease, If possible, collect specimen before patient receives antimicrobials. Collect enough material and an adequate number of samples, e.g. enough blood/serum for more than one set of blood cultures. Avoid contamination: from normal flora, e.g. midstream urine, from non-sterile equipment. Use the correct containers and appropriate transport media. Label specimens properly.

9. Direct examination S ome of the parasites are large enough to be seen with the naked eye. Bacteria and fungi can be seen clearly with the light microscope when appropriate methods are used. Individual viruses can be seen only with the electron microscope, although aggregates of viral particles in cells (viral inclusions) may be seen by light microscopy.

10. Microscopy: Microscopy is an important first step in the examination of specimens M icroorganisms are too small to be seen by the naked eye, and therefore a microscope is an essential tool in microbiology. Bright field microscopy is used to examine specimens and cultures as wet or stained preparations Wet preparations are used to demonstrate: • blood cells and microbes in fluid specimens such as urine, faeces or cerebrospinal fluid (CSF) • cysts, eggs and parasites in faeces • fungi in skin • protozoa in blood and tissues.

11. Microscopy Dark field (dark ground) microscopy is useful for observing motility and thin cells such as spirochetes ( Treponema pallidum, Leptospira interrogans , Borrelia recurrentis ) Phase contrast microscopy increases the contrast of an image; used to observe live specimens Fluorescence microscopy is used for substances that are either naturally fluorescent or have been stained with fluorescent dyes

12. Staining techniques Stains are usually applied to dried material that has been fixed (by heat or alcohol) onto the microscope slide. Samples from specimens themselves, or pure cultures, can be stained. The slide can then be viewed in the light microscope with an oil immersion lens, which improves the resolving power of the microscope. Differential staining techniques include: gram staining, acid fast staining

13. Culture Growth and identification of the infecting agent in vitro is usually the most sensitive and specific means of diagnosis and is thus the method most commonly used. Most bacteria and fungi can be grown in a variety of artificial media, but strict intracellular microorganisms ( eg , Chlamydia , Rickettsia , and human and animal viruses) can be isolated only in cultures of living eukaryotic cells. Bacteria (including mycobacteria and mycoplasmas) and fungi are cultured in either liquid (broth) or on solid (agar) artificial media

14. Culture Liquid media provide greater sensitivity for the isolation of small numbers of microorganisms; however, identification of mixed cultures growing in liquid media requires subculture onto solid media so that isolated colonies can be processed separately for identification. Different types of media can be used for the isolation of microorganisms: Simple media Differential media Selective media Enriched media

15. Culture Chlamydiae and viruses are cultured in cell culture systems, but virus isolation occasionally requires inoculation into animals, such as suckling mice, rabbits, guinea pigs, hamsters, or primates. Rickettsiae may be isolated with some difficulty and at some hazard to laboratory workers in animals or embryonated eggs.

16. Culture Cultures are generally incubated at 35 to 37°C in an atmosphere consisting of air, air supplemented with carbon dioxide (3 to 10 percent), reduced oxygen (microaerophilic conditions), or no oxygen (anaerobic conditions), depending upon requirements of the microorganism. Since clinical specimens from bacterial infections often contain aerobic, facultative anaerobic, and anaerobic bacteria, such specimens are usually inoculated into a variety of general purpose, differential, and selective media, which are then incubated under aerobic and anaerobic conditions

17. Microbial identification Identification of bacteria (including mycobacteria) is based on growth characteristics (such as the time required for growth to appear or the atmosphere in which growth occurs), colony and microscopic morphology, and biochemical, physiologic, and, in some instances, antigenic or nucleotide sequence characteristics. The identification of filamentous fungi is based almost entirely on growth characteristics and colony and microscopic morphology. Identification of viruses is usually based on characteristic cytopathic effects in different cell cultures or on the detection of virus- or species-specific antigens or nucleotide sequences.

18. Serological techniques Serological diagnosis is based on either the demonstration of the presence of specific IgM antibodies or a significant increase in the levels of specific IgG antibodies. Immunoassays are the most commonly used serological assays. Point-of-care tests (POC tests), both for antigens and antibodies, are also becoming more and more common in diagnostic use. Serological assays are useful for many purposes. In primary infections they often provide information about the etiology even after the acute stage when infectious agent or its components can no longer be demonstrated in the samples.

19. Serological techniques They are widely used for screening of blood products for the risk of certain chronic infections, evaluation of the immune status, and need for prophylactic treatments in connection with certain organ transplantations. They are also widely used for epidemiological studies, determination of vaccine-induced immunity, and other similar public health purposes. Serological techniques include: Immunoassays ELISA Immunoflourescent tests Immunoblot

20. Nucleic acid tests Techniques for the detection and quantitation of specific DNA and RNA base sequences in clinical specimens have become powerful tools for the diagnosis of bacterial, viral, parasitic, and fungal infections Nucleic acid tests are used for four purposes. U sed to detect, and sometimes to quantify, specific pathogens in clinical specimens. U sed for identification of organisms (usually bacteria) that are difficult to identify by conventional methods.

21. Nucleic acid tests U sed to determine whether two or more isolates of the same pathogen are closely related (i.e., whether they belong to the same clone or strain). U sed to predict the sensitivity of organisms to a small number of chemotherapeutic agents

22. Molecular Probes for Direct Detection of Pathogens in Clinical Specimens Nucleic acid probes are used for direct detection of pathogens in clinical specimens without amplification of the target strand of DNA or RNA. Such tests detect a relatively short sequence of bases specific for a particular pathogen on single-stranded DNA or RNA by hybridization of a complementary sequence of bases (probe) coupled to a reporter system that serves as the signal for detection. Nucleic acid probes are commercially available for direct detection of various bacterial and parasitic pathogens, including  C. trachomatis, N. gonorrhoeae,  and group A  Streptococcus . 

23. Nucleic Acid Amplification Tests There are several methods for amplification (copying) of small numbers of molecules of nucleic acid to readily detectable levels. These NAATs include PCR, strand displacement amplification, and self-sustaining sequence replication. In each case, exponential amplification of a pathogen-specific DNA or RNA sequence depends on primers whose annealing to the target sequence leads to specific amplification of this sequence. The amplified nucleic acid can be detected after the reaction is complete or (in real-time detection) as amplification proceeds.

24. Next-Generation and Metagenomic Nucleic Acid Tests Next-generation sequencing (NGS) refers to high-throughput methods capable of rapidly sequencing large numbers of DNA molecules in a complex mixture of sequences. Next-generation sequencing has made it possible to rapidly determine the sequence of bacterial and viral genomes and to detect the nucleic acids of pathogens directly in some types of human samples. Whole-genome sequencing (WGS) of bacteria can be used for species identification and potentially to predict antibiotic susceptibility and important virulence factors