Principles of Diagnosis with Medical Microbiology

Microbial infections are diseases that can affect your skin, lungs, brain, blood and other parts of your body. You get them from single-celled organisms multiplying or releasing toxins in your body. Common bacterial diseases include UTIs, food poisoning, STIs and some skin, sinus and ear infections. They’re often treated with antibiotics.

Microbes that cause disease are called pathogens. There are different types of microbes, such as bacteria, viruses, fungi and parasites. Each type of microbe has its own characteristics, modes of transmission and methods of diagnosis and treatment.

Bacteria are living things with only a single cell that can reproduce quickly. There are millions of bacteria that live all around us — in soil or water and on surfaces in our homes and workplaces. There are even millions of bacteria that live on your skin and inside of your body. Most bacteria aren’t harmful, and many are even helpful. They can help you digest food and kill off other harmful forms of bacteria that try to invade your body. But even the helpful ones can hurt you if they grow where they’re not supposed to.

Some examples of bacterial infections are:

• Legionnaires’ disease
• Meningococcal disease
• Q fever
• Strep throat
• Tuberculosis (TB)
• Whooping cough (pertussis)

Viruses are not made up of cells, but they have genetic material (DNA or RNA) surrounded by a protein coat. They always need to infect humans or other living things to create more copies of themselves. Viruses can cause mild illnesses like the common cold or more serious diseases like AIDS or COVID-19.

Some examples of viral infections are:

• Chickenpox
• Cold
• Flu
• German measles
• HIV/AIDS
• COVID-19

Fungi are organisms that feed on organic matter. They can be multicellular (like mushrooms) or unicellular (like yeast). Some fungi live on our skin or inside our body without causing any problems. But some fungi can cause infections, especially in people with weakened immune systems.

Some examples of fungal infections are:

• Athlete`s foot
• Ringworm
• Thrush
• Valley fever

Parasites are organisms that live on or inside another organism (the host) and benefit from it, often at the expense of the host. Parasites can be animals (like worms) or protozoa (single-celled organisms). Some parasites can cause diseases by damaging the host`s tissues, organs or blood cells.

Some examples of parasitic infections are:

• Giardiasis
• Malaria
• Pinworms
• Toxoplasmosis

To cause an infection, microbes must enter our bodies. The site at which they enter is known as the portal of entry. Microbes can enter the body through the four sites listed below:

• Respiratory tract (mouth and nose) e.g. influenza virus which causes the flu.
• Gastrointestinal tract (mouth oral cavity) e.g. Vibrio cholerae which causes cholera.
• Urogenital tract (urethra) e.g. Escherichia coli which causes urinary tract infections.
• Skin e.g. Staphylococcus aureus which causes boils or impetigo.

Once inside the body, microbes may multiply and spread to different tissues or organs, causing damage and inflammation. The body`s immune system tries to fight off the infection by producing antibodies and white blood cells. Sometimes, the immune response can also harm the body`s own cells or cause allergic reactions.

The symptoms and severity of microbial infections depend on many factors, such as:

• The type and number of microbes involved.
• The portal of entry and the site of infection.
• The virulence (ability to cause disease) and resistance (ability to evade treatment) of the microbes.
• The immune status and general health of the host.
• The availability and effectiveness of treatment options.

Microbial infections can be diagnosed by various methods, such as:

• Clinical signs and symptoms.
• Laboratory tests on blood, urine, stool, sputum or other specimens.
• Imaging techniques like X-rays, ultrasound or MRI.
• Molecular techniques like polymerase chain reaction (PCR) or gene sequencing.

Microbial infections can be treated by various methods, such as:

• Antibiotics for bacterial infections.
• Antivirals for viral infections.
• Antifungals for fungal infections.
• Antiparasitics for parasitic infections.
• Supportive care like fluids, painkillers or oxygen therapy.

Microbial infections can be prevented by various methods, such as:

• Vaccination for some diseases like measles, polio or tetanus.
• Hygiene practices like washing hands, covering coughs or sneezes or disinfecting surfaces.
• Food safety practices like cooking food thoroughly, washing fruits and vegetables or storing food properly.
• Water safety practices like boiling water, using filters or purifiers or avoiding contaminated sources.
• Sexual safety practices like using condoms, getting tested or treated for STIs or avoiding multiple partners.
• Travel safety practices like getting vaccinated, taking prophylaxis or avoiding contact with sick people or animals.

Microbial infections are a major cause of morbidity and mortality worldwide. They can affect anyone at any age, but some groups are more vulnerable than others, such as:

• Infants and children.
• Elderly people.
• Pregnant women.
• People with chronic diseases like diabetes, cancer or HIV/AIDS.
• People with weakened immune systems due to medications, transplants or genetic disorders.

Understanding the principles of diagnosis with medical microbiology can help us prevent, detect and treat microbial infections effectively and improve our health and well-being.

Importance of specimen collection and processing in microbiological tests

Specimen collection and processing are crucial steps in the diagnosis of microbial infections. A specimen is any material obtained from a patient that can be examined for the presence of microorganisms or their products. The quality and quantity of the specimen can affect the accuracy and reliability of the microbiological tests. Therefore, it is important to follow some general principles when collecting and processing specimens for microbiological tests:

• The specimen should be collected as soon as possible after the onset of symptoms or signs of infection, preferably before the administration of any antimicrobial agents that may interfere with the detection or growth of microorganisms.
• The specimen should be representative of the site and stage of infection, and should avoid contamination from normal flora or other sources. For example, specimens from respiratory tract infections should be obtained by deep coughing or sputum induction, rather than by saliva or nasal swabs. Specimens from skin or wound infections should be obtained by scraping or aspirating the lesion, rather than by swabbing the surface.
• The specimen should be collected in an appropriate container that is sterile, leak-proof, and labeled with the patient`s name, identification number, date, time, and site of collection. The container should also contain a suitable transport medium that preserves the viability and integrity of the microorganisms during transit to the laboratory. Some transport media may also contain inhibitors or indicators that prevent the growth or enhance the detection of certain microorganisms.
• The specimen should be transported to the laboratory as soon as possible after collection, preferably within 2 hours. If transport is delayed, the specimen should be stored at an appropriate temperature that prevents the overgrowth or death of microorganisms. For example, specimens containing bacteria should be refrigerated at 4°C, while specimens containing viruses should be frozen at -70°C.
• The specimen should be processed in the laboratory according to standard operating procedures that ensure the safety of the personnel and the quality of the results. The processing may include steps such as centrifugation, filtration, staining, inoculation, incubation, identification, and susceptibility testing. The processing should be done in a timely manner to avoid delays in reporting the results to the clinician.

By following these principles, specimen collection and processing can ensure that microbiological tests provide accurate and reliable information for the diagnosis and treatment of microbial infections.

Specimens submitted for microbiological testing require proper handling from the time of collection through all stages of transport, storage, and processing. The following basic principles of specimen collection are fundamental to ensuring appropriate specimen management :

• If possible, collect the specimen in the acute phase of the infection and before antibiotics are administered.
• Select the correct anatomic site for collection of the specimen. Avoid contamination from adjacent tissues or secretions.
• Collect a sufficient quantity of material. Use appropriate collection devices: sterile, leak-proof specimen containers. Use appropriate transport media (anaerobe transport vials, eSwabs for bacterial culture, Cary-Blair for bacterial stool NAT testing, VTM for viral and Chlamydia testing, and urine boric acid transport for bacterial urine cultures).
• Check expiration date before inoculating collection device.
• Properly label the specimen in the presence of the patient (a minimum of two patient identifiers are required) and order appropriately in EPIC. The specific specimen type and source are required in EPIC.
• Send the specimen to the laboratory as soon as possible after collection. If appropriate, decontaminate the skin surface as per hospital guidelines. Allow a contact time of two minutes to maximize the antiseptic effect.

Specimens that do not meet these criteria may be rejected by the laboratory or result in inaccurate or misleading results. Table 1 contains a summary of specimen rejection criteria.

Some specimens may require special handling or processing techniques depending on the type of microorganism or test requested. For example, specimens for anaerobic culture should be collected in anaerobic transport media or syringes with no air bubbles. Specimens for fungal culture should be collected in sterile containers with no preservatives. Specimens for molecular tests such as PCR should be collected in viral transport media or other suitable media that preserve nucleic acids.

The laboratory should provide clear instructions and guidelines for specimen collection and transport to ensure optimal quality and safety. The laboratory should also communicate with the clinicians and other health care workers about any issues or problems with specimen management that may affect the diagnosis and treatment of infectious diseases.

A good quality specimen is essential for accurate and reliable microbiological diagnosis. A specimen that is contaminated, inadequate, or improperly handled can lead to false-negative or false-positive results, which can affect patient care and outcomes. Therefore, it is important to follow the best practices for specimen collection and processing in microbiology. There are four main stages involved in obtaining a good quality specimen for testing:

1. Preparation of the patient: The patient should be informed about the purpose and procedure of the specimen collection, and any questions or concerns should be addressed. The patient should also be instructed to avoid any factors that may interfere with the specimen quality, such as antiseptics, antibiotics, food, or fluids. The patient should be comfortable and relaxed during the collection process, and any sources of pain or discomfort should be minimized. The patient`s identity and medical history should be verified and recorded before collecting the specimen.
2. Collection of the specimen: The specimen should be collected using the appropriate technique, equipment, and container for the type and site of infection. The technique should minimize the risk of contamination from the environment, the patient`s normal flora, or other specimens. The equipment and container should be sterile, labeled, and sealed properly. The specimen should be collected as close as possible to the onset of symptoms or signs of infection, and before starting any antimicrobial therapy. The specimen should also be collected in sufficient quantity and quality to allow for adequate testing and analysis.
3. Processing of the specimen: The specimen should be processed as soon as possible after collection, preferably within 2 hours. The processing may include steps such as centrifugation, filtration, staining, inoculation, or preservation. The processing should be done according to the standard operating procedures and quality control measures of the microbiology laboratory. The processing should aim to preserve the viability and integrity of the microorganisms in the specimen, and to enhance their detection and identification.
4. Storing or transporting the specimen: The specimen should be stored or transported under optimal conditions to maintain its quality until it reaches the microbiology laboratory. The optimal conditions may vary depending on the type of specimen and microorganism involved, but generally they include low temperature (2-8°C), dark environment, and appropriate medium or buffer. The specimen should also be protected from physical damage, leakage, or loss during storage or transport. The specimen should be accompanied by a requisition form that contains relevant information such as patient`s name, identification number, date and time of collection, type and site of specimen, clinical diagnosis, and requested tests.

By following these four stages carefully, a good quality specimen can be obtained for testing in microbiology. A good quality specimen can provide accurate and reliable results that can help in diagnosing microbial infections and guiding appropriate treatment.

Microbiologic evaluations are the methods used to examine the specimens collected from patients with suspected microbial infections. The goal of microbiologic evaluation is to provide accurate, clinically pertinent results in a timely manner. The quality of the specimens submitted to the microbiology laboratory is critical for optimal specimen evaluation. Microbiologic evaluations can be classified into five main categories:

• Direct examination – This involves the observation of the specimen under a microscope or other devices to detect the presence and morphology of microorganisms. Direct examination can also include staining techniques, immunological methods, and molecular methods to enhance the visualization and identification of microorganisms. For example, Gram stain, acid-fast stain, immuno-peroxidase stain, immunofluorescence, and polymerase chain reaction (PCR) are some of the direct examination methods used in microbiology.
• Culture examination – This involves the isolation and growth of microorganisms from the specimen on different types of media. Culture examination can provide information on the number, type, and characteristics of microorganisms in the specimen. Culture examination can also include biochemical tests, antigen detection tests, and molecular tests to further identify and classify the microorganisms. For example, blood agar, chocolate agar, MacConkey agar, catalase test, coagulase test, and MALDI-TOF are some of the culture examination methods used in microbiology.
• Microbial identification – This involves the determination of the genus and species of microorganisms isolated from the specimen. Microbial identification can be based on phenotypic methods (such as morphology, growth characteristics, enzymatic activities) or genotypic methods (such as DNA sequencing, PCR, hybridization). Microbial identification can help in establishing the diagnosis, epidemiology, and treatment of microbial infections. For example, Gram stain, API system, VITEK system, 16S rRNA sequencing, and ribotyping are some of the microbial identification methods used in microbiology.
• Serodiagnosis – This involves the detection and measurement of antibodies or antigens in the serum or other body fluids of patients with suspected microbial infections. Serodiagnosis can provide information on the exposure, immune status, and stage of infection of patients. Serodiagnosis can also include agglutination tests, precipitation tests, complement fixation tests, enzyme immunoassays, and immunoblotting. For example, Widal test, latex agglutination test, ELISA test, and Western blot test are some of the serodiagnosis methods used in microbiology.
• Antimicrobial susceptibility – This involves the testing of microorganisms isolated from the specimen against different types of antimicrobial agents to determine their resistance or sensitivity. Antimicrobial susceptibility can help in selecting the appropriate antimicrobial therapy for patients with microbial infections. Antimicrobial susceptibility can also include disk diffusion tests, broth dilution tests, E-test strips, automated systems, and molecular methods. For example, Kirby-Bauer test, MIC test, VITEK 2 system, and PCR-RFLP are some of the antimicrobial susceptibility methods used in microbiology.

These are some of the microbiologic evaluations that are performed in a microbiology laboratory to diagnose and treat microbial infections. However, these methods may vary depending on the type and source of specimen, the availability and cost of resources, and the clinical relevance and accuracy of results . Therefore, it is important to consult with a microbiologist or an infectious disease specialist before ordering or interpreting microbiologic tests.

One of the main challenges in diagnosing infectious diseases is to identify the specific microorganisms that are responsible for the infection. Traditional methods such as culture, microscopy, and serology may not be sensitive, specific, or rapid enough to detect all the possible pathogens that can cause a given syndrome. Moreover, some pathogens are difficult or impossible to grow in the laboratory, or require special media or biosafety precautions. Therefore, molecular techniques that can directly detect the genetic material of the microorganisms have been developed and applied in clinical microbiology.

Molecular techniques are based on the amplification, hybridization, or sequencing of nucleic acids (DNA or RNA) from the microorganisms. These techniques can offer several advantages over conventional methods, such as:

• Higher sensitivity and specificity: Molecular techniques can detect very low amounts of microbial DNA or RNA in a specimen, and can discriminate between closely related species or strains.
• Faster turnaround time: Molecular techniques can provide results within hours or even minutes, compared to days or weeks for culture-based methods.
• Broader range of detection: Molecular techniques can detect a wide variety of pathogens, including those that are unculturable, fastidious, slow-growing, or emerging.
• Enhanced characterization: Molecular techniques can provide additional information about the microorganisms, such as their virulence factors, resistance genes, subtypes, or phylogenetic relationships.

Some of the most common molecular techniques used in clinical microbiology are:

• Polymerase chain reaction (PCR): This technique uses a pair of synthetic oligonucleotide primers that are complementary to a specific region of the microbial DNA or RNA. The primers anneal to the target sequence and are extended by a DNA polymerase enzyme, resulting in the exponential amplification of the target sequence. The amplified product can be detected by various methods, such as gel electrophoresis, fluorescence, or real-time monitoring. PCR can be used to detect and quantify a single or multiple pathogens in a specimen .
• Nucleic acid hybridization: This technique uses a labeled probe that is complementary to a specific region of the microbial DNA or RNA. The probe hybridizes to the target sequence and can be detected by various methods, such as colorimetric, chemiluminescent, or fluorescent signals. Hybridization can be performed in solution (liquid-phase hybridization) or on a solid support (solid-phase hybridization). Examples of solid-phase hybridization include dot blot, line probe assay, and microarray .
• Nucleic acid sequencing: This technique uses a DNA polymerase enzyme to synthesize a complementary strand of DNA from a template strand of microbial DNA or RNA. The synthesis is terminated by incorporating labeled nucleotides that indicate the identity of the last base added. The sequence of the template strand can be deduced by reading the order of the labeled nucleotides. Sequencing can be performed by various methods, such as Sanger sequencing, pyrosequencing, or next-generation sequencing .

Molecular techniques have revolutionized the diagnosis and management of infectious diseases by providing rapid and accurate identification of pathogens and their characteristics. However, molecular techniques also have some limitations and challenges, such as:

• Cost and complexity: Molecular techniques require specialized equipment, reagents, and personnel that may not be available or affordable in some settings. Molecular techniques also need strict quality control and quality assurance measures to prevent contamination and ensure reliability.
• Interpretation and standardization: Molecular techniques may generate results that are difficult to interpret or compare across different laboratories or platforms. Molecular techniques may also lack validation or standardization for some pathogens or specimens.
• Clinical relevance and utility: Molecular techniques may detect microorganisms that are not clinically significant or causative of the infection. Molecular techniques may also miss microorganisms that are present below the detection limit or that have genetic variations that affect the amplification or hybridization process.

Therefore, molecular techniques should be used in conjunction with other methods and clinical criteria to provide a comprehensive and accurate diagnosis of infectious diseases.

The microbiology laboratory plays a crucial role in the diagnosis, treatment, control and prevention of infectious diseases caused by various types of microorganisms . The microbiology laboratory can provide valuable information to the clinicians and health care workers regarding the identification and characterization of the causative agents of infection, as well as their susceptibility to different antimicrobial agents .

The microbiology laboratory can perform various types of tests and techniques to detect and identify the microorganisms responsible for infection, such as:

• Direct examination of specimens using microscopy, staining, immunofluorescence, or genetic probes .
• Culture examination of specimens using selective or non-selective media, biochemical tests, or molecular methods .
• Serological tests to detect antibodies or antigens in the patient`s serum or other body fluids .
• Antimicrobial susceptibility testing to determine the resistance or sensitivity of the isolated microorganisms to various antimicrobial agents .

The microbiology laboratory can also use advanced molecular techniques such as polymerase chain reaction (PCR) to amplify and detect specific DNA or RNA sequences of the microorganisms, which can improve the speed, sensitivity, and specificity of diagnosis . PCR can also be used to detect mutations or genes that confer resistance to certain antimicrobial agents .

The microbiology laboratory director and staff are often available to offer advice and guidance to the clinicians regarding the differential diagnosis, choice of specimens, optimal tests and techniques, interpretation of results, and selection of appropriate antimicrobial therapy . The microbiology laboratory can also collaborate with other laboratories and public health agencies to monitor and report the epidemiology and trends of infectious diseases and antimicrobial resistance .

Therefore, the microbiology laboratory is an essential partner in the management of infectious diseases, and its role should be recognized and supported by the health care system.

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