Presence or Absence of Microbial Pathogens

The following points highlight the four main steps to determine the presence or absence of microbial pathogens. The steps are: 1. Collection of Samples 2. Handling of Collected Samples 3. Transport 4. General Methods of Laboratory Diagnosis.

Step # 1. Collection of Samples:

The results obtained after diagnosis in clinical laboratories are as good as the quality of the specimen collected for analysis.

1. Methods Used to Collect Samples:

Specimens may be collected by several methods using aseptic technique. Aseptic technique refers to specific procedures used to prevent unwanted microorganisms from contaminating the clinical specimen. Each method is designed to ensure that only the proper material will be sent to the clinical laboratory.

(i) Swab:

Sterile swab is the most common method used to collect specimens from the anterior nares or throat. A sterile swab is a rayon-, calcium alginate, or Dacron-tipped polystyrene applicator. Manufacturers of swabs have their own unique container design and instructions for proper use.

For instance, many commercially manufactured swabs contain a transport medium designed to preserve a variety of microorganisms and to prevent multiplication of rapidly growing members of the population (Fig. 46.3).

However, with the exception of the nares or throat, the use of swabs for the collection of specimens is of little value and should be discouraged for two major reasons:

(1) Swabs are associated with a greater risk of contamination with surface and subsurface microorganisms, and

(2) They have a limited volume capacity (<0.1 ml).

(ii) Needle aspiration:

Needle aspiration is used to collect specimens aseptically (e.g., anaerobic bacteria) from cerebrospinal fluid, pus, and blood. For needle aspiration, stringent antiseptic techniques are used to avoid skin contamination. To prevent blood from clotting and entrapping microorganisms, various anticoagulants (e.g., heparin, sodium citrate) are included within the specimen bottle or tube.

(iii) Intubation:

Intubation [in-into and tuba-tube] is the inserting of a tube into a body canal or hollow organ. For convenience, intubation can be used to collect specimens from the stomach.

In this procedure a long sterile tube is attached to a syringe, and the tube is either swallowed by the patient or passed through a nostril (Fig. 46.4) into the patient’s stomach. Specimens are then withdrawn periodically into the sterile syringe. The most common intubation tube is the Levin tube.

(iv) Catheter:

A catheter is a tubular instrument used for withdrawing or introducing fluids from or into the body cavity. For convenience, urine specimens may be collected with catheters to detect urinary tract infections caused by bacteria from newborns and neonates who cannot give urinary specimen voluntarily.

Three types of catheter are commonly used for urine. The hard catheter is used when the urethra is very narrow or has strictures. The French catheter is a soft tube used to obtain a single specimen sample. Foley catheter is used (Fig. 46.5) if multiple samples are required over a prolonged period.

(v) Clean-catch method:

Clean-catch method is the most common method used for the collection of urine. After the patient has cleaned the urethral meatus (opening), a small container is used to collect the urine. The optimal time to use the clean-catch method is early morning because the urine contains more microorganisms as a result of being in the bladder overnight.

In the clean-catch midstream method, the first urine voided is not collected because ii will be contaminated with those transient microorganisms that normally occur in the lower portion of the urethra.

Only the midstream portion is collected since it most likely will contain those microorganisms that are found in the urinary bladder. If warranted for some patients, needle aspirations also are done directly into the urinary bladder.

(vi) Sputum-cup method:

Sputum is the most common specimen collected in suspected cases of lower respiratory tract infections. Specifically, sputum is the mucous secretion expectorated from the lungs, bronchi, and trachea through the mouth, in contrast to saliva, which is the secretion of the salivary glands. Sputum is collected in specially designed sputum cups (Fig. 46.6).

The sputum cup allows the patient to expectorate a clinical sample directly into the cup. During laboratory diagnosis, the cup can be opened from the bottom to reduce the chance of contamination from extraneous pathogens.

2. Important Concerns during Sample Collection:

Following important concerns should be taken into account during collection of samples from the patient:

(i) The specimen selected should adequately represent the diseased area and also may include additional sites (e.g., liver and blood specimens) in order to isolate and identify potential agents of the particular disease process.

(ii) A quantity of specimen adequate in amount to allow a variety of diagnostic testing should be obtained.

(iii) Attention must be given to specimen collection in order to avoid contamination from the many varieties of micro­organisms indigenous to the skin and mucous membranes.

(iv) The specimen should be forwarded promptly to the clinical laboratory.

(v) If possible, the specimen should be obtained before antimicrobial agents have been administered to the patient.

Step # 2. Handling of Collected Samples:

Immediately after collection the specimen must be properly labeled and handled. The person collecting the specimen is responsible for ensuring that the name, hospital, registration number, location in the hospital, diagnosis, current antimicrobial therapy, name of attending physician, admission date, and type of specimen are correctly and legibly written or imprinted on the culture request form.

This information must correspond to that written or imprinted on a label affixed to the specimen container. The type or source of the sample and the choice of tests to be performed also must be specified on the request form.

Step # 3. Transport:

Speed in transporting the specimen to the clinical laboratory after it has been obtained from the patient is of prime importance. Some laboratories refuse to accept specimens if they have been in transit too long.

Microbiological specimens may be transported to the laboratory by various means. For example, certain specimens should be transported in a medium that preserves the microorganisms and helps maintain the ratio of one organism to another. This is especially important for specimens in which normal microorganisms may be mixed with microorganisms foreign to the body location.

Special treatment is required for specimens when the microorganism is thought to be anaerobic. The material is aspirated with a needle and syringe. Most of the time it is practical to remove the needle, cap the syringe with its original seal, and bring the specimen directly to the clinical laboratory.

Transport of these specimens should take no more than 10 minutes; otherwise, the specimen must be injected immediately into an anaerobic transport vial (Fig. 46.7).

Vials should contain a transport pliedium with an indicator, such as resazurin, to show that the interior of the vial is anaerobic at the time the specimen is introduced. Swabs for anaerobic culture usually are less satisfactory than aspirates or tissues, even if they are transported in an anaerobic vial.

Many clinical laboratories insist that stool specimens (the faecal discharge from the bowels) for culture be transported in special buffered preservatives.

Transport of urine specimens to the clinical laboratory must be done as soon as possible. No more than 1 hour interval should be between the time the specimen is obtained and the time it is examined. If this time schedule cannot be followed, the urine sample must be refrigerated immediately.

Cerebrospinal fluid (CSF) from patients suspected of having meaningitis should be examined immediately by skilled personnel in the clinical microbiology laboratory. CSF is obtained by lumbar puncture under conditions of strict asepsis, and the sample is transported to the laboratory within 15 minutes.

Specimens for the isolation of viruses are iced before transport, and can be kept at 4°C for up to 72 hours; if the sample will be stored longer than 72 hours, it should be frozen at -72°C.

Step # 4. General Methods of Laboratory Diagnosis:

General methods of laboratory diagnosis involve the isolation of microbial pathogens from the clinical samples collected and their proper identification so that the physician may successfully treat the patient. If clinically relevant microbial pathogens are to be isolated and identified, the specimen must be property obtained.

The clinician must ensure that the specimen is removed from the actual site of the infection. In addition, recovery of pathogens may not be possible if insufficient inoculum is taken.

The sample must also be taken under aseptic conditions so that contamination is avoided. Care must also be taken to ensure that metabolic requirements for certain organisms, such as anoxic conditions, are maintained. Once obtained, the sample must be analysed as soon as possible.

Isolation of Microbial Pathogens from Clinical Samples:

Clinical samples collected from the patients may include blood, urine, faeces, sputum, cerebrospinal fluid, or pus from a wound. These samples are inoculated on to the surface of an agar plate medium or a tube of liquid culture medium to carry the primary isolation of the microbial pathogen.

Through the use of various specialized growth media, most microbial pathogens of clinical importance can be grown and isolated in pure culture form. In some cases, small pieces of living tissue may be sampled for culture.

1. Culture media:

Most clinical samples are first grown on general purpose media, media such as blood agar that support the growth of most aerobic and facultatively anaerobic microorganisms. An enriched medium is one that allows metabolically fastidious microorganisms to grow because of the addition of specific growth factors.

This medium is often necessary to enhance the growth of certain microbial pathogens such as Neisseria gonorrhoeae, the causal agent of gonorrhoea. A selective medium is the medium that enhances the growth of certain microbial pathogens while retards the growth of others due to an added media component.

Differential medium, however, is the specialized medium that contains an indicator, usually a dye, that allows differentiation between chemical reactions carried out during growth. Eosine-methylene blue (EMB) agar, for instance, is a widely used differential medium. EMB agar is used for the isolation of gram-negative enteric bacteria.

2. Blood cultures:

The most common bacterial pathogens occurring in blood of diseased individuals include Pseudomonas aeruginosa, enteric bacteria especially E.coli and Klebsiella pneumoniae, and the gram- positive cocci Streptococcas pyrogenes and Staphylococcus aureus. Presence of bacterial pathogens in the blood is called bacteremia, which is uncommon in healthy individuals.

Prolonged bacteremia is generally indicative of systemic infection. Septisemia is another condition found in blood of diseased personnel. Septicemia represents blood infection taken place as a result of the growth of a virulent microorganism entered into the blood from a focus of infection, multiplied, and travelled to various body tissues to initiate new infections.

Blood cultures provide immediate way of isolating and identifying the microbial pathogens of bacteremia and septicemia, and diagnosis therefore depends on careful and proper blood culture. The standard blood culture procedure is to draw 20 ml of blood aseptically from a vein and inject it into two blood culture bottles containing an anticoagulant and an all-purpose culture medium.

One bottle is incubated aerobically and one anaerobically. Blood culture bottles are incubated at 35°C and examined several times each hour for up to five days in most automated systems. Most clinically significant bacteria are recovered within two days, while more fastidious microorganisms may be recovered in three to five days.

Microorganisms from blood cultures are commonly detected by indicators of microbial growth in automated systems, microscopic examination, and subculture. Automated blood culture systems detect growth by monitoring carbon dioxide production and turgidity as often as every 10 minutes.

3. Urine cultures:

Urinary tract infections very commonly occur especially in females. The most common urinary tract pathogens are members of the enteric bacteria, with E. coli accounting for about 90% of the cases.

Other urinary tract pathogens include Klebsiella, Enterobacter, Proteus, Pseudomonas, Staphylococcus saprophyticus, and Enterococcus faecalis. Neisseria gonorrhoeae, the causal agent of gonorrhoea, does not grow in the urine itself, but on the urethral epithelium, and is diagnosed by different techniques.

To culture potential urinary tract pathogens, two media are normally used:

(1) Blood agar as a non-selective general medium and

(2) A medium selective for enteric bacteria, such as MacConkey or eosin-methylene blue agar (EMB).

These specialized enteric media permit the initial differentiation of lactose fermenters from non-fermenters, while the growth of gram-positive bacteria such as Staphylococus spp. (common skin contaminants) is inhibited.

Experienced clinical microbiologists may make a tentative identification of an isolate by observing the colour and morphology of colonies of the suspected pathogen growth on various media. Such an identification must be followed with more detailed tests to make a positive identification.

4. Faecal cultures:

Microbial pathogens present in intestine are isolated from properly collected and preserved faeces. During storage faecal acidity increases, so extended-delay between sampling and sample processing must be avoided. This is especially critical for the isolation of Shigella and Salmonella species, both of which are sensitive to acid pH.

Freshly collected faecal samples are placed in a vial containing phosphate buffer for transport to the lab. Bloody or pus-containing stools as well as stools from patients with suspected foodborne or waterborne infections are inoculated into a variety of selective media (Table 46.2) for isolation of individual bacteria.

Intestinal parasites are identified by observing cysts microscopically in the stool sample or through antigen- detection assays rather than by culture methods. Many laboratories also use a variety of selective and differential media and incubation conditions to identify Escherichia coli O157:H7 and Campylobacter, two important intestinal pathogens generally acquired from contaminated food or water.

5. Wound sample cultures:

Animal bites, burns, cuts, or the penetration of foreign objects normally results in wound infections. Infections associated with wounds must be carefully sampled to isolate the relevant microbial pathogen.

The best sampling method for wound infections is to aspirate pus with a sterile syringe and needle following disinfection of the skin surface. Disinfection of skin surface is necessary before sampling because the wound infections are frequently contaminated with normal skin flora, and samples from such wounds are usually misleading.

Various microbial pathogens are found associated with wound infections. Since some of these pathogens are anaerobes, proper evaluation requires that samples be obtained, transported, and cultured under anaerobic as well as aerobic conditions.

For instance, potential pathogens commonly, associated with purulent discharges from wound infections are Staphylococcus aureus, enteric bacteria, Pseudomonas aeruginosa, and anaerobes from the genera Bacteroides and Clostridium. The main isolation media are blood agar, several selective media for enteric bacteria, and blood agar containing additional supplements and reducing agents for obligate anaerobes.

6. Genitourinary cultures:

Purulent urethral discharge in males is the classical symptom of the sexually transmitted disease gonorrhoea caused by Neisseria gonorrhoeae. The latter is usually found as a gram- negative diplococcus but can be quite pleomorphic. N. gonorrhoeae is isolated primarily on several selective media. One of such selective media used for primary isolation is modified Thayer-Martin (MTM) agar.

This medium incorporates the antibiotics vancomycin, nystatin, trimethoprim, and colistin to suppress the growth of normal microflora, but these antibiotics do not affect N. gonorrhoeae. Inoculated plates are incubated in a humid environment in an atmosphere containing 3-7% CO₂, required for growth of gonococci.

The plates are examined after 24 and 48 hours and tested by the oxidase test because all Neisseria are oxidase-positive. Oxidase-positive gram-negative diplococci growing on selective media are presumed to be gonococci if the inoculum was derived from genitourinary sources, but definitive identification requires determination of carbohydrate utilization patterns and immunological or nucleic acid probe tests.

Identification of Microbial Pathogens:

After isolation and primary identification, definitive identification of microbial pathogens in clinical microbiology laboratories is carried out with the help of:

(1) Microscopic examination of clinical samples,

(2) Growth and biochemical characteristics,

(3) Immunologic techniques,

(4) Bacteriophage typing, and

(5) Molecular techniques.

1. Microscopic examination:

Wet-mount, heat-fixed, or chemically fixed clinical samples can be examined with an ordinary bright-field microscope, phase-contrast, or dark-field microscope. Phase-contrast or dark-field microscopy preferably used for the detection of spirochaetes in skin lesions associated with early syphilis or in blood specimens of people with early leptospirosis.

The fluorescence microscope can be used to identify certain acid-fast microbial pathogens, e.g., Mycobacterium tuberculosis after they are stained with fluorochromes such as auramine-rhodamine.

Many stains that can be used to examine clinical samples for specific microbial pathogens have been described. Two of the more widely used are the Gram-stain and acid- fast stain. Because these stains are based on the chemical composition of cell walls, they are not useful in identifying bacteria without walls.

2. Growth and biochemical characteristics:

Based on growth characteristics on primary isolation media, an unidentified microbial pathogen is usually sub-cultured on to media that are designed to measure one of many different biochemical reactions. The media used are selective, differential, or both. A selective medium contains compounds that inhibit the growth of certain microorganisms.

A differential medium contains an indicator, usually a dye, that allows differentiation between chemical reactions carried out during growth. Eosin-methylene blue (EMB) agar, for example, is a widely used selective and differential medium. EMB agar is used for the isolation of gram-negative enteric bacteria.

Methylene blue dye inhibits the growth of most gram-positive bacteria. Eosin is a dye that responds to changes in pH, going from colourless to black under acidic conditions. EMB agar contains lactose and sucrose, but not glucose, as energy sources. Lactose- fermenting bacteria such as Escherichia coli, Klebsiella and Enterobacter acidify the medium and the colonies appear black with a greenish sheen.

Colonies of lactose non-fermenters, such as Salmonella, Shigella and Pseudomonas, are translucent or pink. Thus, EMB preferentially selects for the growth of gram-negative bacteria and differentiates among several genera of the selected gram-negative bacteria.

Some of the most important biochemical tests are mentioned in Table 46.3. These individual biochemical tests measure the presence or absence of enzymes involved in catabolism of the substrate or substrates in the medium.

Some examples are:

(1) Fermentation of sugars is measured by incorporating pH indicator dyes that change colour on acidification,

(2) Production of hydrogen and/or carbon dioxide during sugar fermentation is assayed by observing gas production either in gas collection vials or in agar,

(3) Hydrogen sulfide (H₂S) production is indicated following growth in a medium containing ferric iron. If sulfide is produced, ferric iron reacts with H₂S to form a black precipitate of iron sulfide, and

(4) Utilization of citric acid, an organic acid with three carboxylic acid groups, is accompanied by a pH increase, and a specific dye in the citric acid test medium changes color as conditions become alkaline.

3. Immunologic technique:

There has been a shift from the multistep methods of growth and biochemical characteristics to unitary procedures with respect to the identification of microbial pathogens in clinical samples. Use of immunologic techniques represents such a shifting.

Immunologic techniques employing detection of antibodies and antigens for identification of microbial pathogens from clinical samples are easy to use, give relatively rapid endpoints, and are sensitive and specific.

The most widely used immunologic techniques available to detect microbial pathogens in clinical samples are:

(1) Agglutination,

(2) Complement fixation,

(3) Enzyme-linked immunosorbant assay (ELISA),

(4) Immunoblotting,

(5) Immunodiffusion,

(6) Immunoelectrophoresis,

(7) Radioimmunoassay (RIA), and

(8) Serotyping.

However, some of the more popular immunologic rapid test kits for viruses and bacteria are presented in Table 46.4.

Perfect interpretation of immunologic tests is essentially based on proper test-selection and timing of sample collection. It is because each individual’s immunologic response to a microbial pathogen is quite variable. For convenience, a single elevated antibody IgM titer usually does not distinguish between active and past infections.

Furthermore, the lack of a measurable antibody titer may reflect either a microorganism’s lack of immunogenicity or an insufficient time for an antibody response to develop following the onset of the infectious disease. Some patients also are immunosuppressed due to other disease processes and/or treatment procedures (e.g., cancer and AIDS patients) and therefore do not respond.

4. Bacteriophage (phage) typing:

Bacteriophages (phages) are viruses that attack members of a particular bacterial species, or strains within a species. Bacteriophage (phage) typing is based on the specificity of phage surface receptors for cell surface receptors.

Only those bacteriophages that can attach to these surface receptors can infect bacteria and cause lysis. On a petri dish culture, lytic bacteriophages cause plaques on lawns of sensitive bacteria. These plaques represent infection by the virus.

In bacteriophage typing the clinical microbiologist inoculates the bacterium to be tested on to a petri plate. The plate is heavily and uniformly inoculated with a cotton swab so that the bacteria will grow to form a solid sheet or lawn of cells. No un-inoculated areas should be left.

The plate is then marked off into squares (15 to 20 mm per side), and each square is inoculated with a drop of suspension from the different phages available for typing. After the plate is incubated for 24 hours, it is observed for plaques. The phage type is reported as a specific genus and species followed by the types that can infect the bacterium.

For example, the series 10/16/24 indicates that this bacterium is sensitive to phages 10, 16, and 24, and belongs to a collection of strains, called a phagovar, that have this particular phage sensitivity. Bacteriophage typing remains a tool of the research and reference laboratory.

5. Molecular techniques:

Molecular techniques are the extremely sensitive techniques available for detecting microbial pathogens. With the application of these techniques, it is now possible to analyse the molecular characteristics of microbial pathogens in a clinical laboratory. Microbial techniques do not depend on pathogen isolation or growth, or on the detection of an immune response to the pathogen.

Since it is often difficult and sometimes impossible to grow and identify microbial pathogens in culture, molecular techniques are now-the-days in wide use.

Though there are a number of molecular approaches (e.g., comparison of proteins; physical, kinetic, and regulatory properties of microbial enzymes; nucleic acid-base composition; nucleic acid hybridization; and nucleic acid sequencing) that help accurate identification of microbial pathogens, the three molecular techniques being widely used are nucleic acid probe hybridization, gas-liquid chromatography, and plasmid fingerprinting.

(i) Nucleic acid probe hybridization:

Nucleic acid probe hybridization is one of the most powerful analytical tools available to clinical microbiologists for the detection and identification of microbial pathogens. Instead of detecting a whole microorganism or its products, nucleic acid probe hybridization detects the presence of specific DNA sequences associated with a specific microorganism.

To identify a microbial pathogen through this technique, the clinical microbiologist must have available a nucleic acid probe for that pathogen. Nucleic acid probes normally consist of a single strand of DNA with a sequence unique to the gene of interest.

The use of DNA probe is based upon the capacity of single-stranded DNA to bind (hybridize) with a complementary nucleic acid sequence present in test specimens to form a double stranded DNA hybrid.

For example, if a microbial pathogen from a clinical specimen contains a strand of DNA complementary to single- stranded DNA probe, the probe sequence hybridizes to the former forming a double-stranded DNA molecule. To detect a reaction, the probe is labelled with a reporter molecule, a radioisotope, an enzyme, or a fluorescent compound that can be detected following hybridization (Fig. 46.8).

Depending on the reporter (radioisotopes are the most sensitive), as little as 0.25 μg of DNA probe per sample can be used for pathogen identification. However, nucleic acid probe hybridization technique may be applied to purified DNA preparations, to bacterial colonies, or to clinical samples such as tissue, serum, sputum, and pus.

Recently, DNA probes have been developed that bind to complementary strands of ribosomal RNA. These DNA:rRNA hybrids are more sensitive than conventional DNA probes, give results in 2 hours or less, and require the presence of fewer microbial pathogens.

DNA probe sensitivity can be increased by over one million-fold if the target DNA is first amplified using the polymerase chain reaction (PCR). DNA:rRNA probes are available or are currently being developed for most clinically important microorganisms.

(ii) Gas-liquid chromatography:

During chromatography a chemical mixture carried by a liquid or gas is separated into its individual components because of processes such a s adsorption, ion-exchange, and partitioning between different solvent phases. In gas-liquid chromatography (GLC), specific microbial metabolites cellular fatty acids, and products from the pyrolysis (a chemical change caused by heat) of whole bacterial cells are analysed and identified.

These compounds are easily removed from growth media by extraction with an organic solvent such as either. The ether extract is then injected into the GLC system. Both volatile and non-volatile acids can be identified. Based on the pattern of fatty acid production, common bacteria isolated from clinical specimens can be identified.

(iii) Plasmid fingerprinting:

A plasmid is an autonomously replicating extra-chromosomal molecule of DNA in bacteria. Plasmid fingerprinting identifies microbial isolates of the same or similar strains; related strains often contain the same number of plasmids with the same molecular weights and similar phenotypes.

In contrast, microbial isolates that are phenotypically distinct possess different plasmid fingerprints. Plasmid fingerprinting of many E. coli, Salmonella, Campylobacter, and Pseudomonas strains and species has demonstrated that this method often is more accurate than other phenotyping methods such as bio-typing, antibiotic resistance patterns, phage typing, and serotyping.

The technique of plasmid fingerprinting involves following five steps:

(i) The bacterial strains are grown in broth or on agar plates.

(ii) The cells are harvested and lysed with a detergent.

(iii) The plasmid DNA is separated from the chromosomal DNA.

(iv) The plasmid DNA is applied to agarose gels and electrophoretically separated.

(v) The gel is stained with ethidium bromide, which binds to DNA, causing it to fluoresce under UV light. The plasmid DNA bands are then located.

Because the migration rate of plasmid DNA in agarose is inversely proportional to the molecular weight, plasmids of a different size appear as distinct bands in the stained gel.

The molecular weight of each plasmid species can then be determined from a plot of the distance that each species has migrated versus the log of the molecular weights of plasmid markers of known size that have been electrophoresed simultaneously in the same gel (Fig. 46.9).