Diagnosis of infectious diseases in clinical microbiological laboratory is attempted through following methods:
- Conventional Methods
Laboratory tests may identify organisms either directly microscopically by stains, growing organisms by cultural techniques and/or detecting their antigens or indirectly by identifying antibodies to the organism.
-Molecular Methods
This techniques Identify organisms by detecting their DNA or RNA by specific technologies.
Conventional technologies for identifying microorganisms usually involve good sampling techniques to assure accurate laboratory diagnosis.
Conventional Methods for Diagnosis of Infectious Diseases
Infectious diseases are common diseases all over the world. A recent World Health Organization report indicated that infectious diseases are now the world’s biggest killer of children and young adults. Infectious diseases in non-industrialized countries caused 45% in all and 63% of death in early childhood.
Causes of Infectious Diseases:
The microbial causes of human diseases are classified into theses groups:
1- Bacteria
2- Viruses
3- Fungi
4- Protoza
5- Chlamydiae
6- Rickettsiae
7- Mycoplasmas.
Infection may be endogenous or exogenous.
1- Endogenous infections: the microorganism (usually a bacterium) is a component of the patient's endogenous flora. Endogenous infections can occur when the microorganism is aspirated from the upper to the lower respiratory tract or when it penetrates the skin or mucosal barrier as a result of trauma or surgery.
2- Exogenous infections: the microorganism is acquired from the environment (e.g., from soil or water) or from another person or an animal.
The ability to control such microbial infections is largely dependent on the ability to detect these etiological agents in the clinical microbiology laboratory (Millar et al., 2003)
Diagnoses of Infectious Diseases
Diagnosis of infectious diseases in clinical microbiological laboratory is attempted through following methods:
- Conventional Methods
Laboratory tests may identify organisms either directly microscopically by stains, growing organisms by cultural techniques and/or detecting their antigens or indirectly by identifying antibodies to the organism.
-Molecular Methods
This techniques Identify organisms by detecting their DNA or RNA by specific technologies.
Conventional technologies for identifying microorganisms usually involve good sampling techniques to assure accurate laboratory diagnosis.
Figure (1): Microbiology laboratory techniques for diagnosis of infectious diseases.
A-Proper Sampling in Clinical Microbiology Laboratory
Specimens selected for microbiologic examination should reflect the disease process and collected in sufficient quantity to allow complete microbiologic examination. The number of microorganisms per milliliter of a body fluid or per gram of tissue is highly variable, ranging from less than 1 to 108 or 1010 colony-forming units (CFU).
Collection of good quality specimens depends on:
-The optimal time of specimen collection.
-The correct type of specimen
-Well collected specimens with minimum contamination from normal flora of the patient or the person collecting the specimen.
-Adequate amounts of each specimens and appropriate number of specimens
-Clearly labeled safe specimens
Optimal Time of Collection of Specimens
The proper time of sampling in clinical microbiology play a crucial role in proper laboratory diagnosis. There are several examples for such proper time. If possible, specimens should be collected before the administration of antibiotics. Above all, close communication between the clinician and the microbiologist is essential to ensure that appropriate specimens are selected and collected and that they are appropriate
-Blood cultures and blood films for malarial parasites are best collected just as the patient’s temperature starts to rise, however, when infective endocarditis is suspected, three blood culture sets collected with 24 hour irrespective of patient, s temperature.
- Specimens for virus isolation are most likely to give positive results when collected during the most acute stages of the disease
-Serology is satisfactory when four fold or greater rising antibody titer is demonstrated in pained sera. The 1st serum sample as early as possible in the disease course. Second in the convalescent stage.
Correct Types of Specimens
Examples:
Bacterial meningitis-------blood cultures, CSF culture
Suspected gonorrhea -------cervical, urethral and rectal swab should be collected rather than high regional swabs.
Well Collected Specimens with Minimum Contamination from the Normal Flora
The good quality of microbiological sample is a need for accurate laboratory diagnosis. The main problem is mixing of samples with normal flora normally resident beside infected tissue. Skin and mucous membranes have a large and diverse endogenou flora; so every effort must be made to minimize specimen contamination during collection. Contamination may be avoided by various means:
- The skin can be disinfected before aspirating or incising a lesion.
- The contaminated area may be bypassed altogether. Examples of such approaches are trans tracheal puncture with aspiration of lower respiratory secretions or supra pubic bladder puncture with aspiration of urine.
It is often impossible to collect an uncontaminated specimen, and decontamination procedures, cultures on selective media, or quantitative cultures must be used.
Specimens collected by invasive techniques, particularly those obtained intra operatively, require special attention. Enough tissue must be obtained for both histopathologic and microbiologic examination.
Adequate Amounts of Appropriate Number of Specimens
The volume of blood for culture from adult -5-10 ml per bottle and in children and neonates 1-5ml per bottle.
-Collection of early morning sputum specimens, and collection of adequate amount of early morning urine specimen for 3 successive days is required for the isolation of Mycobacterium tuberculosis (TB).
-Patients with diarrhea ---at least 2 specimens of stool are collected for culture of Salmonellae spp. or Shigella spp.
-Serological investigations usually require paired sera.
Clearly Labeled and Safe Specimens
Specimens for microbiological investigations should be placed in leak – proof containers, and each container should be enclosed in plastic bag.
The hazards to staff handling leaking container s include acquiring enteric infection from feces, TB from sputum of an open case of pulmonary TB and viruses s such as HCV, HBV, HIV, from leaking blood.
B-Transport of specimens to the laboratory
The specimen must be transported rapidly, in the correct medium, and in conditions that limit growth of any potentially contaminating normal flora. For accurate quantification of the pathogen, additional pathogen growth must be prevented; specimens should be transported to the laboratory immediately or, if transport is delayed, refrigerated (in most cases). Certain cultures have special considerations.
Many pathogenic organisms don’t survive for long in clinical specimens kept at room temperature. Examples include Gonococci, Haemophilus, Bacteroides, anaerobic cocci and most viruses.
On the other hand, some organisms contaminating specimens from the normal flora such as Coliform and Coagulase negative Staphylococci, may rapidly grow in specimen kept at room temp.
-Urine or sputum specimens should reach the laboratory within 2hours of collection when even possible. If delay is expected immediately inoculated into transport media.
-Transport media used:
Stuart’s transport media ----- for pus or swabs for bacterial culture when delays in transport.>1/2hour or when Neisseria infections are suspected. However the inoculated transport media should be sent to the laboratory within 4hours.
Cerebrospinal fluid(CSF) not refrigerated since other wise Meningococci may rapidly die.
Viral transport media is necessary for virus isolation, and also for Chlamydia isolation.
Specimens for virus isolation are kept at –70ºC till time of transferring the appropriate cell line which support growth of the possible virus or Chlamydia.
Figure (2) Laboratory procedures used in confirming a clinical diagnosis of infectious disease with a bacterial etiology.
C- Conventional laboratory Methods for Diagnosis of Infectious Diseases
1-Direct method:
They are Laboratory tests may identify organisms directly (eg, visually, using a microscope, growing the organism in culture)
I- Microscopy
Microscopy can be done quickly, but accuracy depends on the experience of the microscopist and quality of equipment. Regulations often limit physicians' use of microscopy for diagnostic purposes outside a certified laboratory (Siqueira, J. Fet al., 2005).
Most specimens are treated with stains that color pathogens, causing them to stand out from the background, although wet mounts of unstained samples can be used to detect fungi, parasites (including helminth eggs and larvae), vaginal clue cells, motile organisms (eg, Trichomonas), and syphilis (via darkfield microscopy). Visibility of fungi can be increased by applying 10% potassium hydroxide (KOH) to dissolve surrounding tissues and nonfungal organisms (Fredricks and Relman, 1999).
The clinician orders a stain based on the likely pathogens, but no stain is 100% specific. Most samples are treated with Gram stain and, if mycobacteria are suspected, an acid-fast stain. However, some pathogens are not easily visible using these stains; if these pathogens are suspected, different stains or other identification methods are required. Because microscopic detection usually requires a microbe concentration of about 1 × 105/mL, most body fluid specimens (eg, CSF) are concentrated (eg, by centrifugation) before examination.
Types of Commonly Used Stains.
Gram stain: The Gram stain classifies bacteria according to whether they retain crystal violet stain (Gram-positive—blue) or not (Gram-negative—red) and highlights cell morphology (eg, bacilli, cocci) and cell arrangement (eg, clumps, chains, diploids). Such characteristics can direct antibiotic therapy pending definitive identification. To do a Gram stain, technicians heat-fix specimen material to a slide and stain it by sequential exposure to Gram's crystal violet, iodine, decolorizer, and counterstain (typically safranin).
Acid-fast and moderate (modified) acid-fast stains: These stains are used to identify acid-fast organisms (Mycobacterium sp) and moderately acid-fast organisms (primarily Nocardia sp). These stains are also useful for staining Rhodococcus and related genera, as well as oocysts of some parasites (eg, Cryptosporidium).
Although detection of mycobacteria in sputum requires only about 5, 000 to 10, 000 organisms/mL, mycobacteria are often present in lower levels, so sensitivity is limited. Usually, several mL of sputum are decontaminated with Na hydroxide and concentrated by centrifugation for acid-fast staining. Specificity is better, although some moderately acid-fast organisms are difficult to distinguish from mycobacteria.
Fluorescent stains: These stains allow detection at lower concentrations (1 × 104 cells/mL). Examples are acridine orange (bacteria and fungi), auramine-rhodamine and auramine O (mycobacteria), and calcofluor white (fungi, especially dermatophytes).
Coupling a fluorescent dye to an antibody directed at a pathogen (direct or indirect immunofluorescence) should theoretically increase sensitivity and specificity. However, these tests are difficult to read and interpret, and few (eg, Pneumocystis and Legionella direct fluorescent antibody tests) are commercially available and commonly used.
India ink (colloidal carbon) stain: This stain is used to detect mainly Cryptococcus neoformans and other encapsulated fungi in a cell suspension (eg, CSF sediment). The background field, rather than the organism itself, is stained, which makes any capsule around the organism visible as a halo. In CSF, the test is not as sensitive as cryptococcal antigen. Specificity is also limited; leukocytes may appear encapsulated.
Wright's stain and Giemsa stain: These stains are used for detection of parasites in blood, Histoplasma capsulatum in phagocytes and tissue cells, intracellular inclusions formed by viruses and chlamydia, trophozoites of Pneumocystis jiroveci, and some intracellular bacteria.
Trichrome stain (Gomori-Wheatley stain) and iron hematoxylin stain:
These stains are used to detect intestinal protozoa.
The Gomori-Wheatley stain is used to detect microsporidia. It may miss helminth eggs and larvae and does not reliably identify Cryptosporidium. Fungi and human cells take up the stain.
The iron hematoxylin stain differentially stains cells, cell inclusions, and nuclei. Helminth eggs may stain too dark to permit identification.
Disadvantages of Microscopic methods:
(a) Microscopy may suggest an etiologic agent, but it rarely provides definitive evidence of infection by a particular species.
(b) Microscopic findings regarding bacterial morphology may be misleading, because many species can be pleomorphic and conclusions can be influenced by subjective interpretation of the investigator.
(c) Limited sensitivity is because a relatively large number of microbial cells are required before they are seen under microscopy (e.g. 104 bacterial cells/ml of fluid) (Fredricks & Relman, 1999). Some micro-organisms can even require appropriate stains and/or approaches to become visible.
(d) Limited specificity is because our inability to speciate micro-organisms based on their morphology and staining patterns.
II-Culture Methods
Culture is microbial growth on or in a nutritional solid or liquid medium; increased numbers of organisms simplify identification. Culture also facilitates testing of antimicrobial susceptibility (Relman DA., 2002)
Communication with the laboratory is essential. Although most specimens are placed on general purpose media (e g, blood or chocolate agar), some pathogens require inclusion of specific nutrients and inhibitors or other special conditions (Wade W., 2002)
For more than a century, cultivation using artificial growth media has been the standard diagnostic test in infectious diseases. The microbiota associated with different sites in the human body has been extensively and frequently defined by studies using cultivation approaches.
The success in cultivation of important pathogenic bacteria probably led microbiologists to feel satisfied with and optimistic about their results and to recognize that there is no death of known pathogens (Relman, 1992 and Wade W, 2002).
But should we be so complacent with what we know about human pathogens? Making micro-organisms grow under laboratory conditions presupposes some knowledge of their growth requirements. Nevertheless, very little is known about the specific growth factors that are utilized by innumerous micro-organisms to survive in virtually all habitats, including within the human body (Wade, 2002).
A huge proportion of the microbial species in nature are difficult to be tamed in the laboratory. Certain bacteria are fastidious or even impossible to cultivate. Some well-known human pathogens, such as Mycobacterium leprae and Treponema pallidum continue to defy scientists regarding their cultivation under laboratory conditions (Fredricks and Relman, 1999)
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