Diagnosis of infectious disease is initiated by medical history and physical examination. Detailed identification involves the culture of infectious agents (isolated from a patient) followed by their microscopic examination, detecting the presence of substances produced by pathogens, and by directly identifying an organism by its genotype.
A micro-organism may be bacterial, fungal, viral or parasitic in a specimen sample can be detected and identified by any of the below mentioned ways:
1. Direct Examination.
2. Culture of micro-organisms.
3. Serological tests.
4. Molecular diagnostics.
1. Direct Examination:
(i) Microscopic Examination:
Specimen samples can be examined directly under a microscope. For example, motile trophozoites of parasites such as Giardia lamblia and Trichomonas vaginalis can be detected.
a. Dark Field Examination:
Some bacteria cannot be resolved directly under a microscope. Therefore, for such bacteria (e.g., Borrelia burgdorferi, a spirochete which causes Lyme disease) dark field examination (light is reflected or refracted off the surface of object is done.
b. Electron Microscopy:
Detection of viral causes of gastroenteritis such as rotaviruses exploits the electron microscopy technique.
c. Examination of Stained Material:
It is the most useful method for presumptive identification of micro-organisms. There are several staining methods (gram stain, acid fast stain, acridine orange stain, iodine stains) and the most popular is Gram stain which is used to classify bacteria on the basis of their forms, sizes and color reactions.
(ii) Microbial Antigen Testing:
This involves the use of a specific antibody to detect microbial antigens.
For this most frequently used techniques are:
a. Latex particle Agglutination – detection of Cryptococous neofaimans.
b. Immuno-fluorescence assay – Detection of viruses in culture.
c. EIA (Enzyme Immunoassay).
(iii) Genetic Probe Hybridization:
Unique nucleotide sequences within the micro-organisms (DNA or RNA) are detected using nucleic acid probes. This approach is of prime importance in diagnostic microbiology.
The direct examination method for the diagnosis of infectious diseases can be performed in routine clinical lab and it takes around 1-2 hour. It is a Rapid diagnostic method but has very poor sensitivity.
2. Culture of Micro-Organisms:
Culture of micro-organism is the most specific method to establish the presence of a particular pathogen in a test sample.
Micro-organisms can be cultured in:
(i) Artificial Media:
Mycoplasma, bacteria, mycobacteria and fungi can be cultured in –
Liquid artificial media which has greater sensitivity for micro-organisms isolation but it cannot be used for mixed infections diagnosis.
b. Soft Agar Media:
Isolated colonies are identified on the basis of the colony morphology. Bacteria are quantified by the colony forming units (CFU) on an agar plate.
c. Selective Media:
Enhance the growth of specific micro-organisms while retarding growth of other due to addition of antimicrobial agents.
d. Differential Media:
Developed by incorporating one or more carbohydrates along with a suitable pH indicator in media.
e. Enriched Media:
Allows metabolically fastidious organisms to grow and enhance the growth of specific pathogens such as Neisseria gonorrhea.
(ii) Living Cells:
Eukaryotic cell cultures employees –
(iii) Living Hosts:
Embryonated eggs is the best culture media for influenza virus proliferation.
(iv) Can be Identified Phenotypically:
After culturing of micro-organisms them (bacteria) can be identified by phenotypic characters like colony and microscopic morphology, biochemical reactions, growth features, antigenic characteristics, etc.
Culturing of micro-organisms can be performed in clinical and research laboratories within 2-14 days. The advantage of this method is that it can be used for phenotypic drug susceptibility testing but the drawback is that it is time consuming; all micro-organisms cannot be cultured and has poor sensitivity.
3. Serologic Tests:
A number of techniques are exploited in the serologic tests.
A test sample containing a suspected pathogen is reacted with a specific fluorescent antibody and then observed under a fluorescent Microscope. If pathogen contains surface antigens reactive with antibody, the cell will fluoresce.
(ii) ELISA (Enzyme Linked Immunosorbent Assay):
It is used to detect antigens such as virus particles from a blood or fecal sample, antigenic pathogen components or antigenic metabolites in blood, urine and other body fluids.
(iii) Immunoblotting (Western Blotting):
It is one of the most specific serologic methods. It is the main step for confirming infections with Borrelia burgdorferi, HIV after positive ELISA results.
For example, quantitative analysis of C-reactive protein (CRP) or rapid plasma regain (RPR) in the test sample.
Serologic tests are performed in larger sophisticated clinical laboratories and takes 4-6 hours.
4. Molecular Diagnostics:
Over the past several years, the development and application of molecular diagnostic techniques has initiated a revolution in the diagnosis of infectious diseases. This approach replaces biological amplification by enzymatic amplification. Molecular diagnostic enhances the sensitivity, speed and specificity of an etiologic diagnosis.
(i) Target Amplification Systems:
In Target amplification systems, the copy numbers of target nucleic acids are increased via PCR, transcriptional based technologies or strand displacement techniques. Target amplification systems have very rapid kinetics and there is no requirement for a thermocycler.
PCR (Reverse Transcriptase-PCR, nested PCR, multiplex PCR, arbitrary primed PCR, broad range PCR).
b. Transcription Mediated Amplification (TMA):
TMA has been used to detect M.tuberculosis.
c. Nucleic Acid Sequence Based Amplification (NASBA):
NASBA is used to detect and quantify HIV-I infection.
(ii) Probe Amplification Systems:
In Probe amplification systems, the probes hybridize with the amplified target nucleic acid sequences and then target-specific hybridized product is detected.
Ligation Chain Reaction (LCR):
Successful ligation relies on the contiguous positioning and correct base-pairing of the 3′ and 5′ ends of oligonucleotide probes on a target DNA molecule. In this process, oligonucleotide probes are annealed to template molecules in a head-to tail fashion, with the 3′ end of one probe abutting the 5′ end of the second DNA ligase, then joins the adjacent 3′ and 5′ ends to form a duplicate of one strand of the target.
A second primer set, complementary to the first, then uses this duplicated strand (as well as the original target) as a template for ligation. Repeating the process results in a logarithmic accumulation of ligation products, which can be detected by means of the functional groups attached to the oligonucleotides.
(iii) Signal Amplification Systems:
In Signal amplification systems, the concentration of the label attached to the target nucleic acid is increased to increase the signal strength. The Digene hybrid capture system is used to detect human papillomavirus infection.
a. The branched DNA probe is used for the detection of HIV and HCV. Signal amplification systems are of three types.
b. Soluble Enzyme – Antienzyme complexes
c. Coupled Enzyme Cascade systems
d. Avidin-Biotin systems
Molecular diagnostics methods are performed in only sophisticated research and clinical laboratories and takes around 1-2 days. Molecular diagnostics methods have high sensitivity and specificity but can give false or negative results.
The development of rapid, sensitive and specific diagnostic methods for pathogen detection and identification, in both clinical and environmental samples include:
a. Organism cultivation and subsequent
b. Characterization (classical microbiology)
c. Genetic pathogen identification (e.g., real-time PCR)
d. Detection of host immune response by serological tests
e. Visual detection of pathogens in the host by:
ii. Immuno-enzymatic methods
iii. Lateral immuno-chromatography
iv. Immuno-electron microscopy analysis
v. Polypeptide profile analysis (Western-blots, MALDI-TOF/MS)
vi. In-vitro/in-vivo infectivity evaluation.