GENERAL PRINCIPLES OF THE DIAGNOSIS OF INFECTIOUS

DISEASES

Etiologic diagnosis of infection begins with the physical

examination of the patient, formulation of the clinical

diagnosis and in many cases estimation of the

epidemiological circumstances.

Anatomic localization of the infection and the clinical

diagnosis suggest variety of possible etiologic agents.

After accurate interpretation of the clinical and

epidemiological information, the clinician must select the

appropriate specimen, the laboratory tests, and to ensure its

adequate transport to the laboratory in order to preserve the

viability of the microorganisms.

The laboratory diagnosis varies with different pathogens and specimens.

Numbers of methods are used, usually combined as following:

1. Microscopic examination and cultivation of the specimen in nutrient media for isolation of the pathogen in pure culture, followed by identification of the isolated strain by biochemical, immunological or molecular methods and antimicrobial susceptibility testing.

2. Detection of specific antigens of the pathogen in the specimen by immunological methods and

3. Detection of specific antibodies in the sera, body fluids and excreta or detection of cell-mediated immune response to the pathogen by immunological methods.

4. Detection of nucleic acids of the pathogen in the specimen by molecular methods.

Specimen - General Considerations A properly collected specimen is the first important step in the

diagnosis of an infection. The results of diagnostic tests depend on the selection, timing, stage of the patients illness, and method of collection of specimen.

Bacteria can be found in different anatomic sites and in different body fluids and tissues during the course of infectious diseases.

Only sterile equipment and aseptic precaution can be used to avoid contamination of the samples with normal skin flora or saprophytes (e.g. treatment of the injection site with alcohol and iodine solutions).

• Specimens must be taken before or at least three days after

antimicrobial drug therapy.

• The best specimens are infected fluids, excreta and tissue.

Swab should be used only when these specimens are not

available and processed as soon as possible or placed in

transport medium to preserve the viability of the pathogens.

• The specimen must be representative of the infection

localization.

• The specimen must be taken to the laboratory promptly.

• Transport media should be used to maintain the viability of

the anaerobes or microaerophiles or in the case of prolonged

transport.

A properly collected specimen is very important step in the diagnosis of an infection.

The results of diagnostic tests depend upon the selection of specimen, methods of collection, aseptic technique, timing, and transport.

The specimens are collected from different anatomic sites with or without normal bacterial flora.

Methods for identification of infectious agents in clinical specimens

• 1. Microscopic Examination • 2. Cultivation of microorganisms• 3. Innoculation in experimental animals• 3. Serological methods• 4. Molecular-biological methods

Microscopic Examination

• Microscopic identification or so-called direct examination of stained or unstained specimens is a simple and inexpensive method, but much less sensitive than culture for the detection of small number of bacteria.

• Microscopes (with the exception of electron microscope) rely on a light source for their magnification systems.

• The cell shape, cell size, cell arrangement and staining characteristics of microorganisms can be seen clearly with the light microscope when appropriate methods are used.

• Bacteria can be examined in the specimen or in the pure culture, also unstained (in the living state or stained).

• A specimen must contain at least 105 microorganisms in order to see them on the smear under the microscope, using 90-100 X oil immersion objective.

clue cells

•Bright-field microscope• Uses visible light to illuminate the smear. •It is convenient, inexpensive method, suitable for observation of most of bacteria and fungi, usually stained by different methods.

Gram Stain Anthrax

Examination of Stained Preparation

• Gram stain is the single most useful procedure in diagnostic microbiology.

• The etiologic bacteria often are seen in direct Gram smears of specimens in many bacterial infections.

• Except the shape and size of bacteria Gram stain indicates purple-blue microorganisms as Gram-positive and red microorganisms as Gram-negative.

• Gram stain positivity and negativity correspond to the structural differences of the bacterial cell wall.

Examination of Stained Preparation

• In many cases bacteria are seen on Gram stained smears: Gram-positive cocci in pairs, in clusters, in chains or single; Gram-negative rods, etc. This preliminary information, combined with the clinical findings, help for choosing further laboratory diagnostic methods and for antibacterial treatment of the patient before culture results are available.

• For example, the presence of Gram–negative diplococci in the cerebrospinal fluid is highly suggestive of meningitis caused by N. meningitidis. The same morphological type bacteria seen in urethral pus are suggestive of N. gonorrhoeae.

• Specimens that are not suitable for routine Gram staining are throat or stool samples because the pathogen usually cannot be distinguished from the organisms of the normal flora.

• Certain bacteria (e.g. mycobacteria, nocardia, spirochetes and Legionella) stain only feebly or not at all with the Gram staining technique requiring special staining techniques.

• The results from Gram stain are suggestive and

does not permit identification of the species.

• The information of Gram stain combined with the

clinical findings may be useful for the clinicians

before culture results are available.

Dark-field microscope illuminates the object over a dark background and diffracted rays from the specimen make the object visible. Dark-field microscopy is used for observation of unstained, live organisms suspended in liquid, which are too thin to be seen by staining (T. pallidum, V. cholerae, Leptospira etc.).

Leptospira spp. in dark-field

microscopy

Spores in cells of B. anthracis

•Phase-contrast microscope uses brightness differences (light and shade) between thinner and thicker parts of the specimen, since each part of cell has a different composition and reflects light differently.•Unstained, live organisms suspended in liquid may be examined by this method.

• Fluorescent microscopy• It is rapid and good technique to detect pathogenic microorganisms (Treponema

pallidum, Rabies virus, gonococci in vaginal or urethral smear, etc.). • The method can detect bacteria and viruses within the cells, tissues and directly in

the clinical specimens.

Inclusion bodies of C. trachomatis in tissue culture

EM of influenza virus

•Electron microscope uses a beam of electrons instead of beam of light, which permits much greater resolution and thus much higher magnification compared with light microscopy (smallpox, poliomyelitis, Ebola, HFV).

Culture Method

• The ability of microorganism to grow in nutrient media permits in large degree its characterization and identification.

• Bacterial cultivation includes the isolation of bacterial pathogens form patient specimens in pure culture and subsequently identification of the isolated strain by a combination of different methods (e. g., growth characteristics on media, microscopy, biochemical tests, specific antiserum testing, etc.).

Devices for cultivation of microorganisms from clinical specimens

Thermostat for aerobic cultivation

СО2 thermostat for cultivation of microaerophils and tissue cultures

Jars for anaerobic and microaerophil atmosphere

Ламинарен бокс Biohazard за стерилна работа с тъканни култури и опасни микроорганизми

Identification. Pure Culture and Cultural Characteristics

• Identification• Identification of the bacterial species is essential step in the

laboratory diagnosis. Traditionally it relies on the determination of phenotypic characteristics that are observed:

• 1. By microscope examination, using Gram stain of smears from the specimen and the positive broth and other microscope methods; 2. Examination of the culture characteristics of the growing organisms on agar and liquid media under various culture conditions and

• 3. Determination of the metabolic characteristics. Identification of the antigen structure (the serotype), bacteriophage and bacteriocin typing, resistotyping and nowadays the molecular nucleic acid methods are useful for identification at subspecies level and for epidemiological testing.

• The pattern of growth and metabolic characteristics distinguish one microbial species from another, forming the phenotypic basis for identification.

• Classical Determination of Enzymatic and Metabolite Production

• Microorganisms produce different enzymes and metabolic products.

• Its pattern is unique for the species and that permit identification of the bacterial and fungal species.

• The biochemical characteristics and metabolite production of pure cultures have the most widely application as a classical diagnostic tool in the laboratories.

Biochemical Characteristics

Methods for identification of the bacterial species Biochemical identification - Determination of the metabolic characteristics of

different bacteriaPannels for biochemical identification

Instrumentation, Automation and Computer Analysis

• In recent years, new diagnostic systems have been developed.

Instrumentation, automation, and computer analysis has been

applied for identification of bacteria.

• Bacterial families such as Enterobacteriaceae or others have a

great diversity of biochemical activity and their identification

is often difficult and time-consuming procedure.

Instrumentation, Automation and Computer Analysis

• The new diagnostic identification kits and systems are based on biochemical characteristics of cultures, but usually use micromethods for the biochemical identification of bacteria. They are based on commercial identification kits of miniaturized reaction cuvettes with biochemical tests.

• The color reactions of the isolate with the substrates in the tests of the identification kit are red visually or by automated systems. Even using the most reach database, the final diagnosis may need additional testing of biochemical and cultural characteristics of the isolate, which is due to the phenotypic variations of bacterial species.

Automated kits and systems for diagnostic identification of microorganisms

Mini API Phoenix BD

Immunological Methods for Identification and Detection of Microorganisms’ Antigens in the Pure

Culture or in the Specimen

• Bacteria possess many highly specific structures that may be antigenic (capsular polysaccharides, flagellar proteins, exotoxins, and several cell wall components. These antigens, which present on whole bacteria or are at free state as bacterial exotoxins may be demonstrated with specific antigen-antibody reactions in vitro.

• Using the antibodies of known specificity bacterial identification can be made by formation of antigen-antibody complex.

• Polyclonal serum- and monoclonal antibodies of known specificity are used for detection of microorganisms’ antigens in isolated pathogens and directly in the specimens.

• Several immunological methods are used in practice for detecting bacterial antigens in the pure culture, usually for serotyping of the organisms or in the specimens as a direct diagnostic test when urgent diagnosis is needed or the culture method is not available.

Immunological Methods for Identification andDetection of Microorganisms’ Antigens (determination of species, serogroup,

serotype) are:Precipitation Agglutination Immunofluorescention ЕLISA

Slide agglutination for determination of salmonella serogroup

Precipitation Tests

• Precipitation is an antigen-antibody reaction, in which thousands of molecules of soluble antigen and IgG, IgM or IgA antibodies cross-link to form visible aggregates known as precipitates.

• e.g., - streptococcal group antigens from Lancefield classification,

diphtheria toxin

• Agglutination Tests

• Agglutination is an antigen-antibody reaction when particulate antigen is combined with specific and IgG, IgM or IgA antibody cross-link to form visible aggregates known as agglutinates.

• E.g. Salmonella strain, Influenza virus.

Enzyme-Linked Immunosorbent Assay (ELISA)

• The enzyme-linked immunosorbent assay (ELISA) or so-called solid-phase immunoassay is one of the most widely used immunological techniques for detection of antigens or antibodies.

• When the antigen will be detected, the ELISA test uses specific antibodies (capture antibodies) adsorbed to a solid phase (for example, the surface of the wells of the polystyrene or polyvinyl chloride plates).

Enzyme-Linked Immunosorbent Assay (ELISA)

• The patient sample (it may be serum, urine, cerebrospinal fluid, extract from more solid specimen, etc), containing the microbial antigen is added. After the antigen bound by the capture antibody, a second enzyme-labeled antibody of the same antigen specificity is added to the reaction mixture and complex type “sandwich” is formed. Finally, chromogenic enzyme substrate is added and visible color end product is registered, if the antigen exists in the specimen.

Direct Immunofluorescence Test for Antigen Detection (inclusion bodies of Chlamidia, isolated in tissue culture)

Direct Immunofluorescence Test for Antigen Detection

• Direct immunofluorescence is used commonly to detect microbial antigens of organisms, which cultivation is difficult or impossible, in a clinical specimen. Different commercially indirect fluorescent antibody test kits are available.

• Bacteria can be identified in different specimens: Legionella pneumophila or Bordetella pertussis in respiratory tract specimens, Leptospira in fresh blood, CSF or urine, Chlamydia trachomatis or Mycoplasma pneumoniae in genital or conjunctival specimens, Treponema pallidum in tissue fluid from early surface lesions, Rickettsia or Borrelia burgdorferi in ticks or tissue sections, etc.

Enzyme-Linked Immunosorbent Assay (ELISA)

• The enzyme-linked immunosorbent assay (ELISA) or so-called solid-phase immunoassay is one of the most widely used immunological techniques for detection of antigens or antibodies.

• When the antigen will be detected, the ELISA test uses specific antibodies (capture antibodies) adsorbed to a solid phase (for example, the surface of the wells of the polystyrene or polyvinyl chloride plates).

Enzyme-Linked Immunosorbent Assay (ELISA)

• The patient sample (it may be serum, urine, cerebrospinal fluid, extract from more solid specimen, etc), containing the microbial antigen is added. After the antigen bound by the capture antibody, a second enzyme-labeled antibody of the same antigen specificity is added to the reaction mixture and complex type “sandwich” is formed. Finally, chromogenic enzyme substrate is added and visible color end product is registered, if the antigen exists in the specimen.

ELISA for antigen detection:

• ELISA can be used for detection of different microbial

antigens. Detection of Helicobacter pylori – antigen in

stool specimens (test is performed with poly- or

monoclonal antibodies) is appropriate as a diagnostic

test and for control of eradication of H. pylori infection in

patients after antibiotic treatment. Chlamydia antigens in

cervix specimens, streptococcal antigens in extracted

pharyngeal swabs and many virus antigens in different

specimens can be determined. ELISA is very important

for diagnosis of the viral infections.

ELISA for detection of isolated microorganisms or in clinical specimens

Methods for antigen detection in clinical species

ELISA for rotavirus Ag in feces LA for Ag – rotav. in feces, bacteria in CSF

IFT – Ag (Clamidia in specimen from cervix or urethra)

Radioimmunoassay (RIA)

• Radioimmunoassay (RIA) is the same solid-phase

immunoassay diagnostic procedure as ELISA, but RIA

uses radioactive-labeled anti-immunoglobulins instead of

enzyme-labeled anti-immunoglobulins. The RIA is often

more sensitive than an ELISA, but because of problems

associated with radioactive waste, the cost of radiation

measuring instruments, and requirement of special

working areas in the laboratory, the ELISA is more

frequently used diagnostic test.

Хроматографски тестове за доказване антигените на причинителяв клиничния материал (Actim Influenza A&B: A – H1N1, H3N2 and the avian subtypes

H5N3, H7N3, H9N2 and H5N1 and B virus antigen from nasal swab and nasopharyngeal aspirates in 10 min)(също за хламидии, грипни вируси и т.н.)

Step 1: Addition of Step 2: Dipping Step 3: Results  extraction buffer

Antimicrobial Susceptibility Testing

On the basis of clinical impression, the physician selects a probable drug of choice. Before the drug is administered, specimens are obtained for culture and antimicrobial susceptibility testing of the isolated pathogenic strain. It allows the choice of the best antibiotic for the treatment.

Susceptibility testing of the bacterial culture is currently the wide used practical approach for determining antibiotic susceptibilities. They are:- Diffusion Methods

• The results from disc-diffusion methods must be interpreted with caution, because the method does not determine whether a drug is bactericidal or bacteriostatic.

• • - Broth Dilution Methods• The broth dilution test permits determination of minimal

inhibitory concentration (MIC) and minimal bactericidal concentration (MBC).

Determination of antibiotic susceptibility of bacteria

Disc-diffusion method

Е-тест

Determination of the minimal inhibitory concentration (МIC)Broth Dilution Method

Antibodies Detection

• Detection of serum antibodies specific for the antigen suggests that patient is currently or has been previously exposed to the pathogen.

• This diagnostic tool has three limitations: 1. Early in the infection, the antibody titers may be undetectable; 2. The presence of antibodies, which are likely to persist for long periods of time even after infection has been eliminated after successful antibiotic therapy, and 3. Risk exists cross-reactive antibodies to be found.

• So, the laboratory diagnosis on the basis of serological tests is presumptive and does not necessarily prove that an individual is actively infected. For this reason, the results of laboratory tests play a supportive role and diagnosis of infectious disease must be based also on the characteristic clinical findings.

• False positive results, caused by cross-reacting antibodies, represent a major problem for the final and reliable diagnosis. Optimal tests should be highly sensitive and highly specific.

Antibodies Detection

• Diagnoses based on antibody titer are especially useful for some bacterial infections. The antibodies can be detected mainly in serum, but also in other fluids and excretes as CSF, urine, joint fluid, feces, etc.

• During the acute infection, there is usually a rise in the antibodies. IgM isotype is produced first, followed by IgG, IgA or both.

• Antibody titer can be measured as an indicator of disease and convalescent sera obtained 4 weeks apart should be tested. A positive result is usually a fourfold change titer. In some cases a single titer greater than cut-off value may be a positive result.

• High level of specific IgM may be indicative for early acute infection or for intrauterine infection in neonates.

Immunological methods for identification of specific antibodies in sera

Indirect Immunofluorescent Test

• Indirect immunofluorescent tests are used usually to identify specific antibodies in the serum. In this test the known bacterium is fixed onto a slide and patient serum is added. After that the slide is incubated, washed to remove antibodies not bound to antigen, second antibody (fluorochrome labeled anti-human immunoglobulin) is added. Second antibody may be of class IgM, IgG or IgA. This permits determination the isotype of the specific antibodies. It is important for determination the stage of an infection, because IgM is produced early in the course of an infection, while IgG and IgA response comes later, as well as to exclude binding of cross-reactive antibodies.

• Different commercially indirect fluorescent antibody test kits are available for diagnosis of Ehrlichia, Rickettsia, Mycoplasma, Chlamydia, Borrelia, and Treponema pallidum infections.

• The presence of IgM anti-Treponema pallidum antibodies in the blood of newborns may prove in utero infection (congenital syphilis).

ELISA for detection of serum antibodies

Complement-Fixation Test

• Complement fixation test (CF) is the classic test for serologic diagnosis of infectious disease. It can be used for detection of very small amounts of antibody to soluble antigens.

• The complement fixation test is performed in two stages: complement fixation and adding of indicator system.

• Wassermann test is a classic complement fixation test for detection of antibodies against T. pallidum which use cardiolipin antigen, instead of specific treponemal antigen.

Western blot for detection of serum antibodies – confirmation test for HIV, LB etc.

Western blot in LB

Western blot in HIV

Molecular Methods for Detection and Identification of Microorganisms

• Molecular methods can be used for detection and identification of microorganisms that were difficult or impossible to detect, using conventional diagnostic methods. Usually, these organisms are slow-growing (M. tuberculosis) and very fastidious or non-cultivable bacteria (Neisseria gonorrhoeae, Chlamydia trachomatis, Borrelia burgdorferi, Helicobacter pylori, etc) and viruses.

• Now, commercially diagnostic kits using molecular methods are available for identification of some bacteria: Mycobacterium tuberculosis, Neisseria gonorrhoeae, or Chlamydia trachomatis and for identification of some viruses Cytomegalovirus, Enterovirus, Human immunodeficiency virus, Hepatitis C virus, Hepatitis B virus, Herpes simplex virus, Human papilloma virus.

• Molecular methods recently used in practice fall into two major categories:

• 1. nucleic acid probe-based systems and

• 2. nucleic acid amplification systems.

Nucleic Acid Amplification Systems

• Nucleic acid amplification systems assay permits amplification of extremely small amounts of target nucleic acid (bacterial or viral) in the specimen (several hundred DNA copies per milliliter), which can be easy detected after that with different methods.

• Target bacterial or viral DNA or RNA is amplified many times.

• The first amplification technique to be developed is polymerase chain reaction (PCR). Now it is the most widely used molecular diagnostic method, but other amplification methods also exist.

Nucleic Acid Amplification Systems

• PCR is based on the fact that raising the temperature to 90-94˚ C separates the two strands of the DNA chains and lowering the temperature reassociate complementary segments of the DNA. In this analyzes DNA extracted from the clinical specimen along with sequence-specific oligonucleotide primers, nucleotides (4 nucleotide triphosphates - ATP, GTP, CTP, TTP), thermostable DNA polymerase (Taq polymerase), are heated to 90-94˚C to denature and separate the two strands of the target DNA. After that, the temperature in the reaction is lowered to 45-60˚C, which allows annealing of the primers to the target DNA. Each primer is then extended by the thermostable DNA polymerase and the result is twofold amplification of target DNA. This cycle is repeated 30-40 times that yield billions amplification of the target DNA segments,

Amplification methods(PCR, LCR, RT-PCR, multiplex, nested and other variants of PCR)

• If the PCR is performed on RNA targets, it is called reverse

transcriptase PCR. In this case the enzyme reverse transcriptase is

used to transcribe the RNA into complementary DNA for

amplification. This method is used for identification of RNA viruses.

• After amplification the nucleic acid segment can be seen in an

electrophoretic gel, or detected by Southern blot analysis using

labeled DNA probes specific for the segment, or by Northern blot,

which separates and identifies RNA fragments.

• Commercial kits, based on Nucleic acid amplification, are available

for diagnostics of M. tuberculosis, Neisseria gonorrhoeae,

Chlamydia trachomatis, Human papilloma virus, Hepatitis B virus,

Hepatitis C virus, Human herpes viruses (Cytomegalovirus, Herpes

simplex virus), Human immunodeficiency virus.

• Molecular methods becoming increasingly important and they

can be used in addition to conventional antibiotic susceptibility

testing methods.

• Molecular methods promise to make molecular diagnostics an

everyday tool in clinical microbiology laboratories. While these

methods are powerful, it is unlikely that they will replace more

conventional diagnostic methods in clinical microbiology

laboratories. Organisms must be isolated in culture so that they are

available for additional studies.

• Both molecular and phenotypic characterization of organisms will

be necessary for taxonomic purposes, pathogenesis of infection, as

well as for studies of resistance mechanisms, new drug and vaccine

development.