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SERO-IMMUNOLOGICAL REACTIONS
AND BASIC PRINCIPLE
BACTERIAL AGGLUTININS
Agglutination reactions are of proven value in the serum diagnosis of many bacterial, parasitic, rickettsial, and viral diseases. A variety of febrile antigens are prepared specifically for the detection (or exclusion) of serum agglutinins developed during febrile infections. Such detection is accomplished by means of simple, quantitative, rapid slide agglutination tests as well as by means of test tube procedures. These febrile antigens include living or heat-killed bacterial suspensions, and are available commercially. No attempt should be made to prepare these antigens, since all the organisms are highly infectious.
Bacterial antigens exhibit one of two types of agglutination, which depend on the presence or absence of flagellar "H" antigens. When "H" antigen combines with anti-H antibody, flocculation occurs which is caused by entanglement of flagella. This type is easily broken up by vigorous shaking. On the other hand, when no flagella are present as with motile and non-motile bacteria, granular "O" agglutination occurs in the presence of antibodies. These fine, granular clumps are much more difficult to break up. Febrile antigens are used as laboratory sero-diagnostic tools and in such infectious diseases as brucellosis, typhoid and paratyphoid fever, tularemia, Rocky Mountain Spotted Fever, rickettsial infections (typhus fever) and other exotic diseases.
Important tests employed for the determination of above mentioned diseases are:
Widal Test - which is widely used and simple to perform yet is subject to many unreliable interpretation in the diagnosis of typhoid and paratyphoid diseases. There are more than 200 bacterial species that belong to the genus Salmonella that are capable of causing salmonellosis in man and animals. Thus, during infection, it is possible that serum agglutinins maybe developed that are not detectable by Widal Test alone. Approximately 98 % of the Salmonella considered is pathogenic for man and animals belong to five distinct groups: A, B, C (C1 and C2), D, and E (E1, E2, E3, and E4). All Salmonella possess at least two varieties of antigenic structures by which they maybe serologically identified. The "somatic" or "O" antigens are those directly associated with bacterial cell body while the "flagellar" or "H" antigens are mainly associated with the flagella. Flagellar antigens are further subdivided to monophasic (Phase I) and diphasic (Phase II).
Vi antigens are surface antigens peculiar to Salmonella typhi and few other strains. Vi antibodies appear during typhoid fever but tend to disappear soon after recovery. These antibodies are commonly found in carriers.
Widal Test can be done by several method, which include the microscopic tube test and the rapid microscopic slide test. The following factors influence the interpretation of results:
- Cross reactions
- Anamnestic responses that are encountered occasionally. The phenomenon in which a rise in agglutinins to S. typhi may occur in person who has a previous infection or vaccination but whose present illness is unrelated to typhoid.
- Zone reactions
- The stage of the disease is used to determine the significance of elevated titers. Typhoid bacilli can be isolated from the blood, stool and urine before Widal Test show a positive result.
- Antibiotic therapy may depress the titer.
- In vaccinated persons, the "H" and "O" titers are elevated.
The aggltuination test for brucellosis - human brucellosis simulates many other diseases, and clinical diagnosis based on symptoms alone is difficult. For this reason laboratory confirmation is required. Isolation of the organism is the only absolute method but this is out since this is sometime difficult especially if the number of organism is low and if antibodies have been administered. Serologic tests are the ones most preferred - skin test with brucellergen, opsonic index and the most popular is the agglutination test provided with standardized antigens. Human brucellosis is a febrile disease caused by one of closely related species of the genus Brucella (B. abortus, B. melitensis, B. suis). Brucella infections are primarily associated with livestock, such as swine, cattle and goats. Human contract the disease either by direct contact with injected animals or in an indirect manner as a result of consuming milk and its product from infective animals.
In active cases of brucellosis, specific agglutinins usually begin to develop late in the second week of illness, reach significant titers during third and fourth weeks. A cross-agglutination exists between Brucella abortus and Pasteurella tularensis. Brucella agglutinins have been demonstrated in serums of individuals following inoculations of Cholera vaccine. In some instances titers were as high as 1:320. Certain people demonstrate significant titer for as long as one year. The prozone phenomenon is also occasionally observed and has been attributed to a "blocking" property in fresh serum. This inhibiting property is readily remedied by inactivating the serum for 15 minutes at 56 C incubation. Due to variety of clinical symptoms which are common to unrelated diseases (TB, lymphoblastoma and carcinoma), cases of Brucella abortus infections have occurred in which no specific antibodies were determined.
The Weil-Felix Test - this is a serodiagnostic test for typhus fever in which Proteus antigens are used. There are 3 serologic varieties of the bacillus Proteus X which possess antigenic components in common with certain rickettsiae. These species of Proteus X are Proteus OX19, Proteus OXK, and Proteus OX2. A strain of Proteus was first isolated from urine in 1916 by Weil and Felix. The original strain was a non-motile or "O" strain designated as X19, hence, the name. Later two additional strains were found - OX2 and OXK (Kingsbury).
Rickettsial infections that stimulates the formation of antibodies are often referred to as the "Typhus group of fevers." Within this group, Felix has made "provisional" immunologic subgroups based largely upon the antigenic relationships between Proteus X and other rickettsiae. Although agglutinating antigen made from Proteus OX19 is widely used for detecting typhus fever, it is not possible to use it for distinguishing infections caused by R. prowazeki and R. mooseri. Complement-fixation tests, using suitable rickettsial antigen will distinguish the two. It is common procedure in some laboratories to check any serum that reacts in Weil-Felix with complement-fixation test. The factors observed in Widal Test are also applicable in this kind of test.
Newer methods for preparing febrile antigens are being developed in which instead of utilizing killed bacterial suspensions, bacterial extracts absorbed to cholesterol, bentonite, latex and red cells are used and may supersede the classical method just discussed.
GROUP DISEASE ANTIGENS
Typhoid...............................................Typhoid fever...................................................S. typhi, "H" and "O"
Paratyphoid A..................................................S. paratyphoid A, "H" and "O"
Paratyphoid B...................................................S. paratyphoid B, "H" and "O"
Brucella - Tularemia..............................Brucellosis........................................................B. abortus
Tularemia.........................................................P. tularensis
Rickettsial diseases................................Murine and Typhus............................................Proteus vulgaris, OX2
Rocky Mountain Spotted Fever............................Proteus vulgaris, OX19
Tsutsugamushi fever..........................................Proteus vulgaris, OXK
NEXT REACTION: ANTISTREPTOLYSIN O TITER (ASO)
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