Febrile Agglutinins

Febrile Agglutinins Agglutinating antibodies that arise from infections with some microorganisms that induce fever Among bacteria that induce febrile agglutinins are: Rickettsia (typhus ) Brucella (Brucellosis) Salmonella (typhoid fever)

Typhus

Typhus Any of several similar diseases caused by louse-borne bacteria. The causative organism Rickettsia is an obligate parasite and cannot live long outside living cells. Rickettsia is endemic in rodent hosts, including mice and rats, and spreads to humans through mites, fleas and body lice.

Rickettsial Diseases Rickettsias as a group have a worldwide distribution Rickettsias are associated with variety of different vectors and hosts Most types of rickettsiosis are geographic area-specific

Rickettsial Diseases The rickettsiae that are pathogens of humans are subdivided into three major groups based on clinical characteristics of disease: spotted fever group (R. rickettsii ) typhus group (Rickettsia prowazekii ) and scrub typhus group

Rickettsial Diseases Rickettsial diseases are clinically non-specific with many overlapping signs and symptoms Rickettsiosis in humans may be: Subclinical Mild, self-limited Severe, life-threatening Confirmation of rickettsiosis cases requires consideration of clinical, and epidemiological, and laboratory data

Weil-Felix Agglutination Test Developed in 1916 Confirm diagnosis for rickettsial infection Nonspecific rickettsial test based on cross-reacting antibodies Antibodies are produced that cross-react with polysaccharide “O” antigens of some Proteus species P. vulgaris, OX-19 strain P. vulgaris, OX-2 strain P. mirabilis, OX-K strain By the Weil-Felix test, agglutinating antibodies are detectable after 5 to 10 days following the onset of symptoms

Weil-Felix Agglutination Test There are two protocols for the Weil-Felix agglutination reaction: Rapid slide test Tube method (confirmation method)

Routine Laboratory Tests Neutropenia in the acute phase Thrombocytopenia Hypoproteinemia, hypoalbuminemia Elevated ALT, AST, and alkaline phosphatase CPK and LDH often elevated in acute infection

Brucellosis

Brucella spp. Gram negative, coccobacilli bacteria Facultative, intracellular organism Environmental persistence Temp, pH, humidity Frozen and aborted materials (fetuses or placentas) Multiple species Three species (B melitensis, B abortus, B suis) are important human pathogens; B canis is of lesser importance This organism is an aerobic, small, gram-negative coccobacilli that can persist in the environment invariably depending on temperature, pH, and humidity. Brucella spp. can persist indefinitely if frozen or protected in aborted fetuses or placentas. It is a facultative, intracellular pathogen and thus requires prolonged treatment with clinically effective antibiotics.

Transmission to Humans Conjunctiva or broken skin contacting infected tissues Blood, urine, vaginal discharges, aborted fetuses, placentas Ingestion Raw milk & unpasteurized dairy products Rarely through undercooked meat Inhalation of infectious aerosols Pens, stables, slaughter houses Person-to-person transmission is very rare Incubation varies 7-21 days to several months Brucella abortus and B. suis infections often are the result of occupational exposure through contact with infected tissues, blood, urine, vaginal discharge, aborted fetuses, and placentas. Typically, absorption occurs when the bacteria are exposed to areas of abraded or broken skin or the conjunctival sac of the eye. People are often infected with B. suis during the slaughter of swine or assisting in farrowing of infected sows. Brucella melitensis infection is primarily foodborne and results from consuming infected unpasteurized milk or dairy products.

Inhalation of infectious aerosols can also occur while cleaning out an infected animals pen or in a slaughter house. The current vaccines available are strain 19 and RB-51 for B. abortus immunization and Rev-1 for B. melitensis. Self inoculation could occur by a needle stick of a vaccine when handling animals, or when infected tissue or body fluids splash into the conjunctiva of the eye. Conjunctival splashes are more likely to get a larger dose than an injection of a vaccine. Either route of exposure requires antibiotic therapy at treatment levels to prevent disease onset. Person to person transmission is very rare but has been reported as a result of a blood transfusion, bone marrow transplants, breast feeding and sexual contact between lab workers and their spouse. Incubation period in humans is 7 to 21 days up to several months. This often adds to the difficulty in diagnosis due to the latency of clinical signs.

Diagnosis in Humans Isolation of organism Serum agglutination test Blood, bone marrow, other tissues Serum agglutination test Fourfold or greater rise in titer Samples 2 weeks apart Immunofluorescence of organism in clinical specimen PCR Isolation of the brucella organism from blood, bone marrow or other tissues. Brucella is slow growing so cultures must be kept up to 8 weeks. Antibodies appear within 7 to 14 days after infection and identification of B. abortus, B. melitensis, and B. suis is achieved through the serum agglutination test. A four fold increase or greater is considered diagnostic and requires samples taken 2 weeks apart. B. canis requires a specific test as it does not have a smooth lipopolysaccharide cell wall. PCR has begun to gain popularity in the diagnosis of brucellosis due to the high specificity and sensitivity of the test and the quick turn around of results.

Diagnosis Depends on the presence of clinical features and +ve blood or tissue culture and/or detection of raised brucella agglutinins in the blood. Culture: +ve in about 50 -70% of cases. Standard agglutination Test: a titre of 1/160 in non endemic areas & 1/320 in endemic areas are significant.

Typhoid Fever

Definition Causative organism: Typhoid bacillus Typhoid fever is an acute infectious disease of digestive tract caused by typhoid bacillus Causative organism: Typhoid bacillus genus salmonella group D Pathogenicity: endotoxin Resistance: Stable in environment, sensitive to heat, acid, common disinfectants

Salmonella Salmonella  is a Gram-negative facultative rod-shaped bacterium Salmonella are the cause of two diseases called salmonellosis: enteric fever (typhoid), resulting from bacterial invasion of the bloodstream, and acute gastroenteritis, resulting from a food borne infection/intoxication

Antigenicity Salmonellae possess three major antigens: H or flagellar antigen (heat-labile proteins ); O or somatic antigen (heat stable ); and Vi antigen (possessed by only a few serotypes).

Exercise 5 Widal Test

Widal Test To detect the febrile agglutinins to Salmonella species The “O” and “H” antigens are affixed to particles The antigens are adsorbed to differently colored latex particles

Widal test Slide Agglutination Tube agglutination WIDAL test can be performed by two methods Slide Agglutination Tube agglutination Tube agglutination is more accurate in comparison to slide agglutination you can go upto 1:1280 titre but in case of slide you are restricted to 1:320 titre only but it is more rapid result comes within 5 min

Widal test – Slide Method Add the following volumes of serum to a slide 80 µl, 40 µl , 20 µl , 10 µl , and 5 µl Mix the reagent containing the Ags well and add one drop to each drop of serum Mix well Rotate for one minute and then read the result

Widal test – Tube Method Make a series of serum dilutions for each antigen to be tested. Include tubes with 0.5 ml saline for control of each antigen to be used. Use perfectly clean and dry test tubes and prepare dilutions beginning with 1:10 and doubling through 1:320 or so. Add 0.1 ml of serum to 0.9 ml of physiological saline and then dilute serially by mixing 0.5 ml diluted serum with 0.5 ml saline and discarding 0.5 ml from the last tube. From specimen submitted to detect possible rise in titre, prepare a series of 10 dilutions, ending with 1:5120.

Widal test – Tube Method Preparation of antigen Use the killed standardized suspension of S.typhi O, S.typhi H, S.paratyphi AH and S.paratyphi AO. These are commercially available and their preparation should be undertaken as per the instructions of the manufacturers Procedure of test Shake the antigen suspension to ensure even distribution. Add 0.5 ml to each serum dilution and to saline for controls. Incubate the test overnight at 37oC and let tubes stand at room temperature for 15-20 minutes before reading.

Reading the result     In all cases, look first at the control tubes and proceed only if they show satisfactory results. There should be no appreciable sedimentation of the bacteria. Although a fine button may have been deposited at the bottom of the tube, no discernible clearing should have occurred. The antigen suspension should be evenly distributed as a rule. Pick up the individual tubes of each row of the patient’s specimens and look first at the supernatant. When a complete agglutination occurs, practically all the bacteria are removed from the supernatant which appears absolutely clear. When the reaction is negative the suspension should look as turbid as the antigen control is. The in-between reactions can be categorized into +, ++, +++.

Reading the result     Examine the tubes with a hand lens against a dark background End point carpet formation Granular agglutination Floccular agglutination Also look at the supernatant fluid above the aggultinate

Carpet formation

Larger flocules

Widal /reporting Highest dilution showing agglutination If no agglutination report as Salmonella typhi O titer < 1 in 20 Salmonella tyhphi H titer < 1in 20

Widal interpretation A 2-4 four fold rise in titer from the serum tested during the first week and the 2-4 week very significant High titer > 1:160 against O antigen suggest infection High titer > 1:160 against H antigen (develop later in infection but persist for long periods) suggest past infection

Limitations Titers against O and H antigens may be raised in the following diseases/conditions Other salmonellosis Chronic liver disease Immunological disorders rheumatic fever Nephrotic syndrome Ulcerative colitis