Brucella

1. Brucella And other organisms cause febrile reaction

2. OX-19 test Culture & serology Widal test

3. 1st. Brucella • Gram negative small coccobacilli • Non-motile • Non-capsulated • Fastidious ; need special media with co₂ and anaerobic condition called Castañeda medium. • Cause brucellosis disease (undulating fever).

4. Species of Brucella : • Brucella melitensis • Brucella abortus • Brucella suis • Brucella canis Mode of transmission : • Direct contact with the body fluids of infected animals • Consumption of unpasteurized milk products (dairy products) of infected animals • Inhalation of aerosols from infected animals.

5. Diagnosis • Serology test • The objective of this test is to look for antibodies against Brucella, usually IgG is tested as IgM appear & disappear quickly. • The serum agglutination test is the simplest and most widely used testing method. CDC utilizes a test called the Brucella microagglutination test (BMAT), a modified version of the serum (tube) agglutination test (SAT), that can detect antibodies to Brucella species. This test done after 2 weeks (10 days) of fever.

6. 2. Blood Culture : “ the confirmatory test ” • Done in the first week of infection. • Brucella is isolated from a blood culture on Castañeda medium. • Prolonged incubation (up to 6 week) may be required as they are slow-growing, but by modern automated machines, the cultures often show +ve results within seven days. • On gram stain they appear as dense clumps as Gram-negative • coccobacilli and are rather difficult to see.

7. 3. Animal inoculation test. • More tests or follow-up testing may be needed to confirm or rule out brucellosis, as the following : • X-rays. • CT scan or magnetic resonance imaging (MRI). • CSF culture. • Echocardiography.

8. Test & Results • An agglutination test is done by mixing 50ul of sample with 1 drop of reagent. • A normal (negative) result shows no antibodies to Brucella. • Titer about 1:80 or more indicate Brucellosis(serial dilutions). • However, during the first few days to weeks of exposure to antigen, they may be very little antibody production & as brucellosis progresses, more antibodies will be present. • If you suspects brucellosis, you may need to repeat the test every 10 days or 2 weeks after the first test to notify this rise.

9. Brucella antibody ‘IgG’ have long lifespan (1-2) years. Remember • Prozone phenomena is the presence of high antibody titer that lead to Ag block and hence, false negative results are obtained. Dilution will resolve this problem. • This phenomena appears obviously in Brucella serology test.

10. Weil-Felix test OX-19 Test

11. Rickettsiae • Are diverse collection of obigatory intracellular organism. • Gram negative bacteria • Found in ticks, Lice, fleas, mites, chiggers & mammals. • They include the genera Rickettsiae, Ehrlichia, Orientiaand Coxiella. • Cause zoonotic diseases that may disseminate in the blood to many organs. • Reckettsial infection (Typhus Fever) generate heterophilic antibodies can agglutinate some strains of Proteus.

12. Laboratory diagnosis • Rickettsiosis are difficult to diagnose both clinically and in the laboratory. • Culture & isolation. Which require viable eukaryotic host cells, such as antibiotic-free cell cultures, embryonated eggs and susceptible animals as guinea pigs or mice. 2. Serologic test. • OX-19 test ( weil-felix test ). 3. Immunofluorescent antibody technique.

13. Complication of Typhus infection • Lymphocytosis • Leukopenia • Thrombocytopenia • Anemia • In some cases hemoglobin may increase as a result of hemoconcentration • Increase in kidney function tests. • In urine analysis; high RBCs cast and high protein may present

14. Weil Felix test • Also named: Proteus OX-19 test, Typhus test • Is a heterophile agglutination test used to diagnose typhus and certain other rickettsial diseases. • Weil-Felix test is based on cross-reactions which occur between antibodies produced in acute rickettsial infections with antigens of OX (OX  19, OX 2, and OXK) Strains of  non motile Proteus sp.

15. The basis of the test is the sharing of an Alkali stable carbohydrate antigen of rickettsia with certain strains of Proteus. • Serum from endemic typhus usually agglutinate OX-19 & OX-2. • Test is negative in rickettsialpox disease. • The test is done as tube agglutination test along with slide agglutination

16. Procedure 1st : Slide method • On a solid surface (glass slide, tile, card), a small amount (50–100 μL) of the patient’s serum is placed. • A single drop of the desired antigen is added, and the resulting suspension is mixed and then rotated for one minute. • Visible agglutination is indicative of a positive result, and corresponds roughly to a titre of 1:20. • Positive results can be further titrated using the tube method.

17. 2nd : Tube agglutination method • Three tubes containing two-fold dilutions of patient serum are made . • A drop of antigen suspension (OX-19, OX-2, OX K) is added to each tube. • The mixture is incubated at 50–55 °C for 4–6 hours. • A positive tube would show visible flocculation or granulation, which is accentuated when the tube is gently agitated. 1:20 1:40 1:80 1:160 1:320 Start with dillution of 1: 20

18. The titer corresponds to the most dilute tube in the series that still shows positivity. • Generally, a titre of ≥1:80 is considered diagnostic. • A positive test may be due to Rickettsia or Proteus

19. The Weil–Felix test suffers from poor sensitivity and specificity . • As a result, it has largely been supplanted by other methods of serology, including indirect immunofluorescence antibody (IFA) testing, which is the gold standard.

20. Widal test

21. Salmonella • Gram Negative bacilli • The main cause of Typhoid fever • Also implicated in food infection and food poisoning. • There are two species of Salmonella : • Salmonella typhi • Salmonella paratyphi • S. paratyphi A • S. paratyphi B • S. paratyphi C

22. Antigenic structure of Salmonella • H( flagel) antigens • O (somatic) antigens • Vi (Virulence) capsular polysaccharide antigens • Both typhi and paratyphi have two types of antigen: • Somatic ( O ) antigen, and that is thermostable. • Flagellar( H ) antigen, and that is thermolabile. • Salmonella can be isolated from GIT, urine, blood, bile, bone marrow, sputum, food products, and milk. • The confirmatory test for Salmonella is stool culture at the first week of infection.

23. Widal test • Widal test defined as a test involving agglutination of typhoid bacilli when they are mixed with serum containing typhoid antibodies from a person having typhoid fever; used to detect the presence of Salmonella typhi and S. paratyphi . • Use to diagnose the typhoid fever that caused by Salmonella.

24. In diagnosis of typhoid fever, patient serum is tested for salmonella O and H antibodies against Ag suspension. • The test is done after 2-3 weeks of infection or after 10 days of fever. • Principle of test : • Patients suffering from enteric fever would possess antibodies in their sera which can react and agglutinate serial doubling dilutions of killed colored Salmonella antigens in a tube agglutination test.

26. Agglutination procedures Slide method 1st : Qualitative test : • One drop each of patients’ serum samples for the six antigens are placed on the circled card and one drop of each of the six Salmonella antigens are added separately and gently rotated for one minute. • Positive & negative controls are also placed on their specific circles and mixed with drops of Widal TEST antigen suspension ‘O’.

27. Appearance of agglutination gives qualitative results. • To know the titer for each of the antigens, the test is repeated with dilutions of serum.

28. 2nd Semi - Quantitative test serum saline

29. Here, Titer = 160

30. another method for Semi - Quantitative test

31. Reading the results of semi-quantitative method • The control tubes must be examined first, where they should give no agglutination. • The agglutination of O antigen appears as a “matt” or “carpet” at the bottom. • Agglutination of H antigens appears loose, wooly or cottony. • If agglutination was visible, the results were considered positive • The highest dilution of serum that produces a positive agglutination is taken as titer. • The titers for all the antigens are noted.

32. Precipitation • In this test, the result is observed as precipitate rather than visible agglutination • Moreover, in precipitation test; S. typhi Ags are suspended on alcohol & S. paratyphi Ags on formalin rather than latex particles • This test is more sensitive than agglutination test; however it is rarely performed as it is expensive and not frequently available • The test need 18 tubes for the detection of whole Salmonella Ags.

33. Precipitation procedures 20 ul serum + 180 ul saline 10 ul serum + 190 ul saline and Step  1 : 10 1 : 20 8 tubes 8 tubes 100 ul sample + 900 ul Typhi O reagent 100 ul sample + 900 ul Typhi O reagent Step  and 1/10 * 1/20 = 1/200 1/10 * 1/10 = 1/100

34. Repeat step 2 for the reminder of Salmonella Ags - Typhi H - paratyphi A . H - paratyphi A . O - paratyphi B . H - paratyphi B . O - paratyphi C . H - paratyphi C . O Step  We finally get 16 tubes, Centrifuge them at 3,000 rpm for 5 min. Step  tap the bottom of the tube 2 times If the ppt is resuspended, the the results considered positive (Ag-Abrxn) If the ppt still at the bottom, the results considered negative

35. Other Methods: • Typhiod Rapid IgM-Assay • Detects specific IgM and IgG antibodies to S. Typhi • RT-PCR • The PCR technology has an unparalleled sensitivity and specificity for the diagnosis of typhoid