Brucella

1. Brucella And other organisms cause febrile reaction

2. OX-19 test Culture & serology Widal test

3. 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 • Serological 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. Brucellamicroagglutination test (BMAT), a modified version of the serum (tube) agglutination test (SAT), that can detect antibodies to Brucella species - abortus, melitensis or suis. There is no serological test available to detect antibodies to B. canis. • For a diagnosis to be made using serology, two serum samples are required. The first serum sample should be taken when a person is acutely ill (≤7 days after symptom onset); the second serum sample should be drawn 2-4 weeks later to check for a rise in antibodies (a fourfold or greater rise in antibodies would bean an individual is positive for brucellosis). • If submission of paired sera is not possible, a probable diagnosis can be made with a single serum sample.

7. False Positives and Other Concerns About Reliability • There are a few reasons why diagnosing an active Brucella infection can be challenging. • Some other types of bacteria can cause a false positive, which means testing positive for the presence of Brucella when it’s not present. • Some immunizations can cause a test to be positive when there’s no infection. • A positive test doesn’t always mean you have a current infection. It could mean you were exposed to Brucella at some point in the past. It might also mean you have an immunity against this type of bacteria. • If you were recently exposed to the Brucella antigen, there may be too few antibodies to be detected by the test. • More tests or follow-up testing may be needed to confirm or rule out brucellosis.

8. The Rose Bengal test (RBT) • The Rose Bengal test (RBT) is a simple, rapid slide-type agglutination assay performed with a stained B. abortus suspension at pH 3.6–3.7 and plain serum. • It is often used as a screening test in human brucellosis and would be optimal for small laboratories with limited means.   • False-negative reactions occur especially in the early stages of acute infection.

10. Procedure of Rose Bengal Plate Test • Test Serum (0.03 ml) is mixed with an equal volume of antigen on a white tile or enamel plate to produce a zone approximately 2 cm in diameter. • The mixture is agitated gently for 4 minutes at ambient temperature, and then observed for agglutination. • Any visible reaction is considered to be positive. • The test is very sensitive, especially in vaccinated animals, and positive samples should be retested by a confirmatory test such as the CF test or ELISA . • False-negative reactions may occur and can be detected by retesting animals at intervals over a period of at least 3 months.

11. 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. 3. Animal inoculation test.

12. 4- More tests or follow-up testing may be needed to confirm or rule out brucellosis, as the following : • X-rays. X-rays can reveal changes in your bones and joints. • Computerized tomography (CT) scan or magnetic resonance imaging (MRI). These imaging tests help identify inflammation or abscesses in the brain or other tissues. • Cerebrospinal fluid culture. This checks a small sample of the fluid that surrounds your brain and spinal cord for infections such as meningitis and encephalitis. • Echocardiography. This test uses sound waves to create images of your heart to check for signs of infection or damage to your heart.

13. 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.

14. 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.

15. Weil-Felix test OX-19 Test

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

17. 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. • Serologic test. OX-19 test ( weil-felix test ). • Immunofluorescent antibody technique.

18. 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

19. 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.

20. 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

21. 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.

22. 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

23. 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

24. 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.

25. Widal test

26. 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

27. 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.

28. 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. • 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.

29. 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.

31. 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’.

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

33. 2nd Semi - Quantitative test serum saline

34. Here, Titer = 160

35. another method for Semi - Quantitative test

36. 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.

37. Precipitation • In this test, the result is observed as precipitate rather than visible agglutination • Moreover, in precipitation test; S. typhiAgs are suspended on alcohol & S. paratyphiAgs 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.

38. 20 ul serum + 180 ul saline 10 ul serum + 190 ul saline Precipitation procedures 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

39. 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

40. 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