And other organisms cause febrile reaction Brucella And other organisms cause febrile reaction

Typhoid & paratyphoid fever Febrile Tests Brucella Undulating fever Rickettsia Typhus fever Salmonella Typhoid & paratyphoid fever Culture & serology OX-19 test Widal test

Brucella Cause brucellosis disease (undulating fever). 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).

Direct contact with the body fluids of infected animals 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. Brucella is not transmitted to humans via casual contact such as touching. Eating the meat of infected animals is also extremely low-risk, as long as it is fully cooked. Most cases of brucellosis are due to direct contact with the bodily fluids of infected animals. These include urine and blood. It can also be passed on by direct contact with the tissues, placentas, and aborted fetuses of infected animals. The bacteria that cause brucellosis are not passed easily from animals to humans. Also, many people do not come into contact with animals that normally carry Brucella. As a result, testing will likely be ordered when symptoms are present and the person was in a situation where infection could have occurred. People at higher risk than the general population include farm workers and veterinarians. Slaughterhouse workers and meat inspectors also have a higher-than-average risk.

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. Doctors usually confirm a diagnosis of brucellosis by testing a sample of blood or bone marrow for the brucella bacteria or by testing blood for antibodies to the bacteria. To help detect complications of brucellosis, you may have additional tests Centers for Disease Control and Prevention The CDC laboratory has observed that specimens found positive using EIA tests were negative when tested by the BMAT (Brucella microagglutination test). Results of EIA tests must be confirmed by a reference method such as BMAT, which is quantitative and provides evidence of rising antibody titers when paired sera are tested.  BMAT may be obtained by submission of a blood sample to the Special Serology Section of the SLPH using the form in the SLPH section (below) and requesting Brucella-BMA

 Brucella microagglutination 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.

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.

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.

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.

“ the confirmatory test ” Done in the first week of infection. 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. Use of an automated blood culture system  fluorescent sensor technology that allows for fully-automated, walk-away testing using a continuous-monitoring instrument that agitates and incubates BACTEC/F blood culture bottles, resulting in earlier detection of positives. On gram stain they appear as dense clumps as Gram-negative coccobacilli and are rather difficult to see. 3. Animal inoculation test.

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.

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.

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.

Weil-Felix test OX-19 Test

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. Rickettsial infection (Typhus Fever) generate heterophilic antibodies can agglutinate some strains of Proteus.  Heterophile antibodies=multispecific activities

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.

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

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.

Serum from endemic typhus usually agglutinate OX-19 & OX-2. 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 rickettsialpox disease ‘benign febrile illness with rash resembling chickenpox caused by R. akari’

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.

Start with dillution of 1: 20 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

Rickettsia Proteus Difference 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 Rickettsia Proteus Difference Negative positive Culture from ear disharge and urine Deceased (Leukopenia) Increased (Leukocytosis) Total WBCs Lymphocytosis Due to virus-like behavior Neutrophilia WBCs differentiation

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. The Weil–Felix test suffers from poor sensitivity and specificity, with a recent study showing an overall sensitivity as low as 33% and specificity of 46%. 

Widal test

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

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.

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.

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. The antigens used in the test are “H” and “O” antigens of Salmonella Typhi and “H” antigen of S. Paratyphi. The paratyphoid “O” antigen are not employed as they cross react with typhoid “O” antigen due to the sharing of factor 12

Widal test Agglutination Precipitation Qualitative Semi-quantitative Slide method Qualitative Semi-quantitative Precipitation Tube method

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’.

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

2nd Semi - Quantitative test serum saline 1- نعمل 3 صفوف من الانابيب كل صف فيه 8 انابيب 2- نحط في اول انبوبة من كل صف 0.9 مل سلاين مع 0.1 مل سيروم 3- باقي الانابيب كلها نحط فيها 0.5 مل سلاين ونسير ننقل 0.5 مل من الانبوبة للانبوبة اللي بعدها ونعمل ميكس كويس واخر انبوبة نكب 0.5 مل منها عشان تكون الحجوم متساوية في كل الانابيب ونعمل انبوبة ك نيجاتيف كنترول بس بنحط فيها 0.5 سلاين 4- ع الصف الاول من الانانبيب بنحط ع كل انبوبة نقطة من اوه انتيجين و ع الصف التاني ع كل انبوبة نقطة من اتش انتيجين و ع الصف التالت ع كل انبوبة نقطة من الاي اتش انتيجن تم استبعاد البي اتش انتيجن ” ما عرفت السبب ” 5 – نوضعهم في الحضانة ع درجة 37 طول الليل وبعدها نقرأ النتائج

The titre of the patient serum using Widal test antigen suspensions is the highest dilution of the serum sample that gives a visible agglutination. The sample which shows the titre of 1:100 or more for O agglutinations and 1:200 or more for H agglutination should be considered as clinically significant (active infection). Example: In the above figure, titre is 160. Demonstration of 4-fold rise between the two is diagnostic. H agglutination is more reliable than O agglutinin. Agglutinin starts appearing in serum by the end of 1st week with sharp rise in 2nd and 3rd week and the titre remains steady till 4th week after which it declines. Here, Titer = 160

another method for Semi - Quantitative test Pipette one drop of isotonic saline into the first reaction circle and then place 5, 10, 20, 40, 80 ul of the test sample on the remaining circles. Add to each reaction circle, a drop of the antigen which showed agglutination with the test sample in the screening method. Using separate mixing sticks, mix the contents of each circle uniformly over the reaction circles. Rock the slide gently back and forth, observe for agglutination macroscopically within one minute. 80 μl corresponds to 1/20 dilution, 40 μl to 1/40, 20 μl to 1/80, 10 μl to 1/160 and 5 μl corresponds to 1/320 titre.

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.

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.

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  ممكن اعمل من تخفيف 1 : 10 حجوم 10 و 90 لكن بحاول اكبر الاحجام لانه الانبوبة الواحدة راح يتحضر منها 8 أنابيب and 1/10 * 1/10 = 1/100 1/10 * 1/20 = 1/200

tap the bottom of the tube 2 times 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 Tap = انقر في الانبوبة اللي تطلع + للتأكيد نحركها مرة تالتة اذا الراسب ذاب معناها + أكيد ومهما عملنا مش راح يرجع يتكون الراسب If the ppt is resuspended, the the results considered positive (Ag-Ab rxn) If the ppt still at the bottom, the results considered negative

Other Methods: Typhiod Rapid IgM-Assay RT-PCR 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