1. And other organisms cause febrile reaction
Brucella
And other organisms cause febrile reaction

2. Typhoid & paratyphoid fever
Febrile Tests
Brucella
Undulating fever
Rickettsia
Typhus fever
Salmonella
Typhoid & paratyphoid fever
Culture & serology
OX-19 test
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.
هو من zoonetic disease لأنه ينتقل من الانسان للحيوان
Cause brucellosis disease (undulating fever) or
(Malta fever).

4. 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.
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.
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.

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.
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

6. On gram stain they appear as dense clumps as Gram-negative
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.

7. CT scan or magnetic resonance imaging (MRI). CSF culture.
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. 
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.

8. Results
An agglutination test is done by mixing 50ul of sample with one drop of reagent.
A normal (negative) result shows no antibodies to Brucella.
Titer about 1:80 or more indicate Brucellosis.
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 adiverse 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.
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.
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. 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 and by 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’

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.
Start with dillution of : 20

18. 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

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.
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%. 

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
Salmonella infection may spread from the intestines to the blood stream, and then to other body sites.
Practically salmonella divided into 2 groups :
1- enteric fever group – typhoid & paratyphoid
2- food poisioning group ( enteric fever )
Typhoid fever :- high fever, delirium (هذيان الحمى ) , tender abdomen, enlarged spleen, constipation rather than vomiting or diarrhea
Paratyphoid C
Paratyphoid C is a rare infection, generally seen in the Far East. It presents as a septicaemia with metastatic abscesses. Cholecystitis is possible in the course of the disease. Antibodies to paratyphoid C are not usually tested and the diagnosis is made with blood cultures.

22. Antigenic structure of Salmonella
H( flagel) antigens
O (somatic) antigens
Vi (Virulence) resemble 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.
( H ) antigen strongly immunogenic
Vi (Virulence) inhibit phagocytosis , resist complement activation, and bacterial lysis ( poorly immunogenic )

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.
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

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

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
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

29. 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

30. 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.

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 ul saline
10 ul serum ul saline
and
Step 
1 : 10
1 : 20
8 tubes
8 tubes
100 ul sample ul Typhi O reagent
100 ul sample 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

34. 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

35. Other Methods: Typhiod Rapid IgM-Assay RT-PCR
Detects specific IgM antibodies to S. Typhi
RT-PCR
The PCR technology has an unparalleled sensitivity and specificity for the diagnosis of typhoid
Salmonella have a vaccine called TAB vaccine