Symptoms usually begin within 10 days of being bitten. The most common symptoms of scrub typhus include fever, headache, body aches, lymphadenopathy and sometimes maculopapular rashes and an escher at the site of bite.

The most common signs are similar to a variety of other infectious diseases like typhoid fever, malaria, leptospirosis and dengue fever etc. Thus Laboratory-based methods are more reliable and precise in the diagnosis of scrub typhus. Scrub typhus can be diagnosed in the laboratory by one of the following methods:

Direct Detection

Detection in tissue

When stained with Giemsa, Macchiavello or Gimenez stains, aggregations of bacterial particles can be detected under light microscope.
Skin biopsies from the center of petechial lesion can be examined by immunofluorescence, immune-enzyme methods and histochemical methods.

PCR Based Detection

Polymerase Chain Reaction (PCR) based detection methods utilize genetic markers such as 56 kDa tsa, GroEL, 16s RNA and 47 kDa HtrA to detect specifically the target organism in the specimens like skin biopsy, necrotic tissue and blood mononuclear cells.

Culture and Isolation

It is expensive and laborious procedure. Orientia tsutsugamushi can’t be cultured in artificial media. It can be isolated and cultivated by inoculating in the peritoneum of mice & guinea pigs, in the yolk sac of embryonated chicken egg and in cell culture. This should be performed under BSL-3 facilities.

Serological Tests

1. Weil Felix Reaction

It is a heterophile agglutination test in which orientia antibodies are detected using antigens of certain non-motile Proteus strains. The basis of the test is sharing of an alkali stable lipopolysaccharide (LPS) antigen by some orientia/rickettsiae and by certain strains of Proteus (P. vulgaris OX19 and OX2 & P. mirabilis OX K). OX K agglutinin is found only in scrub typhus.

Weil Felix test is done as macroscopic agglutination. Serum dilutions of 1:10 to 1:640 are made to which equal amounts of antigens are added. The tubes are incubated in a water bath at 37 degree Celsius for 2 hours followed by incubation at 4 degree Celsius overnight. Complete agglutination is shown by complete clearing of the supernatant fluid and the formation of smaller masses in the bottom of the tube.

2. Rapid lateral-flow Assays

Most of the commercially available rapid test kits for scrub typhus diagnosis are based on both IgM and IgG detection. These usually use single strip (IgG, IgM), where the Kp r56 protein is conjugated to gold particles as the indicator system.

3. Indirect Fluorescence Assay (IFA)

IFA has been considered as the gold standard and most commonly used test for serologic detection of scrub typhus due to its higher sensitivity and specificity. Karp, Kato and Gilliam’s are the most frequently used antigens.

Known antigen is immobilized on a glass slide. Test serum is added over the smear. If specific antibodies are present in the serum, the antigen-antibody complex is formed. The serum is washed off and a secondary antihuman immunoglobulin conjugated to a fluorochrome is added. Upon examination under fluorescence microscope, bacteria will only be visible if they have been bound by the antibodies from the patient’s serum.

4. Enzyme-Linked Immunosorbent Assay (ELISA)

The 56-kDa protein (located on the outer membrane of O. tsutsugamushi) is highly reactive with patient sera and therefore preferred for use in the diagnosis of scrub typhus.

5. Other Tests

Indirect immunoperoxidase, a modification of the standard IFA method, can be used with a light microscope. Latex agglutination tests are also available.

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The Mantoux test is a qualitative, skin test to screen in vivo sensitization by Mycobacterium tuberculosis either due to active infection or past infection. It is also used to check the prophylaxis and efficacy of BCG vaccination. Mantoux test is a routine screening procedure for children, healthcare workers, individuals at high risk of being infected and individuals who are suspected of being infected with Tuberculosis.

Mantoux Test doesn’t distinguish between an active and a latent infection; nor does it provide a definitive diagnosis. If positive reaction occurs, additional tests such as sputum smear, culture and chest X-rays etc are necessary to establish a diagnosis of an active TB infection.

Principle of Mantoux Test

Mantoux test is based on a delayed type hypersensitivity reaction (Type IV) to test for individauls cell mediated immunity against Mycobacterium tuberculosis. Mycobacterial antigen is available in the form of Purified Protein Derivative (PPD). 5 units of PPD (0.1 ml) is injected intradermally with 26,27 or 30 gauze needle. The results are read in 48-72 hours for induration (elevated hardened area). Erythema (redness) is not significant.

After injection, cytokines are released by memory Th1 cells to attract macrophages and granulocytes which cause induration and erythema. Delayed hypersensitivity reaction begin after 5-6 hours and reach peak at 48-74 hours.

Requirements

1. Graduated 1ml syringe
2. PPD or Tuberculin
3. Spirit swab

Procedure of Mantoux Test

1. Bring PPD reagent to room temperature.
2. The preferred site for injection is dorsal surface of the forearm, about 4cm below the elbow joint. Select an area free of barriers (e.g. scars, sores).
3. Disinfect the site of injection and allow to dry.
4. Draw up just over 0.1 ml of PPD by using 1 ml syringe. Remove excess PPD to make exactly 0.1 ml and remove air from the syringe if present.
5. Using 27 g needle to inject the PPD intradermally to make the deposition wheel, in the diameter of 6 to 8 mm which will rise up to the point of needle.
6. Mark the area of injection with indicator.
7. Read the result after 48-72 hours for induration.

Results and Interpretation

After 48-72 hours of administration of PPD, reaction should be measured in millimeters of induration (elevated hardened area). Erythema (redness) is not significant, it is thus not measured.

According to Center for Disease Control (CDC), interpretation of Mantoux test depends on two factors:

• Measurement in millimeters (mm) of the induration
• Person’s risk of being infected with TB and progression to disease if infected

Induration of ≥5 mm is considered positive in

1. Human immunodeficiency virus (HIV)-infected persons
2. Recent contacts of TB case patients
3. Persons with fibrotic changes on chest radiograph consistent with prior TB
4. Patients with organ transplants and other immunosuppressed patients

Induration of ≥10 mm is considered positive in

1. Recent immigrants (i.e., within the last 5 years) from countries with a high prevalence of TB
2. Injection drug users
3. Residents and employees of the high-risk congregate settings like; prisons and jails, nursing, hospitals and other health care facilities, residential facilities for patients with AIDS and homeless shelters
4. Mycobacteriology laboratory personnel
5. Persons with the clinical conditions that place them at high risk; silicosis, diabetes mellitus, chronic renal failure, some hematologic disorders (e.g., leukemias and lymphomas), other specific malignancies (e.g., carcinoma of the head, neck, or lung)
6. Infants, children, and adolescents exposed to adults at high risk for developing active TB

Induration of ≥15 mm is considered positive in

1. Persons with no known risk factors for TB

Limitations of Mantoux Test

Mantoux Test doesn’t distinguish between an active and a latent infection. If positive reaction occurs, additional tests such as sputum smear, culture and chest X-rays etc are necessary to establish a diagnosis of an active TB infection.
Several factors can lead to false-positive or false-negative skin test reactions.

False Positive Reactions

Due to the test’s low specificity, most positive reactions in low-risk individuals are false positives. Some major causes of false positive Mantoux Test are:

• Infection with nontuberculous mycobacteria (NTM)
• BCG vaccination.
• Administration of incorrect antigen.
• Incorrect interpretataion of results.

False Negative Reactions

Some people have a negative reaction to the TST even though they have been infected with M. tuberculosis. A false-negative reaction can be caused by many things:

• Concurrent viral infection (e.g., measles, mumps, chicken pox, HIV)
• Concurrent bacterial infection (e.g., typhoid fever, brucellosis, typhus, leprosy, pertussis)
• Concurrent fungal infection
• Chronic renal failure
• Low protein states (e.g., severe protein depletion, afibrinogenemia)
• Diseases affecting lymphoid organs (e.g., Hodgkin’s disease, lymphoma, chronic leukemia, sarcoidosis)
• Immunosuppressive drugs (e.g., medical steroids)
• Children aged 6 months or less or elderly patients (i.e., immature or waning immunity)
• Stress (e.g., surgery, burns, mental illness, graft-versus-host reactions)
• Incorrect storage or handling of antigen or results that are not measured or interpreted properly
• Vaccinations using live virus; or
• Recent TB infection.

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Principle

S. aureus produces two types of coagulases: bound and free. Slide coagulase test is done to detect bound coagulase, whereas tube coagulase test is done to detect free coagulase. Both tests utilize rabbit plasma treated with anticoagulant to interrupt the normal clotting mechanism.

Bound Coagulase

It is also known as clumping factor. It is attached to bacterial cell wall and reacts directly with fibrinogen. This is shown by formation of visible mass. it doesn’t require coagulase reacting factor (CRF).

Free Coagulase

It is an extracellular enzyme (released from the cell). It converts fibrinogen to fibrin by activity of coagulase reacting factor (CRF) in plasma. This is detected by appearance of fibrin clot in the tube coagulase test. It is usaually recommended to do tube coagulase test on all ‘slide-coagulase-negative’ staphylococci.

Procedure

Slide Test

1. Place two separate drops of saline on a slide.
2. Using a sterile inoculating loop, emulsify one or two colonies of organism in one drop to make thick suspension of bacteria.
3. Add a loopful of plasma to both the suspension and saline drop and mix gently.
4. Look for immediate coarse clumping of the mixture within 10-15 seconds.

Tube Test

1. Dilute the plasma 1:10 with saline.
2. Take 2 test tubes and add 0.5 ml of diluted plasma to each.
3. Inoculate a tube with bacterial colonies to make a cloudy suspension. Alternatively, add about 5 drops of thick 18-24 hours broth cultures.
4. Incubate both tubes at 35 degree celcius for 1 to 4 hours in water bath.
5. Afterward, examine both tubes for presence or absence of clots.

Results and Interpretation

Slide Coagulase Test: The formation of clumps within 10-15 seconds is positive test result. Saline and plasma mixture should show no clumping.

Tube Coagulase Test: A positive coagulase test is represented by any degree of clotting, from a loose clot suspended in plasma to a solid clot. If negative, the plasma remains a liquid.

Positive coagulase test is shown by: Staphylococcus aureus, S. pseudintermedius, S. intermedius, S. schleiferi, S. delphini, S. hyicus, S. lutrae etc.

Negative coagulase test is shown by: Staphylococcus epidermidis, S. saprophyticus, S. warneri, S. hominis, S. caprae etc.

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