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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Methods for Testing SARS-CoV-2 for Covid-19 Diagnosis

There are 2 main kinds of tests for SARS-CoV-2. One type involves detection of the virus itself (viral RNA or antigen) and the other type involves detection of the human immune response to infection (antibodies or other biomarkers).

Standard confirmation of acute SARS-CoV-2 infections is based on the detection of unique sequences of virus RNA by nucleic acid amplification tests (NAATs), such as reverse-transcription polymerase chain reaction (RT-PCR). The viral genes targeted so far include the N, E, S and RdRP genes.

Reverse-Transcription Polymerase Chain Reaction (RT-PCR)

Polymerase chain reaction (PCR) is a molecular technique which allows production of million copies of a specific DNA sequence from initially smallest sample, within few hours.

Reverse transcription polymerase chain reaction (RT-PCR) is a laboratory technique combining reverse transcription of RNA into DNA (called complementary DNA or cDNA) and amplification of specific DNA targets using polymerase chain reaction (PCR). This is achieved by monitoring the amplification reaction using fluorescence, a technique called real-time PCR or quantitative PCR (qPCR).

Principle of RT-PCR

The PCR involves the primer mediated enzymatic amplification of DNA. PCR is based on using the ability of DNA polymerase to synthesize new strand of DNA complementary to the offered template strand.

Reverse transcription PCR (RT-PCR) is used when the starting material is RNA. As viruses like SARS-CoV-2 contain RNA as their genetic material, RT-PCR is used. In this method, A sample is collected from the parts of the body where the COVID-19 virus gathers, such as nasopharyngeal or oropharyngeal swab. RNA is extracted removing undesired components by chemical treatment. This extracted RNA is a mix of the person’s own genetic material and, if present, the virus’s RNA. RNA is first transcribed into complementary DNA (cDNA) by reverse transcriptase from total RNA or messenger RNA (mRNA). The cDNA is then used as the template for the PCR reaction.

RT-PCR can be performed by two methods: one-step or a two-step assay. One-step assays combine reverse transcription and PCR in a single tube and buffer, using a reverse transcriptase along with a DNA polymerase. In two-step assays, the reverse transcription and PCR steps are performed in separate tubes, with different optimized buffers, reaction conditions, and priming strategies.

Components of RT-PCR

The RT-PCR reaction requires the following components:

1. RNA Template

   The single stranded RNA (ssDNA) of interest, separated from the sample.

2. Primers

   A primer is a short nucleic acid sequence that provides a starting point for DNA synthesis. It helps to provide 3′-OH group to add the first nucleotide. Primer for reverse transcription anneals to the template mRNA strand and provide reverse transcriptase enzymes a starting point for synthesis. These are complementary to the 3’ ends of the sense and anti-sense strands of the target sequence.

3. Reverse Transcriptase

   Reverse transcriptase is an RNA-dependent DNA polymerase, catalyzing DNA synthesis using RNA as the template.

4. DNA Polymerase

   Usually a thermostable Taq polymerase that can function at a temperature optimum of about 70°C and does not rapidly denature at high temperatures (98° C).

5. Deoxynucleotide triphosphates

   Single units of the bases A, T, G, and C (dATP, dTTP, dGTP, dCTP) provide the energy for polymerization and the building blocks for DNA synthesis.

6. Buffer system

   Includes magnesium and potassium to provide the optimal conditions for DNA denaturation and renaturation; also important for polymerase activity, stability and fidelity.

7. Thermocycler

   A thermal cycler (also known as a PCR machine or thermocycler) is a laboratory instrument that heats and cools samples in repetitive cycles to facilitate DNA or RNA amplification through the polymerase chain reaction.

Stages of RT-PCR

The process of the PCR is subdivided into three stages: denaturation, annealing and elongation. RT–PCR is a variation of PCR which use the same process except that RT–PCR has an added step of reverse transcription of RNA to DNA at first to allow for amplification. The first cycle is reverse transcription to synthesize cDNA. The second cycle is initial denaturation. During this cycle reverse transcriptase is inactivated. The next 40 to 50 cycles are the amplification program, which consists of three steps: (1) denaturation, (2) annealing, (3) elongation.

1. Reverse Transcription:

   Reverse transcriptase enzyme synthesizes a complementary DNA (cDNA) strand with nucleotides, extending from the primer. It occurs between 40°C and 50°C, depending on the properties of the reverse transcriptase enzyme utilized. This is one time reaction and the product is mRNA:cDNA hybrid. After the reverse transcription, the mRNAs are hydrolyzed and single-stranded cDNAs are then replicated by the DNA polymerase during a first temperature cycle.

2. Denaturation:

   All the component mixture is heated to 94°C for 15-30 seconds. This causes breakdown of hydrogen bonds and double stranded cDNA is denatured to single strands. These will act as templates for the production of the new strands of DNA.

3. Annealing:

   The reaction temperature is rapidly lowered to 54-60°C for 20-40 seconds. Primers bind to the target DNA sequences and initiate polymerization.

4. Elongation/Extension:

   This step usually occurs at 72-80°C (most commonly 72°C). The DNA Taq polymerase enzyme sequentially adds bases to the 3′ end of primer, extending the DNA sequence in the 5′ to 3′ direction. DNA polymerase will add about 1,000 bp/minute under optimal conditions.

The result of one cycle of PCR is two double-stranded sequences of target DNA, each containing one newly made strand and one original strand. Upon denaturation, these new fragments also serve as templates, Each cycle doubles the previous number.As the cycles are repeated, more and more copies are generated and the number of copies of the template is increased exponentially.

Detection in Real-Time RT-PCR (rRT-PCR/RT-qPCR)

Real Time Real-time reverse-transcription PCR (rRT-PCR) is the technique of collecting data throughout the PCR process as it occurs, thus combining amplification and detection into a single step. This is using fluorescent dyes that yield increasing fluorescent signal in direct proportion to the number of PCR product generated. Fluorescent reporters used in real-time PCR include double-stranded DNA (dsDNA)- binding dyes, or dye molecules attached to PCR primers or probes that hybridize with PCR product during amplification. This value is usually referred to as cycle threshold (Ct), the time at which fluorescence intensity is greater than background fluorescence. Greater the quantity of target DNA in the sample, there will be significant increase in fluorescent signals earlier, yielding a lower Ct.

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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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A gravid female roundworm can lay upto 2,50,000 eggs per day. This accounts for about three eggs per mg of feces. At this concentration, the eggs can be readily seen by the microscopic examination of saline emulsion of feces. Two types of eggs are liberated from the female worm of Ascaris lumbricoides:

• Fertilized eggs
• Unfertilized eggs

Feces sample may show both fertilized and unfertilized eggs, or either type alone.

Fertilized Eggs

The fertilized eggs are laid by females after inseminated by mating with a male. These are embryonated and develop into the infective eggs.

Morphology and Features

Unfertilized Eggs

The unfertilized eggs are laid by uninseminated female. Thes are non-embryonated and cannot become infective.

Morphology and Features

The post Ascaris lumbricoides (Roundworm) Eggs: Morphology, Characteristics and Identification appeared first on LaboratoryTests.org.

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.

The post Coagulase Test : Types, Principle, Procedure, Interpretation and Examples appeared first on LaboratoryTests.org.

S.N	Characteristics	Entamoeba histolytica	Entamoeba coli
Trophozoite
1.	Structure
2.	Size	12-30 µm	20-40 µm
3.	Motility	Progressive. Hyaline, fingerlike pseudopodia	Sluggish, nondirectional Granular pseudopodia
4.	Nucleus	1, difficult to visualize in unstained smear	1, often visible in unstained smear
5.	Karyosome	Sharp central	Eccenteric
6.	Pheripheral Chromatin	Fine and dispersed	Coarse and Clumped
7.	Cytoplasmic Inclusions	RBCs, leukocytes and tissue debris but no bacteria	Bacteria and other material but never RBCs
Cysts
1.	Structure
2.	Size	10-15 µm	15-25 µm
3.	Shape	Usually spherical	Usually spherical, may be oval, triangular or other shaped
4.	Nucleus	Mature Cyst=4 nuclei	Mature Cyst=8 Nuclei
5.	Peripheral Chromatin	Fine and dispersed	Coarsely granular or clumped
6.	Chromatoid Bodies	Rounded ends	Filamentous, thread like, pointed ends
7.	Glycogen Mass	Visible in uninucleate stage	Large and visible in binucleate stage

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Human Chorionic Gonadotropin (hCG)

Human chorionic gonadotropin (hCG) is a hormone produced by the developing placenta shortly after fertilisation. In normal pregnancy, hCG can be detected in urine as early as 7 to 10 days after conception. Levels of hCG rise rapidly, frequently exceeding 100mIU/mL by the first missed menstrual period, and peaking at 100,000-200,000 mIU/mL about 10-12 weeks into pregnancy. The appearance of hcG soon after conception and it’s subsequent rise in concentration during early gestational growth make it excellent marker for the early detection of pregnancy.

Principle of Urine Pregnancy Test

Most of the urine pregnancy test kits are based on lateral-flow technology. Most of them qualitatively detect the presence of hCG in urine specimen at the sensitivity of 25 mIU/mL. The test uses two lines to indicate results. The test line utilizes a combination of antibodies including a monoclonal hCG antibody to selectively detect elevated levels of hCG. The control line is composed of goat polyclonal antibodies and colloidal gold particles.

The assay is conducted by adding a urine sample to the sample well of the test device and observing the formation of colored lines. The sample migrates via capillary action along the membrane to react with the colored conjugate.

Positive samples react with the specific antibody-hCG-colored conjugate to form a colored line at the test line region of the membrane. Absence of this colored line suggests a negative result. A colored line will always appear in the control line region if the tests has been performed properly.

Specimen Collection

Collect urine into a clean, dry container. The first morning urine specimen is preferred since it generally contains the highest concentration of HCG; however, urine specimens collected at any time of the day may be used. Refrigerate specimens at 2° to 8°C (36° to 46°F) for up to 72 hours, if the testing is not performed immediately. If samples are refrigerated, bring them to room temperature before testing.

Procedure

1. Allow the Pregnancy Test Strip and urine sample to reach room temperature (15-30°C) before opening the foil pouch.
2. Remove the Pregnancy Test Strip from the pouch and use it as soon as possible.
3. Place the test device on clean and level surface. Hold the dropper vertically and transfer 3 full drops to the specimen well and start the timer. Avoid air bubble formation.
4. Wait for the colored line(s) to appear. Read the result after 5 minutes. Do not read the result after 15 minutes.

Results and Interpretation

POSITIVE: Two coloured lines appear. One line should be in the Control region (C) and another line should be in the Test region (T). This means there is a strong possibility that patient is pregnant.

NEGATIVE: One coloured line in the Control region (C). No apparent colored line appears in the Test region (T). This means patient is either not pregnant or has tested too early.

INVALID: The result is invalid if the Control Line (C) fails to appear. Insufficient volume of urine or incorrect procedure are the most likely reasons for an invalid result.

Limitations

1. In cases where very high levels of HCG are present (>500,000 mlU/ml) a false negative result can occur due to a “Prozone” effect. If pregnancy is still suspected, simply dilute specimen 1:1 with deionized water and retest.
2. If a urine sample is too dilute (ie: low specific gravity) it may not contain a representative level of HCG. If pregnancy is still suspected, a first morning urine sample should be obtained and retested 48 hours later.
3. A number of conditions other than pregnancy, including trophoblastic disease and certain non-trpohoblastic neoplasms including testicular tumors, prostate cancer, breast cancer and lung cancer cause elevated levels of hCG. Therefore, the presence of hCG in urine should not be used to diagnose pregnancy unless these conditions have been ruled out.
4. Drugs containing hCG may interfere with the test, and produce misleading results.

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