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RAPID TESTS: AGGLUTINATION AND IMMUNOLOGY TESTING

RAPID AGGLUTINATION LATEX TESTS - from blood:

- ASLO;

- CRP;

- RF.


HEPATITIS: A, B, C, D and E:

- HEPATITIS A VIRUS - from blood;

- HEPATITIS B VIRUS - from blood ;       

- HEPATITIS C VIRUS - from blood;

- HEPATITIS D VIRUS - from blood;

- HEPATITIS E VIRUS - from blood, stool. 


SYPHILIS - treponema pallidum; from blood and other fluids:

- VDRL;

- RPR;

- TPHA.

 

HIV TESTING - from blood, oral fluid, urine.


TROPONIN LEVEL TESTING - from blood. 


HELICOBACTER PYLORI DETECTION: 

- ANTIBODY DETECTION - from blood;

- ANTIGEN TEST - from stool.

 

FAECAL OCCULT BLOOD (FOB) TEST - from stool.


GIARDIA LAMBLIA - ANTIGEN DETECTION - from stool.


IMMUNOGLOBULIN E - from blood.


CORONAVIRUS - from blood, nose/throat swab. 


ASLO, CRP, RF: rapid agglutination tests

Serology - Immunology

Agglutination Kits & Serology Reagents: there are many options of latex serology kits on the market. Common serological tests to detect in serum:
- ASLO/ASO: Anti-streptolysin-O;
- CRP: C-Reactive Protein;
- RF: Rheumatoid Factor.

The latex assays are available in different kit sizes, with positive and negative controls provided. The full kits include all the required consumables. They use the common agglutination method for efficient, reliable testing, to ascertain the causes of inflammation, rheumatoid disorders and streptococcal infections. Latex agglutination testing is sometimes referred to as latex serology. 
Agglutination occurs when an antigen comes into contact with its corresponding antibody. The rapid test kits are used to identify streptococcal infections, levels of C-reactive protein that would indicate high levels of inflammation in the body and rheumatoid factors that would indicate a diagnosis of rheumatoid arthritis.

Latex serology tests are used to ascertain whether specific antigens or antibodies are present in a sample. This is done by applying the samples to the latex beads. If the suspected substance is present in the sample, the latex beads clump together (agglutinate). The results of latex serology tests can be obtained within 15 minutes to an hour, allowing a rapid diagnosis to be made, which is obviously a significant advantage.
 

These slide agglutination assay tests are used for the qualitative and semi-quantitative determination of ASLO, CRP and RF in human serum. The simple, five-step procedure provides easy-to-read results in 2 minutes. No initial dilution of patient samples is required. Kits usually include ASLO/CRP/RF latex reagents, reactive control, nonreactive control, 100 disposable stirrer pipettes and disposable cards. The latex reagent does not require additional preparation. MATERIAL REQUIRED: automatic pipettes, mechanical rotator, adjustable at 100 r.p.m, laboratory alarm clock.

QUALITY CONTROL: Positive and negative controls should be run daily following the steps outlined in the Qualitative Test, in order to check the optimal reactivity of the reagent. The positive control should produce clear agglutination. If the expected result is not obtained, you must not use it. 

ASO/ASLO: slide agglutination test for the qualitative and semi-quantitative detection of streptolysin antibodies in human serum. Analytical sensitivity: 200 (+/-50) IU/mL. 
CRP: slide agglutination test for the qualitative and semi-quantitative detection of C-reactive protein in human serum. Analytical sensitivity: 6 mg/L. 
RF: slide agglutination test for the qualitative and semi-quantitative detection of rheumatoid factors in human serum. Analytical sensitivity: 8 IU/mL. 

SOURCES OF ERROR: Bacterial contamination of controls and specimens as well as freezing and thawing of the ASLO/CRP/RF-latex reagents may lead to false positive results. Traces of detergent in the test cards may give false positive results. Preferably the used cards would not be reused. If cards are reused, wash them first under tap water until all reactants are removed and then with distilled water. Allow to air dry, avoiding the use of organic solvents as they may impair the special finish on the slide. 
The ASLO/CRP/RF-latex antigen must not be used beyond its expiry date because a prolonged storage can affect the sensitivity of the suspension. Do not allow reagents in the kit to get in contact with skin or mucous membranes.


Reagents are stable if stored at 2-8ºC up to expiration date. Allow the suspension to reach to room temperature and mix gently prior to use. Do not freeze. Frozen reagents could change the functionality of the test. Reagent and Controls are ready for use and stable until the expiry date stated on the label. Do not use haemolysed, lipaemic or contaminated serum for testing. Hemoglobin (<10 g/L), bilirubin (<20 mg/dL) and lipemia (<10 g/L) do not interfere. Other substances (rare) may interfere. SAMPLES: must be fresh, clear serum. After the clear serum has been separated it may be stored at 2-8ºC for up to one week or longer periods at -20ºC, before testing. Undiluted samples should be used. Do not use plasma since fibrinogen can form non-specific agglutination. The sensitivity of the test may be reduced at low temperatures. The best results are achieved at 15-25ºC. 


PROCEDURAL STEPS for the QUALITATIVE TEST
1. Bring the test reagents and samples to room temperature. 
2. Transfer 50µl/1 drop of the patient's serum into one of the circles of the test card. Dispense 1 drop of positive control and 1 drop of negative control into two additional circles. 
3. Gently shake the suspension of the latex reagent (ASLO/CRP/RF), then using the pipette, aspirate dropper several times to obtain a thorough mixing. Add 1 drop/50µl reagent of the suspension to the same test circle with the patient's serum and to those with the controls. 
4. Using the stirrers, mix the serum and the latex reagent and spread them. Mix the contents of each circle while spreading over the entire area enclosed by the ring. Use separate stirrers for each mixture. 
5. Rotate the test card/slide for 2 minutes by means of a mechanical rotator (100r.p.m.) and observe immediately under a suitable light source for any degree of agglutination. 

READING THE LATEX SLIDES:

- Nonreactive: smooth suspension with no visible agglutination, as shown by the negative control. 
- Reactive: any degree of agglutination visible macroscopically (best to check under a suitable light). 

The delays in reading the results may generate in overestimation of the antibody present.


For the SEMI-QUANTITATIVE TEST:

- Dilute the sample to be tested following the 2-fold dilutions, as follows: 1/2, 1/4, 1/8, 1/16, 1/32. Follow the other steps as in the quantitative test.

How to make the dilutions: it is up to the biologist, for as long as it is correctly diluted mathematically. 

Example of dilution 1/2: 50µnon-diluted sample + 50µL dilution liquid (physiological serum/distilled water), then transfer 1 drop/50µL from the ASLO/CRP/RF latex reagent over the mixture. The other steps are as in the quantitative test.   

Reading is the same for both the Qualitative and for the Semi-Quantitative Test:
- Negative/Non-reactive: no agglutination; 
- Positive/Reactive: visible agglutination.  

The titer of the specimen for the Semi-Quantitative test is reported as the highest dilution that shows reactivity. The next higher dilution should be negative. If the highest dilution tested is reactive, repeat the test using the next dilution. 
The results obtained for the Semi-Quantitative Test do not replace the fully-automated quantitative tests available on the market. The levels obtained following the Semi-Quantitative Test procedure are considered only "approximate", not exact values, hence the naming of "Semi-Quantitative Test". 

The approximate ASLO/CRP/RF levels (IU/mL) present in the sample may be obtained by multiplying the titer of the last positive dilution by the minimum detectable unit (analytical sensitivity) corresponding to each reagent type. 


Reading the Semi-Quantitative tests:

ASLO: 
- 1/2 dilution shows reactivity => ASLO level: 2 x 200 = 400 IU/mL
- 1/4 dilution shows reactivity => ASLO level: 4 x 200 = 800 IU/mL
1/8 dilution shows reactivity => ASLO level: 8 x 200 = 1600 IU/mL
- 1/16 dilution shows reactivity => ASLO level: 16 x 200 = 3200 IU/mL

1/4 reactive and 1/8 non-reactive => ASLO level: 800-1600; ASLO>800 and <1600.

CRP: 
- 1/2 dilution shows reactivity => CRP level: 2 x 6 = 12 mg/L
- 1/4 dilution shows reactivity => CRP level: 4 x 6 = 24 mg/L
1/8 dilution shows reactivity => CRP level: 8 x 6 = 48 mg/L
- 1/16 dilution shows reactivity => CRP level: 16 x 6 = 96 mg/L

1/2 reactive and 1/4 non-reactive => CRP level: 12-24; CRP>12 and <24.

RF: 
- 1/2 dilution shows reactivity => RF level: 2 x 8 = 16 IU/mL
- 1/4 dilution shows reactivity => RF level: 4 x 8 = 32 IU/mL
1/8 dilution shows reactivity => RF level: 8 x 8 = 64 IU/mL
- 1/16 dilution shows reactivity => RF level: 16 x 8 = 128 IU/mL.

1/8 reactive and 1/16 non-reactive => RF level: 64-128; RF>64 and <128.

ASLO

Antistreptolysin O (ASO/ASLO)
 
- ASLO/ASO latex kit test: an indirect agglutination kit for the detection of anti-streptolysin-O in human serum using latex particles coated with streptolysing-O. 

ASO titer is adviced for the diagnosis of post infectious glomerulonephritis, scarlet fever, streptococcus infections and othersThe Streptolysin O is antigenic and cause hemolysis of RBCs. So there is antibody formation (Antistreptolysin-Ab) in the blood. 
Anti-Streptolysin O (ASO or ASLO) is the antibody made against streptolysin O, an immunogenic, oxygen-labile hemolytic toxin produced by most strains of group A and many strains of groups C and G streptococci. 
In the course of streptococcal infections, the extracellular products of the bacteria act as antigens to which the body responds by producing specific antibodies. Streptolysin O (ASLO) is one of two hemolysins (the other being Streptolysin S) produced by virtually all strains of Streptococcus pyogenes.  

Principle:
Patient serum with dilution is mixed with a fixed amount of streptolysin O- Ag and a reaction takes place. Then add Ab-coated RBC. There will be hemolysis in the tube, where streptolysin O is free.

Antistreptolysin (ASO) leading to hemolysis
Antistreptolysin (ASO) leading to hemolysis

The result is reciprocal of the highest dilution where hemolysis starts. Units are international units (IU).

Antistreptolysin (ASO) titer procedure
Antistreptolysin (ASO) titer procedure

Normal value: below 200.
Definitive value: 400 or more.
Rising titer from 200-250 is significant.

ASLO-Latex Test: The assay is performed by testing a suspension of latex particles coated with streptolysin O antigen against unknown serum. The presence or absence of visible agglutination indicates the presence or absence of ASLO in the samples tested. 
REAGENT COMPOSITION ASLO-Latex Antigen. Suspension of polystyrene latex particles coated with stabilized streptolysin O in a buffered saline solution.
CONTROL + Human serum with an ASLO activity > 200 IU/mL.
CONTROL - Animal serum with an ASLO activity < 100 IU/mL. 


Precautions: Components of different human origin have been tested and found to be negative for the presence of antibodies anti-HIV 1+2 and anti-HCV, as well as for HBsAg.  Do not allow to contact with skin or mucous membranes.  

EXPECTED VALUES: 95% of healthy adults have ASLO titers of 200 IU/mL or less, the highest titers been found in school children with titers up to 250 IU/mL. Since a single ASLO determination does not provide much information unless it is high, titrations at bi-weekly intervals for 4 to 6 weeks of the doubtful cases are advisable to follow the evolution of the disease.
CLINICAL SIGNIFICANCE: Elevated ASLO serum titers occur in response to infection with hemolytic streptococci of Group A, C and G, producers of streptolysin O, an extracellular protein of enzymatic character with strong antigenic properties. Immunochemical assay of these specific antibodies to streptococcal metabolites provide valuable information to establish a diagnosis of streptococcal infections (acute rheumatic fever, glomerulonephritis). ASLO testing has a high diagnostic value on a tentative diagnosis made on the basis of case history and clinical findings. 
ANALYTICAL PERFORMANCE: The minimum detectable unit (analytical sensitivity) is of approximately 200 IU/mL (± 50 IU/mL). 
LIMITATIONS OF PROCEDURE: Positive reactions do occur in conditions other than glomerulonephritis, in which the production of ASLO is especially high. In scarlet fever, early and acute periods of rheumatoid arthritis, healthy carriers, complicated and no complicated tonsillitis and various streptococcal infections increased ASLO levels have been found. Biologically false negative reactions can occur in early infections and during the early years of life (from six months to 2 years). The sensitivity of the test may be reduced at low temperatures. The best results are achieved at 15-25ºC. Delays in reading the results may result in over-estimation of the antibody present. 

CRP

C-reactive protein (CRP)  

Definition
    1. CRP is γ-globulin (in the γ-region); is found in various inflammatory diseases. This is also called acute-phase protein. 
    2. CRP is produced in the liver.
    3. CRP name is derived from the reaction with streptococcal capsular (C) polysaccharide. 

C-Reactive Protein (CRP)                                                                C-Reactive Protein (CRP)

CLINICAL SIGNIFICANCE: C-reactive protein is an acute phase protein present in normal serum, which increases significantly after most forms of tissue injuries, bacterial and virus infections, inflammation, and malignant neoplasia. CRP contributes to non-specific defense by complement activation and accelerating phagocytosis. CRP testing has a high diagnostic value on a tentative diagnosis made on the basis of case history and clinical findings. 

Principle:
1. CRP is opsonin and it activates the complement system and ultimately leads to lysis.  

C-Reactive protein (CRP) leads to lysis by the activation of complement                         C-reactive protein (CRP) leads to lysis by the activation of complement 

C-Reactive protein (CRP) role in Complement activation                                      C-reactive protein (CRP) role in Complement activation

2. Serum of the patient (CRP) + Somatic C polysaccharide of pneumococci mixed and gives rise to a precipitate.
3. Practically CRP is injected into the rabbit when the anti-CRP antibody is produced. Now take serum of the patient (CRP) + Mix anti-CRP. This will give precipitation. 
                                                                              
CRP + Anti CRP = Precipitation

C-Reactive Protein (CRP) formationC-Reactive Protein (CRP) formation 

Causes of CRP
    1. Produced in various bacterial diseases.
    2. Produced by injured myocardial muscle in myocardial infarction.
    3. Positive in acute and chronic rheumatic fever.

CRP is also positive in:
    1. Sydenham's Chorea.
    2. Myocardial infarction and negative in angina.
    3. Many malignancies.
    4. Rheumatoid arthritis.
    5. Gout.
    6. Viral infection like Viral Hepatitis.
    7. Bacterial Pneumonia.
    8. Active Tuberculosis.
    9. Lepromatous Leprosy.
    10. Acute Tonsillitis, Scarlet fever, and Mumps.

PRINCIPLE: CRP-Latex Test is a rapid slide agglutination procedure, developed for the direct detection and semi-quantitation of C-reactive protein (CRP) in serum. The assay is performed by testing a suspension of latex particles coated with anti-human CRP antibodies against unknown serum. The presence of a visible agglutination indicates an increase of the CRP level above the upper limit of the reference interval in the samples tested. 
REAGENT COMPOSITION - CRP-Latex Reagent: Suspension of polystyrene latex particles coated with specific anti-human C-reactive protein antibodies in a buffered saline solution. CONTROL + Human serum with a CRP concentration > 15 mg/L. CONTROL - Animal serum with a maximum concentration of human CRP of 1 mg/L. Precautions: Components of different human origin have been tested and found to be negative for the presence of antibodies anti-HIV 1+2 and anti-HCV, as well as for HBsAg. However, the controls should be handled cautiously as potentially infectious. 
EXPECTED VALUES: While the C-reactive protein concentration is generally below 5 mg/L in the sera of healthy adults, in a number of disease states these values often exceeded within 4 to 8 hours after an acute event and reach levels up to 500 mg/L. Since an elevated CRP level is always associated with pathological changes, determination of CRP is of great value in diagnosis, treatment and monitoring of inflammatory conditions. 
ANALYTICAL PERFORMANCE The minimum detectable unit (analytical sensitivity) is of approximately 6 mg/L (5-10 mg/L). Hemoglobin (<10 g/L), bilirubin (<20 mg/dL) and lipemia (<10 g/L) do not interfere. Rheumatoid factors (>100 IU/mL) interfere. Other substances may interfere. 

LIMITATIONS OF PROCEDURE  The presence of rheumatoid factors (RF) in a serum sample may cause false positive reactions. The sensitivity of the test may be reduced at low temperatures. The best results are achieved at 15-25ºC. Delays in reading the results may result in over-estimation of the CRP concentration. In case of positivity, use the titration/dilution procedure, as presented above, at the "ASLO/CRP/RF" section.   

RF

Rheumatoid Factor (RF)

The diagnosis of Rheumatoid arthritis is based on clinical assay, but the laboratory and radiological tests are useful to confirm clinical diagnosis and to evaluate the seriousness and the phase of the disease. Rheumatoid Factor (RF) in serum is one of the main clinical markers for rheumatoid arthritis. RF is a term used to describe a group of antibodies (IgM, IgG and IgE) directed towards the antigenic site on the Fc portion fragment of the human and animal body's own IgG antibodies. 

Rheumatoid Factor Testing: clinical testing supplies designed to identify the presence of Rheumatoid Factor (RF) in patient blood samples and aid in diagnosing rheumatoid arthritis. RF-latex serology kit test is an indirect agglutination kit for the detection of Rheumatoid Factor (RF) in human serum using latex particles coated with human IgG. 

PRINCIPLE: RF-Latex Test is a rapid agglutination procedure, developed for the direct detection and the semi-quantitation on a slide of rheumatoid factors (RF) in serum. Imuno Latex RF is a very sensitive test and is performed by testing a suspension of polystyrene latex particles coated with purified and stabilized human gamma globulin IgG and suspended in glycine buffer pH 8.2, against unknown serums. The reagent is based on an immunological reaction between human IgG bound to biologically inert latex particles and rheumatoid factors in the test specimen. The presence or absence of a visible agglutination indicates the presence or absence of RF in the samples tested. 

When serum containing rheumatoid factors is mixed with the latex reagent, visible agglutination occurs. The
 suspension of latex is mixed on a test card with serum containing elevated rheumatoid factor levels; a clear agglutination is seen in 2 minutes. 

The RF latex reagent sensitivity has been adjusted to detect a minimum of 8 IU/mL of rheumatoid factors according with the WHO International Standard without previous sample dilution. The regular kit contains: suspension of latex, positive control serum, negative control serum, stirrers, test card, instructions for use.
REAGENT COMPOSITION - RF-Latex Reagent: Suspension of polystyrene latex particles coated with human gamma globulin in a buffered saline solution. CONTROL + Human serum with an activity equivalent to appr. 25 IU/mL. CONTROL - Animal serum with an activity < 5 IU/mL. Precautions: Components of different human origin have been tested and found to be negative for the presence of antibodies anti-HIV 1+2 and anti-HCV, as well as for HBsAg.
EXPECTED VALUES: Of those patients with a clinical diagnosis of rheumatoid arthritis approximately 70-80% are seropositive for rheumatoid factor. Positive results were shown for nearly all patients with variants of rheumatoid arthritis. A positive result can be expected in less than 5% of healthy individuals, while in the population aged 60 years and older as many as 30% may be seropositive using latex tests for the detection of rheumatoid factor. 
CLINICAL SIGNIFICANCE: Rheumatoid factors are found in the sera of most patients with rheumatoid arthritis as well as in a variety of other diseases. Rheumatoid factors testing has a high diagnostic value on a tentative diagnosis made on the basis of case history and clinical findings. 
ANALYTICAL PERFORMANCE: The minimum detectable unit (analytical sensitivity) is of approximately 8 IU/mL (6-16 IU/mL).  
LIMITATIONS OF THE PROCEDURE: Positive reactions do occur in conditions other than rheumatoid arthritis such as mononucleosis, hepatitis C, syphilis, various other infections and in elderly patients. When tested by the quantitative test, however, most of these specimens give very low results. 

Rapid immunological tests - from blood, stool etc.

RAPID KIT TESTS

    These are commonly used in immunological diagnosis. The most frequent tests performed as rapid kit tests are for detecting: helicobacter pylori antigen (from stool) and antibody (from blood); hepatitis antibody/antigen (from blood); others: giardia lamblia antigen (from stool), troponin antibody (from blood), detection of parasites (from stool) and many others (from other fluids as well). 

    Serological detection techniques emphasize antibody presence. They are highly used in clinical labs as immunological rapid tests, rapid kit tests that can also identify antigenic presence. The most common technique that such rapid kits use is the immunochromatography method of detection. A certain amount of blood (upon kit specification) is released on the cassette which may already have the reagent added. In some cases (kit specification) an amount of reagent is also added, along with the blood (serum/whole/venepuncture blood, upon kit specification).

    The quick test devices are designed for the in-vitro diagnosis use only; the cassette has a sample hole made of the material enabling the reagent to proceed. The membrane that constitutes the test device has been formed ultimately once the specific antigens/antibodies are passed through the test band space as well as the monoclonal antibodies specific to various pathogens under analysis are passed through the front cover. 

    The antibody reaction takes part in the end section of buffer membrane contenting the golden resultant. In case the antigens are present in the patient's sample, it dissolves in the solution included in the sampling bottle then proceeds by using the mixture form of chromatographically antigen-antibody-antigen golden particles towards test space (T) in order to form a visible line. Thus, a visible line appearance in the T space confirms the positive result in the detection of the pathogen. Otherwise, this visible line shall not appear. Unless this visible line is seen in T space, the result is negative. However, there is a color line every time in the control space (C). This control line is a procedural indicator. It confirms that the sufficient sample solution is dropped, the sample expands in the test properly and reagent is under control. 





HEPATITIS: A, B, C, D, E

HEPATITIS can be caused by A (HAV), B (HBV), C (HCV), D (HDV) or E (HEV) viruses.


Hepatitis A virus:

- highlighting acute-phase anti-HAV IgM antibodies by ELISA method and other methods (transaminases go up as well); acute-phase antibodies from the debut and last for about 10 weeks;

- after 10 weeks: anti-HAV IgG that lasts until the end of life, their presence reflecting either disease or vaccine. 


Hepatitis B virus:

- serological markers associated with hepatitis B virus infection:

        - AgHBs - HBV infection; from incubation until 3-4 months after infection;

        - AgHBe - along with HBV DNA they appear in serum immediately after AgHBs; indicators of active virus replication;

        - anti-HBc antibodies - undefined persistence; anti-HBc IgM are serum detectable a bit before the clinical debut, along with transaminases rise; indicators of acute phase;

        - anti-HBe antibodies - self-limiting infections;

        - anti-HBs antibodies - 4-6 months after infection; on curing; virus replication stop markers;

- HBV DNA copy-number = most sensible marker of active HBV replication. 


Hepatitis C virus: 

- anti-HCV antibodies - from blood; 

- RT-PCR to count HCV RNA: most accurate indicator. 


What is the difference between hepatitis A, hepatitis B, and hepatitis C? 
Hepatitis A, hepatitis B, and hepatitis C are liver infections caused by three different viruses. Although each can cause similar symptoms, they are spread in different ways and can affect the liver differently. Hepatitis A is usually a short-term infection and does not become chronic. Hepatitis B and C can also begin as short-term, acute infections, but in some people, the virus remains in the body, resulting in chronic disease and long-term liver problems.

Hepatitis D virus: 

- anti-HDV IgM antibodies - from blood; 

- HDV RNA in serum.


Hepatitis E virus: 

- anti-HEV antibodies - from blood; 

- RT-PCR to detect the HEV RNA in blood and stool.

HEPATITIS A VIRUS - detection from blood

HEPATITIS A VIRUS (HAV)

OVERVIEW

Hepatitis A: inflammation of the liver, causing mild to severe illness. It is a highly contagious, short-term liver infection, caused by the hepatitis A virus (HAV). When the liver is inflamed or damaged, its function can be affected. Heavy alcohol use, toxins, some medications, and certain medical conditions can cause hepatitis, but it is often caused by HAV. Hepatitis A is vaccine-preventable.


HAV is a member of the Picornaviridae family . HAV is primarily transmitted from person-to-person by the fecal-oral route either through person to person contact or consumption of contaminated food or water. The disease is closely associated with unsafe water or food, inadequate sanitation, poor personal hygiene and oral-anal sex. Although hepatitis A is not ordinarily a sexually transmitted disease, the infection rate is high among men who have sex with men due to oral-anal contact. HAV is found in the stool and blood of people who are infected. HAV is spread when someone unknowingly ingests the virus - even in microscopic amounts - through close personal contact with an infected person or through eating contaminated food or drink.  

Almost everyone recovers fully from hepatitis A, with a lifelong immunity. However, a very small proportion of people infected with HAV could die from fulminant hepatitis. Safe and effective vaccines are available to prevent hepatitis A. Unlike hepatitis B and C, hepatitis A does not cause chronic liver disease, but it can cause debilitating symptoms and rarely fulminant hepatitis (acute liver failure), which is often fatal.

Hepatitis A occurs sporadically and in epidemics worldwide, with a tendency for cyclic recurrences. Epidemics related to contaminated food or water can erupt explosively. They can also be prolonged, affecting communities for months through person-to-person transmission. 

GEOGRAPHICAL DISTRIBUTION

Geographical distribution areas can be characterized as having high, intermediate or low levels of HAV infection. However, infection does not always mean disease, because infected young children do not experience any noticeable symptoms. Infection is common in low- and middle-income countries with poor sanitary conditions and hygienic practices, and most children (90%) have been infected with the HAV before the age of 10 years, most often without symptoms. Infection rates are low in high-income countries with good sanitary and hygienic conditions.

Disease may occur among adolescents and adults in high-risk groups, such as people who inject drugs, men who have sex with men, people travelling to areas of high endemicity and in isolated populations. In developed countries, large outbreaks have been reported among people experiencing homelessness. In middle-income countries and regions where sanitary conditions are variable, children often escape infection in early childhood and reach adulthood without immunity.    

TRANSMISSION/EXPOSURE
 
HAV is primarily transmitted by the faecal-oral route; that is when an uninfected person ingests food or water that has been contaminated with the faeces of an infected person. In families, this may happen through dirty hands when an infected person prepares food for family members. 
The fecal-oral route of transmission can happen through:
- close person-to-person contact with an infected person
- sexual contact with an infected person
- ingestion of contaminated food or water
Waterborne outbreaks, though infrequent, are usually associated with sewage-contaminated or inadequately treated water. The virus can also be transmitted through close physical contact (such as oral-anal sex) with an infectious person.
Although viremia occurs early in infection, bloodborne transmission of HAV is known to be uncommon.  
Can a person spread HAV without having symptoms?
Yes. Many people, especially children, have no symptoms but can still spread the infection. A person can transmit HAV to others up to 2 weeks before symptoms appear. 

How is hepatitis A spread?
The HAV, found in the stool and blood of infected people, is spread when someone ingests the virus. 
- Person-to-person contact
Hepatitis A can be spread from close, personal contact with an infected person. Hepatitis A is very contagious, and people can even spread the virus before they feel sick. 
- Eating contaminated food or drink
Contamination of food with the HAV can happen at any point: growing, harvesting, processing, handling, and even after cooking. Although uncommon, foodborne outbreaks have occurred in the developed countries from people eating contaminated fresh and frozen imported food products.   
What should I do if I think I have been exposed to hepatitis A virus?
If you think you have been exposed to the hepatitis A virus, call your health professional or your local or state health department as soon as possible, ideally within 2 weeks. A health professional can decide next steps based on your age and overall health.
Can I prevent infection after an exposure to the hepatitis A virus?
A single shot of the hepatitis A vaccine can help prevent hepatitis A if given within 2 weeks of exposure. Depending upon your age and health, your doctor may recommend immune globulin in addition to the hepatitis A vaccine.
If I have had hepatitis A in the past, can I get it again?
No. Once you recover from hepatitis A, you develop antibodies, protecting you for life.   
How long does hepatitis A virus survive outside the body?
The hepatitis A virus can survive outside the body for months. Heating food and liquids to high temperatures can kill the virus.

SYMPTOMS

The incubation period of hepatitis A is usually 14-28 days. The symptoms can last up to 2 months and can be varied: 10-15% of symptomatic people have prolonged or relapsing disease for up to 6 months.
Symptoms of hepatitis A range from mild to severe and can include fever, malaise and/or fatigue, loss of appetite, diarrhea, nausea, abdominal discomfort (e.g.: stomach pain), dark-colored urine and jaundice (a yellowing of the eyes and skin). Not everyone who is infected will have all the symptoms. 
Adults have signs and symptoms of illness more often than children. The severity of disease and fatal outcomes are higher in older age groups. Infected children under 6 years of age do not usually experience noticeable symptoms, and only 10% develop jaundice. Hepatitis A sometimes relapses, meaning the person who just recovered falls sick again with another acute episode. This is normally followed by recovery. 
Most people with hepatitis A do not have long-lasting illness. The best way to prevent hepatitis A is to get vaccinated. Among older children and adults, infection is typically symptomatic. Symptoms usually occur abruptly and can include the following: fever, fatigue, loss of appetite, nausea, vomiting, abdominal pain, dark urine, diarrhea, clay/light-colored stool, joint pain, jaundice. Most (70%) of infections in children younger than the age of 6 are not accompanied by symptoms. When symptoms are present, young children typically do not have jaundice; most (>70%) older children and adults with HAV infection have this symptom.  
People who get hepatitis A may feel sick for a few weeks to several months but usually recover completely and do not have lasting liver damage. In rare cases, hepatitis A can cause liver failure and even death; this is more common in older people and in people with other serious health issues, such as chronic liver disease. 
Not everyone with hepatitis A has symptoms. Adults are more likely to have symptoms than children. If symptoms develop, they usually appear 2 to 7 weeks after infection. Symptoms usually last less than 2 months, although some people can be ill for as long as 6 months. 

WHO IS AT RISK?

Anyone who has not been vaccinated or previously infected can get infected with HAV. In areas where the virus is widespread (high endemicity), most hepatitis A infections occur during early childhood. Although anyone can get hepatitis A, certain groups of people are at higher risk for getting infected and for having severe disease if they do get hepatitis A.
Risk factors include: 
- poor sanitation;
- lack of safe water;
- living in a household with an infected person;
- being a sexual partner of someone with acute hepatitis A infection;
- use of recreational drugs;
- sex between men;
- travelling to areas of high endemicity without being immunized;
- people with occupational risk for exposure;
- people with chronic liver disease, including hepatitis B and C;
- people with HIV.
The last 2 are people at increased risk for severe disease from hepatitis A infection.

INCUBATION PERIOD FOR HAV: 28 days (range: 15-50 days). 

DIAGNOSIS

How is hepatitis A diagnosed?

A doctor can determine if you have hepatitis A by discussing your symptoms and ordering a blood test that can tell whether you have been recently infected with the virus that causes hepatitis A.  
Cases of hepatitis A are not clinically distinguishable from other types of acute viral hepatitis. Specific diagnosis is made by the detection of HAV-specific immunoglobulin IgM antibodies in the blood. Additional tests include reverse transcriptase polymerase chain reaction (RT-PCR) to detect the hepatitis A virus RNA and may require specialized laboratory facilities.
The case definition for acute hepatitis A:
- clinical criteria: an acute illness with a discrete onset of any sign or symptom consistent with acute viral hepatitis (e.g., fever, headache, malaise, anorexia, nausea, vomiting, diarrhea, abdominal pain, or dark urine)
AND
a) jaundice or elevated total bilirubin levels ≥ 3.0 mg/dL, OR
b) elevated serum alanine aminotransferase (ALT) levels >200 IU/L
AND
c) the absence of a more likely diagnosis


LABORATORY CRITERIA FOR DIAGNOSIS
Confirmatory laboratory evidence:
- Immunoglobulin M (IgM) antibody to hepatitis A virus (anti-HAV) positive, 
OR
- Nucleic acid amplification test (NAAT; such as polymerase chain reaction [PCR] or genotyping) for hepatitis A virus RNA positive.

CASE CLASSIFICATION - CONFIRMED
- A case that meets the clinical criteria and is IgM anti-HAV positive+ OR 
- A case that has hepatitis A virus RNA detected by NAAT (such as PCR or genotyping ) OR
- A case that meets the clinical criteria and occurs in a person who had contact (e.g., household or sexual) with a laboratory-confirmed hepatitis A case 15-50 days prior to onset of symptoms 

CAN HEPATITIS A BECOME CHRONIC?
No. Hepatitis A does not become a chronic, long-term infection. 

CAN SOMEONE BECOME RE-INFECTED WITH THE HEPATITIS A VIRUS?
No. Immunoglobulin G antibodies (IgG) to the hepatitis A virus, which appear early in the course of infection, provide lifelong protection against the disease.  

Hepatitis A virus detection:

- highlighting acute-phase anti-HAV IgM antibodies by ELISA method (transaminases go up as well); acute-phase antibodies appear from the debut and last for about 10 weeks;

- after 10 weeks: anti-HAV IgG that lasts until the end of life, their presence reflecting either disease or vaccine. 

HAV IgG/IgM Rapid Test example: The onsite HAV IgG/IgM Rapid Test is usually a lateral flow chromatographic immunoassay for the qualitative detection and differentiation of antibodies (IgG and IgM) to Hepatitis A virus in human serum, plasma or whole blood. It is intended to be used as a screening test and provides a preliminary test result to aid in the diagnosis of HAV infection.

The presence of anti-HAV IgM in blood samples suggests an acute or recent HAV infection. In most infected individuals, anti-HAV IgM rapidly increases in titer over a period of 4-6 weeks post infection and then declines to non-detectable levels within 3 to 6 months. Anti-HAV IgG can be detected at the onset of symptoms and remain elevated throughout an individual's life. Protective immunity from an infection with HAV is indicated by an anti-HAV IgG level  20-33 mIU/mL, though the presence of anti-HAV IgG  20-33 mIU/mL does not necessarily ensure protection from a future HAV infection. A patient without protective levels of anti-HAV IgG (<20-33 mIU/mL) is considered a risk of acquiring an HAV infection. 

The OnSite HAV IgG/IgM Rapid Test can usually be performed within 15 minutes by minimally skilled personnel without the use of laboratory equipment. Test principle: the test strip in the cassette device consists of: 1) a burgundy coloured conjugate pad containing HAV antigens conjugated with colloidal gold (HAV conjugates) and a control antibody conjugated with colloidal gold; 2) a nitrocellulose membrane strip containing two test lines (G and M lines) and a control line (C line). The G line is pre-coated with mouse anti-human IgG for detection of anti-HAV IgG. The M line is pre-coated with mouse anti-human IgM for detection of anti-HAV IgM. The C line is pre-coated with a control antibody. 

When an adequate volume of test specimen and sample diluent is dispensed into the sample well and buffer well, respectively, the specimen migrates by capillary action across the test strip. If anti-HAV IgG is present in the specimen, it will bind to the HAV conjugates. The immunocomplex is then captured on the membrane by the pre-coated mouse anti-human IgG forming a burgundy coloured G-line, indicating an HAV IgG positive test result. If anti-HAV IgM is present in the specimen it will bind to the HAV conjugates. The immunocomplex is then captured on the membrane by the pre-coated mouse anti-human IgM forming a burgundy coloured M line, indicating an HAV IgM positive test result.

Absence of any test lines (G or M) suggests a negative result. The test contains an internal control (C line) which should exhibit a burgundy coloured line of the immunocomplex of the control antibodies, regardless of colour development of the test lines (G and M). If no control line (C line) develops, the test result is invalid and the specimen must be retested with another device.


TREATMENT

There is no specific treatment for hepatitis A. Recovery from symptoms following infection may be slow and can take several weeks or months. Hospitalization is unnecessary in the absence of acute liver failure. Therapy is aimed at maintaining comfort and adequate nutritional balance, including replacement of fluids that are lost from vomiting and diarrhea.


HOW IS HAV INFECTION PREVENTED?

Vaccination with the full, two-dose series of hepatitis A vaccine is the best way to prevent infection. Hepatitis A vaccines are available for use in people 1 year of age and older. 
Immune globulin can provide protection against hepatitis A, both pre- and postexposure.

PREVENTION

Improved sanitation, food safety and immunization are the most effective ways to combat hepatitis A.
The spread of hepatitis A can be reduced by:
- adequate supplies of safe drinking water;
- proper disposal of sewage within communities; and
- personal hygiene practices such as regular handwashing before meals and after going to the bathroom. 

HEPATITIS A VACCINATION


WHO SHOULD BE VACCINATED AGAINST HEPATITIS A?
Children
- All children aged 12-23 months
- Unvaccinated children and adolescents aged 2-18 years old
People at increased risk for HAV infection
- International travelers
- Men who have sex with men
- People who use injection or noninjection drugs 
- People with occupational risk for exposure
- People who anticipate close personal contact with an infected person
- People experiencing lack of hygienic conditions
People at increased risk for severe disease from HAV infection
- People with chronic liver disease 
- People with human immunodeficiency virus infection
Other people recommended for vaccination
- Pregnant women at risk for HAV infection or severe outcome from HAV infection
- Any person who requests vaccination
Vaccination during outbreaks
- Unvaccinated people in outbreak settings who are at risk for HAV infection or at risk for severe disease from HAV

What if an infant receives the first dose of hepatitis A vaccine at an age younger than 12 months?
Although no known harm is associated with giving hepatitis A vaccine to infants, the hepatitis A vaccine dose(s) administered prior to 12 months of age might result in a suboptimal immune response, particularly in infants with passively acquired maternal antibody. Therefore, hepatitis A vaccine dose(s) administered at <12 months of age are not considered valid doses.  
The two-dose hepatitis A vaccine series should be initiated when the child is at least 1 year of age.  
How long does protection from hepatitis A vaccine last?
The exact duration of protection against hepatitis A virus infection after vaccination is unknown.
Anti-HAV has been shown to persist for at least 20 years in most people receiving the 2-dose series as infants <2 years of age, those vaccinated with a 3-dose series as young children (aged 3-6 years), and adults receiving the entire vaccine series during adulthood.  
Can hepatitis A vaccine be administered concurrently with other vaccines?
Yes. Hepatitis B, diphtheria, poliovirus (oral and inactivated), tetanus, typhoid (oral and intramuscular), cholera, rabies, and yellow fever vaccines can be given at the same time that hepatitis A vaccine is given.
In studies among young children, simultaneous administration of hepatitis A vaccine did not affect the immunogenicity or reactogenicity of diphtheria-tetanus-acellular pertussis; inactivated polio; measles, mumps, rubella (MMR); hepatitis B; and Haemophilus influenzae type b vaccines.  
Can a patient receive the first dose of hepatitis A vaccine from one manufacturer and the second (last) dose from another manufacturer?
Ideally, doses of vaccine in a series come from the same manufacturer; however, if this is not possible or if the manufacturer of doses given previously is unknown, providers should administer the vaccine that they have available. The dose should be considered valid and does not need to be repeated.
What should be done if the second (last) dose of hepatitis A vaccine is delayed?
The second dose should be given as soon as possible. Even if the second dose is delayed, the first dose does not need to be repeated.   

IMMUNE GLOBULIN

What is immune globulin?
Immune globulin is a substance made from human blood plasma that contains antibodies, which are the body's natural defense against infection. Injections of immune globulin may be given under certain circumstances, like when someone is too young to get vaccinated or can't get vaccinated because of a previous, life-threatening reaction to the hepatitis A vaccine or vaccine component. Unlike the hepatitis A vaccine, immune globulin does not provide long-term protection against infection. 


POSTEXPOSURE PROPHYLAXIS FOR HEPATITIS A

What should be done when a case of hepatitis A is found in a setting providing services to children or adults (e.g., a school, hospital, office setting, corrections facility, or homeless shelter)?
- Postexposure prophylaxis (PEP) is not routinely indicated when a single case occurs in a school or work setting and the source of infection is outside of the setting. 
- When a person who has HAV infection is admitted to a hospital, staff members should not routinely be administered PEP; instead, appropriate infection control practices should be emphasized. 
- PEP should be administered to people who have close contact with index patients if an epidemiologic investigation indicates HAV transmission has occurred among students in a school or among patients or between patients and staff members in a hospital. 
- PEP should be considered for all previously unvaccinated residents and employees when a confirmed hepatitis A case occurs in a setting where close personal contact occurs regularly and hygiene standards are difficult to maintain (e.g., correctional facility, homeless shelter, various facilities). In a setting containing multiple enclosed units or sections (e.g., prison ward), PEP administration should be limited only to people in the area where there is exposure risk.

Do immunocompromised people require additional protection after being exposed to someone with hepatitis A?
Yes. People who are immunocompromised or have chronic liver disease and who have been exposed to hepatitis A virus within the past 2 weeks and have not previously completed the 2-dose hepatitis A vaccination series should receive both immune globulin and hepatitis A vaccine simultaneously in a different anatomic site (e.g., separate limbs) as soon as possible after exposure. When the dose of hepatitis A vaccine administered is the first dose the exposed individual has received, a second dose should be administered 6 months after the first for long-term protection.

Should pregannt women at increased risk for exposure to the hepatitis A virus (HAV) receive postexposure prophylaxis?  
Women with increased likelihood of exposure to HAV during pregnancy can be administered immune globulin. There has been no observed increase in maternal or infant adverse events after hepatitis A vaccination or IG administration in pregnancy. 

HEPATITIS A AND INTERNATIONAL TRAVEL 

Who should get the hepatitis A vaccine before travelling internationally?
All unvaccinated people, along with those who have never had hepatitis A, should be vaccinated before traveling to countries where hepatitis A is common. Travelers to countries where hepatitis A is common are still at risk. International travelers have been infected, even though they regularly washed their hands and were careful about what they drank and ate. Those who are too young or can't get vaccinated because of a previous, life-threatening reaction to the hepatitis A vaccine or vaccine component should receive immune globulin.

How soon before travel should I get the hepatitis A vaccine?
You should get the first dose of hepatitis A vaccine as soon as you plan international travel to a country where hepatitis A is common. The vaccine will provide some protection even if you get vaccinated closer to departure. For older adults (age >40 years), people who are immunocompromised, and people with chronic liver disease or other chronic medical conditions the health-care provider may consider, based on several factors, giving an injection of immune globulin at the same time in different limbs. 

What should I do if I am traveling internationally but cannot receive hepatitis A vaccine?
People who are allergic to a vaccine component or are younger than 6 months should receive a single dose of immune globulin before traveling to a country where hepatitis A is common. Immune globulin provides effective protection against hepatitis A virus infection for up to 2 months, depending on the dosage given. If you are staying longer than 2 months, you can get another dose of immune globulin during your visit for continued protection against hepatitis A.