4. chapter 4 serology of viruses.pdf

Chapter 4 Serology for viral infection Viruses  Obligate Intracellular Parasites  only demonstrate characteristics of life while “inside” a host cell  Outside a host cell, inert, no enzyme or other activity  Inside a host cell – viral Nucleic Acid (DNA or RNA) takes over the c...

Chapter 4 Serology for viral infection Viruses  Obligate Intracellular Parasites  only demonstrate characteristics of life while “inside” a host cell  Outside a host cell, inert, no enzyme or other activity  Inside a host cell – viral Nucleic Acid (DNA or RNA) takes over the cell and directs the cell to produce new virus particles (replication) HIV Serology  HIV weakens the immune system, making it difficult to fight off infections.  If left untreated, it can lead to AIDS (acquired immune deficiency syndrome).  HIV is passed on in blood and other body fluids through sexual contact or infected needles.  HIV can be passed on to your baby during pregnancy, birth or breastfeeding if it's not treated.  Laboratory method – Rapide test, ELISA , Rt-PCR Common HIV antibody test A. Enzyme Linked Immunosorbent Assays (ELISA)  Indirect ELISA B. Rapid Tests C. Western Blot Rapid Assay: tests Principle  An antigen is coated on the strip with positive control.  Up on addition of serum /plasma or whole blood depending on the test procedure , there will be reaction between antigen and antibody if present in the sample.  Postive results : two/three colored bars (one for the control & the other for the patients that if the kite screens the two sero types HIV-1 and HIV-2)  Negative: single colored bar ( positive control only )  Invalid result : without having any line. Previous HIV kit Algorithm Then, KHB HIV test algorithm Blood Sample Test 1 (KHB) Non-reactive Reactive Report Negative Test 2 (Stat Pak) Non Reactive Reactive Report Positive Test 3 (Unigold) Reactive Result Non-reactive Result Report Positive Report Negative Next to KHB kit, Wanti beijing HIV test algorithm Current one Immunoassay HIV 1/2 Ab kit current HIV rapid tests kits SD-Bioline-HIV-rapid-diagnostic-test Possible-results-for-the-three-line-SD-Bioline-HIV-rapid- diagnostic-test-RDT-that Positive Negative Read results in 10 -12 minutes  Western blot assay  Western blot assay is the most widely accepted confirmatory assay for the detection of HIV.  In the western blot procedure, purified HIV-1 viral antigens are electrophoresed on sulfate polyacryamide gel and the separated polypeptides are then transferred on to sheets of nitrocellulose paper incubated with the serum specimen.  Any antibody that binds to the separated peptides present on the nitrocellulose paper is detected by a secondary antihuman antibody, conjugated to enzyme substrate.  Antibody specificities against known viral components are considered true positive results  laboratory markers are routinely used to monitor patients with HIV infection for disease progression and guide their treatments o peripheral blood CD4 T-cell count - Flowcytometry o CD4+ T cell counts are below 200/µL being categorized as progressing to AIDS o HIV-1 RNA level, or “viral load - Rq PCR o highly sensitive; it is able to detect HIV RNA o HIV RNA levels become detectable about 10 days after infection  Serological tests are not reliable in detecting HIV infection in children younger than 18 months of age ??? Why Serology of Dengue fever  INTRODUCTION  Dengue is a mosquito-borne Viral disease found in tropical and sub-tropical regions around the world.  It is transmitted by the bite of infected female Aedes Aegypti mosquito  which is a day biting mosquito  can breed in small collection of water.  Rainy season increase risk of DF.  Dengue virus (DEN) is an RNA virus belong to the genus Flavi virus, family Flavi viridae.  spherical with a diameter of 50nm.  There are four antigenically distinct serotypes of the virus that cause dengue  DEN-1, DEN-2, DEN-3,DEN-4  infection with one type usually gives lifelong immunity to that type  Dengue Fever(DF)  high fever (40°C/ 104°F)  severe headache  pain behind the eyes  muscle and joint pains  nausea, vomiting  swollen glands or rash  Self limiting Symptoms usually last for 2–7 days  Laboratory diagnosis of dengue Detection of the virus (cell culture) Detection of antigens. Detection of viral RNA(within 24–48 hours) Detection of anti-dengue antibodies. Antigen detection  NS1(glycoprotien) secreted by the virus  NS1 antigen can be detected with rapid kits within a few hours.  Used in field settings and provide results in less than an hour.  It Can be also detected by ELISA  NS1 produces a very strong humoral response.  Many studies have been directed at using the detection of NS1 to make an early diagnosis of dengue virus infection.  high concentrations of these antigens could be detected in patients with both primary and secondary dengue infections up to nine days after the onset of illness.  Antibody Detection  IgM antibodies are the first immunoglobulin isotype to appear.  Are detectable in 50% of patients by days 3-5 after onset of illness, increasing to 80% by day 5 and 99% by day 10  Anti-dengue IgM positivity suggests a recent dengue infection.  IgM levels peak about two weeks after the onset of symptoms and then decline generally to undetectable levels over 2–3 months.  Anti-dengue serum IgG is generally detectable at low titres at the end of the first week of illness, increasing slowly thereafter, with serum IgG still detectable after several months, and probably even for life.  IgG positivity may only indicate infection at an indeterminate time in the past.(unless tittered)  The Flavi virus genera share epitopes inducing cross-reactive antibodies  Leading to great difficulty in differentially diagnosing flavi viral infections.  Rapid antibody test 1.Add 10μl serum, plasma or whole blood to sample well. 2. Add 2 drops buffer to sample READ RESULTS AFTER 15 MINUTES well. C No C Secondary IgM antibodies IgM dengue IgG detected IgG C C Primary Secondary IgM IgM dengue dengue IgG IgG Source: Monica cheesbrough part II. 2006 IgM Result IgG Result Possible Interpretation Positive Negative Current infection Positive Positive Current infection negative Positive Past infection Negative Negative Too soon after initial exposure for antibodies to develop or symptoms due to another cause Serology of hepatitis tests Introduction  Hepatitis is a generic term referring to an inflammation of liver.  It can be caused by several viruses  Some viruses primarily infect the liver and are called hepatotrophic viruses.  These includes hepatitis A, B, C, D, E  Hepadnaviridae family Hepatitis A virus  RNA virus  Spread by feco-oral route  Blood exposure (rare) (e.g.injecting drug use, transfusion)  Has an incubation period of approximately one month  Doe not cause chronic liver disease  Has vaccine (formalin-killed HAV) Hepatitis B virus  DNA virus  Spread by- blood and contaminated needle - Sexual contact - perinatal route-during delivery  It has an incubation period of approximately 3 months.  Major cause of morbidity and mortality throughout the world(350 million chronic and 1.2 million death)  It is more infectious than HIV  The risk of HBV transmission is 6 to 30%  licensed Vaccine( recombinant HBsAg) Hepatitis C virus  RNA virus  account 90% of transfusion associated hepatitis  Only 1 to 3 percent of cases progress to a chronic state.  While, in super-infections results a greater risk of developing chronic liver disease with an accelerated progression toward cirrhosis, liver decompensation, and hepatocellular carcinoma Hepatitis D virus  Defective virus that replicates in only HBV infected cells  RNA virus  Similar route of transmission with HBV Hepatitis E virus  RNA virus  Has feco- oral route of transmission  causes sever infection in pregnant ladies when changes in the immune response occur  Markers for hepatitis B virus 1. Hepatitis B surface antigen (HBsAg)  The first marker to appear, becoming detectable 2 to 10 weeks after exposure to HBV.  levels peak during the acute stages of infection, then gradually decline as the patient develops antibodies to the antigen.  found in noninfectious spherical and tubular particles that lack viral DNA and circulate freely in the blood.  Serum HBsAg usually becomes undetectable by 4 to 6 months after the onset of symptoms in patients with acute hepatitis B.  In patients with chronic HBV infection, HBsAg remains elevated for 6 months or more.  HBsAg is thus an indicator of active infection and is an important marker in Detecting initial infection, monitoring the course of infection and progression to chronic disease, and screening of donor blood. 2. Hepatitis B core antigen (HBcAg)  It is the inner core protein of HBV  It is located on the surface of the nucleo-capsid core (the inner most layer of the hepatitis b virus)  HBcAg is not secreted, does not circulate in the blood.  It is not detectable in serum  But antibodies produced against HbcAg i.e. (anti-HBc) can be detected  novel biomarker that has an important role in CHB, because it correlates with serum HBV DNA 3. Hepatitis Be antigen (HBeAg)  secreted protein;  appears shortly after HBsAg (during acute infection) and disappears shortly before HBsAg in recovering patients.  It may be elevated during chronic infection.  usually reliable diagnostic indication of active infection.  Serological test for HBsAg 1. Reverse passive latex agglutination  Principle: The latex particle coated with anti HBsAg is mixed with the patient serum and observed for the occurrence of agglutination  If erythrocytes are employed for the procedure. The processes called reveres passive hemagglutination.  2. Chromatographic technique  It is a qualitative detection of HBsAg in serum or plasma  The membrane is pre coated with antiHBsAg on the test line region of the strip  Serum or plasma migrates up ward by capillary action to reach the test and control areas and forms a colored line.  3. ELISA  Serological markers for hepatitis B are most commonly detected by ELISA  Principle – wells of a micro titer plate coated with mouse monoclonal antibody to HBsAg  If patient’s serum or plasma contains HBsAg, it bind with the solid phase antibody and anti- HBsAg conjugated with horse reddish peroxidase (HRPO)is added.  The substrate for HRPO is O-phenylene diamine (OPD) added on the preparation.  The presence of HBsAg in patient’s serum is detected by the color change after the addition of specific substrate. 4. PCR Molecular test (q PCR) - a confirmatory test that detects HBV DNA. Antibody testing for HCV  The diagnosis of hepatitis C infection is usually made serologically by detecting anti-HCV IgG in serum.  Antibody is detectable 6–8 weeks after infection.  Detecting anti-HCV antibody is also used to screen donor blood.  ELISA and rapid anti-HCV antibody tests to diagnose hepatitis C and screen blood for HCV are commercially available.  which use recombinant and synthetic antigens developed from the conserved domains of the C, NS3, NS4, and NS5 proteins. HAV, HDV, HEV  Hepatitis A can be diagnosed serologically by detecting HAV-specific IgM antibody which appears in the serum at the onset of jaundice and persists for about 10 weeks.  Antigen and antibody tests are available to diagnose hepatitis D and hepatitis E, but the assays are expensive and usually performed only in specialist laboratories.