3. chap 3 serology of bacterial infection.pptx

Chapter 3. Common Serologic Tests for Bacterial infection Syphilis serology 1 Menber 08/11/2024 Syphilis syphilis is a sexually transmitted disease caused by the spirochete Treponema pallidum cxcs Gram negative motile Spi...

Chapter 3. Common Serologic Tests for Bacterial infection Syphilis serology 1 Menber 08/11/2024 Syphilis syphilis is a sexually transmitted disease caused by the spirochete Treponema pallidum cxcs Gram negative motile Spirochaetes Do not grow in artificial medium  Cannot be seen by light microscopy because they are very thin (0.15 μm), long 5-15 μm. 2 Menber 08/1 1/20   but can be seen by  Dark field microscopy  Phase contract technique  Can be stained by  Silver impregnation  Fluorescent antibody technique  Sensitive to penicillin 3 Menber 08/11/2024  Epidemiology The incidence of syphilis decreased due to penicillin in the 1940s There are more than 70,000 new cases of syphilis each year Syphilis, chronic and slowly progressive, is the third most common sexually transmitted disease. 4 Menber 08/11/2024  Mode of Transmission Direct sexual contact (90 – 96%) Blood transfusion Via placenta from infected pregnant mother faetus causes congenital syphilis. accidental contact E.g. Medical personnel. Source of T. pallidum: Primary and secondary syphilis lesions. 5 Menber 08/11/2024  Clinical Features of Syphilis / Symptoms and signs of Acquired syphilis Incubation Period: 10 – 90 days (average – 21 days) 6 Menber 08/11/2024 1- Primary syphilis:  most often associated with a single painless chancre  develops after 2-10 weeks  a well defined indurated painless ulcer mainly on the genitalia (90%) and extra-genital on Lips (5-10%). In female, chancre occurs in the cervix. The chancre is painless and exudate （ discharge) is formed in the centre. 7 Menber 08/11/2024  This fluid is highly infectious and examination by dark field microscopes shows Spirochaetes. Primary chancre heals spontaneously without treatment within 3-8 weeks. Serological tests for Syphilis are positive in 80% cases. 8 Menber 08/11/2024  2- Secondary Syphilis: Happens After 6-8 weeks of primary chancre: Disseminated secondary stage develops. Muco-cutaneous lesion occurs e.g. Skin rash, mucosal ulcers, condylomata on genitalia, Lymphadenopathy, fever, headache malaise. 9 Menber 08/11/2024  Secondary syphilis is highly infectious  Serological tests for syphilis becomes almost uniformly positive. Secondary Stage may follows by the following: a) Cured spontaneously b) Early latent c) Late Latent d) Tertiary stage 10 Menber 08/11/2024  After the secondary syphilis symptoms subsides, the disease enters a latent stage Latent Stage: is defined by a positive serology in the absence of clinical disease Early latent syphilis is latent syphilis where infection occurs within the past 12 months. Late latent syphilis is latent syphilis where infection occurs more than 12 months ago 11 Menber 08/11/2024 3- Tertiary Syphilis: After 2-20 yrs, tertiary stage develops produces Gummatous Lesions (contains a mass of dead and swollen fiber-like tissue). perforation of the palate (Roof of the mouth Skin Bone Joints Charcoat’s joints 12 Menber 08/11/2024  Tertiary or late syphilis After 2-20 yrs, tertiary stage develops Tertiary stage is not infectious. is classified into  gummatous syphilis cardiovascular syphilis, and neurosyphilis. 13 Menber 08/11/2024  Congenital Syphilis: most distressing and dangerous form of Syphilis.  Occurs in 2nd and 3rd trimesters  10% 18 – 22 weeks  50% >23 weeks  Untreated 1o and 2o syphilis in pregnancy affects almost 100% fetuses with 50% premature delivery or fetal death  Early latent syphilis - 40% premature delivery or fetal death  May remain infectious to fetuses for many years 14 Menber 08/11/2024   Early Congenital syphilis: a) Skin rash b) Mucocutaneous lesions c) Hepatospleenomegaly d) Lymphadenopathy  Late Congenital Syphilis: a) Deafness b) Bone sclerosis, Arthritis c) Damage of Mental development and other neurological symptoms. d) Stillbirths 15 Menber 08/11/2024  Laboratory Diagnosis of Syphilis The Common Methods serologic tests for syphilis are important because Unlike most bacteria, T. pallidum subspecies cannot be readily isolated or sustained in cell culture a specimen source is not available in the latent and late stages, since lesions are absent. 16 Menber 08/11/2024 8   Two classes of serologic tests Non-treponemal Treponemal 17 Menber 08/11/2024  Serologic Tests for Syphilis: Non-Treponemal Assays Principle: T. pallidum infection leads to the production of reagin Reagin – Antibodies to substances released from cells damaged by T. pallidum This antibody is IgM and IgG antibodies The substances are lipoidal as well as lipoprotein-like material 18 Menber 08/11/2024 9  Reagin reacts with cardiolipin Cardiolipin – a phospholipid component of certain eukaryotic and prokaryotic membranes  Non treponemal test does not confirm T. palladium infection. 19 Menber 08/11/2024  Serologic Tests for Syphilis: Non-Treponemal Assays Examples of non-treponemal tests: Rapid Plasma Reagin (RPR) Venereal Disease Research Laboratory (VDRL) 20 Menber 08/11/2024 10  A. VDRL In the VDRL test, heat-inactivated serum (to inactivate complement) is reacted with freshly prepared cardiolipin cholesterol - lecithin antigen and the resulting flocculation is read microscopically using 10x objective Reactive tests are quantified to obtain the antibody titer (double dilution is used). 21 Menber 08/11/2024  a) VDRL The VDRL test is a slide microfloculation test. The antigen, which is an alcohol solution Contain 0.03% cardiolipin  0.21% lecithin, and 0.9% cholesterol, is suspended in a buffered saline solution. Before testing, the serum must be heat inactivated at 560 C for 30 minutes. 22 Menber 08/11/2024   In activated serum should be reheated at 560C for 10 minutes if tested more than 4 hours after the original in activation. Cerebrospinal fluid is also an appropriate fluid for testing. 23 Menber 08/11/2024  false-negative results if you have had syphilis for less than three months and in late-stage syphilis. false-positive results: HIV Lyme disease Malaria  pneumonia systemic lupus erythematosus IV drug use tuberculosis 24 Menber 08/11/2024   The advantage of the VDRL test It can be performed on serum or cerebrospinal fluid (CSF). The disadvantages of the VDRL test:  The antigen must be prepared fresh daily  The test needs to be read with the aid of a microscope, and  Serum specimens must be heat inactivated before testing Result reporting  Specimens exhibiting medium and /or large flocculation particles are reported as reactive. Those with small particles are reported as weakly reactive While those with complete dispersion of antigen particles or slight roughness are reported as nonreactive. Sera exhibiting slight roughness should be quantitated to check for the prozone phenomenon. 25 Menber 08/11/2024  b) RPR 18-mm circle card test macroscopic, used to screen for syphilis. Antigen suspension containing In the RPR test, the cardiolipin cholesterol lecithin antigen has added to it choline chloride choline chloride :-to eliminate the need to heat inactivate serum EDTA to enhance the stability of the suspension charcoal particles as a visualizing agent. 26 Menber 08/11/2024  In the test, the RPR antigen is mixed with unheated If antibodies are present, they combine with the lipid particles of the antigen, causing them to agglutinate.  The charcoal particles coagglutinate with the antibodies and show up as black clumps against the white card  If antibodies are not present, the test mixture is uniformly gray. 27 Menber 08/11/2024  Serologic Tests for Syphilis: Non-Treponemal Assays  RPR and VDRL are agglutination assays Charcoal Cardiolipin Reagin Serum or CSF 28 Menber 08/11/2024 11  Non-Treponemal Tests: Advantages Rapid turn around time – Minutes Inexpensive No specialized instrumentation required Usually revert to negative following therapy Can be used to monitor response to therapy 29 Menber 08/11/2024 12  Non-Treponemal Tests: Limitations Results are subjective Non-specific False positive results can result from other infectious or non- infectious conditions EBV, Lupus, etc. Limited sensitivity in early/primary syphilis and in late/latent syphilis Problematic for high volume laboratories 30 Menber 08/11/2024 13  Non-Treponemal Tests: Limitations, continued Possibility for prozone effect High levels of antibody may inhibit the agglutination reaction To identify prozone, labs must serially dilute samples Undilute 1:2 1:4 1:8 1:16 31 Menber 08/11/2024 14  RPR strip and casssete test Syphilis Test Strip/cassete is A rapid, qualitative test  Detects antibodies (IgG and IgM) to Treponema Pallidum (TP) in whole blood, serum and plasma. 32 Menber 08/11/2024  Principle The Biopanda Syphilis Rapid Test strip/ Cassette is a qualitative membrane based immunoassay for the detection of TP antibodies (IgG and IgM) in whole blood, serum, or plasma.  In this test recombinant Syphilis antigen is immobilized in the test line region of the strip/ cassette.  After specimen is added to the specimen well it reacts with Syphilis antigen coated particles in the test strip/ cassete.  This mixture migrates chromatographically along the length of the test and interacts with the immobilized Syphilis antigen. The double antigen test format can detect both IgG and IgM in specimens. If the specimen contains TP antibodies, a coloured line will appear in the test line region, indicating a positive result. If the specimen does not contain TP antibodies, a coloured line will not appear in this region, indicating a negative result. To serve as a procedural control, a coloured line will always appear in the control line region, indicating that proper volume of specimen has been added 33 and Menber membrane wicking has occurred. 08/11/2024  Procedure 1.Bring the test strip and sample to be tested to room temperature prior to testing. 2. Label the strip with the patient’s identifier. 3. Using the sample loop provided, or a suitable micropipette, place one loop of serum or plasma (5 microliters) onto the test strip in the area just below the arrows. If testing a whole blood sample, use two loops (10 microliters). 4. Alternately, the end of the test strip with the arrows may be dipped into the sample for 1 second, only to the level of the edge of blue tape with arrows (about 7 mm). 34 Menber 08/11/2024  5. Drop the strip, with arrows pointing down, into the test tube containing the running buffer. Keep the tube vertical so that the running buffer comes in contact with only the portion of the assay strip below the arrows. 6. After 15 minutes, read the results. Do not read test results after 30 minutes. 8. Record the number of lines observed. 35 Menber 08/11/2024  Limitations of the Test 1. The assay is designed to detect antibodies to Treponema pallidum in human serum, plasma or whole blood. Other body fluids or pooled specimens may not give accurate results. 2. The test procedure, precautions and interpretation of results for the test must be followed strictly. 3. No test provides complete assurance that a sample does not contain low levels of antibodies to Treponema pallidum such as those present in very early stages of infection. A negative result at any time does not preclude the possibility of infection with syphilis. 4. As with all diagnostic tests, the test result must always be consistent with clinical findings.  5. Persons with a past or current non-venereal Treponema infection (Yaws, Pinta) may give positive reactions with this test. 6. Persons with history of Treponema pallidum infection, even though 36cured,Menber 08/11/2024 will give positive reactions. 37 Menber 08/11/2024  Serologic Tests for Syphilis: Treponemal Assays Principle: Infection leads to production of specific antibodies directed against T. pallidum Treponemal tests detect IgG or total IgM/IgG antibodies directed against T. pallidum 38 Menber 08/11/2024 15  Treponemal Assays  Fluorescent treponemal antibody (FTA-ABS)  Treponema pallidum particle agglutination (TP-PA) FTA-ABS TP-PA www.mastgrp.biz 39 Menber 08/11/2024 16  2.2.1 FTA-ABS  The FTA-ABS test is an indirect fluorescent-antibody technique, the antigen used is T.pallidum subsp (Nichols strain).  The patient’s serum is diluted 1:5 in sorbent (an extract from cultures of the nonpathogenic Reiter treponeme) to remove group treponemal antibodies that are produced in some person in response to nonpathogenic treponemes.  The absorbed serum is layered on a microscope slide to which T.pallidum has been fixed.  If the patient’s serum contains antibody, it coats the treponeme. 40 Menber 08/11/2024   FICT-labeled anti-human immunoglobulin is added and combines with the patient’s antibodies resulting in FICT stained spirochetes that are visible when examined by a fluorescence microscope.  The mean sensitivities of the FTA-ABS during primary syphilis and late latent are 84% and 96%, respectively; while the sensitivities during secondary and recent latent syphilis are 100%.  The mean specificities are 97%.  Until recently, FTA-ABS was considered the “gold standard” serological test for laboratorial diagnosis of syphilis. 41 Menber 08/11/2024  b) Treponema pallidum hemagglutination assay or TPHA. TPHA is an indirect hemagglutination assay used for the detection and titration of antibodies In the test, RBC are sensitized with antigens from T. pallidum. The cells then aggregate on the surface of a test dish if exposed to the serum of a patient with syphilis. It is used as a confirmatory test for syphilis infection.  A negative test result shows a tight button or spot of red blood cells on the surface of the test dish. 42 Menber 08/11/2024  Treponemal Assays: Advantages  High Specificity  Possibly higher sensitivity during early and late syphilis stages compared to non-treponemal tests  Newer Methods High throughput High reproducibility/precision 43 Menber 08/11/2024 20  Treponemal Assays: Limitations  Remain positive despite treatment Cannot be used to monitor response to therapy Subjective interpretation requiring technician expertise to read Expensive instrumentation Higher cost/test 44 Menber 08/11/2024 21  3.2. Agglutination Tests for Febrile Diseases 45 Menber 08/11/2024  Febrile Diseases When any pathogenic microb invades the human Body → Immune response →natural → adaptive → CMI → Ab mediated The host and microbial factors influence the rate of antibody formation  the type and amount of antibodies and the persistence of antibody in the circulation. 46 Menber 08/11/2024  antibodies produced in response to certain pathogenic microorganism are febrile agglutinins. The microbs that elicits the production of febrile agglutinin is characterized by presence of persistent fever & frequently difficult to grow in laboratory cultures. Some of the causative agents of febrile diseases are salmonella species, rickettsial and brucella abortus. 47 Menber 08/11/2024  Salmonella Serological Diagnosis 48 Menber 08/11/2024  Typhoid and Paratyphoid Fever An infectious feverish disease caused by the bacterium Salmonella typhi and less commonly by Salmonella paratyphi The etiological agent is Salmonella species; it occurs in human only. Some times it is termed as enteric fever since they colonize the intestine Salmonella of medically important species are S.typhi (typhoid fever), S.paratyphi A and B (paratyphoid fever). 49 Menber 08/11/2024  Salmonella typhi Rod shaped, flagellated, aerobic, Gram -ve bacilli. Refrigeration and freezing could slow their growth. Pasteurizing and food irradiation kill Salmonella for commercially-produced foodstuffs Foods prepared in the home from raw eggs can spread salmonella if not properly cooked before consumption. 50 Menber 08/11/2024  Typhoid and paratyphoid fever is transmitted through ingestion of contaminated food or water.  Typhoid or enteric fever is a clinical syndrome characterized by  fever, headache,  splenomegaly,  leucopenia & cough. 51 Menber 08/11/2024 How does the bacteria cause disease ? Ingestion of contaminated food or water Salmonella typhi Carried by white blood cells into the liver, spleen, and bone marrow Multiply and reenter the bloodstream (Clinical illness) Bacteria invade the gallbladder, biliary system, and the lymphatic tissue of the bowel and multiply in high numbers Then pass into the intestinal tract (can be identified for diagnosis in cultures from the stool) Typhoid ulcers can cause perforation and hemorrhage 52 Menber 08/11/2024  Its incubation period ranges from 7 to 14 days The gall bladder is the site of persistent intestinal infection Identification of salmonella Salmonella species can be identified based on their antigenic structure They have three different antigenic structures 53 Menber 08/11/2024 a) O- antigen (somatic antigen) It is lipopolysaccharide of the outer membrane it is heat and alcohol stable antigen. Salmonella is divided in to five distinct serogroups (A-E) on the basis of somatic antigen. b)H-antigen (flagellar antigen) H-antigen is protein, which makes the perithrchous flagella. It is heat and alcohol labile  Salmonella is further subdivided in to more than 1200 serotypes on the basis of flagellar antigens. c)Vi- Antigen: This is the antigen that determines the virulence, the ability to cause disease, of the organism. 54 Menber 08/11/2024  Preparation of antigen suspension a) Preparation of O antigen Procedure 1. Suspend the bacteria from an agar culture in saline 2. heat for 30 minute at 100 o C to remove the flagella. 3. Centrifuge to separate the bacteria from the detached flagella. 4. Re-suspend the bacteria in saline. 55 Menber 08/11/2024  Alternatively 1. Remove the flagella by mixing a dense saline suspension of the bacteria with an equal volume of absolute ethanol 2. Incubate for 20 hr at 37 oC 3. Dilute the suspension with saline 56 Menber 08/11/2024  b) Preparation of H antigen (flagellar antigen) Procedure 1. Inoculate bacteria from a single colony in to a broth and incubate for 6hrs 2. View a drop of the culture in a wet film to confirm that most of the bacteria are motile and therefore sufficiently flagellated for the tests. 3. Kill the culture by adding formaldehyde to a concentration of 0.2% and incubate for several hours at 370C 57 Menber 08/11/2024  Widal test Widal test is a serological test, which is commonly used to diagnose typhoid and paratyphoid fever The patient’s serum is tested for O and H antibodies 58 Menber 08/11/2024  Rapid slide (Screening) test 1. Clean the glass slides supplied in the kit well and wipe it free of water. 2. Place one drop of undiluted test serum in each of the first circle (1to4) and one drop of positive control serum in each of the last two circles. 3. Place one drop of antigen O,H 59 Menber 08/11/2024  4. Mix the contents of each circle with separate applicator stick and spread to fill the whole area of the individual circle. 5. Rotate the slide for one minute and observe for agglutination. If agglutination is visible, quantitative estimation of the titer of the appropriate antibodies should be done 60 Menber 08/11/2024 Tube agglutination method Procedure 1. Take a set of 8 clean dry test tubes for each serum to be tested. 2. Place 1.9ml of saline in tube 1 and 1 ml of saline in other tuber (2-8) 3. Transfer 0.1 ml of undiluted serum to tube 1. Mix thoroughly. The resultant dilution of serum is 1:20. 61 Menber 08/11/2024   Further dilutions are done in the following a) Transfer 1ml of the diluted serum from tube 1 and place in tube 2 this leads to 1:40 dilutions in tube 2 b) Repeat the transfer process for tube 7 after mixing. c. Leave 1 ml of saline in tube 8 at the ‘saline control’ Note. Tube 1 has a serum dilution of 1:20, 1:40 (2), 1:80 (3), and 1:160 (4), 1:320 (5), 1:640 (6) & 1:1280 (7). 5. Add one drop of appropriate antigen in each (use only that antigen suspension which has given a positive reaction in the screening test). 62 Menber 08/11/2024  Note: Each antigen (O, H) will require a series of tubes for determine the titer of their corresponding antibodies. 6. Mix well and incubate overnight (16-18 hrs) at 370C. 7. Examine agglutination macroscopically. 63 Menber 08/11/2024  Interpretation 1. Only a titer above 1:80 should be considered as significant. 2. A rise in titer (done each week) is considered to be definite evidence of infection. 3. A single test result is considered of diagnostic value only when it is usually high (above 160). 64 Menber 08/11/2024  Rickettsial Disease 65 Menber 08/11/2024  Rickettsial Disease they are obligate intercellular gram negative bacteria Rickettsiae resemble viruses unable to survive as free-living org... intercellular They are about the size of the large viruses and can just be seen with the light microscope. 66 Menber 08/11/2024  Unlike viruses  rickettsiae contain both RNA and DNA  multiply by binary fission  they have cell wall that contains muramic acid and enzyme. 67 Menber 08/11/2024  Based on their antigenic structure, the genus rickettsia has been divided into three main groups a) typhus group R. prowazeki, R. typhi b) scrub typhus group R. tsutsugamushi c) spotted fever group R.conori, R.siberica, R.rickettsi 68 Menber 08/11/2024  Transmit ion Man is an accidental host of rickettsia species except R. prowazeki; they live in intestinal tract of louse, fleas, ticks and mites  Reservoirs host include, dogs, rats, mice, rodents Rickettsial disease can be acquired by inhaling of dried infected vector faces, through damaged skin bite of an infected vector ticks, mite, etc. 69 Menber 08/11/2024  Sign and symptom High continuous fever severe head ach body pains marked weakness enlarged spleen 70 Menber 08/11/2024 Laboratory diagnosis Embryonated egg inoculation technique used for culturing viruses can also be used for isolating rickettsiae however it require costly materials and performed in reference laboratory. Serology: In rickettsial infection, specific IgM antibodies are produced followed by IgG response in the later stages. 71 Menber 08/11/2024 Weil-felix reaction A Weil Felix test is a type of agglutination test The reaction is based on similarity of particular antigenic determinant An antigenic cross reaction activity between few serotypes of Proteus spp (non-motile) and Rickettsia spp is considered as the Weil Felix test principle. A cross-reaction between the OX antigens (OXK, OX 2 and OX 19) Proteus species strains along with the antibodies produced in acute rickettsial infections forms the basis for the Weil Felix test interpretation. 72 Menber 08/11/2024  P. vulgaris OX19 antigen reacts with antibodies to the typhus group (TG) P. mirabilis OX-K antigen reacts with antibodies to the scrub typhus group (STG), and  Both P. vulgaris OX2 and OX19 antigens react with antibodies to the spotted fever group (SFG)  In other word, Proteus antigen is used to detect rickettsial antibody. 73 Menber 08/11/2024  Weil-felix reactions in Rickettsial infection Weil-Felix Reaction Organisms OX-19 OX-2 OX-K TYPHUS GROUP R.prowazeki +++ +/- - R.typhi +++ +/- - SCRUB TYPHUS GROUP R.tsutsugamclshi - - +++/- SPOTTED FEVER +/++ +/++ - GROUP +/++ +/++ - R. conori +/++ +/++ - R.conoripijperi +/++ +/++ - R.siberica R.rickettsi +++ frequent and large antibody response - no antibody response 74 Menber 08/11/2024 Note: False negative reactions are common in scrub typhus. False positive reactions may occur in Proteus infections,  relapsing fever, brucellosis and other acute febrile illnesses. 75 Menber 08/11/2024  Serology for H. pylori 76 Menber 08/11/2024 77 Menber 08/11/2024 78 Menber 08/11/2024 Specimen for Lab diagnosis of H. pylori 79 Menber 08/11/2024 80 Menber 08/11/2024 81 Menber 08/11/2024 82 Menber 08/11/2024 83 Menber 08/11/2024 84 Menber 08/11/2024  H.pylori Ag test kit H.Pylori Ab test kit 85 Menber 08/11/2024 86 Menber 08/11/2024 87 Menber 08/11/2024 88 Menber 08/11/2024 89 Menber 08/11/2024 90 Menber 08/11/2024  Thank You ! 91 Menber 08/11/2024