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1 Ondřej Zahradníček To practical of VLLM0421c
Searching for microbes Part VII. Complementfixing test and neutralization Ondřej Zahradníček To practical of VLLM0421c

2 Content of this slideshow
Dynamics of titre Complement and its properties Complement fixing test (CFT), its principle Trouble shooting in CFT Examples of CFT use in practice Neutralization reaction – principle Individual neutralization reactions ASO and its importance

3 Dynamics of titre

4 Interpretation of serological reactions
Antigen detection: it is a direct method. Positive result means presence of the microbe in the pacient‘s body Antibody detection: it is an indirect method. Some ways how to assess, when the microbe met the body: Amount of antibodies (titre) and mostly its changes during the time (dynamics) Class of antibodies: IgM/IgG (More in J08) (Avidity of antibodies)

5 Dynamics of titre 1 first pacient‘s visit 2 after 2 – 3 weeks Absolute amount of antibodies is not the most sure information: some patients are poor antibody-producers, etc. Dynamics of titre: better, means how the response gets changed during the time (usually during two or three weeks) 2 1

6 Why the titre alone is not sufficient
Sometimes a patient with low reactivity has a low titre even in an accute phase On the contrary, a very reactive patient has a high titre long after the reaction

7 Pair sera and non-pair sera
Pair sera = first specimen is kept in the refrigerator until the second comes to the lab (cca 10–14 days), and thed examined together. 4-fold increase is told to be significant under such circumstances Other situations (second specimen is examined separately): an accidental error should be taken into account. So even 4-fold increase is not certain, it is recommended to have better proof.

8 Dynamics of titre – more aspects
A special situation is so named seroconversion – there are no antibodies in the first specimen (it is too soon), but there are antibodies in the second one. Such a finding is more sure than the four-fould increase Sometimes titre decrease is found instead of increase (a subaccute infection) titre value does not correspond to infection activity. Often the highest amount of antibodies comes after the end of disease.

9 Examples of various effects of titre dynamics:
1 – 2: seroconversion 3 – 4: titre elevation 5 – 6: titre decrease

10 Complement and its properties

11 The Complement part of non-specific humoral immunity
a complex cascade system

12 Complement-fixing test (CFT)
Complement = one component of immunity reaction For CFT, we use animal (guinea-pig) complement. The patient‘s complement is inactivated before the reaction Complement is not able to get bound to isolated antigen Complement is not able to get bound to isolated antibody Complement is only able to get bound to the COMPLEX antigen – antibody When sheep RBCs are used as antigen and rabbit antibodies against them as antibody, then after binding of complement haemolysis occurs. We can see this in a simple task in the practical session.

13 Complement and its properties

14 Complement fixing test (CFT), its principle

15 Tale 1 There was a curious park guard.
He wanted to know the true relations between a boy and a girl that used to visit his park. Are they a couple, or they aren‘t? He knew, there is one only bench in the park. When one wanted to embrace somebody else somewere, one had to do it there. So, he placed parts of a plant (globules with hooklets, see next slide) on the bench with hope, that the couple would catch them on their clothes

16 The plant

17 However – how to ascertain…
…when both the girl and the boy used another exit? Then the guard realized, that during a moment his niece and her boy-friend will come to him, and he was sure, that on the way through the park, they will certainly use the bench for embracing. And so he made a plan: when his niece and her boy-friend will have globules on them, it means, that the first couple was no true couple, as it did not catch the globules first.

18 What to learn from the tale
Today we have to learn complement fixing test, quite a complicated test. Not only that we use complement to visualize antigen-antibody complex, but also two more parts of the reaction: the indicator couple (niece and boy-friend). This couple consists of indicator antigen (sheep RBC) and indicator antibody (amboceptor = rabbit antibody against sheep RBC)

19 CFT principle Patient serum is mixed with laboratory antigen (or laboratory animal serum with patients specimen in direct CFT). Complement is added. It binds in positive case (it is only able to bind when a complex Ag-Ig is present) In the 2nd phase, we add indicator system (sheep RBCs + amboceptor). In positive reaction indicator system remains intact. In negative reaction the indicator system is haemolysed

20 CFT – principle

21 Complement – how it reacts with the indicator system
The haemolysis requires presence of sheep (not rabbit) antibodies, amboceptor and complement. One of components missing or replaced  no haemolysis. Sheep RBC + amboceptor without complement  no haemolysis Sheep RBC + complement without amboceptor  no haemolysis Rabbit RBC + complement + amboceptor  no hemolysis Sheep RBC + complement + amboceptor  haemolysis

22 Use of CFT CFT is used for diagnostics of many (mainly viral) pathogens CFT, like other serological reactions, may be used for antigen detection or antibody detection For simplification, we shall only speak about antibody detection in this practical So, we think about a laboratory antigen being mixed with patient‘s serum (where we search for antibodies

23 Trouble shooting in CFT

24 Problems existing in CFT
Too much complement: false negative results. What to do? Titrate the complement to asses the proper amount Something in serum binding the complement itself (anticomplementarity component): false positive results. What to do? Perform anticomplementarity test – like normal course of CFT, but without antigen (A situation like a homeless man sweeping the plant globules from the bench, even when the boy did not come into the park because he was ill)

25 Titration of complement
For the reaction, we need an amount of guinea-pig complement that is neither too small nor too big That is why we test, what amount of complement is just able to perform haemolysis of a specified amount of red blood cells with amboceptor Too big amount of complement  false negativity (too many plant globules  some of them remain for niece&boy-friend)

26 Anticomplementarity test

27 Examples of CFT use in practice

28 Clinical situation A A patient with long term respiratory problems, a few clinical signs, the most probable diagnosis: atypic pneumonia Atypic pneumonia may be caused by many respiratory viruses, but also several bacteria (Mycoplasma, Chlamydia) Eventual mycoplasmal/chlamydial etiology would mean effect of antibiotics. In viral etiology antibotics would have no effect

29 Respiratory pathogens
The whole seropanel belongs to one patient. We have six respiratory pathogens, each in two rows (acute speciemen, reconvalescent specimen). First collumn = the anticomplementarity test Then we have seven dilutions of sera, i. e. dilution 1 : 5 in 2nd collumn, 1 : 10 in 3rd etc., with coeficient two. Besides viruses, a bacterium Mycoplasma pneumoniae is in the panel, too (difficult culture)

30 Clinical situation B We have three patients with suspicion for tick-borne encephalitis, all of them with neurologic symptoms and anamnesis of being bitten by a tick Tick borne encephalitis is a disease quite common in central Europe. Although it has worse course in adults (mostly seniors), people tend to vaccinate rather their childrens and not their parents.

31 Tick-borne encefalitis
We test antibodies again, now against tick-borne encefalitis. positive control in the first row in 2nd and 3rd row the first patient in 4th and 5th row the second patient in 6th and 7th row the third patient Each patient has two rows (accute serum and the reconvalescent one) In the first collumn, we have anticomplementarity tests again, and then sera dilutions, starting from 1 : 4 (continued: 1 :8, 1 : 16, 1 : 32, 1 : 64 etc.)

32 Clinical situation C We have several patients that should be screened for presence of antibodies againts toxoplasmosis (Toxoplasma gondii is a tissue parasite, cat is the definitive host) Seronegativity means that the person never met the infection*. Seropositivity should be studied in more details (one more sampling, eventually ELISA reaction for immunoglobin class assessment) *Or the infection is so fresh that the antibodies had no time do be created.

33 Toxoplasmosis The seropanel belongs to a positive control (1st row) and three patients (2nd to 7th row) We search for antibodies against toxoplasmosis. There are anticomplementarity tests in the first collumn, and then dilution by geometric row starting from 1 : 8. Each patient has only one row (we do not follow titre dynamics)

34 Neutralization reaction – principle

35 Tale 2 Once there was a killer toxin, and the toxin wanted to kill a red blood cell That toxin had in also character of an antigen, that chalenges the body to produce antibodies And when the toxin prepared for killing the RBC, an antibody, crossed his way, bound to it and did not allow him killing The red blood cell was very happy, and it sedimented to the bottom with other RBCs.

36 What to learn from the tale
Today, we have also neutralization reaction This reaction is important in viruses and bacterial toxins, that can be directly neutralized by a corresponding antibody The whole bacterium is rarely neutralized like that Majority of neutralization application is in virology. An exception is the most common serological reaction at all – ASO reaction

37 Neutralization reactions: general principle
There are many ways, how antibodies do work. One of them is direct neutralizing effect This effect is rarely present in whole bacteria. On the other hand, it may be observed in whole viruses, and in bacterial toxins Nevertheless, sometimes antibodies neutralize some characteristic of the whole bacteria, e. g. motility of Treponema in Nelson‘s test

38 Neutralization schematically
Antibody (Ig) prevents an effect of a toxin/virus to a cell / red blood cell + Cell in a tissue culture or a red blood cell Cell in a tissue culture or a red blood cell Toxin or virus Antibody Toxin or virus

39 Examples of neutralization reactions
Neutralized Object Reaction Bacterial toxin (haemolysin) RBC haemolysis ASO Virus RBC agglutination HIT Cell metabolic effect VNT

40 Individual neutralization reactions

41 ASO Principle: The antibody blocates the haemolytical effect of the toxin (streptolyzin O) on the RBC. Positive is blocation of haemolysis (as in CFT, but for a different reason) The microtitration plate is composed of a positive control and seven patient The titre above 250 is supposed to be risky for an autoimmune disease.

42 Course of serum dillution – ASO
Common course (dillution with reometric row, coefficient 2) would be too rough, we need a more detailed one. In fact, it is a geometric row too, but the coefficient is 1,2 and not two as usually

43 HIT Haemagglutination Inhibition Test: Pay attention, it is NOT an agglutination reaction, it is a neutralization! Antibody neutralizes the aggregation of RBCs due to viruses. So: Potato-like shape = negative response. Dense round target = positive response Example of use: We can read HIT results for tick-borne encephalitis. In each patient an accute and a reconvalescent serum is evaluated Interpretation of accute vs. reconvalescent sera is of course the same as in any other serological reaction

44 Remember: HIT is not an agglutination reaction, it is neutralization of viral agglutination HIT differs from ASO reaction mostly by the fact, that the RBCs are not haemolyzed, but agglutination. But the fact, that a specific antibody blocates the reaction is valid in both of the HIT for detection of antibodies against tick borne encephalitis (unlike ASO) is again a typical „indirect diagnostic“

45 HIT for tick-borne encephalitis: example of a clinical situation
We have several patients with suspicion for tick borne encephalitis, already tested using complementfixing Now we have decided to use an independent test to check the results

46 VNT (do not confuse with TNT  )
Virus Neutralization Test Cell culture uses to be dammaged by a virus. The dammage is visible as a change of colour from original yellow to changed red (pH is changed) Antibodies, if present, may prevent this viral action on the cell culture, so the colour remains yellow titre = last well with unchanged colour

47 VNT – clinical situation
Patient R. S., 35 years, has chronical pain in chest. Cardiological examination showed suspition for inflamation of heart muscle (myocarditis) As coxackieviruses are common causative agent of myocarditis, it was decided to perform test of antibodies against these viruses

48 VNT – example of use in coxsackieviruses
The whole panel belongs to one patient examination. Odd rows = accute serum, even rows = reconvalescent rows. Every two rows = one coxsackievirus (B1 to B6) First collumn has dillution 1 : 5 (then 1 : 10, 1 : 20…) Last collumn = controls. When there are six yellow and six red wells here, everything is OK. titre is the last well with unchanged (yellow) collour. When two coxsackieviruses have a significant (at least four-fold) increase of titre, it might be a co-infection, but it is more likelly that the coxsackievirus with the lower titre has a cross-reaction only

49 ASO and its importance

50 What is the antistreptolyzin O and why we attempt to detect it
After every streptococcal infection antibodies are produced, often including antibodies against streptococcal toxin – streptolysin O. Nevertheless, sometimes after infection the antibodies increase instead of decreasing. Antibodies are bound to some structures of the host organism (autoimmunity), so a „circulus vitiosus“ starts to run In such a situation, paradoxically the antibodies are worse than the pathogen that challenged the antibody response to protect us.

51 Remember: ASO is not an indirect diagnostics reaction, despite the fact that we search for antibodies. The aim is not to get a pathogen, but to assess the antibodies themselves, as they may be dangerous Indication for ASO examination: suspicion for so named „late sequellae“ of streptococcal infection: accute glomerulonephritis, or rheumatoid fever

52 Rheumatic Fever

53 Accute glomerulonephritis
Diffuse inflammatory cellular infiltration and mesangial hypercellularity (Hematoxylin and Eosin Staining: original magnification X 200)

54 Acute glomerulonephritis II

55 ASO examination principle: haemolysis neutralization
In Czech Republic, abbreviation ASLO is used for the same thing as ASO in English

56 The End

57 Tick borne encephalitis virus
Tick borne encephalitis often infects children, serious symptoms are rather typical for adults. Despite that adults rarely let themselves vaccinated. In the first phase it has flu-like symptomas, in the second meningeal or cerebral symptomas. Letality of infection is 1–5 %. It is a typical arbovirus (=arthropode borne virus), rodents are source Diagnostics is mostly indirect.

58 More flaviviral encephalites and fevers
Besides Central-European tick borne encephalitis we have more tick borne encephalites. Russian spring-summer encephalitis, is another subtype to the Central-European, less related is the scotish „louping ill“ and Omsk haemorrhagic fever. Also there exist Japanese encephalitis, transmitted by mosquitoes of genus Culex. Related is also West Nile fever, also mosquito transmitted. It is likely that is is present even in Czechia around Lanžhot

59 Tick – Ixodes ricinus

60 Virus of tick borne encephalitis

61 Toxoplasma gondii It is a protozoon; cats are its source, but people having dogs are in higher risk (dogs use to have cat faeces in their fur) Majority of infections in immunocompetent persons is asymptomatic, or only temporarily enlarged lymphonodes are observed. Ocular form is dangerous Infection of foetus is dangerous, too, especially in 1st trimester

62 Toxoplasma gondii

63 Down: Toxoplasma cyst in brain
Toxoplasma life cycle Down: Toxoplasma cyst in brain

64 Toxoplasma gondii

65 Toxoplasma – life cycle

66 In some persons, toxoplasma retinitis may occur…

67 Letality and mortality
Letality is the ratio between the persons dying for the disease and the total of infected persons Mortality, on the other hand, is the average number of persons dying for a disease (usually counted per inhabitants and one year)

68 Coxsackieviruses: survey of family Picornaviridae
Family Picornaviridae contains mostly following viruses important for humans: enteroviruses, (name shows their way of transmission, but they cause infection mostly outside intestine!) further classified into polioviruses – viruses of poliomyelitis coxsackieviruses and echoviruses newer enteroviruses 68, 69, 70 and 71 rhinoviruses – viruses of common cold virus of hepatitis A

69 Coxsackieviruses – more info
There exist coxsackieviruses A1–A22, A24 and B1–B6 Diagnostic can be done by virus isolation on newborn mice or tissue cultures Indirect diagnostic is difficult because of cross-reactions; nevertheless, it is used in coxsackieviruses of B group in suspicion for myocarditis

70 Coxsackieviruses – pathogenicity
CNS: aseptic meningitis (majority of types) herpangine (A types, mostly A4) hand-foot-mouth disease (A16) respiratory infections (all types) myocarditis and other muscle disease (B types) lymphadenitis (all types) relation of some types of diabetes mellitus (B group)?

71 Check-up questions Why patient's own complement is not used for CFT and guinea pig complement is used instead? What type of errors is caused by anticomplementarity of serum? What type of errors is caused by too big amounts of used complement? Why 2-fold increase of titre cannot be considered significant? Why it is recomendable to use pair sera when using reactions like CFT? What is the meaning ot the term „seroconversion“? In what clinical situations ASO diagnostics is rational? Why it is not suitable to classify ASO as „indirect diagnostic reaction for microbial detection“, althougth it is a method of antibody detection? Some viruses are unable to agglutinate RBC – how does the fact influence HIT diagnostic? Which is a Czech abbreviation for ASO? Why neutralisation reactions are rare in bacteriology? And one more 

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