Searching for microbesPart VII.

Complementfixing test and neutralization

Ondřej ZahradníčekTo practical of VLLM0421c

zahradnicek@fnusa.cz

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

Dynamics of titre

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)

Dynamics of titre• 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)

1 first pacient‘s visit

1

2 after 2 – 3 weeks

2

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

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.

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.

Examples of various effects of titre dynamics:

• 1 – 2: seroconversion• 3 – 4: titre elevation• 5 – 6: titre decrease

Complement and its properties

The Complement

• part of non-specific humoral immunity

• a complex cascade system

http://img.tfd.com/dorland/thumbs/complement.jpg

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.

Complement and its properties

http://web.indstate.edu/thcme/micro/comp_fix.gif

Complement fixing test (CFT), its principle

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

The plant

http://www.ordinace.cz/clanek/lopuch-vetsi-lopuch-plstnaty/

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.

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)

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

CFT – principle

Complement – how it reacts with the indicator systemThe 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

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

Trouble shooting in CFT

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)

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)

Anticomplementarity test

Examples of CFT use in practice

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

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)

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.

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.)

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.

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)

Neutralization reaction – principle

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.

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

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

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

Toxin or virus

Toxin or virus

Antibody

+ –Cell in a tissue culture or a red blood cell

Examples of neutralization reactions

Neutralized Object Reaction

Bacterial toxin (haemolysin)

RBChaemolysis

ASO

Virus RBC agglutination

HIT

Virus Cell metabolic effect

VNT

Individual neutralization reactions

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.

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

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

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“

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

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

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

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

ASO and its importance

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.

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

Rheumatic Feverhttp://mednote.co.kr

Accute glomerulonephritis

www.ispub.com

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

Acute glomerulonephritis II

iws.ccccd.edu

ASO examination principle: haemolysis neutralization

In Czech Republic, abbreviation ASLO is used for the same thing as ASO in English

The End

http://web.uct.ac.za/depts/mmi/stannard/emimages.html

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.

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

Tick – Ixodes ricinus

http://www.presse.uni-wuppertal.de/archiv/output/okt98

Virus of tick borne encephalitis

http://vietsciences.free.fr/khaocuu/nguyenlandung/virus01.htm

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

Toxoplasma gondii

http://fullmal.hgc.jp/tg/icons/Toxo_ultrastructure.gif

Toxoplasma life cycle

Down: Toxoplasma cyst

in brain

http://web.indstate.edu/thcme/micro/parasitology

http://www.antoranz.net/CURIOSA/ZBIOR3/C0311/03-QZC08043-3_Toxoplasma.jpg

Toxoplasma gondiihttp://webdb.dmsc.moph.go.th/ifc_nih/applications/pics/Toxoplasma.jpg

http://www.smittskyddsinstitutet.se/upload/Analyser/ToxoplasmaSB.jpg

Toxoplasma – life cycle

http://www.dpd.cdc.gov/dpdx/images/ParasiteImages/S-Z/Toxoplasmosis/Toxoplasma_LifeCycle.gif

In some persons, toxoplasma retinitis may occur…

http://web.indstate.edu/thcme/micro/parasitology

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 100 000 inhabitants and one year)

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

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

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)?

Check-up questions1. Why patient's own complement is not used for CFT and guinea

pig complement is used instead?2. What type of errors is caused by anticomplementarity of serum?3. What type of errors is caused by too big amounts of used

complement?4. Why 2-fold increase of titre cannot be considered significant?5. Why it is recomendable to use pair sera when using reactions like

CFT?6. What is the meaning ot the term „seroconversion“? 7. In what clinical situations ASO diagnostics is rational?8. Why it is not suitable to classify ASO as „indirect diagnostic

reaction for microbial detection“, althougth it is a method of antibody detection?

9. Some viruses are unable to agglutinate RBC – how does the fact influence HIT diagnostic?

10. Which is a Czech abbreviation for ASO?11. Why neutralisation reactions are rare in bacteriology?12. And one more