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Comparison Between Haemagglutination Inhibition and Complement Fixation Tests in Detecting Antibodies Responses Following Influenza Viral Infection

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Comparison Between Haemagglutination Inhibition and Complement Fixation Tests in Detecting Antibodies Responses Following Influenza Viral Infection Egypt. Acad. J. Biolog. Sci., 4(1): 35-38 (2012) G. Microbiology Email:[email protected] ISSN: 2090-0872 Received: 20/7/2012 www.eajbs.eg.net Comparison between Haemagglutination Inhibition and Complement Fixation Tests in Detecting Antibodies Responses Following Influenza Viral Infection Muthana, A.K. Al-zobaei Medical Microbiology, Microbiol. Dept. Virology, College of Medicine, Univ. Anbar. ABSTRACT Objectives: To evaluate sensitivity and specificity of HI and CF tests for detecting antibody responses following naturally occurring influenza A and B infections. Methods: One hundred and twenty blood samples were drawn from one hundred patients with flue-like illness and twenty apparently healthy control subjects and tested at Central Health Laboratory in Baghdad/ Iraq using HI and CF tests for detecting three currently circulating influenza A and B strains during the period from the 1st of January 2008 to the 1st of June 2009. Results: 78-80% of the patients with flu-like illness responded to a t least one of three influenza virus antigens as measured by HI, while 34-35% of those patients showed a response by CF test. Conclusions: HI test has a higher sensitivity and lower specificity than CF test for detecting antibody responses from patients with influenza A and B viral infections. Keywords : Influenza viruses, HI, CF, Influenza serology INTRODUCTION SD. (1986), whereas CF test based on Several studies have clearly fixation of complement by antigen- demonstrated that HI is a more sensitive antibody complexes. A known amount of method than CF test for detecting complement is included in the test and antibody responses to naturally occurring residual complement is detected by the influenza virus A and B infections, addition of sheep RBCs sensitized with Julkunen, I. et al. (1985); Zigler T, et al specific hemolysin. Absence of (1983); Masurel A.et al (1983). hemolysis indicates fixation complement Haemagglutination Inhibition test detects and the presence of an antigen-antibody antibodies to strain-specific reaction, Pyhala R. and Kleemola M haemagglutinins, whereas CF test mainly (1976); Sever JL (1962). detects antibodies to type specific Because HI Assays for influenza nucleoproteins, Julkunen, I. et al.(1985). virus antibodies are not widely available, Haemagglutinins are used as antigens in clinicians often whether CF is an influenza virus, this making HI, the acceptable method for assessing the method of choice for measuring influenza infection responses of their influenza virus infection, Madora et patients. Although the point assays to al.(1983); Palmer D.F And Whaley S.D monitor the influenza virus infections, (1986); Pyhala R. and Kleemola M. search of the National Library Medicine (1976); Sever J.L (1962). database did not identify any reports The key test is HI, which depends directing comparing CF and HI for on the ability of the virus to agglutinate detecting influenza virus infection induce human group 'O' or chicken erythrocyte antibodies, MMWR, (2005). and inhibition of this process by specific The aim of this study was antibody. Diagnosis required at least a conducted to evaluate the HI and CF tests fourfold rise in antibody titer, Julkunen, for measuring antibody response for I. et al. (1985) ;Palmer DF And Whaley influenza virus infection and to provide 36 Muthana, A.K. Al-zobaei comparative data for dissemination for units (8HA units) were used as antigen and inquiring clinicians. reference antisera were included in each test batch. Complement fixation test was MATERIALS AND METHODS carried out by standard procedures using One hundred and twenty blood microtiter methods, Sever, JL. (1962). samples were drawn from one hundred Prior to CF testing, the sera were treated patients with flu-like illness in addition to with cholera filtrate for 18 hours, and twenty apparently healthy subjects as a subsequently heated at 56◦C for 60 minutes control group during a period between1st to inactivate complement and the CF of January 2008 to the 1st of June 2009 in antibody titers were taken as the highest Central Health Laboratory in Baghdad serum dilution which gave 50% fixation of /Iraq. Three ml of venous blood were taken complement. aseptically from each patients and transferred to clean and dry plane tube RESULTS (without anticoagulant) for serum 80% of the patients exhibit an collection after clotting and centrifuged antibody response to influenza the blood at 2500 rpm for 5 minutes pooled A/Brisbane (H1N1) antigen (Table 1) serum was kept in Eppendorf tubes at -20c. and influenza A/Brisbane (H3N2) to be used. antigen (Table 2) and 78% of those Three strains of influenza viruses patients exhibit an antibody responses to currently causing infections in the world influenza B (table 3) using HI test in were used: These were influenza contrast to 18-20% of those patients A/Brisbane/59/2007 (H1N1)-like antigen, exhibit negative result using the same test influenza A/Brisbane/10/2007 (H3N2)-like and this result was statistically significant antigen and influenza B/Florida/4/2006- (p<0.01), whereas the responses rates like antigen. The Haemagglutination were markedly low using CF test and inhibition (HI) antibody test was carried only 34% patients showed a respond to out by standard procedures, using the influenza A/Brisbane (H1N1) as showed microtiter methods, Masurel, A. and in (Table 1), 35% patients showed Heijtink AA. (1983). Prior to HI testing, the sera were respond to influenza A/Brisbane (H3N2) treated with cholera filtrate for 18 hours using the same test (Table 2 ), and only and subsequently heated at 56◦C for 60 21% of the patients respond to influenza minutes, in HI test eight haemagglutination B/Florida/4/2006 like antigen (Table 3). Table 1: Sensitivity and specificity of HI and CF tests for detecting influenza A /Brisbane/59/2007 (H1N1)-like antigen. Test used Complement fixation Total + _ Haemagglutination inhibition + 31 49 80 _ 3 17 20 Total 34 66 100 Sensitivity = 91.2% Sensitivity = 38.7% Specificity = 25.7% Specificity = 85% Table 2: Sensitivity and specificity of HI and CF tests for detecting influenza A /Brisbane/10/2007 (H3N2)-like antigen. Test used Complement fixation Total + _ Haemagglutination inhibition + 35 45 80 _ 0 20 20 Total 35 65 100 Sensitivity = 100% Sensitivity = 43.7% Specificity = 30.8% Specificity = 100% Comparison between Haemagglutination Inhibition and Complement Fixation 37 Out of 80% patients exhibiting HI 20% of the patients not response to HI (table response to influenza A/Brisbane (H1N1), 2). Out of 35% patients exhibiting HI only 31 patients showed CF response to response to influenza B, only one patient these strains (table 1), at the same time out of showed CF response to influenza B and 22 of the 20 patients non exhibiting response to the patients exhibiting non HI influenza B, these strains, only three of them showed CF 21 of them response to influenza B (Table3). response. Out of 80 patients exhibiting an HI All healthy control sera were negative for HI (H3N2), only 35 patients showed CF and CF test against three strains of influenza response to these strains, at the same time viruses. Table 3: Sensitivity and specificity of HI and CF tests for detecting influenza B /Florida/4/2006 -like antigen. Test used Complement fixation Total + _ Haemagglutination inhibition + 35 43 78 _ 1 21 22 Total 36 64 100 Sensitivity = 97.2% Sensitivity = 44.9% Specificity = 32.8% Specificity = 95.4% DISCUSSION group was consistent with other studies, Several studies have shown that the Julkunen, I., et al. (1985); Madora H., et al. incidence of antibodies against three (1983); Pyhala R. and Kleemola M.(1976) currently strains of influenza viruses can be who reported that this test was more measured by sero-epidemiological studies sensitive for detecting antibody responses to using blood samples collected from patients naturally occurring influenza A and B with flu-like illness MMWR (2009); viruses and also chosen of CF test in this MMWR (2008). Sera collected and treated research support HI in diagnosis of influenza with 56◦C to inhibit complement and this viral infection, Julkunen, I., et al. (1985); finding was agreement with, Okuno, Y. et al. Zigler T., et al. (1983) ;Azad TM., et al. (1990). (2004). Results in the present study showed Detection of antibody responses to that Iraqi persons were infected with naturally and experimentally occurring influenzaA/Brisbane/59/2007(H1N1)-like influenza A and B viruses using HI and CF antigen, influenza A/Brisbane /10/2007 test was consistent with other reports, Zigler, (H3N2)-like antigen and influenza T. et al. (1983); Madora H., et al. (1983). B/Florida/4/2006-like antigen but the titer of From this study we can conclude that antibody was differ for each strain in spite of HI testing is superior to CF testing for the same HA units (8HA units) used in this detection antibody responses of patients test as reported by previous studies, Madora, infected with influenza viruses and it's clear H. et al. (1983); Pyhala R. and Kleemola that the CF assays give false negative (1976). and this might be interpreted that antibody responses results for the majority of each of influenza virus isolates were patients infected with influenza viruses and inhibited with different titers of antibodies in influenza A/Brisban/59/2007 (H1N1)-like HI and CF test and this was provided an antigen, influenza A/Brisban/10/2007 evidence for prevalence strains of influenza (H3N2)-like antigen and influenza virus in the world and this was in agreement B/Florida/4/2006 like antigen was circulating with the result reported by WHO, MMWR, strains during 2008-2009 season. (2009); MMWR, (2008). Who indicated that these strain was circulating in population REFERENCES during 2008-2009 season. Azad TM., Mohammad h.
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