Heterophile infectious mononucleosis antigen used in the latex diagnostic test. I. A method of antigen isolation and its serologic activity

The aim of this study was to elaborate a method of heterophile mononucleosis antigen preparation useful for latex coating. This antigen was isolated from bovine red blood cells stroma by the technique of Schwarzweiss and Tomcsik with author's own modification, in which introductory extraction of erythrocytes stroma ++ was performed by means of trichloracetic acid, aqueous extraction and elution of active substance with 80% ethanol. Besides of heterophile antigen preparation obtained by the method of Schwerzweiss and Tomcsik (preparation S-T) two serologically++ active preparations were obtained (fraction I and IV), which ability to inhibit PBD agglutinating reaction and bovine red blood cells haemolysis was 16 and 8 times lower, respectively, than S-T preparation. The preparation of heterophile mononucleosis antigen obtained differed in latex coating efficacy. In order to prepare latex reagent MZ-I (from fraction I) a solution of preparation of 125 micrograms/ml concentration was used, for MZ-II (from fraction IV)--50 micrograms and for MZ-III (from preparation S-T)--15 micrograms/ml. The reagent MZ-I showed, the highest activity in agglutinating test with human serum containing heterophile mononucleosis antibodies while two others reacted with 2-4 times lover serum dilutions. Similar differentiated reactivity with these reagents was found in latex test with 15 sera from patients suspected of having infectious mononucleosis.     

Use of heterophilic mononucleosis antigen in the latex diagnostic test. III. Diagnostic field studies

The aim of this study was to evaluate usefulness of latex coated with a preparation of heterophilic mononucleosis antigen for the detection of antibodies present during a course of infectious mononucleosis. Studies were performed in district serological laboratories. Sanitary-Epidemiological Stations in conditions of routine diagnostics. Studies were performed on 656 blood serum samples collected from individuals with a clinical suspicion of infectious mononucleosis. Latex and Paul-Bunnell-Davidsohn (PBD) tests were run in parallel. Out of 154 blood serum samples which contained antibodies detected by PBD test in diagnostically significant titer of 1:56 or higher, 151 were reactive in latex test in the dilution 1:5 or higher, while in the remaining three cases agglutination appeared with undiluted serum only. Out of 268 serum samples tested in latex test 167 reacted in 1:5 titer - diagnostically accepted as a significant or in higher titers. The sensitivity of latex test amounted to 98.1% and specificity was 96.9% as compared to reference Paul-Bunnell-Davidsohn test. The results of our study suggest the possibility of replacing Paul-Bunnell-Davidsohn test by latex test performed as a method providing the possibility of determination of the presence of heterohilic antibodies in blood serum samples and the follow up of their dynamics.     

Limitations of immunofluorescence tests in the diagnosis of infectious mononucleosis

The relative value of heterophil agglutinins (HA) and of specific EBV antibodies in the diagnosis of infectious mononucleosis (IM) was assessed in 108 cases of the disease and in 280 controls. Among the 108 cases 93 were HA-positive by sheep cells in at least one of their sera, while 15 were HA-negative by the same test. Among the 280 controls false-positive HA tests were not encountered except in eight cases with the horse cell microtitre tests. With one of the two slide tests at least two false-positive tests and 12 false-negative tests were also found but these sera had low titres in microtitre tests. The HA life-span was found to be unexpectedly long in a few cases, sheep cell HA lasting up to 8 to 10 months and horse cell HA up to 21 to 23 months.Many false-positive tests may therefore not be true false-positives and may result from the persistence of HA following unrecognized mononucleosis months before. Virtually all cases of IM had (or developed) antibodies to Epstein-Barr virus, viral capsid antigen (EBV-VCA), whereas only half of the controls were EBV-VCA-positive. The comparative analysis of nonspecific and specific test results in mononucleosis allows the following conclusions: (1) horse cell microtitre tests and the monospot test are more sensitive than sheep cell microtitre tests and the monotest; (2) false-negative results are occasionally seen with the latter tests but not with the former; (3) more false-positive results, however, are probably seen with the former tests; and (4) specific EBV-IgM and EBV-EA antibody tests are useful in the diagnosis of selected borderline cases of mononucleosis.     

EBV与HCMV传染性单核细胞增多症患儿的实验室指标比较

目的观察EB病毒（EBV）与人类巨细胞病毒（HCMV）感染所致的传染性单核细胞增多症（IM）患儿超敏C-反应蛋白（hs-CRP）、白细胞（WBC）计数、嗜中性粒细胞比值（N）、异常淋巴细胞（异淋）、嗜异性抗体和血清酶的变化。方法选择70例确诊有EBV病毒感染且具备传染性单核细胞增多症临床特点的患儿（A组）进行实验室检测指标分析及总结;并与37例HCMV相关传染性单核细胞增多症患儿（B组）进行比较。结果与EB组（A组）比较,HCMV组（B组）感染患儿hs-CRP水平、肌酸激酶同功酶（CK-MB）、丙氨酸氨基转移酶（ALT）、外周血WBC计数、异型淋巴细胞增高程度较低（P〈0.05）,嗜异性抗体常为阴性,两组N值差异无统计学意义（P〉0.05）。结论 EBV与HCMV感染所致的传染性单核细胞增多症患儿的实验室指标变化不同,应重视IM患儿的实验室检查以辅助诊断。     

Angioimmunoblastic lymphadenopathy after infectious mononucleosis.

Angioimmunoblastic lymphadenopathy occurred in a 46-year-old man 16 months after an episode of infectious mononucleosis induced by Epstein-Barr (EB) virus. The features of infectious mononucleosis included fever, pharyngitis, lymph gland enlargement, hepatosplenomegaly, hyperbasophilic mononuclear cells, and IgM antibodies to EB virus, although heterophile antibodies were not detected. The illness was severe and prolonged and included an asymptomatic measles virus infection. Over a year later massive enlargement of the lymph nodes led to a biopsy, which showed a diffuse infiltration with lymphoid cells and a proliferation of arborising small vessels typical of angioimmunoblastic lymphadenopathy. In spite of corticosteroids, levamisole, chlorambucil, and radiotherapy, no remission occurred, and serious infections led to death 18 months after the onset. Viral infections with EB virus and measles virus associated with pre-existing or subsequent immunological changes probably resulted in the appearance of angioimmunoblastic lymphadenopathy.     

Cytotoxic lymphocytes in infectious mononucleosis.

Seven patients with a clinical diagnosis of infectious mononucleosis (IM) and detectable heterophil antibodies were found to have peripheral blood lymphocytes that were cytotoxic for lymphoid cells containing Epstein-Barr virus from a patient with Burkitt''s lymphoma. The cytotoxic lymphocytes persisted in the peripheral circulation for up to 45 days. Patients who had had IM 1 to 5 years previously lacked such cytotoxic lymphocytes. Patients who had signs and symptoms of IM but no detectable heterophil antibodies lacked cytotoxic lymphocytes. The lymphocytes of one patient with IM showed progressive diminution of cytotoxic ability after prednisone treatment.     

Formation of Paul-Bunnell antibodies by cultures of lymphocytes from infectious mononucleosis.

Short-term cultures of peripheral blood lymphocytes obtained from 20 infectious mononucleosis patients 2–4 weeks after the onset of the disease were studied for formation of heterophile antibodies. In studying pooled supernatant fluids of lymphocytes from three patients cultured for 3–20 days, lytic antibodies for red blood cells of bovine (BRBC) and sheep (SRBC) origin were demonstrated. These hemolysins were shown to be of IgM nature and Paul-Bunnell specificity. Subsequently, plaque-forming cell (PFC) assays were performed with lymphocyte cultures of 15 patients. Significant numbers (60–750/2 × 10⁷ cells) of PFC secreting antibodies against BRBC were demonstrated in lymphocyte cultures of 12 patients. The number of PFC apparently reached its peak after 5 to 10 days of culturing. No or a very few PFC were observed in the lymphocytes that were not cultured or in lymphocytes cultured for 3 weeks or longer. Lymphocyte cultures prepared in a similar fashion from normal individuals or patients suffering from sore throat and submandibular lymphadenopathy of other than infectious mononucleosis origin did not produce PFC. Production of lytic zones by antibodies to BRBC secreted by PFC was inhibited by preincubation of lymphocytes of infectious mononucleosis patients with solubilized Paul-Bunnell antigen but not with other heterophile antigens, indicating that antibodies involved in the PFC formation are of Paul-Bunnell specificity. An increased number of PFC against BRBC were obtained in two of three lymphocyte cultures after cultivation with BRBC or solubilized Paul-Bunnell antigen.     

Usefulness of the latex test for rapid detection of Salmonella O antigen in bacterial culture fluids and clinical specimens. III. Diagnostic field studies]

The aim of this study was to evaluate a latex reagent prepared in our laboratory for a routine diagnosis of Salmonellosis in humans. Liquid cultures in selenite broth (SF) (18-24 hr), previously inoculated with faeces samples of individuals suspected of being infected with Salmonella were subjected to the study. In these cultures, after 15 min. of heating at boiling temperatures, group antigens of Salmonella with an aid of polyvalent latex reagent A-E and monovalent reagents B, C1, C2, D, and E were searched. The results of latex test were compared to the results obtained by routine bacteriological examination. Studies performed in 13 laboratories of Sanitary Epidemiological Stations included 5246 faeces samples. Out of these samples 1835 (35%) reacted with monovalent latex reagent and 1897 (36.2%) samples were positive, for Salmonella by culture technique and belonged to 14 genera of group B, C1, D, and E. S. enteritidis was the most frequently isolated and encountered for 98.6% of all isolated strains. Latex test with A-E reagent was positive in 2246 (42.8%) of culture samples in SF medium, of which 1736 were positive by culture and 510 samples were negative for Salmonella in routine bacteriological examination. The samples positive in culture and with A-E latex reagent reacted in 97.2% with one monovalent reagent. Out of bacteriologically negative samples and reacting with A-E latex reagent 28.8% were positive with monovalent latex reagents. In summary, we can conclude that latex test used in a survey studies can be an usefull test in addition to routine bacteriological examination, since after 18-24 hr it allows with high credibility of 95% to confirm or exclude Salmonella in a tested sample. Such a procedure due to a shortening of routine diagnostic course brings significant savings. Moreover, latex test makes possible rapid detection of mixed infections with Salmonella of different serological groups. The use of extremely carefully, properly prepared selenite broth constitutes a basic condition for agreement between results of latex test and routine bacteriological investigation.     

Viral antibodies in infectious mononucleosis

Abstract Patients with Epstein-Barr virus (EBV) infectious mononucleosis (IM) usually develop heterophilic antibodies and some autoantibodies. Antibodies to rubella, measles, adeno-, entero-, herpes simplex, cytomegalo- and varicella-zoster viruses were titrated in sera from IM patients and matched healthy controls using the complement fixation test (CFT) and the haemagglutination inhibition test. Except for herpes simplex virus and cytomegalovirus, the IM sera had significantly higher arithmetical and geometrical mean antibody titres and showed in most cases higher antibody prevalences in the CFT. The titre rise was most pronounced for rubella and measles antibodies, between 2- and 3-fold. There were no cases of very high titres occasionally seen in IM. The IM sera had higher total IgG serum levels than the controls, 17.27 g/1 and 11.8 g/1, respectively ( P < 0.001). The present data show that in addition to previously reported high levels of some autoantibodies and of heterophilic antibodies, there is a more general increase in IgG antibodies to commonly occurring viruses. This increase is most likely due to the polyclonal activation of B-lymphocytes following the binding of EBV to the complement receptor CR2 (CD21). When due consideration is given to the possible occasional occurrence of a false positive rubella IgM test, the raised antibody-titres will most likely not interfere with routine diagnostics.     

The use of the latex agglutination reaction for the diagnosis of a Brucella infection

The authors have developed the optimum conditions for the preparation of antigenic diagnosticum based on latex manufactured in the USSR. To sensitize latex with the diameter of microspheres equal to 0.83 microns, Brucella polysaccharide was used in a dose of 100 micrograms/ml. As stabilizer, polyvinylpyrrolidone at a concentration of 0.1% was used. The specificity and sensitivity of the diagnosticum were studied in analysis of serum samples taken from 102 healthy donors and patients with infectious diseases of nonbrucellar etiology and from 120 patients with different forms of brucellosis. The specificity of the diagnosticum was found to be 94.1% and its sensitivity, 77.5%. Comparative study of the latex agglutination test with other serological tests showed that the former test is highly effective both in acute and chronic forms of the disease. A high degree of correlation between the agglutination test, Coombs' test, the passive hemagglutination test and the latex agglutination test was established (r = 0.83, 0.72 and 0.62, respectively).     

Reactivity of Paul-Bunnell type heterophile antibody in sera from infectious mononucleosis patients with the surface of lymphoid cells carrying Epstein-Barr virus genomes.

The possible presence of Paul-Bunnell (PB) antigen on Epstein-Barr virus (EBV)-transformed lymphocytes were investigated. Of 23 EBV genome-positive lymphoblastoid cell lines tested all but one absorbed PB type antibody from the serum of an infectious mononucleosis patient. The one EBV-negative B cell line tested did not absorb the heterophile antibody. PB antibody, purified by an immunoadsorbent procedure using beef cell antigen, reacted with the EBV producer P3HR-1 cell line in an indirect membrane immunofluorescence test and was shown to be IgM antibody. Titers of heterophile agglutinin and reactivity with the cell surface were reduced to the same degree by absorption with beef cell antigen but not with guinea pig kidney antigen. PB antibody was distinct from IgM antibody against the EBV-determined membrane antigen, since the latter was not absorbed by beef cell antigen. PB antibody was also reactive with other EBV-positive B cell lines (QIMR-WIL, NC-37, and Raji) which were free of surface IgM. No reaction occurred with the nonproducer P3HR-1 line, a null cell line, and two T cell lines. The results suggest the presence of PB antigen on most EBV-transformed B lymphocytes, and its appearance in each of the transformed lymphocytes of patients with acute infectious mononucleosis.     

Evaluation of the usefulness for the latex agglutination test for serodiagnosis of Mycoplasma pneumoniae infections

The usefulness of latex agglutination test prepared in our laboratory for the diagnosis of M. pneumoniae infections was assessed. A total of 628 serum samples obtained from patients with respiratory tract infections were tested by complement fixation test and by latex test, from among them 274 serum samples were additionally tested by ELISA--Ig A/--IgG/--IgM and by immunoelectroprecipitation test. The highest sensitivity and specificity was displayed by the latex test in relation to ELISA when determining mycoplasmal antibodies of IgM class (respectively 82.1% and 89.6%) and to the complement fixation test (81.0% and 89.0%). Positive latex test results in our investigations were associated only with the presence of IgM antibodies and were not dependent on the IgA and IgG antibody classes. The latex agglutination test may be used in routine serodiagnosis of mycoplasmosis under condition that the results obtained in this test will be confirmed by the complement fixation test or ELISA.     

Spontaneous splenic rupture in infectious mononucleosis: a review.

Spontaneous rupture of the spleen is a rare complication of infectious mononucleosis (IM) occurring in 0.1-0.5 percent of patients with proven IM [1]. Although splenectomy has been advocated as the definitive therapy in the past, numerous recent reports have documented favorable outcomes with non-operative management. A review of the literature suggests that non-operative management can be successful if appropriate criteria, such as hemodynamic stability and transfusion requirements are applied in patient selection. We report the case of a 36 year old man with infectious mononucleosis who had a spontaneous splenic rupture and who was successfully managed by splenectomy. Based on review of the literature, an approach to management of a spontaneously ruptured spleen secondary to IM is suggested.     

Lymphocyte subpopulations and reactivity during and after infectious mononucleosis

T- and B-lymphocyte numbers, as well as lymphocyte reactivity to mitogens in vitro, were studied and correlated to other laboratory tests during the acute phase of infectious mononucleosis (30 patients) and up to one year thereafter. During the acute disease an absolute increase in both T- and B-lymphocyte numbers was recorded, the relative increase in B-lymphocytes occurring at the start. B-lymphocyte numbers fell after the second week and T-lymphocyte numbers after the fourth week of disease. Lymphocyte activation was impaired in all patients during the acute phase and was still significantly impaired for some mitogens after 6-9 months. Very few correlates between lymphocyte tests and other laboratory and clinical parameters were found.     

Therapeutic efficacy of various formulations of cycloferon in the treatment of infectious mononucleosis in children

One hundred twenty six children at the age of 4 to 7 years with infectious mononucleosis were observed. The subjects under the complex therapy were treated with cycloferon suppositoria (1st group), intramuscular cycloferon (2nd group) or placebo (3rd group). The results demonstrated similar therapeutic efficacy of the parenteral cycloferon and rectal suppositoria (a novel cycloferon formulation).     

Cytology and immunocytology of infectious mononucleosis in fine needle aspirates of lymph nodes

Two cases of infectious mononucleosis with atypical clinical presentations were initially diagnosed by fine needle aspiration (FNA) of lymph nodes and subsequently confirmed by serologic studies. The cytologic features that allowed recognition included a high percentage of cells with relatively abundant cytoplasm that stained pale to deep blue using a Giemsa-type stain. Many of the cells had plasmacytoid features. The cells ranged in size from small lymphocytes to large immunoblastic forms. Other features included mitotic figures and occasional binucleated forms. Immunologic studies showed a mixture of B and T cells, with many of the cells having a cytotoxic/suppressor phenotype. The features seem to be relatively characteristic and distinct from those of malignant processes that could be aspirated in lymph nodes. Recognition of infectious mononucleosis by FNA in these cases allowed for confirmation by serologic studies, thereby avoiding the need for an excisional biopsy. These cases show that FNA of lymph nodes may shed light on the nature of the process underlying the lymphadenopathy in selected cases of clinically atypical infectious mononucleosis.     

儿童传染性单核细胞增多症嗜异性凝集抗体与EBV-VCA-IgM检测的比较

目的比较分析外周血异型淋巴细胞（AL）、血清嗜异性抗体（HA）及EBV-VCA-IgM在儿童传染性单核细胞增多症（IM）诊断中的意义。方法对238例儿童IM进行AL计数、AVITEX-IM乳胶凝集法检测HA、ELISA法检测EBV-VCA-IgM,分析比较此3种检验方法的阳性率。结果（1）82．35％的患儿外周血AL〉10％,其中71．94％在病程第2周AL〉10％。（2）HA阳性者占48．85％,HA阳性率〈3岁（14．55％）明显低于≥3岁（53．90％）,P〈0．05。（3）EBV-VCA-IgM 163例阳性（75．81％）,各年龄组EBV-VCA-IgM阳性率差异无显著性。（4）72例同时检测血清EBV-VCA-IgM和HA,EBV-VCA-IgM阳性者58例（80．56％）,HA阳性者36例（50％）,EBV-VCA-IgM与HA均为阳性者33例（45．83％）,EBV-VCA-IgM的阳性检出率明显高于HA。结论EBV-VCA-IgM的阳件率明昂高干HA的阳件塞罩期殛时拱奉台EBV抗体对于及早、快速、准确诊断IM十分必要。     

Isolation and partial characterization of the heterophile antigen of infectious mononucleosis from bovine erythrocytes

The heterophile antigen (Paul-Bunnell antigen, PBA) of infectious mononucleosis was isolated by extraction of an aqueous suspension of bovine erythrocyte stromata with chloroform-methanol (2:1). The upper aqueous layer contained gangliosides, PBA, and a high-molecular-weight glycoprotein. PBA and gangliosides were separated from the high-molecular-weight glycoprotein by extraction of lyophilized upper layer with chloroform-methanol solvents. Separation of PBA from gangliosides was carried out by chromatography on DEAE-cellulose with chloroform-methanol solvents. PBA appeared to be a minor glycoprotein component of the erythrocyte membrane and had both hydrophobic and hydrophilic properties. It was soluble in either organic or aqueous solvents. On SDS-polyacrylamide gel electrophoresis, it migrated as a single component that stained for protein with Coomassie blue, for carbohydrate with periodic acid-Schiff reagent, and for lipid with oil red 0; it had an apparent molecular weight of 26,000. It was composed of 62% protein with major amino acids: glutamic acid, proline, glycine, isoleucine, leucine, and threonine (158, 116, 98, 90, 85, and 82 residues per 1,000 residues, respectively). Carbohydrate content was 9.2% with major sugar constituents: sialic acid, galactosamine, and galactose. Serologic activity of PBA was destroyed by pronase but not by trypsin.     

Screening for plant lectins by latex agglutination tests

The latex agglutination test has been applied as a detection system for lectins, the method being especially useful in locations where the dependence on blood for hemagglutination tests could be minimised. The binding of various glycoproteins and sugars individually to the latex particles facilitated the agglutination with lectins having varying sugar specificities. The glycoproteins used were ovalbumin, horseradish peroxidase, porcine mucin and fetuin, while N-acetylglucosamine, N-acetylgalactosamine comprised the sugars used for binding to latex. The sensitivity of the latex agglutination tests was comparable with that of hemagglutination tests. Sugar binding specificity of the lectins could also be determined by inhibition of the agglutination in the presence of corresponding free sugars. The method proved to be useful in screening crude seed extracts for the presence of lectins.