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Laboratory-based diagnosis of brucellosis--a review of the literature. Part II: serological tests for brucellosis.

Brucellosis is a common zoonotic disease transmittable to humans from infected animal reservoirs. Malta, Rock, Gibraltar, Cyprus or Mediterranean fever, Bang's disease, intermittent typhoid or typho-malarial fever, undulant fever, etc. are just various synonyms for brucellosis. Patients suffering from this disease show unspecific symptoms, e.g. fever, chills, malaise, arthralgia, headache, tiredness and weakness. Human brucellosis may be caused by four of totally six genetically and phenotypically closely related Brucella species, i.e. B. melitensis, B. abortus, B. suis and B. canis. Although many organ systems may be involved, brucellosis is rarely fatal. Therapeutic failure and relapses, chronic courses and severe complications like bone and joint involvement, neurobrucellosis and endocarditis are characteristic for the disease. A definite diagnosis requires the isolation of Brucellae from blood, bone marrow or other tissues. However, cultural examinations are time-consuming, hazardous and not sensitive. Thus, clinicians often rely on the indirect proof of infection. The detection of high or rising titers of specific antibodies in the serum allows a tentative diagnosis. A variety of serological tests has been applied, but at least two serological tests have to be combined to avoid false negative results. Usually, the serum agglutination test is used for a first screening and complement fixation or Coombs' test will confirm its results. As Brucella ELISAs are more sensitive and specific than other serological tests, they may replace them step by step. This review will summarize advantages and disadvantages of the serological techniques used in clinical laboratories for indirect verification of human brucellosis.

Agglutination Tests↗

Diagnostic efficacy of stool antigen test (HPSA), CLO test and serology for the detection of Helicobacter pylori infection.

BACKGROUND: The diagnosis of Helicobacter pylori infection was initially being made through invasive methods but now non invasive methods have been developed to make the diagnosis easier. The present study was done to evaluate the diagnostic efficacy of a two non invasive tests i.e. Helicobacter pylori Stool antigen test (HpSA) and Helicobacter pylori IgG serology with an invasive method i.e. Campylobacter like organism (CLO) gel test. METHODS: The study was conducted in the gastroenterology unit of Pakistan Medical Research Council Research Centre Karachi. Adult patients with gastroduodenal disease were selected for study and their medical history was recorded. Endoscopy was done on all patients and the antral biopsy sample was tested for H. pylori using CLO test. Serology (IgG) was done elsewhere using ELISA and titers of over 50 units were recorded as positive. HpSA was done to determine the presence of H. pylori antigen in stool. RESULTS: Out of 43 patients 34 (79%) were males and 9 (21%) females. The main presenting symptom was epigastric pain in 74% cases. Although H. pylori IgG antibody titers of over 50 were taken as positive but for this study titres of over 100 were taken as significant for comparison with other tests. CLO test was positive in 26 (60.5%) cases, H. Pylori antibody titers of over 100 IU were present in 33 (76.7%) cases and HpSA in 21 (48.8%). Using CLO test as the gold standard the sensitivity of serology was 81% and that of HpSA 65% with a 29% and 76% specificity respectively. CONCLUSION: In our setting CLO test is still the best diagnostic test for H. Pylori detection. Both non invasive tests i.e. serology and stool HpSA are less sensitive than CLO but amongst each other both are equally sensitive.

Adult↗

Card agglutination test for trypanosomiasis (CATT) end-dilution titer and cerebrospinal fluid cell count as predictors of human African Trypanosomiasis (Trypanosoma brucei gambiense) among serologically suspected individuals in southern Sudan.

The diagnosis of human African trypanosomiasis (HAT) due to Trypanosoma brucei gambiense relies on an initial serologic screening with the card agglutination test for trypanosomiasis (CATT) for T. b. gambiense, followed by parasitologic confirmation in most endemic areas. Unfortunately, field parasitologic methods lack sensitivity and the management of serologically suspected individuals (i.e., individuals with a positive CATT result but negative parasitology) remains controversial. In Kajo-Keji County in southern Sudan, we prospectively collected sociodemographic and laboratory data of a cohort of 2,274 serologically suspected individuals. Thirty-three percent (n = 749) attended at least one follow-up visit and HAT was confirmed in 64 (9%) cases. Individuals with lower initial CATT-plasma (CATT-P) end-dilution titers had lowest risks (10.4 and 13.8/100 person-years for 1:4 and 1:8 titers, respectively) that significantly increased for higher dilutions: relative risks = 5.1 (95% confidence interval [CI] = 2.6-9.5) and 4.6 (95% CI = 2.8-9.8) for 1:16 and 1:32 titers, respectively. The cumulative yearly risk was also high (76%) in individuals found with 11-20 cells in the cerebrospinal fluid, but this involved only eight patients. Adjustment for potential confounders did not affect the results. In conclusion, treatment with pentamidine should be considered for all serologically suspected individuals with a CATT-P end-dilution titer >/= 1:16 in areas of a moderate to high prevalence of HAT.

Adolescent↗

Acute Helicobacter pylori infection in an infant, associated with gastric ulceration and serological evidence of intra-familial transmission.

Reports of the clinical picture and laboratory findings associated with naturally acquired acute H. pylori infection are rare and, although family studies have suggested person-to-person transmission, there have been no accounts of serologically proven intra-familial spread. This report describes the clinical and histological features associated with acute H. pylori infection in an infant and includes serological evidence for the possible transmission of this organism within his direct family. The infant, who presented with acute vomiting and severe haemetemesis, was found on endoscopy to have two shallow ulcers in the gastric antrum. Evidence of H. pylori infection was provided by the urease test, histology, and serology, the latter two tests showing the infection to be acute. Serologically, the patient's mother was shown to have an established H. pylori infection prior to her son, his twin brother to have acquired the infection shortly before the patient, and his father to have become infected some 63 days after the patient. Information on the acute presentation of H. pylori in children and evidence of the spread of H. pylori within family members is presented.

Acute Disease↗

Application of the proteolytic enzyme papain in routine platelet serology.

The use of proteolytic enzymes is well established in red cell serology. These enzymes modify some antigen structures and remove sialic acid from the red cell membrane. Enzyme-sensitive structures have also been identified on the platelet membrane. The effect of papain, a proteolytic enzyme used widely in red cell serology, on the detection of various platelet alloantibodies was examined to determine its usefulness in platelet serology. Antisera with the specificities anti-HPA-la, -2b, -3a, -4a, -5a, -5b, and -Naka were examined. HLA antibodies were also included. All sera were tested by a solid-phase red cell adherence technique in parallel with untreated platelets (UP) and platelets treated with papain (PP) for 15 minutes at 37 degrees C. The reactivity of anti-HPA-2b was eliminated and that of anti-HPA-3a was either eliminated or almost eliminated with PP. Antisera specific for the other alloantigens tested reacted similarly or more strongly with PP compared with UP. These findings were confirmed by flow cytometry. The reactivity of HLA antibodies with PP was generally enhanced. Inactivation by papain of platelet alloantigens in the HPA-2 and HPA-3 systems, but not in other systems, may assist in resolving mixtures of platelet alloantibodies. Also, detection of weak antibodies of other specificities may be enhanced. The use of PP may be a simple and useful serologic tool for investigating platelet alloantibodies.

Journal Article↗

Indeterminate HTLV serologic results in U.S. blood donors: are they due to HTLV-I or HTLV-II?

Current screening tests to detect human T-lymphotropic virus type I (HTLV-I) in volunteer blood donors commonly yield indeterminate HTLV serologic results (mostly isolated gag reactors). To assess the significance of indeterminate HTLV serologic results in U.S. blood donors, we compared 56 persons who had such serologic patterns with 30 HTLV seropositive blood donors and with HTLV seronegative controls. Polymerase chain reaction assays showed that none of the 56 individuals with indeterminate HTLV serologic results were infected with HTLV-I or HTLV-II, while all 30 HTLV seropositive blood donors were infected with either HTLV-I (in 15) or HTLV-II (in the other 15). The seroindeterminate blood donors were also different from the HTLV seropositive blood donors and more like HTLV seronegative controls in their demographic characteristics and the presence of HTLV risk factors. These results are evidence that volunteer blood donors with isolated and persistent gag seroreactivity in the United States are unlikely to be infected with HTLV-I or HTLV-II.

Adolescent↗

An investigation of Helicobacter pylori using culture, histopathological and serological examination methods and its antimicrobial sensitivities.

OBJECTIVES: In this study, the determination of Helicobacter pylori (H. pylori) by culture, histopathological and serological methods in cases of endoscopically diagnosed as duodenitis and duodenal ulcer (DU), a comparison of their relative advantages, and its antibiotic sensitivities were investigated. METHODS: Helicobacter pylori was investigated using 3 methods (culture, histopathological and serological examination) in 50 patients (25 diagnosed with duodenitis and 25 with DU) at the Department of Gastroenterohepatology, Istanbul Haydarpasa Numune Hospital, Turkey between December 2000 and February 2001. An investigation into its antibiotic sensitivities to amoxicillin, clarithromycin, metronidazole and azithromycin by disc diffusion methods and to amoxicillin and clarithromycin by E-test were investigated. RESULTS: Helicobacter pylori bacteria were observed in Gram stained preparates prepared from biopsy material in 34 out of 50 patients (68%), and were able to be produced in active culture in all these cases. Histopathological examination revealed the presence of H. pylori in 80% cases of DU and 60% cases of duodenitis; anti-CagA(IgG) was positively determined in 88% DU cases and in 60% duodenitis cases. There was a significant difference between the 2 groups in terms of diagnosis by histopathological and serological methods. The difference between the 2 groups produced in active culture in 84% cases of DU cases and 52% of duodenitis was statistically significant (p=0.0322). Using the E-test and disc diffusion methods, 8.8% of the strains that reproduced in culture were resistant to and 91.2% were sensitive to clarithromycin. All strains were determined to be sensitive to amoxicillin: 17.6% of the strains were determined to be resistant to metronidazole, 11.7% to azithromycin. CONCLUSION: It was observed that Gram staining is a rapid and reliable method of pre-diagnosis for H. pylori; that histopathological examination methods are of considerable importance in diagnosis; and that the investigation of the positivity of anti-CagA(IgG) will be a guide in the identification of virulent strains in particular. In addition, it was also concluded that since serological examination does not require invasive measures, this will pose an advantage. The culture method can be applied with the aim of diagnosis in cases identified as DU using endoscopy, and that in cases resistant to treatment it can be applied for the purpose of determining antimicrobial sensitivity. E-test and disc diffusion methods exhibited a rather good correlation, for which reason the disc diffusion method can be used in the determination of antimicrobial sensitivity in H. pylori strains.

Anti-Bacterial Agents↗

Treponema denticola infection is not a cause of false positive Treponema pallidum serology.

It has long been assumed that parodontal disease can be a cause of false positive results in syphilis serology, but so far there are no definitive data supporting this hypothesis. In this study we tested 250 serum samples obtained from blood donors. All of them were negative when routinely screened for antibodies against Treponema pallidum. Then, all these samples were tested by immunoenzymatic (ELISA) and Western Blot (WB) assays to investigate reactivities against T. denticola. Thirteen samples showed a strong positivity when tested by both methods. When tested by WB against T. pallidum no sample met the positivity criteria. Nevertheless, bands with molecolar weights of about 30-35 KDa (endoflagellar core antigens) were recognized. All the 13 subjects serologically T. denticola positive underwent oral clinical and radiological observation: all showed a very poor parodontal status (CPSS > 103). Eleven crevicular fluid samples out of the total of 13 patients were T. denticola positive by Real Time PCR carried out using a LightCycler system. In this study we demonstrated that the presence of T. denticola in the crevicular fluid samples obtained from patients with a severe periodontal status and/or a positive serology against T. denticola is not a cause of false positive results in syphilis serology.

Antibodies, Bacterial↗

The place and role of serologic methods in detecting Helicobacter pylori infection.

The aim of the study was to determine the place and role of serologic methods in detecting Helicobacter pylori (H. pylori) infection, on the basis of estimated enzyme-linked immunosorbent assay (ELISA) and complement fixation test (CFT) sensitivity and specificity. A total of 549 patients were included in the study. ELISA and CFT as serologic methods were compared with invasive methods (rapid urease test--CLO test, culture, histology). The sensitivity of serologic methods was above 90%, and their specificity was around 80%. Study results confirmed the value, reliability and usefulness of serologic methods in the detection of H. pylori infection.

Adult↗

Celiac disease serology in dermatitis herpetiformis. Which is the best option for detecting gluten sensitivity?

BACKGROUND: Dermatitis herpetiformis (DH), a well-established gluten-sensitive skin disorder presenting variable degrees of enteropathy, constitutes a very useful model in order to assess the utility of the celiac disease (CD)-related serology in patients with mild intestinal damage. OBJECTIVE: Our aim was to explore comparatively the performance of a panel of CD-related serologic tests in patients with DH. METHODS: We assessed a series of 18 consecutive patients with skin biopsy proven DH presenting the overall spectrum of intestinal damage ranging from normal mucosa (n = 6) to total villous atrophy (TVA) (n = 6) through partial villous atrophy (PVA) (n = 6). Sera were obtained from all patients while consuming a gluten containing diet. Serologic tests were antiendomysial, anti-tissue transglutaminase and antigliadin antibodies, and newly developed tests detecting both antibody isotypes (IgA and IgG) against deamidated synthetic gliadin-derived peptides (a-GDP). RESULTS: Serologic tests had a variable behaviour depending on the degree of enteropathy. While the majority of tests detected patients with TVA, only 50% of those with normal histology had positive assays. Patients with PVA had discordant results. Classical CD-specific tests were positive in only some patients with mild damage while all of them were identifed by a single assay detecting both isotypes of a-GDR CONCLUSION: The detection of a-GDP antibodies was the most reliable tool in order to identify gluten sensitivity in DH patients presenting a wide range of intestinal damage. Further studies should explore if these findings can be extrapolated to patients with CD having mild enteropathy.

Adolescent↗

[Possibilities and limits of serological diagnosis in pig stocks in the case of Mycoplasma hyopneumoniae infection].

When conducting their investigations to diagnose infectious diseases in swine, practitioners are often forced to use reduced numbers of animals in their samples in order to minimize costs for farmers. A cross-sectional study was conducted approximating such field conditions to show the limits of interpretation with reduced sample sizes in case of Enzootic Pneumonia. Compared with other respiratory pathogens, Mycoplasma hyopneumoniae, the etiologic agent of Enzootic Pneumonia spreads very slowly, mainly when animals are in direct contact. Furthermore, the interpretation of serological results is difficult because several weeks must usually pass for serological reactions to become apparent. Serological testing is normally used to confirm a clinical diagnosis by detecting an increase in antibodies against the etiologic agent. Samples are collected at the beginning of disease and four to six weeks later. An increasing number of serological positive animals in a herd is usually interpreted as spread of infection. The ,,true" prevalence we observed in our investigation was used to make a statistical analysis describing the probability of detecting an increasing prevalence from 0.07 to 0.33 with a reduced sample size. We showed that the probability of detecting an increase of two seropositive animals was 44% if 5 samples per group were analysed. When only 3 samples were analysed per group, this probability decreases to 21%. Compromise must be found between epidemiological necessary and financially acceptable sample size; this could be a minimum of 10 samples per age group.

Animals↗

[The role of serology in the diagnosis of Helicobacter (Campylobacter) pylori infection].

The purpose of the communication is to review the different aspects of the Helicobacter (Campylobacter) pylori infection. The first part of the communication is devoted to the description of the different gastric pathologies induced by the Helicobacter pylori infection and to the different methods used for the detection of this infection. Today a consensus assesses a causal role to Helicobacter pylori in the development of chronic active gastritis (or type B gastritis), in the pathogenesis of duodenal ulcer, and a major contributing factor in the development of peptic ulcer disease. The possible role played by this bacterium in the development of non-ulcer dyspepsia is still unclear. H. pylori infections can be detected using different methods including invasive methods--requiring an endoscopy (e.g.: culture of the micro-organism, urease test, microscopy) and non-invasive methods (e.g.: breath test, serology). Each of these methods has advantages but also some disadvantages, and none shows an absolute sensitivity and specificity. The second part of the presentation analyses the results obtained with a serologic method using a specific fractioned and purified antigenic complex extracted from Helicobacter pylori. This report demonstrates a good correlation with the other detection methods. Serology appears also as a useful tool for the therapeutical monitoring of infected patients. Serological results must however be interpreted in the light of the complete clinical examination of the patient.

Antibodies, Bacterial↗

Serologic and blood culture survey of Trypanosoma cruzi infection in four canine populations of southern Louisiana.

Blood culture and serologic testing were used to study the prevalence of Trypanosoma cruzi infection in a group of 85 dogs from southern Louisiana rural environment. These dogs were known to have been in contact with wild mammalian hosts of the hemoflagellate. Results were compared with blood culture and serologic test results in 103 dogs from a rural environment and with limited known wild mammalian T cruzi host contact. Serologic test results for the 188 dogs from the rural environment were compared with results for 176 dogs from an urban animal shelter and for 100 household pet dogs from an urban southern Louisiana environment. Blood culture was not performed on urban dogs. Culture results were negative in all dogs from rural environments. Serologic evidence of infection was obtained for 4 of the 85 (4.7%) dogs of rural environment with known host contact. Of 176 dogs from the animal shelter, 4 (2.3%) had high antibody titer to T cruzi, and 11 others had low titer (less than 2 adjusted ELISA units [aEU]). Two and 4 dogs of the housed urban and rural groups, respectively, had antibody titer to T cruzi that was less than 2 aEU. Results indicate that prevalence for exposure to T cruzi antigen is higher in dogs with high potential contact with the vector and wild mammalian hosts of T cruzi, whether they are from rural or urban environment. Furthermore, results indicate that similar studies on high-risk human populations may be indicated.

Animals↗

[False positive serological tests for Borrelia burgdorferi in patients with primary antiphospholipid syndrome].

Serological tests for Borrelia burgdorferi were carried out in a series of 27 patients with primary antiphospholipid syndrome (PAPLS). The results showed a 14% prevalence of false positive Lyme disease serology in this patient population. In patients with PAPLS and false positive serologic tests for Borrelia burgdorferi neurological involvement was significantly more common (p = 0.012, Fisher's exact test) than in patients with PAPLS without that finding. We insist on the possibility that patients with PAPLS may have false positive serological tests for Borrelia burgdorferi as measured by IFI with the resulting diagnostic error. In these cases, confirmation by other techniques such as ELISA or immunoblotting is recommended.

Adult↗

Comparison of stool microscopy and serology (enzyme linked immunosorbent assay) in epidemiology of amebiasis.

Stools from 634 individuals from Varanasi were examined for Entamoeba histolytica (EH). Serology was done in these subjects by enzyme linked immunosorbent assay (ELISA) employing filter paper technique. Stools were positive for EH in 16.9%, and serology in 15.9%. Both the tests were positive in only 5.2%. In 72.4% both the tests were negative. In 11.7% of stool positive cases, serology was negative, and in 10.7% with positive serology stool examination did not reveal EH. A majority (92.5%) of stool positive subjects had only cysts. Additional parasites were detected in 15.3%.

Amebiasis↗

[Serological study of the presence of Actinobacillus pleuropneumoniae antibodies serotype 2 and 9 on pig-breeding farms in the provinces of North Brabant and Overijssel (The Netherlands)].

Serological studies for the presence of CFT antibodies to Actinobacillus pleuropneumoniae, both to serotype 2 and serotype 9, were done in twenty-five sows on each of thirty pig-breeding farms in the provinces of North Brabant and Overijssel. Antibodies to serotype 2 and/or serotype 9 were detected in one or several sows on forty-two out of sixty farms. Similar findings were recorded on eighteen and twenty-four farms respectively in the provinces of Overijssel and North Brabant; the number of herds showing positive reactions to serotype 2 was sixteen and twenty-three in these provinces respectively, that showing positive reactions to serotype 9 being six and five respectively. An average proportion of nineteen per cent of the samples taken on the farms having serologically positive animals was positive for serotype 2, and twelve per cent for serotype 9. Only one of the twenty-five animals on twelve farms was found to be positive; serotype 2 was present in ten cases and serotype 9 in two cases. A marked effect of the size of the herds on the serological status of the farms was not observed in the present studies. This was also true of the purchasing policy; there was no difference between farms which had no longer purchased any breeding pigs for an average period of seventeen years and for one year respectively. Clinical symptoms of an infection due to Actinobacillus pleuropneumoniae were only reported as occurring on two farms having serologically positive animals.

Actinobacillosis↗

Serological and allergic reactions of ewes after simultaneous vaccinations with two living attenuated strains of Brucella and Salmonella.

Four groups of 50 4-6-month old ewe-lambs were vaccinated with 1 or 2 living attenuated vaccines administered simultaneously. One hundred ewe-lambs received B melitensis strain REV 1 vaccine subcutaneously and 100 others received Rev 1 vaccine conjunctivally. In each group, 50 ewe-lambs simultaneously received the S abortusovis strain Rv6 vaccine via a contralateral subcutaneous route. Reactions to Brucella antigens (estimated by allergic intrapalpebral reaction to brucellin, complement fixation test and Rose Bengal plate test) or to S abortusovis antigens (agglutination of O and H antigens) were recorded. Agglutinating titers against Salmonella antigens increased in all animals vaccinated with Abortusovis Rv6, whatever the route of the associated Rev1 vaccination. Rv6 vaccination did not interfere with serological or allergic responses to Rev 1 vaccination 1 month after vaccination. Six months after vaccination, no interference was observed with serological reactions to Rev 1 vaccination, which were still positive in about 10% of subcutaneously vaccinated ewes and negative in all conjunctivally vaccinated ewes. However, 6 months after vaccination, the frequencies of allergic responses to Rev 1 vaccine were significantly decreased by Rv6 vaccination in the Rev 1 subcutaneously vaccinated (45 versus 24%, P less than 0.05), but not in the Rev 1 conjunctivally vaccinated groups (12 versus 17%). According to these serological and allergic criteria, 1), association of the 2 vaccines does not appear to interfere with detection of Brucellosis more than by using a single anti-Brucella vaccination; 2), immunogenicity of each vaccinal valence was demonstrated after simultaneous vaccination; and 3), anti-Brucella serological and allergic responses remained in any case lower after conjunctival vaccination than after subcutaneous Rev 1 injection.

Allergens↗

Molecular characterization of serologic recognition sites in the human HLA-A2 molecule.

The localization of the amino acid residues involved in the serologic specificity of the HLA-A2 molecule has been investigated using a combination of site-directed mutagenesis, DNA-mediated gene transfer, indirect immunofluorescence and flow cytometry techniques. Synthetic oligonucleotides were designed to introduce individual and combined amino acid substitutions in both the alpha 1 (positions 9, 43, and the highly polymorphic cluster of residues from aa 62 to 83) and alpha 2 (positions 107, 152, and 156) domains to investigate the effect of the specific mutation on the recognition of the molecule at the surface of transfected human and mouse cell lines by a panel of mAb that recognize monomorphic or polymorphic determinants in MHC class I molecules. At least three non-overlapping serologic epitopes were identified. Mutations in the highly polymorphic region at aa 62 to 66 completely eliminated binding of mAb MA2.1 (A2/B17 cross-reactive). Mutation at position 107 resulted in complete loss of binding of the A2/Aw69-specific mAb PA2.1 and MA2.2 and partial loss of mAb BB7.2 binding. The recognition by other allotypic mAbs was not affected by these mutations and they therefore represent at least a third serologic epitope. Mutations at positions 152 and 156, known to be important for T cell recognition, did not affect serologic recognition. Introduction of residues of HLA-B7 origin in the polymorphic segment spanning aa 70 to 80 created a molecule carrying the -Bw6 supertypic determinant as demonstrated by mAb SFR8-B6 binding.

Amino Acid Sequence↗