ELISA Diagnostic Test Research Articles

Early trans-plasma membrane responses to Tobacco mosaic virus infection

Early trans-plasma membrane behavior after in vivo mechanical inoculation of Nicotiana tabacum with Tobacco mosaic virus (TMV) was investigated and compared to virus quantification in leaf tissues. To identify early events related to virus/host interaction, the systemic virus TMV was used to infect lower leaves and tests were carried out on upper leaves which were not directly infected. Non-invasive microelectrodes were used to estimate trans-plasma membrane electron transport and membrane potential after artificial inoculum of virus, monitoring the plant for the following 15 days. Virus infection was assessed by ELISA and quantified by quantitative RT-PCR. Collected data showed that after 2-day post-inoculation (dpi), TMV was able to modify membrane parameters: transient hyperpolarization of trans-membrane potential was observed until 10 dpi, while redox activity in infected samples was higher compared to control until end of tests. Conversely, ELISA diagnostic test was not able to reveal the virus presence in tobacco leaves until 6 dpi, while leaf symptoms were manifested after 13 dpi.

Histopathological and diagnostic study of Toxoplasmosis in human and sheep by using ELISA in Kut city

This study is designed to diagnosis blood Toxoplasmosis in human (women and men) and sheep (male and female) by using Elisa test; as well histopathological examination of heart, liver and lung of sheep were also done, internal organs were also obtained heart, liver and lung. Women diagnosis was performed by collection 96 samples from aborted women ageing from 15-45 years and divided into three groups; first group 15-25 years involved 50 sample, second group 25-35 years include 37 samples; and the third group 35-45 years included 9 samples. The results of Elisa test showed six positive of IgG, (two for each group) and one positive IgM for the second group, a significant (P<0.05) difference in the first and third groups between IgG and IgM was found. The total percentage of the infection 4, 8.1and 22.2 for the first, second and third groups respectively, and Total of 96 blood samples were collected from men with different chronic diseases and were divided into four groups, (first, second and third) each containing 25 samples and suffering from Thalasemia, renal failure and cancer respectively; of them 21 samples collected from normal men as fourth control group. The results of Elisa diagnostic test indicated 9, 9, 7 and 2 positive of IgG for first, second, third and fourth groups respectively, and 1, 2, 0 and 1 positive of IgM. The total percentage of Toxoplasmosis infection 38.4, 44, 28 and 14.28 for the first, second, third and fourth groups respectively, with a difference between them except the second group appeared significantly P(0.05). 92 samples of sheep blood were collected from male 46 and 46 female. The results of Toxoplasmosis showed one case positive IgG for female and one for male only. The histopathological examination of sheep positive case with chronic infection revealed pathological changes in the organs, the heart showed multiple variable sizes of parasite cysts embedded and scatter between cardiac muscle fibers, in addition to aggregation of inflammatory cells around the blood vessels forming nests of inflammatory cells with congestion of blood vessels in liver and lung. In conclusion that the infection with Toxoplasmosis in Kut city revealed the percentage of infection in men more than the aborted women, also it reported few cases of infection in sheep.

Trends in HIV-2 Seroprevalence at the National Reference Center of HIV from 2005 to 2014 in Lome, Togo

Background: In Togo, the HIV/AIDS epidemic is characterized by the circulation of the 2 subtypes of HIV. Thus, patients infected with HIV-2 are diagnosed and monitored in the care centers. Objective: To document the trend of HIV-2 prevalence over a decade of activities of the National Reference Center for HIV tests and screening (CNR-VIH). Methods: A cross sectional study was carried out from the screening data archived from January 2005 to December 2014 at the CNR-VIH, a laboratory located in the Sylvanus Olympio University Hospital (CHU SO) Lome. The sampling consisted of adults and children outpatients or those who were hospitalized in CHU SO, subjects presenting for voluntary testing, pregnant women and patients or samples referred for HIV confirmation. All samples were tested for HIV-1 and HIV-2 infections by combining ELISA and rapid diagnostic tests. Result: During the decade, 34,077 subjects were screened for HIV infection. The overall prevalence of HIV infection was 20.70% (7055/34077). In 10 years, the prevalence of HIV infection in CNR-VIH decreased significantly from 35.40% CI95% [34.50% - 36.20%] in 2005 to 14.20% CI95% [13.60% - 14.70%] in 2014 (p = 0.03). The prevalence of HIV-1, HIV-2 and dual HIV1&HIV-2 was respectively 20.40%, 0.23% and 0.07%, with annual prevalence of HIV-2 between 0.07% and 0.39%. The differences between the HIV-2 prevalence over the decade were not statistically significant (p > 0.15). A 4-year-old child from mother-to-child HIV-2 transmission was diagnosed. Sixty-five percent of adult patients were over 40 years of age with an average age of 43.5 ± 11.3 years. Conclusion: Data from the National Reference Center for HIV Tests in Togo over the last ten years confirm the existence of a weak epidemic of HIV- 2 infection with a tendency towards stability.

A promising new ELISA diagnostic test for cattle babesiosis based on Babesia bigemina Apical Membrane Antigen-1.

Babesiosis due to Babesia bigemina is a relevant tick-borne disease, affecting cattle worldwide. Many surface proteins of the pathogen including the Apical Membrane Antigen 1 (AMA-1) - have been analysed for vaccine and diagnostic purposes. This study focused on B. bigemina AMA-1 and on its use for the assessment of diagnostic tests. After bioinformatic analyses, AMA-1 codifying region was amplified and cloned into an expression vector used to induce protein synthesis in Escherichia coli cells. AMA-1 was purified by affinity chromatography and used to set up the best condition for an ELISA protocol. Bovine field sera positive to B. bigemina were used to evaluate the presence of anti-AMA-1 antibodies. In order to verify the assay specificity, sera positive to Babesia bovis or to the piroplasm Theileria annulata were also included. Significant differences were obtained between sera negative to both B. bigemina and B. bovis and samples positive to B. bigemina, to B. bovis or to both pathogens. No significant reaction was observed with T. annulata positive sera. The results showed that AMA-1 protein is suitable to be used as antigen in diagnostic assays for babesiosis diagnosis in cattle, as it does not show any cross reaction with anti-T. annulata antibodies.

Estudio epidemiológico de paratuberculosis bovina en hatos lecheros del sur de Nariño, Colombia

Introducción: la paratuberculosis (PTBC), también llamada enfermedad de Johne, es un trastorno gastrointestinal crónico de rumiantes domésticos y silvestres causado por Micobacterium avium subespecie paratuberculosis (MAP). Está distribuida mundialmente y genera un alto impacto en la ganadería, debido a que disminuye la producción, se pierde potencial genético por reemplazos tempranos de animales infectados y se incrementa la mortalidad. Objetivos: evaluar la presencia de anticuerpos anti-MAP en fincas lecheras del sur de Nariño y describir su distribución de acuerdo con características poblacionales. Métodos: se realizó un estudio transversal descriptivo en 958 vacas mayores de 2 años en 16 fincas lecheras. Para determinar la presencia de anticuerpos, se usó la prueba diagnóstica de Elisa indirecto (kit comercial Svanova®). La seropositividad fue asociada con las variables de raza, edad, ubicación, condición corporal, número de lactancias y estadio clínico mediante la prueba estadística de chi cuadrado. Resultados: se encontraron 15 fincas (94 %) con al menos un animal positivo y 77 vacas (8 %) con anticuerpos anti-MAP. Se estableció una asociación significativa (p < 0,05) con la condición corporal del animal. Conclusiones: en los principales municipios lecheros se encontraron vacas con anticuerpos anti-MAP y no se encontró relación entre edad, raza, ubicación y estatus clínico con la seroprevalencia-MAP, pero sí con la condición corporal.

ELISA diagnostic test systems, serodiagnosis of arboviral infections

There are developed and tested by ourselves ELISA kits for serodiagnostics of most endemic to Russia arboviral infections and some imported tropical diseases. Their application allowed in 1999 to set the value WN virus in the etiology of outbreaks ofWN fever in the southern region of Russia, then in Voronezh and Tula regions. From 1999 to 2014, according to the data of Russian Service for Surveillance on Consumer Rights Protection and Human Wellbeing in the Russia there were diagnosed 2284 cases of WNfevers and 1725 cases of CCHF. In 2012-2014 specific IgG and neutralizing antibodies to the WN virus were detected in the population of the Southern and Black earth regions of Russia. Data of the examination of residents ’ sera from Tatarstan, Tver, Ryazan, Kaluga, Kursk and Vologda regions were proved to be negative. In 2000 there were revealedfirst cases of CCHF in the Volgograd region, Kalmykia and Dagestan also. The application of ELISA-IgM kits allowed to reveal cases of Sindbis, Batai, Tahyna, Inkoo, sandfly fevers, Uukuniemi and Bhanja. During the period of2009-2014 there were verified 190 imported cases of dengue fever, Chikungunya, WNfever, sandfly fevers, and Japanese encephalitis among persons returning from travel in tropical countries.

Recombinant AgB8/1 ELISA test vs. commercially available IgG ELISA test in the diagnosis of cystic echinococcosis

The diagnosis and clinical management of cystic echinococcosis (CE) rely on imaging and serology, the latter still having a complementary role as its accuracy in assessing cyst viability is unsatisfactory. We used an experimental IgG ELISA test based on the recombinant antigen rEgAgB8/1 cloned from Echinococcus granulosus to differentiate active from inactive/cured CE infection, comparing its performance to that of a commercially available ELISA test used routinely in our hospital laboratory. Both tests were performed on sera from 88 patients with hepatic echinococcal cysts, grouped according to cyst stage based on ultrasonographical morphology, and on 17 patients surgically treated for echinococcosis and 18 patients with nonparasitic hepatic cysts included as controls. Tests' performances did not differ significantly, but the overall concordance between tests drastically dropped when groups were analysed separately. Further longitudinal studies should evaluate whether these discrepancies reflect the different ability of either test to predict the evolution of cysts over time. Although the recombinant-AgB8/1-based ELISA test seems to have no clinical advantage over the commercially available ELISA test in the assessment of hepatic CE cyst viability, the easiness of production and reproducibility of high-quality recombinant antigens makes rEgAgB8/1 a valid candidate for use in CE ELISA diagnostic tests.

Tempering the risk: Rift Valley fever and bioterrorism

Rift Valley fever virus (RVFV) is an arthropod-borne pathogen that primarily affects ruminants in eastern and sub-Saharan Africa first described following an outbreak on a farm in Kenya in 1931. Periodic outbreaks of RVFV since that time have resulted in significant losses to the African livestock industry as well as large numbers of infections in some of the most impoverished human populations. In one 2006/2007 outbreak across Kenya, Somalia and Tanzania alone, there were an estimated 145 000 human cases, and the ban imposed on imports after the 1997/1998 outbreak in Somalia led to a collapse of the vital livestock industry. Previously ignored, it is only in the past decade that the international community has started to take an increased interest in the disease. This followed the recognition of its potential to spread beyond the confines of the African continent after a large outbreak in Saudi Arabia in 2000. There has also been acknowledgement of the widespread presence of arthropod vectors capable of transmitting RVFV in many nonendemic regions of the world. This has led to a range of increased efforts in better understanding the virus and developing tools to predict outbreaks, combat the disease and limit its spread (Anyamba et al. 2010; Pepin et al. 2010). However, a more longstanding, parallel interest in the disease has also developed internationally; one centred around the biosecurity implications of the virus. The United States for instance, included RVFV as a candidate pathogen in its offensive biological weapons programme; a programme officially closed in 1969 (Borio et al. 2002). In more recent times, the classification of the virus as a potential bioterrorism agent has spurred investment and activity, particularly in the area of vaccine development and diagnostics (Borio et al. 2002; Sidwell & Smee 2003). While biosecurity interest has contributed to this increased funding over the past few decades, most notably from military sources such as the US Army Medical Research Institute of Infectious Diseases (USAMRIID), it may have acted as an impediment to international collaboration, with research being restricted to fewer, more expensive laboratories. After the signing of the US Patriot Act of 2002 and the classification of RVFV as a ‘select agent’, visiting experts and scientific collaborators are, for instance, now required to provide fingerprints, signed affidavits and be registered with intelligence services before working with the pathogen. Such measures are likely to act as a disincentive amongst scientists wanting to study the virus and could ultimately serve to drive experts to dedicate their efforts to other pathogens with fewer working restrictions (Animal & Plant Health Inspection Service, Centre for Disease Control & Prevention 2005, 2011). These restrictions have also been applied in parts of Europe as well, with national legislation such as the Anti-terrorism, Crime and Security Act 2001 of the UK, which also includes RVFV as a potential bioterrorism agent. For comparison and contrast, we include the current lists of biological agents and toxins around which bioterrorism legislation has been passed in the US and UK in Table 1. Focus on US policy internationally stems from its greater leadership role within the global community and the influence and impact its decisions have on people and institutions far beyond its borders. With large numbers of laboratories worldwide affected by US policy either directly through funding or indirectly as a result of political influence, restrictions have also resulted in the transfer between laboratories of RVFV samples for culture also becoming constrained and increasingly expensive. This undermines efforts to lower the industrial production costs of existing vaccines and of commercial kits for virus neutralisation and ELISA diagnostic tests (currently the prescribed tests for international livestock trade) (World Organization for Animal Health 2008). Expertise and experience thus tends to remain confined to a limited number of laboratories and companies by and large located in high income countries where investigation of the disease is neither a significant economic or health priority nor considered sufficiently profitable for drug companies. The resulting monopolies on expert technical knowledge and skills not only delays progress in developing new therapies

An atypical biotype I Actinobacillus pleuropneumoniae serotype 13 is present in North America

Atypical Actinobacillus pleuropneumoniae serotype 13 strains present in North America are described here for the first time. Different from serotype 13 strains described in Europe, North America strains are biotype I and antigenically related to both, serotypes 13 and 10. Chemical and structural analysis of the capsular polysaccharide (CPS) and lipopolysaccharide (LPS) of a representative strain revealed that the CPS is almost identical to that of the reference strain of serotype 13, having a slightly higher degree of glycose O-acetylation. However, it produces an O-PS within the LPS antigenically and structurally identical with that of the reference strain of A. pleuropneumoniae serotype 10. The O-PS was characterized as a homopolymer of 1,2 linked β-d-galactofuranosyl residues, a structure unrelated to that of the O-PS produced by the reference strain of serotype 13. Strains from Canada and United States are antigenically, phenotypically and genotypically similar. Animals infected by one of these strains induced antibodies that were detected by a LPS-based ELISA diagnostic test using either the homologous antigen or that of serotype 10. Based on the LPS and toxin profile, these strains might be misidentified as A. pleuropneumoniae serotype 10.

Field evaluation of commercial Immunoglobulin M antibody capture ELISA diagnostic tests for the detection of Japanese encephalitis virus infection among encephalitis patients in Nepal

Japanese encephalitis (JE) is a devastating disease with high rates of death and disability that occurs particularly in resource-limited, rural regions of Asia. Simple, accurate and inexpensive diagnostics tests are vital for quantifying the burden of illness. This field study evaluated two commercial JE immunoglobulin M antibody capture (MAC) ELISA kits using samples from routine JE surveillance. Positive (n=132) and negative (n=218) sera were randomly selected from patient samples collected as part of JE surveillance in Nepal in 2005. Samples were tested in a national public health laboratory with commercial kits produced by XCyton and Inverness (Panbio). Results were compared with those of the research lab-based reference standard, the Armed Forces Research Institute of Medical Sciences JE MAC ELISA. Positive and negative predictive values and 95% confidence intervals were 90% (82-95%) and 85% (79-89%) for Panbio1, 94% (88-98%) and 89% (87-93%) for Panbio2, and 84% (77-90%) and 96% (92-98%) for XCyton kits, respectively. Sensitivities of Panbio1, Panbio2, and XCyton kits were 71% (63-79%), 80% (72-87%), and 93% (88-97%); specificities were 95% (91-98%), 97% (94-99%), and 89% (85-93%), respectively. Overall percent agreement was 86% for Panbio1 and 91% for both Panbio2 and XCyton. Both commercial kits had good predictive values when single serum samples from encephalitis cases were tested in a national laboratory. Either kit can be used in similar JE-endemic settings where co-transmission of dengue virus, a flavivirus which has strong cross-reactivity with JE, is limited. These results can inform decisions by countries and the World Health Organization laboratory networks on national-level use of these kits for JE surveillance.

Different levels of humoral immunoreactivity to different wheat cultivars gliadin are present in patients with celiac disease and in patients with multiple myeloma

BackgroundImmunity to food antigens (gliadin, cow's milk proteins) is in the centre of the attention of modern medicine focused on the prevention of diseases, prevention which is based on the use of appropriate restriction diet. Detection of the enhanced levels of the immune reactions to antigen(s) present in food is from this point of view of great importance because there are reports that some of health disturbances, like celiac disease (CD) and some premalignant conditions, like monoclonal gammopathy of undetermined significance (MGUS), were vanished after the appropriate restriction diets.It is well known that gliadin is toxic to small bowel mucosa of relatively small population of genetically predisposed individuals, who under this toxic action develop celiac disease (CD). As the quantity of immunogenic gliadin could vary between different wheat species, the first aim of this work was to determine the percentage of immunogenic gliadin in ten bread wheat cultivars and in three commercially grown durum wheat cultivars. The second part of the study was initiated by results of previous publication, reporting that sera of some of multiple myeloma (MM) patients showed the presence of elevated levels of anti-gliadin IgA, without the enhanced levels of anti-gliadin IgG antibodies, determined with commercial ELISA test. It was designed to assess is it possible to reveal is there any hidden, especially anti-gliadin IgG immunoreactivity, in serum of mentioned group of patients. For this purpose we tested MM patients sera, as well as celiac disease (CD) patients sera for the immunoreaction with the native gliadin isolated from wheat species used for bread and pasta making in corresponding geographic region.ResultsGliadin was isolated from wheat flour by two step 60% ehanolic extraction. Its content was determined by commercial R5 Mendez Elisa using PWG gliadin as the standard. Results obtained showed that immunogenic gliadin content varies between 50.4 and 65.4 mg/g in bread wheat cultivars and between 20 and 25.6 mg/g in durum wheat cultivars.Anti-gliadin IgA and IgG immunoreactivity of patients' sera in (IU/ml) was firstly determined by commercial diagnostic Binding Site ELISA test, and then additionally by non-commercial ELISA tests, using standardized ethanol wheat extracts -gliadin as the antigen.In both patients groups IgA immunoreactivity to gliadin from different cultivars was almost homogenous and in correlation with results from commercial test (except for one patient with IgA(λ) myeloma, they were more then five times higher). But, results for IgG immunoreactivity were more frequently inhomogeneous, and especially for few MM patients, they were more then five times higher and did not correlate with results obtained using Binding Site test.ConclusionResults obtained showed different content of immunogenic gliadin epitopes in various species of wheat.They also point for new effort to elucidate is there a need to develop new standard antigen, the representative mixture of gliadin isolated from local wheat species used for bread production in corresponding geographic region for ELISA diagnostic tests.

Isolation of OspC Recombinant Proteins and FlaB (f-FlaB) Fragments from Western-Siberian Borrelia garinii NT29 Isolates and Investigation of Their Immunochemical Properties

The structural OspC and FlaB proteins of the Lyme disease (LD) agent are known to be the main antigens that induce a humoral immune response at the initial stage of the disease. It is the purpose of this paper to obtain the recombinant OspC protein and a fragment of FlaB protein (f-FlaB) from the western Siberian Bor- relia Garinii NT29 isolates and to evaluate the possibility of their use for LD diagnosis. Encoding regions of the ospC and f-FlaB genes were amplified using PCR, inserted into pREB expressive vectors and cloned in the BL21 and C-600 E. coli strains, respectively. The recombinant OspC and f-FlaB proteins were purified by affin- ity chromatography on Ni-NTA sepharose 6B. Their ability to bind serum antibodies from patients with LD was then tested using western-blot and ELISA methods. The results of analyses allow one to consider these proteins to be promising components for the elaboration of diagnostic tests for LD. A prototype of the ELISA diagnostic test was designed on the basis of OspC and FlaB recombinant antigens. This test system was shown to provide satisfactory parameters of diagnostic specificity (70%) and sensitivity (85%).

Purification of prostaglandin D synthase by ceramic- and size exclusion chromatography

Prostaglandin D synthase (L-PGDS) is a major glycosylated polypeptide in cerebrospinal fluid (CSF). The overexpression of L-PGDS in inflamed bovine mammary glands indicates its role as biomarker. No diagnostic tool for the quantitative detection of L-PGDS in cows has been reported. Immunometric ELISA tests might help to identify inflamed bovine tissue. The isolation of pure bovine L-PGDS, which is required for the generation of monoclonal antibodies, is an important prerequisite for a diagnostic ELISA test. Our goal was to identify a suitable technique to generate pure L-PGDS from bovine substrates. In the present study a two-step method for the purification of bovine CSF using ceramic hydroxyapatite chromatography followed by size exclusion chromatography is described. Subsequently, the identification of bovine L-PGDS was demonstrated by Western blot analysis and the high grade of the pure product was shown by 2-D PAGE. The yield of purified L-PGDS was 6.8 mg/l bovine CSF. L-PGDS from bovine CSF is shown to consist of multiple isoforms identical in molecular mass and p I values to those in previously described secretions of inflamed bovine mammary glands. In addition, the method was successfully applied to the purification of L-PGDS from human CSF.

Purification and characterization of a recombinant listeriolysin O expressed in Escherichia coli and possible diagnostic applications

The secreted pore-forming toxin listeriolysin O (LLO) is an essential virulence factor that allows the food-borne bacterial pathogen Listeria monocytogenes to escape from the phagocytic vacuole and reach the host cytosol. This protein belongs to the group of cholesterol-binding sulfhydryl-activated toxins, expressed by a large number of Gram-positive bacteria. A protocol for large-scale expression and purification of recombinant LLO was previously optimized. By a simple two-step purification method, we achieved a high-level LLO synthesis (4.5 mg l −1 of cell culture) in a hemolytically active form (1.2×10 6 HU mg −1 of protein). This procedure can solve the problem of LLO isolation from L. monocytogenes cultures which is a difficult task, mainly owing to the low levels of toxin released in the culture media. Here we report the characterization of toxin properties and its preliminary application in an ELISA diagnostic test for listeriosis.

Lipopolysaccharide O-Antigen Antibody-Based Detection of the Fish Pathogen Flavobacterium psychrophilum

Several yellow-pigmented species within the family Flavobacteriaceae are commonly associated with diseases in fish and are difficult to speciate due to their fastidious, slow-growing nature and cross-reactive antigens. Here we report the development of specific, antibody-diagnostic tests for Flavobacterium psychrophilum, the aetiological agent of rainbow trout fry syndrome and bacterial cold water disease. A unique antigen from F. psychrophilum, the lipopolysaccharide (LPS) O-polysaccharide (O-PS), formed the basis for the antibody test. LPS O-PS was purified and conjugated to keyhole limpet haemocyanin and bovine serum albumin for the generation of rabbit immune sera and the development of antibody-based diagnostic tests. Rabbit polyclonal anti-O-PS serum was highly specific for F. psychrophilum, without the need for prior cross-absorption with related bacteria and was the basis of an effective ELISA diagnostic test. Antibodies were purified from rabbit anti-O-PS serum and adsorbed onto coloured latex beads for the development of a specific, bead agglutination assay for F. psychrophilum.

Diagnosis of footrot in goats: application of ELISA tests for response to antigens of Dichelobacter nodosus

Goats are an important natural host for footrot and are infected with Dichelobacter nodosus that have virulence characteristics similar to those of sheep strains. However, the humoral response of goats to D. nodosus antigens and the possibility of a serological diagnosis of footrot in goats have not been studied. With the aim of evaluating a diagnostic ELISA test, we investigated the primary immune response of goats to experimental and natural infection, the memory response in recovered animals, and the transfer and persistence of colostral antibodies in kids. Footrot stimulated the goat’s immune system and, as in sheep, under-running lesions were the primary stimulus for production of anti- D. nodosus antibodies. The immune response could be detected in ELISA using either fimbrial or outer membrane protein (KSCN) antigens of D. nodosus. Antibody titres resulting from infection declined quickly after recovery and reached pre-infection levels within 3–4 months. Previously affected animals, however, mounted a memory response when injected with purified D. nodosus antigens. Antibody levels attained after anamnestic challenge were correlated with the maximum levels attained during infection, and were therefore indicative of the infection status. Anti- D. nodosus antibodies were also transferred to kids via colostrum, but these antibodies did not persist and therefore were unlikely to interfere with the diagnostic ELISA after 3 months of age. Though these ELISA tests were highly specific, their sensitivity was rather low. Therefore, they are only suitable for a herd diagnosis of footrot in goats and are dependent on the development of advanced under-running infections in a proportion of affected goats.

Selection for antigenic diversity of Tams1, the major merozoite antigen of Theileria annulata.

Tams1, the major merozoite/piroplasm surface antigen of Theileria annulata has the potential to be a component of a diagnostic ELISA test and be included in a recombinant subunit vaccine. However, the observation that this antigen displays diversity could constrain these applications. In this paper we have extensively characterized Tams1 diversity at the DNA level, using a PCR/sequencing strategy. Up to 44 alleles have been cloned and sequenced. The comparison of these alleles has identified regions of sequence conservation, variability and hyper-variability. Computer analysis of these alleles has indicated that positive selection may operate on certain regions of Tams1. Expression and Western blot analysis of selected alleles has indicated that sequence diversity is reflected in altered antigenicity and a continuum of relatedness and antibody cross recognition may exist. The possible function of the sequence conservation and polymorphism within Tams1 is discussed in relation to protein structure, host cell invasion and immune evasion.

Evaluation of Alpha-1 Elastase Proteinase Inhibitor as an Indicator of Neonatal Sepsis 1428

Alpha-1 Elastase proteinase inhibitor (A-1-EPI) is a neutrophil constituent released from azurophilic granules during cell activation. Previous studies have suggested the alpha-1-elastase ELISA diagnostic test may be sensitive and specific in predicting neonatal sepsis. We performed a prospective study on 216 neonates admitted to Wilford Hall Medical Center nursery undergoing a sepsis evaluation from April 1, 1996 to January 31, 1997. Infants were assigned a group classification based on presence/absence of clinical signs, diagnosis and duration of antibiotic use. Elastase levels were drawn at time of evaluation and 12-24 hours after initiation of antibiotic therapy. Group definitions are summarized in the table below.

Tuberculosis antigen A60 serodiagnosis in tuberculous infection: Application in extrapulmonary and smear‐negative pulmonary tuberculosis

An ELISA diagnostic test for tuberculosis antigen A60 (TBA60) IgG/IgM was used in a tertiary referral hospital in Taiwan. From June 1992 to December 1993, serum samples obtained from 907 patients were analyzed for TBA60 IgG and IgM titres. The final diagnosis of these patients was confirmed by microbiological study and clinical follow up for 18-24 months. Among 147 patients with active pulmonary tuberculosis, IgG was positive in 112 (76.2%), IgM was positive in 14 (9.52%). Among 90 patients with active extrapulmonary tuberculosis, IgG was positive in 53 (58.9%), IgM was positive in 9 (10%). Among 153 patients with inactive tuberculosis, IgG was positive in 28 (18.3%), IgM was positive in 1 (1.6%). Among 517 patients with nontuberculous disease, IgG was positive in 50 (9.7%), IgM was positive in 3 (0.6%). In this study population with 26% (237/907) active tuberculous infection rate, the TBA60 ELISA IgG had a diagnostic sensitivity of 69.6% and a specificity of 92.1%. These results indicate a positive predictive value of 67.9% and a negative predictive value of 89.2%. The sensitivity of IgM was 10.5% and specificity, 99.4%. The serum IgG tire had good correlation with the extent of pulmonary TB had a higher percentage of IgG seropositivity (83.9%) than those with smear-negative pulmonary TB (70.6%) and extrapulmonary TB (58.9%). In 50 cases with active tuberculosis, follow-up examinations were carried out one month after treatment. In 18 cases with initially negative IgG and IgM titres, 13 showed elevation of serum IgG titres into positive level, one had positive seroconversion of IgM which was the only serological marker indicating active infection. Therefore, 77.8% (14/18) gained diagnostic benefit from follow-up serological examination. It was concluded that TBA60 IgG and IgM ELISA is a useful test when diagnosing tuberculosis. This test also assists in the clinical judgement of tuberculosis when used as an adjunct to symptoms and sputum smear, and for monitoring therapeutic response at the commencement of treatment.

Detection of Two Anaerobic Periodontopathogens in Children by Means of the BANA and ELISA Assays

The mouths of young children become colonized by a variety of bacteria, but there have been only a few studies that have sought the presence of periodontopathic species in this population. Almost all of these studies used culturing techniques rather than the newer detection methodologies for various periodontopathogens. Studies in adults have shown that Treponema denticola and Porphyromonas (Bacteroides) gingivalis can be detected in dental plaque by use of the BANA and ELISA diagnostic tests. In the present study, plaque samples from four subgingival sites in each of 157 children (aged from two to 18 years) were tested for BANA hydrolysis with a BANA reagent card, and for T. denticola and P. gingivalis with an ELISA assay. Anaerobic periodontopathogens hydrolyzing the BANA substrate were found to be present in at least one of four plaque samples in 88 children (56%). T. denticola and/or P. gingivalis were detected by ELISA in at least one plaque sample in each of 135 children (86%). This study shows that children are widely colonized by these micro-organisms. A higher proportion of Black children than Caucasian children was colonized by these BANA-positive organisms. Also, children having a parent with a documented history of periodontal disease were more likely to be BANA-positive than were children of parents with unknown periodontal status.