Negative Serum Samples Research Articles

Evaluation of the Korean Isolate-1 Tachyzoite Antigen for Serodiagnosis of Toxoplasmosis

To evaluate the usefulness of the Korean Isolate-1 (KI-1) antigen for serodiagnosis of toxoplasmosis, antigen profiles of KI-1 tachyzoites were analyzed in comparison with RH tachyzoites by SDS-PAGE and immunoblotting. ELISA was performed on latex agglutination (LA)-positive and negative serum samples using KI-1 and RH antigens. Immunoblotting of the KI-1 antigen showed multiple antigen bands with molecular sizes of 22-105 kDa. Among them, 1 and 6 common bands were noted against a KI-1-infected and a RH-infected human serum, respectively, which represented differences in antigenic profiles between KI-1 and RH tachyzoites. However, all 9 LA-positive human sera were found positive by ELISA, and all 12 LA-negative sera were negative by ELISA; the correlation between the ELISA titers and LA titers was high (r = 0.749). Our results suggest that tachyzoites of KI-1 may be useful for serodiagnosis of human toxoplasmosis.

Using improved technology for filter paper-based blood collection to survey wild Sika deer for antibodies to hepatitis E virus

Recent reports from Japan implicated wild Sika deer ( Cervus nippon) in the zoonotic transmission of hepatitis E to humans. Seroprevalence studies were performed to determine if imported feral populations of Sika deer in Maryland and Virginia posed a similar risk of transmitting hepatitis E virus (HEV). Hunters collected blood on filter paper discs from freshly killed deer. The discs were desiccated and delivered to a collection point. The dried filters were weighed to estimate the amount of blood absorbed and were eluted and collected in one tube via a novel extraction system. The procedure was quantified and validated with negative and positive serum and blood samples obtained from domestic Sika deer before and after immunization with HEV recombinant capsid protein, respectively. None of the 155 tested samples contained antibody to HEV, suggesting that Sika deer in these populations, unlike those in Japan, do not pose a significant zoonotic threat for hepatitis E. However, the new method developed for collecting and eluting the samples should prove useful for field studies of many other pathogens.

Incidental discovery of high systemic lupus erythematosus disease activity associated with cytomegalovirus viral activity

We try to find the association of cytomegalovirus (CMV) infection and anti-beta2 glycoprotein 1 autoantibodies (anti-beta2 GP1), a key antibody in antiphospholipid syndrome (APS), among systemic lupus erythematosus (SLE) and cerebral vascular accident (CVA) patients. This retrospective study enrolled serum samples obtained from 87 SLE and 97 CVA patients who have been checked for the existence of anti-beta2 GP1. First, the prevalence rate of anti-CMV IgG and IgM in patients with and without anti-beta2 GP1 were compared. Second, the prevalence of anti-CMV IgG and IgM were compared between SLE and CVA patients. Last, this study analyzed the clinical characteristics and disease activity in SLE patients with positive anti-CMV IgM and IgG. No difference existed in the prevalence rate of anti-CMV IgG and IgM between positive or negative anti-beta2 GP1 serum samples in both SLE and CVA patients. However, the prevalence of anti-CMV IgM was significantly higher in the SLE group than in the CVA group. Severity of clinical features and SLEDAI scores were considerably higher in patients with positive anti-CMV IgM than in SLE patients with negative anti-CMV IgM. Very impressively, all IgM-positive SLE samples (9/9) carrying highest levels of anti-CMV IgG, indicated reactivation of the latent CMV infection. Hence, it suggests that CMV reactivation might contribute toward the disease flare in some SLE patients. In future, a prospective and longitudinal study is stongly indicated.

Development and evaluation of a competitive ELISA for estimation of rabies neutralizing antibodies after post-exposure rabies vaccination in humans

Currently three tests are approved for the estimation of neutralizing antibodies after rabies vaccination: the mouse neutralization test (MNT), the rapid fluorescent focus inhibition test (RFFIT), and the fluorescent antibody virus neutralization (FAVN) test. Performance of these tests requires a lot of expertise and is generally carried out in reference laboratories and, hence, they are not available to many people. The aim of the present study was to develop and evaluate a competitive ELISA (C-ELISA) for estimation of neutralizing antibodies in order to make this testing more widely available. The C-ELISA was designed based on competition between a murine neutralizing monoclonal antibody (Mab) and the antibodies in serum of vaccinated people. The test was initially standardized using known negative and known positive serum samples for determining the optimal dilution of the Mab as well as the cut-off value (%) for ascertaining the level of inhibition. Nine hundred and ninety serum samples were tested from 250 people who had been administered purified chick embryo cell vaccine (PCECV). Serum samples were collected on days 0, 14, 30 and 90 post-vaccination, and were tested by C-ELISA. All the serum samples that were positive by RFFIT were also positive by C-ELISA. The titers obtained with C-ELISA were marginally higher than the RFFIT titers, but a significant correlation was noted between the two tests (r=0.897). None of the negative controls were detected to be positive for rabies antibodies by either of these tests. Therefore the C-ELISA was found to be 100% specific and sensitive in comparison to RFFIT. Further, the initial rise and fall of antibody titers on different days post-vaccination was comparable for both tests. The C-ELISA described herein can be used to quantify rabies neutralizing antibody levels after vaccination. This test is simple and can be conveniently used under field conditions for monitoring seroconversion after post-exposure rabies vaccination. Moreover it does not require handling of infectious virus by the end user.

Seroprevalence of Antibodies against Porcine Teschovirus 1 in Lithuania

The objective of the present work was to study the serological status and epidemiological situation of porcine teschovirus 1 (PTV-1) on swine farms in different Lithuanian regions by using virus neutralization test and virus isolation. Clinical, epidemiological, serological and virological examinations were performed for the diagnosis of Teschen disease (porcine enterovirus encephalomyelitis, TE). Swine blood serum samples (n = 1680) were collected on 28 farms (60 samples per farm) of 8 regions in 2003. For virus isolation 29 samples of pathological material were taken from the brains of pigs (2 - 4 months of age) that died suddenly on seven swine farms in five regions. Epidemiological and clinical examination revealed no signs of porcine enterovirus encephalomyelitis on 32 Lithuanian swine farms. PTV-1 was not isolated and identified in the PK-15 cell culture. Antibodies against PTV-1, detected by using VNT, were found on all investigated farms and in all age groups. Negative serum (2.3%, 39 from 1680) samples were found in 15.5% (16 from 103) of 2-4-month-old pigs and in 4.7% (23 from 494) of 4-6-month-old ones. The positive serological and negative epidemiological, clinical and virological results suggest that less virulent or avirulent PTV-1 strains were spread on Lithuanian swine farms.

Development of an Indirect ELISA for the Detection of Antibodies against Peste-des-petits-ruminants Virus in Small Ruminants

Peste des petits ruminants (PPR) is an acute, febrile, highly contagious and economically important viral disease of small ruminants. A polyclonal antibody based indirect ELISA was developed for detection of antibodies to PPR virus in the serum samples of goats and sheep using purified PPR viral antigen propagated in Vero cell culture. A threshold (cut-off) value was set as twice the mean of the negative population based on the distribution of known negative serum samples in respect of PPR virus antibodies in the test. A total of 1544 serum samples from goats and sheep were screened by indirect ELISA and competitive ELISA. The indirect ELISA compared very well with competitive ELISA, with a high degree of specificity (95.09%) and sensitivity (90.81%). When compared with virus neutralization test, the present assay had 100% specificity and 80% sensitivity. With serum samples, the assay could clearly differentiate animals from the infected population from uninfected ones. These results suggest that the indirect ELISA may be a good alternative tool to competitive ELISA for seroepidemiological surveys.

A comparative indirect ELISA for the detection of henipavirus antibodies based on a recombinant nucleocapsid protein expressed in Escherichia coli

The indirect ELISA is a simple and useful method for detection of pathogen-specific antibodies in animal sera. However, non-specific or background binding is often a problem, especially when recombinant proteins from Escherichia coli are used. In this study, a comparative indirect ELISA in which the total reactivity and the background binding were determined simultaneously on the same ELISA plate was reported. The background was determined by incubation of the test sera with excess free antigen to block specific binding. The sample was considered positive only when its total reactivity reading was higher than a pre-determined cut-off value and the ratio of the total reactivity to the background reading was more than 2.0. Using this approach, an antibody assay for henipaviruses using a recombinant Nipah virus nucleocapsid protein expressed in E. coli was developed. A total of 919 negative serum samples were tested in this assay and the specificity was 95.8%. In addition, eight positive experimental serum samples all tested positive. The use of recombinant protein as the ELISA antigen, instead of inactivated virus antigens, will be of significant advantage for countries where there is no facility of Biosafety level 4 to handle this group of zoonotic viruses.

A New HBsAg Screening Assay Designed for Sensitive Detection of HBsAg Subtypes and Variants

The design of a new HBsAg screening assay, the Hepanostika HBsAg Ultra is based on the use of monoclonal antibodies raised against native wild-type HBsAg and reactive with HBsAg in which the common ‘a’-determinant is modified by site-directed mutagenesis of four of the cysteine moieties. The design was checked using the same cysteine variants and samples from patients known to be infected with HBsAg variants. The results found were compared with other state-of-the-art commercial screening assays. The design of the Hepanostika HBsAg Ultra enabled detection of all variant HBsAg-positive samples in contrast to the other commercial assays. An additional 980 samples were tested to assess the specificity and sensitivity of the Hepanostika HBsAg Ultra. Screening of presumed negative serum and plasma samples resulted in a specificity of 100%. This makes the Hepanostika HBsAg Ultra the first screening assay with a design able to detect HBsAg variants with high sensitivity and specificity.

A new recombinant protein-based ELISA for the diagnosis of malignant theileriosis of sheep and goats

Tick-borne diseases of small ruminants are of highly economic importance in many countries. Malignant theileriosis of sheep and goats caused by Theileria lestoquardi is considered among the most important of these diseases and constitutes an obstacle to the sheep industry in countries like the Sudan. Here the application of a newly discovered surface protein of T. lestoquardi (Clone-5) in an enzyme-linked immunosorbent assay (ELISA) and the potentials of the application of the test in epidemiological surveys and diagnosis are described. Clone-5 contains a predicted number of 20 antigenic determinant sites and two polypeptides derived from the protein were recombinantly produced, purified and tested with control serum samples in both ELISA and Western Blot. One of the polypeptides was further used in validation experiments that involved the testing of negative and positive field serum samples collected from an area that had witnessed an outbreak of malignant theileriosis in Northern Sudan. ELISA, based on this recombinant protein, demonstrated a satisfactory performance with a calculated sensitivity and specificity of 94.6 and 88%, respectively, when countertested with a standard indirect fluorescent antibody test (IFAT). Moreover, no cross-reactions could be demonstrated against Theileria species (China) nor Cowdria spp. This test is recommended for further field validation experiments.

Evaluation of a Western Blot Method for the Detection of Yersinia Antibodies: Evidence of Serological Cross-Reactivity between Yersinia Outer Membrane Proteins and Borrelia burgdorferi

Yersinia enterocolitica and Yersinia pseudotuberculosis have been identified as causative organisms of reactive arthritis in humans. We evaluated a Western blot assay which uses Yersinia outer membrane proteins as antigens for the detection of Yersinia antibodies as a replacement for the complement fixation (CF) assay. Clinical agreement, sensitivity, and specificity were determined by testing 19 positive and 21 negative serum samples by the CF assay, Western blot assay, and enzyme-linked immunosorbent assay (ELISA). The CF assay and ELISA were compared to the Western blot assay, which was the reference method used in this study. Sera with antibodies that could potentially cross-react with Yersinia were also tested by the Western blot assay. The agreement, sensitivity, and specificity of the CF method were 61%, 26%, and 95%, respectively; and those for the ELISA were 89%, 95%, and 82%, respectively. The prevalences of Yersinia antibodies in 50 healthy donors were 6% for immunoglobulin G (IgG), 2% for IgA, and 2% for IgM. Sera positive for Bartonella henselae, Brucella, Chlamydia pneumoniae, and Rickettsia rickettsii antibodies showed cross-reactivity by the Western blot assay. The highest cross-reactivity was observed with Borrelia burgdorferi; 5 of 11 (45%) specimens were cross-reactive by the IgM-specific assay. Overall, the Western blot assay performs acceptably and is more sensitive than the CF assay, warranting replacement of the CF assay in the laboratory. Due to the evidence of cross-reactivity, particularly with B. burgdorferi, which can cause an oligoarthritis similar to reactive arthritis, the diagnosis of reactive arthritis should be based on clinical findings and complete serologic analysis of the potential causative infectious pathogens.

Lipopolysaccharide-Based Enzyme-Linked Immunosorbent Assay for Experimental Use in Detection of Antibodies toLawsonia intracellularisin Pigs

An enzyme-linked immunosorbent assay (ELISA) for Lawsonia intracellularis was developed and compared with a whole-cell antigen-based immunofluorescence antibody test (IFAT). The antigen-containing lipopolysaccharide (LPS) was derived from Percoll gradient purified cultures of L. intracellularis by using a modification of the Westphal hot phenol procedure. The antigen was bound directly to polystyrene 96-well microtiter plates, and the assay was performed in an indirect ELISA format. Specificity and sensitivity values based on 80 known positive and 80 known negative serum samples from controlled experimental trials were 93.7% and 88.7%, respectively. Serological results from a controlled L. intracellularis challenge exposure study confirmed the high specificity and sensitivity of this assay (100% and 99.5%, respectively). Comparisons between the LPS ELISA and the IFAT in detecting anti-Lawsonia antibodies in this controlled study revealed significantly more LPS ELISA-positive pigs than IFAT-positive pigs on days 21, 28, 35, and 42 (P = 0.003, 0.030, 0.002, and 0.006, respectively). This indirect ELISA (LPS ELISA) test is an improved method of detecting antibodies in pigs soon after exposure to L. intracellularis, regardless of isolate type (vaccine or wild type) in experimental studies. The LPS ELISA may be used as a tool to support future research trials on vaccine efficacy and to further understand the immune response induced by L. intracellularis.

Anti-VHE IgM en casos de infección por el virus hepatitis E

Hepatitis E virus is the main enterically transmitted non A non B hepatitis agent. The host IgM response in the acute infection phase is short lived. Therefore, only IgG antibodies against E virus are usually investigated. To measure IgM antibodies against virus E in serum samples. IgM antibodies against virus E were measured by ELISA in 35 positive and 18 negative serum samples for IgG antibodies against hepatitis virus E, without evidence of infection with hepatitis A, B or C virus. Measurement of the same antibodies in 25 additional samples positive for IgM antibodies against hepatitis A virus but without study for hepatitis virus B or C. IgM antibodies against virus E were detected in 12 of the 35 samples positive for IgG antibodies (34%) and in five of the 25 samples positive for antibodies against virus A (20%). An acute hepatitis E virus infection was detected in 34% of samples positive for IgG antibodies against this virus. The absence of IgM antibodies in the rest of the IgG positive samples could be due to an old or a recent virus E infection in the stage of antibody titer reduction. The detection of IgM antibodies against virus E in samples positive for virus A antibodies, could be due to co infection by virus A and E or a cross reaction of assays.

Assessment of antibodies to rabbit haemorrhagic disease virus in fox serum as an indicator of infection in sympatric rabbit populations

To assess the value of foxes as indicators of rabbit haemorrhagic disease virus in sympatric populations of rabbits. Serum samples from 341 foxes in central western New South Wales were tested for exposure to rabbit haemorrhagic disease virus using antibody cELISAs. Selected samples were tested for viral antigens by ELISA, viral nucleic acids by reverse transcriptase PCR and viral infectivity by rabbit inoculation. Antibodies against rabbit haemorrhagic disease virus were first detected in foxes 4 months after rabbit haemorrhagic disease was observed in rabbits in the same area. There was evidence of exposure of foxes to the virus in two subsequent years. A proportion of antibody positive, inconclusive and negative serum samples from foxes gave false positive results in an antigen ELISA for rabbit haemorrhagic disease virus. These serum samples were negative for viral nucleic acid by reverse transcriptase PCR and for infectious virus by rabbit inoculation. Liver samples from foxes were negative for viral antigens. Antibodies against rabbit haemorrhagic disease virus in fox serum can serve as an index of the occurrence of rabbit haemorrhagic disease in rabbit populations. Some fox serum samples exhibit false positive reactivity in an antigen ELISA for rabbit viral haemorrhagic disease virus.

Evidence for natural desensitization to mosquito salivary allergens: mosquito saliva specific IgE and IgG levels in children

There are few epidemiologic data on the prevalence of mosquito allergy, although local reactions to mosquito bites are common. To investigate the prevalence of mosquito allergy in children by measuring serum levels of mosquito saliva specific IgE and IgG antibodies that correlate well with the size of mosquito bite local reactions. Using enzyme-linked immunosorbent assays to measure mosquito (Aedes vexans) saliva-specific antibodies, we investigated sensitization to mosquito bites in 402 children aged 1 month to 18 years and correlated mosquito saliva specific IgE and IgG levels with age and sex. Twenty-three serum samples from infants who had never been exposed to mosquitos were used as negative controls. Mean levels of mosquito saliva specific IgE and IgG were lowest in the 23 negative control serum samples. In the 402 samples from children who may have been exposed to mosquitos, mean saliva specific IgG levels were higher in boys than in girls (P < .008). Levels of IgE and IgG correlated with each other (P < .001). A significant inverse correlation was found between age and both IgE and IgG levels. IgE levels peaked at the age of 6 to 12 months of age, and IgG levels peaked at 1 to 6 months of age. Levels of IgE and IgG antibodies gradually declined after the age of 5 years. Based on the presence of mosquito saliva specific antibodies, exposed infants and young children are at increased risk of having allergic reactions to mosquito bites. Antibody levels decline throughout childhood and adolescence, suggesting that natural desensitization may occur.

A Multilaboratory Evaluation of a Commercial Enzyme-Linked Immunosorbent Assay Test for the Detection of Antibodies against Mycobacterium Avium Subsp. Paratuberculosis in Cattle

Five laboratories participated in a study to evaluate sources of variation in results from an enzyme-linked immunosorbent assay (ELISA) for antibodies against Mycobacterium avium subsp. paratuberculosis. Each laboratory repeatedly tested duplicates of a negative, positive (P), and high-positive (HP) serum sample, which were supplied by the United States Department of Agriculture: Animal and Plant Health Inspection Service: Veterinary Services, National Veterinary Services Laboratories, Ames, IA, on all 96-well microtiter plates when routinely testing other samples for M. avium subsp. paratuberculosis antibodies. These 3 sera were aliquoted and sent to the 5 participating laboratories. This study focused on variation in test results because of assay reagents and laboratory techniques and did not account for biologic variability associated with the time course of infection in cattle. Overall, results from 868 microtiter plates were used in the study. For each sample a sample-to-positive (S/P) ratio was calculated according to the manufacturer's directions. The S/ P ratio for the P sample ranged from 0.06 to 1.039 (mean = 0.466 and 0.484 for wells 1 and 2, respectively) and those for the HP sample ranged from 2.446 to 8.727 (mean = 4.027 and 3.980 for wells 1 and 2, respectively). The majority of the variation in S/P ratio for the P sample was attributed to kit lot (37.5%), followed by random (unexplained) error (27.0%), laboratory (18.3%), and kit lot by laboratory (11.9%). By eliminating plates in which the separation between negative and positive control ODs was less than 0.4, the proportion of variation attributed to laboratory was reduced markedly. These results confirm that there is variability in M. avium subsp. paratuberculosis ELISA results and that several sources contribute to the observed variability. The study gives a relative estimate of the contribution of various sources to the overall variability observed in the M. avium subsp. paratuberculosis ELISA results with kit lot being a primary contributor. Similar data for other ELISA tests for antibodies to M. avium subsp. paratuberculosis or other antigens also should be developed.

Antigenic relevance of F protein in chronic hepatitis C virus infection

The hepatitis C virus (HCV) F protein is a recently described, frameshift product of HCV core encoding sequence of genotype 1a. Its function and antigenic properties are unknown. Using enzyme-linked immunosorbent assay, we assessed the prevalence of anti-F antibodies in 154 patients chronically infected with HCV, 65 patients with other liver diseases, and 121 healthy controls. For this purpose, we expressed a highly purified HCV F recombinant protein from HCV genotype 1a in Escherichia coli. Because the F protein shares the 10 first amino acids with the core protein, the anti-HCV F response was also assessed by a F recombinant protein deleted of its 10 first amino acids [Δ(1-10)-F]. Ninety-six (62%) of the 154 HCV serum samples reacted with the complete F recombinant protein, whereas 39 (25%) showed a weaker anti-Δ(1-10)F reactivity and 150 (97%) had anti-core antibodies. No reactivity against F, Δ(1-10)F, or core was detected in any of the controls. To exclude a potential cross-reaction of anti-F antibodies with anti-core antibodies, a specific enzyme-linked immunosorbent assay was performed for anti-core antibodies. The specificity of anti-F antibodies was confirmed using an F synthetic peptide. The prevalence of anti-F antibodies did not correlate with HCV RNA serum level, genotype, or stage of liver disease. Sequence analysis from 8 anti-F–-positive and 5 anti-F–-negative serum samples did not reveal any particular difference potentially accounting for their respective anti-F responses. In conclusion, the F protein elicits specific antibodies in 62% of individuals chronically infected with HCV; such anti-F response does not seem to be affected by the F sequence heterogeneity. (Hepatology 2004;40:900-909).

Detection of hepatitis A virus RNA in serum during the window period of infection.

Hepatitis A virus (HAV) infection is the leading cause of clinically apparent viral hepatitis in many parts of the world, including developed and developing countries. Only limited information is available regarding the seronegative viremic window that follows HAV infection, and no systematic search has been reported for HAV RNA positive, IgM anti-HAV negative serum samples during hepatitis A outbreaks. To determine the proportion of HAV infected individuals among (i) children who were tested negative for anti-HAV antibodies during hepatitis A outbreaks which occurred in a public school (n = 157) and a child care center (n = 38); (ii) subjects (n = 46) initially classified as acute non-A-C hepatitis patients after clinical examination and serological tests (sporadic cases). Reverse transcription (RT)-PCR was performed to detect the presence of HAV genome in serum samples collected from anti-HAV negative, susceptible subjects. HAV RNA was detected in 19/157 (12%) and 5/38 (13%) anti-HAV negative children from the public school and child care center, respectively. Twelve (26%) out of the 46 acute hepatitis patients (sporadic cases) were also HAV RNA positive. From nine of these 12 patients, a second blood sample was obtained 18-34 days after the first one: all nine had seroconverted to IgM anti-HAV, and their serum transaminases had reached elevated levels (mean ALT, 418; mean AST, 241). Detection of HAV RNA before IgM anti-HAV seroconversion may be used as an early diagnosis method during hepatitis A outbreaks. HAV RNA testing should also help to elucidate acute hepatitis cases of unknown etiology.

Serum biomarkers of hepatitis B virus infected liver inflammation: a proteomic study.

Hepatitis B virus (HBV), a serious infectious and widespread human pathogen, represents a major health problem worldwide. Chronic HBV infection has a very high risk of evolving into hepatocellular carcinoma. Although considerable progress was made during the recent past, the pathogenesis of HBV infection is still elusive and a definite diagnosis of HBV infected liver information still relies on biopsy histological test. In this report, we used proteomics technology to globally examine HBV infected serum samples aiming at searching for disease-associated proteins that can be used as serological biomarkers for diagnosis and/or target proteins for pathogenetic study. By comparing with normal and HBV negative serum samples, we found that at least seven proteins were significantly changed in HBV infected sera. These greatly altered proteins were identified to be haptoglobin beta and alpha2 chain, apolipoprotein A-I and A-IV, alpha1-antitrypsin, transthyretin and DNA topoisomerase IIbeta. The alteration of these proteins is displayed not only in quantity but also in patterns (or specificity), which can be correlated with necroinflammatory scores. In particular, apolipoprotein A-I presents heterogeneous change in expression level with different isoforms and alpha1-antitrypsin produces evidently different fragments implying diverse cleavage pathways. These unique phenomena appear specific to HBV infection. A combination simultaneously considering the quantities and isoforms of these proteins could be a useful serum biomarker (or index) for HBV diagnosis and therapy.

An immunochromatographic serological assay for the diagnosis of Mycobacterium tuberculosis

A rapid serological test for tuberculosis (TB) infection was designed using antigens specific to Mycobacterium tuberculosis. Tuberculosis infection, TB vaccination and exposure to environmental Mycobacteria cannot be distinguished using skin tests based on tuberculin protein derivatives. The standard diagnostic techniques such as skin tests, X-rays and DNA techniques are time consuming, expensive, and not practical for screening large populations. We used the 38, 63, 64, 14, 59-kDa antigens of M. tuberculosis to develop a rapid immunochromatographic test kit. This study evaluates the diagnostic potential of the rapid test kit using TB positive and TB negative serum samples from various hospitals in India. The samples were obtained from patients infected with or exposed to bacteria and viral pathogens. The results demonstrated that the combination of antigens improved the diagnostic specificity and sensitivity. The specificity of the test was 99.42% with sensitivity of 98.52% ( n=241). In case of multiple infections, the specificity was 93.15% with a low sensitivity of 73.52% ( n=141). The test kit may offer an improved alternative to purified protein derivative (PPD). This rapid TB test kit may be a useful tool for first-line testing of suspected cases, epidemiological studies and in designing a quality health system to reduce health hazards in resource-poor countries.

Fluorogenic RT–PCR assay (TaqMan) for detection and classification of bovine viral diarrhea virus

A single tube fluorogenic RT–PCR-based ‘TaqMan’ assay was developed for detection and classification of bovine viral diarrhea virus (BVDV). TaqMan–PCR was optimized to quantify BVD virus using the ABI PRISM 7700 sequence detection system and dual-labeled fluorogenic probes. Two different gene specific labeled fluorogenic probes for the 5′ untranslated region (5′ UTR) were used to differentiate between BVD types I and II. Sensitivity of the single tube TaqMan assay was compared with two-tube TaqMan assay and standard RT–PCR using 10-fold dilutions of RNA. Single tube TaqMan assay was 10–100-fold more sensitive than the two-tube TaqMan assay and the standardized single tube RT–PCR. Specificity of the assay was evaluated by testing different BVD virus strains and other bovine viruses. A total of 106 BVD positive and negative pooled or single serum samples, field isolates and reference strains were tested. Quantitation of cRNA from types I and II BVD virus was accomplished by a standard curve plotting cycle threshold values ( C T) versus copy number. Single tube TaqMan–PCR assay was sensitive, specific and rapid for detection, quantitation and classification of BVD virus.