Detection of eastern equine encephalomyelitis virus and Highlands J virus antigens within mosquito pools by enzyme immunoassay (EIA). I. A laboratory study

Enzyme immunoassays (EIAs) producing either chromogenic or fluorogenic end products were developed and evaluated for detection of eastern equine encephalomyelitis (EEE) and Highlands J (HJ) viruses in pools of Aedes triseriatus mosquitoes. Overnight incubation of the mosquito samples in the EIA significantly enhanced the sensitivity of the test. Both the EEE and HJ EIAs were sensitive, readily detecting one infected mosquito in a pool with 99 noninfected, and specific, distinguishing homologous from the alternate alphavirus and other arboviruses. By 3 days post-infection after intrathoracic inoculation, EEE virus was isolated from 100% (30/30) of the mosquitoes examined. Concurrently, EEE virus antigen was detectable by EIA in 100% (30/30) of examined mosquitoes and by indirect fluorescent antibody (IFA) technique in 77% (23/30) of the examined mosquito head squash preparations.     

A comparative study of the infection thresholds of Thogoto virus in Rhipicephalus appendiculatus and Amblyomma variegatum

Infection thresholds of Thogoto virus in 2 ixodid tick species, Rhipicephalus appendiculatus and Amblyomma variegatum, were compared. Thogoto virus has been isolated from naturally infected R. appendiculatus and A. variegatum in Central Africa, where both commonly parasitize the same hosts. No significant difference was found between the infection thresholds of Thogoto virus in the 2 species. The percentage of nymphs of both species infected by feeding on viremic hamsters was directly correlated with the time between host inoculation and tick engorgement. The blood titers in infected hamsters increased each day during the 3-4 day viremia until the hamsters died. The percentage of nymphs infected correlated with the viremic titer on the final day of engorgement (the time of maximum blood uptake). The 5% infection threshold for nymphs of R. appendiculatus and A. variegatum was estimated as 10(2.8) and 10(2.7) plaque forming units (PFU)/ml viremia, respectively. The prevalence of infection approached 100% for blood titers greater than 10(6.3) PFU/ml and greater than 10(7) PFU/ml, respectively. The apparent bloodmeal size of the 2 species differed by 8-fold and suggested that, in terms of the number of PFU ingested, R. appendiculatus was more susceptible than A. variegatum to per os infection by Thogoto virus.     

Screening of Blood Donors for Antibody to Human Immunodeficiency Virus Type I by Sensitive Particle Agglutination Assay

A particle agglutination (PA) assay for antibody to human immunodeficiency virus type I (anti-HIV) was used to screen blood donors and to test leukemia and hemophilia patients. Results by PA showed complete agreement with the results of two kinds of enzyme immunoassay (EIA), and the false-positive rate was the same as or lower than with the EIAs. The sensitivity of PA was 10- to 100-fold higher than that of the EIAs. This simple sensitive assay takes less time and fewer personnel than the EIAs, and has been used for massive screening of blood donors in our blood center.     

Experimental infection and protection of lambs with a minute plaque variant of Rift Valley fever virus

The injection of a single dose containing 5 X 10(3) plaque forming units (PFU) of a minute plaque variant of Rift Valley fever virus (RVFV) into two susceptible lambs resulted in no detectable viremia, pyrexia or clinical signs of disease. Immunization with the minute plaque variant induced neutralizing antibody as early as seven days postinoculation; however, no complement fixing antibodies were detected. Lambs immunized in this manner were protected when challenged with an infectious dose containing 1 X 10(3) PFU of wild type RVFV. These data indicate that the minute plaque variant may hold promise as a candidate live virus animal vaccine.     

Improved detection of acute parvovirus B19 infection by immunoglobulin M EIA in combination with a novel antigen EIA

BACKGROUND AND OBJECTIVES: Although parvovirus B19 is a significant blood product contaminant, few methods other than polymerase chain reaction (PCR) have been developed to detect the presence of the virus. MATERIAL AND METHODS: A B19 antigen enzyme immunoassay (EIA) has been developed and the sensitivity of detection is ascertained using dilutions of the B19 capsid protein VP2 and 10-fold dilutions of B19 viraemic serum. Once the assay cut-off was established, a panel of viraemic donations (n = 70) was screened by the antigen EIA. The B19 immunoglobulin M (IgM) and IgG status of these specimens was also determined. During screening of blood donor units by quantitative PCR, 70 individuals were identified with levels of B19 DNA greater than 10(6) IU/ml at the time of blood donation. RESULTS: The sensitivity of the B19 antigen EIA was estimated to be equivalent to between 10(8) and 10(9) IU/ml B19 DNA or 1-10 pg/ml of recombinant capsid protein. B19 detection was significantly enhanced when viraemic specimens were pretreated with a low pH proprietary reagent. Unlike other virus-detection assays, detection of the B19 antigen was not affected by the presence of B19 IgM or IgG antibodies. In addition, the assay was capable of detecting all three genotypes of human erythrovirus. Combined specimen analysis by the B19 antigen assay and a B19 IgM assay facilitated the detection of 91% of acute B19 infections in the test population. CONCLUSION: In combination with B19 IgM detection, application of the B19 antigen EIA is a flexible and efficient method of detecting recent B19 infection and can be used as an alternative to PCR.     

Development of EIA for detection of Chlamydia trachomatis in genital specimens

A double antibody sandwich enzyme immunoassay (EIA) for chlamydial antigen detection was developed using a monoclonal antibody against lipopolysaccharide (LPS) of Chlamydia trachomatis as a coating antibody. Polyclonal rabbit antiserum against partially purified antigen from elementary body (EB) antibody and horse-radish peroxidase conjugated goat anti-rabbit antibody were used as the primary and secondary antibody respectively. The developed EIA could detect protein of partially purified EB at the lowest concentration of 250 ng/ml. The assay was evaluated against the cell culture (CC), DNA hybridization assay (PACE2 system: Gen-Probe, San Diego, CA, USA) and a commercial enzyme immunoassay (kEIA) (Bioquest, NSW, Australia). The sensitivity, specificity, positive and negative predictive values of the developed EIA (dEIA) were 87, 96.2, 80, 97.7 for the specimens from females and 90.9, 90.7, 71.4, 97.5 for the specimens from males repectively. Cross reaction was not found with Escherichia coli, Acinetobacter anitratus, beta-Streptococcus group A, Enterobacter spp, Enterococcus, Lactobacillus spp, Neisseria spp, but it was found with Candida albicans and herpes simplex virus type 1. The developed EIA can be applied successfully for both genders, particularly males. The cost per test is less than those for CC, kEIA and PACE2.     

Detection of murine typhus infected fleas with an enzyme-linked immunosorbent assay

An enzyme-linked immunosorbent assay (ELISA) for the detection of Rickettsia typhi antigen in homogenates of pooled or individual laboratory infected fleas is described. The assay uses a double sandwich technique, employing a pool of monoclonal antibodies to capture the antigen and a hyperimmune rabbit serum for antigen detection. Using pools of R. typhi infected Xenopsylla cheopis, Ctenocephalides felis, and Leptopsylla segnis, the sensitivity of the ELISA was compared with direct fluorescent antibody examination of individual fleas for rickettsiae and with rickettsial titers determined by plaque enumeration on primary chicken embryo fibroblasts (PFU). Pooled samples with less than 4 PFU of viable rickettsiae gave ELISA results which were not significantly above background. Both ELISA OD and ELISA titer (last dilution giving an OD that was 2 SD above the control) of a 1:10 dilution of homogenate (4 fleas/ml) were linearly related to rickettsial titer up to 10(6.8) PFU/sample. Multiple freeze-thaws of pools of infected fleas led to a rapid loss of ELISA sensitivity. ELISA assays on single fleas demonstrated large individual variability in rickettsial content. This was independent of the number of days postinfectious feeding or the mean number of PFU/flea (10(1.7-6.9) found for pooled fleas in the same cohort. The sensitivity and ease of performance of ELISA should make it usable under field conditions.     

Application of one step RT-PCR assay for detection of flavivirus RNA in mosquitoes

The conditions of one step RT-PCR method for detection of virus RNA in field-collected mosquitoes, and preservation period of infected mosquitoes for one step RT-PCR were examined. We compared several virus RNA extraction methods with artificially contaminated mosquito pools with dengue virus (DV), Japanese encephalitis virus (JEV), and yellow fever virus (YFV) with a known amount of plaque forming unit (PFU) to establish the condition of one step RT-PCR. In this study, most effective RNA extraction method was ISOGEN-LS extraction combined with supernatant of centrifuged mosquito homogenates. Detection limit of one step RT-PCR using flavivirus universal primer in ten mosquitoes/tube (pool) was 10 PFU of DV, JEV and YFV, 1 PFU of each viruses using species-specific primer respectively, in one hundred mosquitoes/tube, 100 PFU/tube using universal primer pairs, 10 PFU/tube using species-specific primer pairs respectively. Dengue virus infected single mosquito was mixed with 99 un-infected mosquitoes, and tested by one step RT-PCR. We could detect single infected mosquito in pools containing 99 un-infected mosquitoes. Aedes aegypti and Aedes albopictus were inoculated intrathoracically with a mouse-adapted strain of dengue-1 virus and were kept up to 30 days at different temperature. Then examined by one step RT-PCR to determine the appropriate mosquito handling method and the condition of transportation. Positive result was obtained up to 30 days after the mosquito died naturally. These results suggested that we could detect flavivirus RNA tested not only from live mosquitoes but also dead mosquitoes as well, and could apply one step RT-PCR as a rapid, specific, and highly sensitive tool for flavivirus surveillance.     

Replication of Rift Valley fever virus in the sand fly Lutzomyia longipalpis

Rift Valley fever virus was shown to replicate in Lutzomyia longipalpis after intrathoracic inoculation. Viral titers peaked at approximately 4 days postinoculation [mean titer = 10(4.0) plaque forming units (PFU)] and remained relatively constant through day 7. A minimum of 6 of 326 sand flies transmitted virus by bite to susceptible hamsters after 5-9 days of extrinsic incubation. Viral titers of sand flies exposed per os declined steadily through day 9. None of 378 flies that had ingested approximately 10(4.0) PFU of virus transmitted virus when refed on susceptible hamsters.     

An EIA method on single donor solubilized HLA antigens for the identification of anti-HLA antibodies

An enzyme immunoassay (EIA) method based on solubilized human leucocyte antigens (HLA) derived from single donor platelets is described. The EIA results on these solubilized single donor HLA antigens (SDszHLA) correlated well with the complement dependent cytotoxicity (CDC) results on the lymphocytes of the same donors and also with the panel reactivity (PRA) in CDC. A concordancy rate of 78% was found for individual HLA specificities. The EIA+/CDC? (‘false positive’) discrepancies were more pronounced than EIA?/CDC+ (‘false negative’) discrepancies and varied for the different donors. To confirm discrepancies, our method was compared with a commercial PRA-STAT EIA method (based on secreted soluble HLA antigens). The same discrepancies between CDC and PRA-STAT EIA were found and are probably due to the higher and different sensitivity (e.g. non complement fixing antibodies) of EIA methods. A SDszHLA EIA method allows the identification of HLA specificities of HLA-antisera. The possibility of using individual and selected donors for the production of SDszHLA allows the directed search for well defined HLA specificities in order to confirm anti-HLA specificities found in other anti-HLA screening methods. An individualized HLA panel can be established with the support of blood banks that have HLA typed blood and platelet donors.     

Current testing strategies for hepatitis C virus infection in blood donors and the way forward

Screening tests for blood donations are based upon sensitivity,cost-effectiveness and their suitability for high-throughput testing.Enzyme immunoassay(EIAs)for hepatitis C virus(HCV)antibodies were the initial screening tests introduced.The"first generation"antibody EIAs detected seroconversion after unduly long infectious window period.Improved HCV antibody assays still had an infectious window period around 66 d.HCV core antigen EIAs shortened the window period considerably,but high costs did not lead to widespread acceptance.A fourth-generation HCV antigen and antibody assay(combination EIA)is more convenient as two infectious markers of HCV are detected in the same assay.Molecular testing for HCV-RNA utilizing nucleic acid amplification technology(NAT)is the most sensitive assay and shortens the window period to only4 d.Implementation of NAT in many developed countries around the world has resulted in dramatic reductions in transfusion transmissible HCV and relative risk is now<1 per million donations.However,HCV serology still continues to be retained as some donations are serology positive but NAT negative.In resource constrained countries HCV screening is highly variable,depending upon infrastructure,trained manpower and financial resource.Rapid tests which do not require instrumentation and are simple to perform are used in many small and remotely located blood centres.The sensitivity as compared to EIAs is less and wherever feasible HCV antibody EIAs are most frequently used screening assays.Efforts have been made to implement combined antigen-antibody assays and even NAT in some of these countries.     

Antibodies to a novel antigen in acute hepatitis C virus infections

BACKGROUND: Conformational viral proteins potentially play an important role in the immunobiology of acute hepatitis C virus (HCV) infection and may enable earlier antibody detection. MATERIALS AND METHODS: HCV RNA was detected using nucleic acid testing. Early antibody production was evaluated using three enzyme immunoassays (EIAs) containing antigenic proteins not present in licensed EIAs. Respectively, these contained: (1) multiple-epitope fusion antigen (MEFA) 7.1-NS3/4a, (2) F and Core, and (3) E1/E2 proteins. NS3/4a is a conformational antigen retaining protease and helicase enzymatic activities. MEFA 7.1 contains the linear epitopes used in licenced EIAs, including the latest EIA-3.0, in combination with genotype 1-3 specific epitopes. Forty-two RNA positive, EIA-3.0 negative samples, including two persistently serosilent cases, were used to evaluate these research EIAs. As controls, 54 EIA-3.0 negative/RNA negative and three HCV RNA+/antibody positive specimens were included. RESULTS: Only the MEFA 7.1-NS3/4a EIA was positive in seven (17%) of the 42 HCV RNA + specimens, in all three EIA-3.0 positive controls but in none of 54 EIA-3.0 negative/HCV RNA negative controls. Notably, six of the seven (86%) specimens had evidence of active hepatitis (ALT > 210 IU/l). The two serosilent cases were research EIA negative. CONCLUSION: A novel EIA with conformational and linear epitopes detected HCV antibodies in 17% of viraemic specimens missed by the standard reference EIA-3.0. Our research EIA appears to detect HCV antibodies closer to the initiation of acute hepatitis. Given that the average RNA-positive, antibody-negative window period is 56.4 days, this 17% yield would translate into a 10-day earlier detection of antibodies.     

Evaluation of an antigen‐capture EIA for the diagnosis of hepatitis E virus infection

An enzyme immunoassay (EIA) has been developed for hepatitis E virus (HEV) antigen (HEV‐Ag) detection and marketed in China. This study aimed to evaluate the sensitivity of the assay and assess the value of HEV‐Ag detection in the diagnosis of HEV infection in comparison with HEV RNA detection. Using serial dilutions of a genotype 4 HEV strain, significant correlation was found between the EIA (S/CO) and HEV RNA (IU/mL) concentration in the range 10^3.5 to 10^0.5 IU/mL HEV RNA, the Pearson correlation coefficient r approached 0.97. The EIA detection limit was 54.6 IU/mL, compared to 24 IU/mL for HEV RNA using real‐time RT‐PCR. In clinical samples from hepatitis E patients, the HEV‐Ag and HEV RNA positivity rates were 55.6% (65/117) and 60.7% (71/117) in sera and 76.7% (56/73) and 84.9% (62/73) in stools, and the concordance of these two markers was 77.8% in sera and 80.8% in stools. In serum samples, the HEV‐Ag positivity rate and the concordance between HEV‐Ag and HEV RNA were inversely proportional to the presence of anti‐HEV antibody. The presence of anti‐HEV IgG could reduce the S/CO of the HEV‐Ag EIA. These results reveal a significant correlation between the detection of HEV‐Ag and HEV RNA. The sensitivity of the HEV‐Ag EIA was lower than real‐time RT‐PCR but could be higher than conventional nested RT‐PCR. Therefore, the detection of HEV‐Ag in serum and faeces is valuable for the diagnosis and prognosis of HEV infection in developing regions where real‐time RT‐PCR is not available.     

Differential replicative fitness of the four dengue virus serotypes circulating in Colombia in human liver Huh7 cells

Dengue has been a significant public health problem in Colombia since the simultaneous circulation of the four dengue virus serotypes. The replicative fitness of dengue is a biological feature important for virus evolution and contributes to elucidating the behavior of virus populations and viral pathogenesis. However, it has not yet been studied in Colombian isolates. This study aimed to compare the replicative fitness of the four dengue virus serotypes and understand the association between the serotypes, their in vitro infection ability, and their replication in target cells. We used three isolates of each DENV serotype to infect Huh-7 cells at an MOI of 0.5. The percentage of infected cells was evaluated by flow cytometry, cell viability was evaluated by MTT assay, and the pathogenicity index was calculated as a ratio of both parameters. The replicative fitness was measured by the number of viral genome copies produced using quantitative PCR and the production of infectious viral progeny was measured by plaque assay. We showed that Huh-7 cells were susceptible to infection with all the different strain isolates. Nevertheless, the biological characteristics, such as infectious ability and cell viability, were strain-dependent. We also found different degrees of pathogenicity between strains of the four serotypes, representative of the heterogeneity displayed in the circulating population. When we analyzed the replicative fitness using the mean values obtained from RT-qPCR and plaque assay for the different strains, we found serotype-dependent behavior. The highest mean values of replicative fitness were obtained for DENV-1 (log 4.9 PFU/ml) and DENV-4 (log 5.28 PFU/ml), followed by DENV-2 (log 3.9 PFU/ml) and DENV-3 (log 4.31 PFU/ml). The internal heterogeneity of the replicative fitness within each serotype could explain the simultaneous circulation of the four DENV serotypes in Colombia.     

Evaluation of influenza virus detection by direct enzyme immunoassay (EIA) and conventional methods in asthmatic patients

Throat gargle specimens of fifty-seven acute asthmatic patients (age range 18-40 years) were collected for the study. Thirty-four patients were found influenza virus positive in acute asthma cases. Influenza virus was isolated by conventional culture method on MDCK cell-line and by enzyme immunoassay test (EIA). The EIA negative specimens were retested after virus amplification on MDCK cell-line. Virus shedding and virus surface receptors assay was carried out to determine influenza virus titre. Airway functions were measured by spirometry. A good relationship was observed between the degree of airflow limitation and presence of influenza virus infection (p < 0.001; r = 0.85). A comparable difference in % FEV1 was observed in relation to the symptoms. The patients with greater viral antigen load had lower % FEV1. Two specimens, which were EIA negative, turned out to be positive after amplification on MDCK cell-line. The sensitivity was 98% and specificity was 100%. It was concluded that EIA method is a useful diagnostic tool as it detects influenza viral antigen quickly as compared to conventional methods.     

Detection of yellow fever virus in serum by enzyme immunoassay

Yellow fever (YF) virus is present in patient's blood during the acute phase of illness. Virus isolation and identification provide a potential method of early diagnosis, but available techniques are slow and require specialized materials and equipment. An alternative approach is direct detection of YF antigen in serum by means of an enzyme-linked immunosorbent assay (ELISA). An antigen-capture ELISA was developed, which used anti-YF antibodies, immobilized on a solid phase (polystyrene plates), to capture YF virus from serum samples. After addition of the virus-containing sample, anti-YF detecting antibody conjugated to alkaline phosphatase was added to detect viral antigen. Trials with various capture and detecting antibodies in systems employing purified YF 17D virus, led to the selection of: 1) two capture antibodies (pooled human serum containing high titer YF IgM antibodies and a type-specific YF monoclonal antibody), and 2) a detecting antibody conjugate consisting of monoclonal antibody broadly cross-reactive with all flaviviruses, purified by affinity chromatography, and conjugated to alkaline phosphatase. The limit of sensitivity in tests against purified YF 17D virus diluted in buffer or normal human serum was 10(3.0) - 10(3.6) PFU/0.05 ml or 0.007-0.029 microgram viral protein/0.05 ml. Sera obtained at intervals from rhesus and cynomolgus monkeys after infection with a wild YF virus strain were tested. The limit of sensitivity of the assay applied to viremic monkey serum was similar (approximately 3.5 log10PFU/0.05 ml).(ABSTRACT TRUNCATED AT 250 WORDS)     

Experimental studies on the transmission cycle of Thogoto virus, a candidate orthomyxovirus, in Rhipicephalus appendiculatus

Thogoto (THO) virus, a candidate orthomyxovirus, replicated in and was transmitted by larvae, nymphs, and adults of the brown ear tick, Rhipicephalus appendiculatus. Larvae fed on viremic hamsters (10(7-8) PFU/ml blood) acquired an average of 10(2.5) PFU per tick. Following engorgement the titer dropped to 10(1.9) PFU on day 2 but increased by day 6 to 10(3.3) PFU. Virus survived transstadially in these ticks as demonstrated by the fact that, on day 10, newly moulted nymphs contained, on average, 10(3.5) PFU/tick. When 10 such infected nymphs were placed on a hamster a fatal infection of the animal developed involving a viremia of 10(6.7) PFU/ml blood. Another group of 6 infected nymphs did not elicit a detectable viremia in a hamster, or cause death. However the animal seroconverted to THO, virus indicating that virus transmission had occurred. Following acquisition of THO virus at the larval stage, virus was detected in adult ticks 138 days later. Uninfected nymphs fed on viremic hamsters acquired an average of 10(4) PFU/nymph. No virus was detected in the nymphs 4 days post-engorgement. Virus was, however, recovered by 6 days post-engorgement (10(4.7) PFU/nymph). Virus persisted transstadially as shown by the presence of an average of 10(3.4) PFU in newly moulted adults. Three groups of these infected adults (5-6 ticks/group) induced viremia in hamsters with blood titers of the order 10(2.8-3.5) PFU/ml. Virus persisted in engorged adults for up to 66 days following nymphal engorgement.(ABSTRACT TRUNCATED AT 250 WORDS)     

Transstadial and horizontal transmission of Rift Valley fever virus in Hyalomma truncatum

We exposed Hyalomma truncatum and Rhipicephalus appendiculatus to Rift Valley fever (RVF) virus in order to assess the possible role of these ticks as enzootic/epizootic RVF vectors. The virus replicated in H. truncatum after intracoelomic inoculation, and a minimum transmission rate of 17% was achieved after 15 days intrinsic incubation. The virus persisted at least 58 days in these ticks. Virus was also shown to pass transstadially from inoculated H. truncatum nymphs to adults, with peak viral titers reaching 10(3.5) plaque-forming units (PFU) in adult males after they were provided with bloodmeals. Virus was recovered from adult females 121 days after they were inoculated as nymphs. Viral titers peaked in inoculated male ticks after dropping off a host (mean titer = 10(4.3) PFU). RVF virus was not detected in pools of eggs and larval progeny from 11 infected female H. truncatum. H. truncatum larvae and nymphs did not become infected after ingesting greater than 10(2.0) PFU while feeding on a RVF viremic hamster. The number of infected specimens declined rapidly after RVF virus was inoculated into R. appendiculatus adults, and virus was undetectable 12 days post-inoculation.     

Dose-related effect of acetylsalicylic acid on replication of varicella zoster virus in vitro.

Cultivation of human embryonic lung (HEL) cells in media containing acetylsalicylic acid (ASA) at 100 micrograms/ml and maintenance at this level after inoculation with either cell-free varicella zoster virus (VZV) or virus-infected cells resulted in a 2- to 4-fold increase in yields of cell-free virus released by sonication. The degree of enhancement was dependent upon multiplicity of infection and time of harvest. Enhanced viral yields were not consistently accompanied by an increase in the number of infected cells, nor was VZV plaque formation in HEL indicator cells significantly increased in the presence of ASA at 100 micrograms/ml. In the presence of ASA at 500-1000 micrograms/ml, VZV plaque formation was inhibited; this inhibition was partially reversible, depending on concentration and period of exposure to ASA. These findings may bear on the apparent association between ASA ingestion and the development of Reye syndrome after infection with varicella virus.     

Clinical application of polymerase chain reaction to diagnose Clostridium difficile in hospitalized patients with diarrhea.

BACKGROUND & AIMS: Clostridium difficile is a common cause of diarrhea in hospitalized patients and is associated with significant morbidity and cost. The current diagnostic standard, enzyme immunoassay (EIA), has low sensitivity, leading to duplicate testing and empiric treatment. We sought to show the usefulness and potential cost effectiveness of polymerase chain reaction (PCR) amplification of toxin B gene for diagnosis of C. difficile-induced diarrhea. METHODS: A total of 148 stool samples from academic and community-based hospitals were sent for EIA testing and were evaluated prospectively for the presence of toxin B gene by PCR. Results were compared with EIA regarding sensitivity, specificity, and predictive values. Medical charts were reviewed to determine the following: (1) number of EIAs sent per admission, (2) number sent within a 24-hour time period, and (3) how caregivers practiced based on EIA results. RESULTS: The mean age of 130 patients was 55 years. EIA and PCR were positive in 6.8% and 13.6% of patients, respectively. EIA sensitivity was 40%, specificity was 98%, and positive and negative predictive values were 80% and 91%, respectively. The cost of the PCR was $22/sample. Empiric treatment for C. difficile was given unnecessarily in 42% of EIA-negative results. Thirty percent of patients had 3 or more EIAs sent during their hospital admission. Of patients with multiple samples sent, 57% had more than 1 sample sent in a 24-hour period. CONCLUSIONS: Many physicians do not conform to practice guidelines regarding recommended diagnosis and empiric treatment of C. difficile. Toxin B gene PCR represents a more sensitive and potentially cost-effective method to diagnose C. difficile-induced diarrhea than EIA and should be considered for use as an alternative diagnostic standard.