MRL Diagnostics Research Articles

Comparative Seroepidemiologic Analysis of Chlamydophila Pneumoniae Infection using Microimmunofluorescence, Enzyme Immunoassay and Neutralization Test: Implications for Serodiagnosis

A seroepidemiologic study using the microimmunofluorescence (MIF) technique was conducted to determine the prevalence of Chlamydophila pneumoniae IgG antibodies among 205 healthy Singapore university undergraduates using the MRL Diagnostics MIF test kit. The overall seroprevalence was 35.1% with significantly higher seropositivity rates among males than females (48.2 vs. 18.7%, P<0.001). A comparative study using the Labsystems MIF test kit was conducted on sera from 192 students. Using the MRL MIF test as the reference, the sensitivity and specificity of Labsystems MIF test were 92.6 and 87.9%, respectively. A total of 78 samples comprising 15 MIF-negative and 63 MIF-positive samples were also tested for complement-independent neutralizing antibodies in vitro. All the 78 samples and 11 additional MIF-negative samples were also tested for IgM, IgG and IgA against C. pneumoniae by enzyme immunoassay (EIA) using the Labsystems EIA test kit. None of these 89 samples were seropositive for IgM. The percentages of IgG and IgA seropositivity increased with increasing grades of MIF-positivity. Among the IgG seropositive samples, 69.1% were also positive for IgA, suggesting that a high proportion of infected individuals also had IgA antibodies denoting chronicity. Neutralizing antibodies were detected in 22.2% of MIF-positive sera, but only in 6.7% of MIF-negative sera. 26.4 and 34.2% of samples which were IgG and IgA seropositive respectively also exhibited neutralizing activity. The percentages of MIF-positive sera with neutralizing activity increased with the grade of MIF positivity, i.e. 0% (1+), 7.1% (2+), 18.8% (3+), and 63.6% (4+). High-grade MIF positivity (particularly with MRL MIF kits) may represent a useful serologic marker of predictive value for neutralizing activity.

Performance of Three Microimmunofluorescence Assays for Detection of Chlamydia pneumoniae Immunoglobulin M, G, and A Antibodies

The microimmunofluorescence (MIF) test is considered the "gold standard" for laboratory diagnosis of acute and chronic Chlamydia pneumoniae infection. The performance of a MIF test based on C. pneumoniae antigen from Washington Research Foundation (WRF) was compared with those of assays from Labsystems (LAB) and MRL Diagnostics (MRL) by investigation of sera from three groups of patients: group I, 83 sera from 28 patients with atypical pneumonia; group II, 37 sera from 16 patients with acute C. pneumoniae or Chlamydia psittaci respiratory tract infection confirmed by PCR or culture; group III, 100 sera from 100 persons enrolled in the Copenhagen City Heart Study. The accordance among the results of the WRF assay and the two commercial assays was excellent for the immunoglobulin M (IgM) antibody detection rate (98%). The accordance in detection rates for IgG and IgA antibodies in sera from patients with acute infections was acceptable (87 and 88%), and in sera from group III, it was excellent (95 and 97%). The determinations of endpoint titers were reproducible with <1 dilution step difference for all three methods, except that the mean IgM antibody titer found by the LAB assay was almost 2 dilution steps higher than that found by the other two methods. Although the three assays use different C. pneumoniae strains as antigens, the detection rates and IgG and IgA endpoint titers were similar. The difference in endpoint titers of IgM antibodies is of no major concern, as the diagnosis of acute C. pneumoniae infection rests on the presence of IgM antibodies, not on their level.

Clinical evaluation of commercial serological test for Bartonella infection

We evaluated the usefulness of a serological diagnostic kit (Bartonella IFA IgG, IgM; MRL Diagnostics) for Bartonella henselae infection. Of the 110 healthy individuals, 107 (97.3%) were with titers being less than 1:64 for IgG antibody to B. henselae, 2 were with titers being 1:64 and 1 with 1:128, IgM antibody to B. henselae was negative in all individuals. Serological diagnosis of cat scratch disease (CSD) using indirect fluorescence antibody (IFA) methods (in-house and diagnostic kit) was made in either elevated titers of IgM (> or = 1:20) or IgG (> or = 1:256) antibodies, or a four-fold rise in IgG titer between acute and convalescent sera. Of the 18 individuals with serological diagnosis of CSD by in-house IFA method in 26 CSD clinical diagnosed patients, 15 (83%) were compatible with the results of the diagnostic kit, whereas 3 (17%) were not compatible. Of the 8 without serological diagnosis, 1 (13%) was serologically diagnosed as CSD, and the others were negative. Overall, the serological diagnosis was made in 16 of 26 (62%). The specificity and sensitivity of this kit were 100% and 62%, respectively. The cross-reaction between B. henselae and Bartonella quintana was observed in sera from controls and patients. Our results show that the diagnostic kit as well as in-house method is an useful tool for the serological diagnosis of cat scratch disease.

Evaluation of four methods for detection of immunoglobulin M antibodies to dengue virus.

Dengue has become hyperendemic in many islands of the Caribbean region. The performance in a diagnostic laboratory of four commercial assays for detection of immunoglobulin M (IgM) antibodies was evaluated. Sera from 62 patients with dengue virus infection were studied. These included 18 patients from whom dengue virus type 2 was isolated in a 1997 outbreak (specimens collected a mean of 14 days after onset of symptoms), 8 patients with dengue hemorrhagic fever (mean time after onset, 11 days), and 36 patients in whom dengue was previously confirmed by serology (mean time after onset, 10 days). Thirty serum specimens from blood donors in a country where dengue is not endemic were used as negative controls. The methods evaluated were two IgM-capture enzyme-linked immunosorbent assays (ELISA) (MRL Diagnostics, Cypress, Calif., and PanBio, Queensland, Australia), a dot ELISA dipstick assay (Integrated Diagnostics, Baltimore, Md.), and a rapid immunochromatographic assay for dengue IgG and IgM (PanBio IC). IgG antibodies were also detected by an ELISA method (MRL Diagnostics). The sensitivities of the four assays were as follows: MRL Diagnostics IgM ELISA, 98.4%; PanBio IgM ELISA, 85.5%; Integrated Diagnostics IgM dot ELISA, 96. 8%; and PanBio IC, 83.9%. The specificities of all tests were 100%. Evidence of secondary dengue was found in all patients with dengue hemorrhagic fever and in 83% of the remaining patients. The MRL Diagnostics IgM ELISA appears to be more sensitive than the PanBio IgM ELISA, and this may be significant when IgM titers are low, particularly in patients with secondary dengue infections. The dot ELISA dipstick assay is equally sensitive and may be more appropriate for use in laboratories with lower workloads.

Evaluation of the MRL diagnostics dengue fever virus IgM capture ELISA and the PanBio Rapid Immunochromatographic Test for diagnosis of dengue fever in Jamaica.

We evaluated two new commercial dengue diagnostic tests, the MRL Diagnostics Dengue Fever Virus IgM Capture ELISA and the PanBio Rapid Immunochromatographic Test, on serum samples collected during a dengue epidemic in Jamaica. The MRL ELISA method correctly identified 98% (78 of 80) of the samples as dengue positive, while the PanBio test identified 100% (80 of 80). Both tests were 100% (20 samples of 20) specific.

Evaluation of the indirect hemagglutination assay for diagnosis of acute leptospirosis.

Serology plays an important role in the diagnosis of leptospirosis. Few laboratories have the resources and expertise to perform the microscopic agglutination test. There is a need for rapid and simple serological tests which facilitate the early diagnosis of leptospirosis, while antibiotic therapy may be most effective. A commercially available indirect hemagglutination assay (IHA; MRL Diagnostics, Cypress, Calif.) was evaluated with multiple serum specimens from 107 patients being investigated for leptospirosis. By using a combination of enzyme-linked immunosorbent assay (ELISA) methods for immunoglobulin M (IgM) and IgG antibodies and the microscopic agglutination test, 54 patients were found to have leptospirosis and 53 were found not to have leptospirosis. The sensitivity of IHA for the detection of acute leptospirosis was 100%, the specificity was 94%, the positive predictive value was 95%, and the negative predictive value was 100%. IHA was negative when 13 antinuclear antibody-positive sera, 24 serum specimens from patients with syphilis, and 16 serum specimens false positive by the Venereal Disease Research Laboratory test were tested. IHA was shown to detect both IgM and IgG classes of antibodies in human sera. Serum specimens from 27 dogs investigated for leptospirosis were studied: 3 samples gave nonspecific hemagglutination, but for all remaining samples, the results of IHA and an IgM ELISA were concordant. Performance of IHA was simple, and IHA requires no specialized equipment. It represents a useful assay for laboratories which require a leptospiral diagnostic capability but lack the expertise to perform specialist investigations.

Evaluation of commercial slides for detection of immunoglobulin G against Bartonella henselae by indirect immunofluorescence.

Four commercial slides were compared with in-house slides for the detection of immunoglobulin G (IgG) against Bartonella henselae in 58 healthy persons from a rural region by an indirect immunofluorescence assay. MRL-BA slides (MRL Diagnostics, USA) and Virion slides (Virion, Switzerland) with agar-derived Bartonella henselae showed IgG titers of > or = 1:256 in 44.8% and 51.7%, respectively, whereas Bion slides (Bios, Germany), MRL-Vero slides (MRL Diagnostics), and in-house slides with cell-associated Bartonella henselae showed such titers in 3.4%, 5.1% and 3.4%, respectively. The MRL-Vero slides (Bartonella IgG substrate slides, MRL Diagnostics) were further evaluated with 26 patients with cat scratch disease, 20 patients with lymphadenopathy not due to cat scratch disease, 100 blood donors from an urban area, and 120 blood donors from a mixed urban/rural area. In our mixed urban/rural population the IgG titer of 1:256 had a sensitivity of 84.6% and a specificity of 93.4% for the serodiagnosis of cat scratch disease. Seroprevalence was higher in blood donors from the mixed area (50.8%) than from the urban area (37%). MRL-Vero slides were considered useful for the serodiagnosis of cat scratch disease by indirect immunofluorescence and have replaced our in-house system. However, patients with low IgG titers should be retested three to four weeks after initial sampling to demonstrate a possible rise of IgG titers in paired sera.

Evaluation of a new commercial microimmunofluorescence test for detection of antibodies to Chlamydia pneumoniae, Chlamydia trachomatis, and Chlamydia psittaci.

A new commercial test for chlamydial serology, the MRL-Micro-Immunofluorescent Test (MRL; MRL Diagnostics, USA) was compared with the standard microimmunofluorescence test (MIF) using sera from 246 patients. Chlamydia pneumoniae immunoglobulin G (IgG) antibodies were detected in 46.3% (MIF) and 64.2% (MRL) of sera and Chlamydia trachomatis IgG in 23.2% (MIF) and 25.2% (MRL); Chlamydia psittaci IgG antibodies were found with the MRL in 1% of the sera from a general population and in 17.3% of preselected sera with elevated complement fixation titers. Titers were usually higher with the MRL. IgG titers of > or = 1:512 were detected in only 2% of sera using the standard MIF but in 30% using the MRL. In 16 sera from three Chlamydia pneumoniae culture-positive patients, the diagnosis of acute infection could be confirmed serologically in one with the MRL test but in none with the MIF test, indicating a higher sensitivity of the MRL.

Widely used, commercially available Chlamydia pneumoniae antigen contaminated with mycoplasma.

Mycoplasma contamination was detected in a widely used commercially available Chlamydia pneumoniae antigen preparation. Contamination was studied with a mycoplasma group-specific 16S rRNA polymerase chain reaction (PCR) and sequence analysis. Several lots of the purified C. pneumoniae antigen from the Washington Research Foundation appeared to be contaminated with the same Mycoplasma species, which appeared to be closely related to M. arginini. Antigen slides prepared for the detection of chlamydia antibodies by MRL Diagnostics were contaminated with the same Mycoplasma sp. Chlamydia antigen slides from Labsystems OY and two chlamydia complement fixation reagents (Virion International Distribution Ltd and Behring Werke) were not contaminated. It is concluded that commercially available C. pneumoniae antigens may contain mycoplasma antigens as well. Although the impact of such mycoplasma contamination on the results of chlamydia serology may not be significant, routine screening of all antigen preparations obtained by tissue culture before their distribution and use is recommended.