False-Positive IgM Antibody Tests for Cytomegalovirus in Patients with Acute Epstein-Barr Virus Infection

 The diagnosis of acute cytomegalovirus (CMV) infection is frequently based on a positive IgM result. False-positive reactions due to interfering infections may exist. Between August 1998 and May 1999, 62 patients were found to be IgM positive and IgG negative with the Axsym assay (Abbott, Germany). Serological testing for Epstein-Barr virus (EBV) was performed in these patients to detect any cross-reactivity due to acute mononucleosis. Additionally, the results of the CMV Axsym was evaluated in 40 patients with acute EBV infection. The results suggest that the CMV-IgM Axsym assay shows a lack of specificity due to acute EBV infection. Precautions must be taken when CMV-IgM Axsym results are interpreted. It seems necessary to confirm equivocal results with another technique and to take into account other clinical and biological observations.     

Coccidioidomycosis

Coccidioidomycosis is a largely self-limited fungal respiratory illness. However, the infrequent case of progressive or disseminated disease can be devastating. As international travel to and from endemic areas increases, physicians unfamiliar with the disease may be called upon to recognize and treat serious coccidioidal infections. The major risk factors for dissemination are race and immunosuppression. The most common sites of dissemination are the skin, lymph nodes, bone and meninges. Diagnosis is aided by investigation of the patient's clinical history, delayed-type hypersensitivity skin test reaction, serologic testing, and recovery of organisms from infected tissue or secretions for direct examination and culture. Fungicidal agents are not available, fortunately, fungistatic therapy allows many patients to recover. The availability of both intravenous/intrathecal and oral agents now allows more therapeutic flexibility in the treatment of this disease.     

Comparative Accuracy of the InBios Scrub Typhus Detect IgM Rapid Test for the Detection of IgM Antibodies by Using Conventional Serology

This study investigated the comparative accuracy of a recombinant 56-kDa type-specific antigen-based rapid diagnostic test (RDT) for scrub typhus for the detection of IgM antibodies by using conventional serology in well-characterized serum samples from undifferentiated febrile illness patients. The RDT showed high specificity and promising comparative accuracy, with 82% sensitivity and 98% specificity for samples defined positive at an IgM indirect immunofluorescence assay positivity cutoff titer of ≥1:1,600 versus 92% and 95% at ≥1:6,400, respectively.     

Coccidioidomycosis of the tongue

A Spanish-speaking farm worker with type 2 diabetes mellitus presented to our institution with an ulcerating tongue lesion. He was initially evaluated for possible squamous cell carcinoma; however, histologic examination revealed coccidioidomycosis. Coccidioidal titers were elevated and consistent with disseminated disease, although the patient had no systemic symptoms and clinical evaluation was negative. He was ultimately treated with intravenous and oral antifungal medication, which was followed by improvement of his tongue lesion.     

Serology of coccidioidomycosis.

Serologic tests have assisted in the diagnosis and prognosis of coccidioidomycosis for a half-century. The causative agent, Coccidioides immitis, is a dimorphic fungus existing in a hyphal form with arthroconidia in nature and in the usual culture. The arthroconidia represent the inhaled infective forms which in vivo and under special laboratory conditions form spherules which endosporulate. The culture filtrate/autolysate (coccidioidin) from the hyphal phase has provided antigens of suitable reliability for currently used serologic tests. These tests are primarily to determine the two major antibody responses: the early immunoglobulin M (IgM) response is useful in the diagnosis of acute primary coccidioidomycosis. Later, IgG is produced and usually outlasts the IgM, persisting in chronic coccidioidomycosis. The IgM is detectable by tube precipitin, a corresponding immunodiffusion, or latex particle agglutination tests. The pertinent antigen(s) is heat stable and pronase resistant and appears to be largely carbohydrate, mainly mannose with some 3-O-methyl mannose. The IgG detectable in the serum and other body fluids by complement fixation and a corresponding immuno-diffusion is useful in diagnosis, and its quantitation provides an indicator of progression of disease (increasing titer) or regression (decreasing titer). The pertinent antigen appears to be a heat-labile, pronase-sensitive protein which in an unreduced form has a molecular weight of 110,000. A third very useful serologic procedure is the exoantigen test for identification of putative cultures of C. immitis.     

Coccidioidomycosis in hematopoietic stem cell transplant recipients.

Coccidioidomycosis is an endemic fungal infection of the desert southwestern United States that can cause devastating disseminated infection in immunocompromised persons. Clinical coccidioidomycosis, which is caused by Coccidioides species, has been well characterized in patients who have had solid organ transplants, but it has rarely been described in patients who have received a hematopoietic stem cell transplant (HSCT). We report the experience of 121 consecutive HSCT recipients at a single tertiary care institution in an endemic area. One patient had fatal disseminated coccidioidomycosis after receiving an allogeneic transplant, and 2 patients had pulmonary infection before successful autologous HSCT; 1 of these 2 had a reactivation of coccidioidal infection after HSCT but was treated and survived. Coccidioidomycosis was not commonly identified in HSCT recipients, even in the endemic area. A prospective evaluation is required to address the optimal use of coccidioidal serologic tests, antifungal protocols, and secondary prophylaxis in these patients.     

Coccidioidomycosis of the female genital tract

Female genital tract involvement is a rare manifestation of disseminated coccidioidomycosis; to our knowledge, only ten patients have previously been described in the English literature. We describe a patient who seems to be unique in that she developed female genital tract coccidioidomycosis and coccidioidal peritonitis after chemotherapy for Hodgkin's disease. Coccidioidomycosis of the female genital tract is usually manifest as granulomatous endometritis and/or granulomatous tubo-ovarian disease with peritonitis. The diagnosis of coccidioidomycosis was unsuspected clinically in all 11 reported cases (including our patient); initial diagnosis was made by biopsy or culture in all 11 patients. In eight of the reported cases of female genital tract coccidioidomycosis (including our patient), clinical improvement occurred after treatment with surgery or antifungal chemotherapy; three patients died of disseminated coccidioidomycosis.     

Circulating heavy IgM in IgM nephropathy.

IgM nephropathy (IgMN) causes nephrotic syndrome and is characterized by IgM mesangial deposits. It is speculated that these deposits are derived from circulating IgM aggregates or immune complexes, either of which would have a molecular weight heavier than that of normal IgM. To test this hypothesis the sera of 11 patients with IgMN, five patients with nephrotic syndrome of other etiologies, and 13 normal controls were analysed for such heavy IgM. The serum samples were passed over a Biogel A5M molecular sieve column and the fractions were tested for IgM concentration by enzyme linked immunosorbent assay (ELISA). The column effluent from the void volume to the IgM peak was divided into four equal regions, and the average IgM concentrations in each region were compared. The IgMN group had significantly higher IgM concentrations than normal controls in the heaviest region (0.81 +/- 0.84 vs. 0.32 +/- 0.17 micrograms/ml; P = 0.01) and in the lightest region (95.8 +/- 59.5 vs. 46.3 +/- 41.2 micrograms/ml; P = 0.02). Although the IgMN group appeared to have about double the IgM levels of the nephrotic control group in all four regions, this was only significant in the lightest (19S) region. In serum samples from two IgMN patient methods known to break antigen antibody bonds eliminated the heavy IgM; in one case we used gel filtration in potassium thiocyanate and in another ultracentrifugation at pH 2.8. In addition, the heavy IgM in this second patient exhibited complement fixation activity in a sandwich ELISA for IgM-C3 complexes. We conclude that IgMN patients have circulating heavy IgM, which by preliminary studies probably consists of complement fixing IgM immune complexes.     

Rh Serology - Coordinator's report

168 Rh specific monoclonal antibodies (Mabs) were evaluated in 43 laboratories. Red cells from 63 Rh variant phenotype donations were circulated in panels to evaluators. Circulation was organised to ensure adequate duplicate testing. Evaluators were asked to test each Mab at 3 dilutions in two specified techniques against normal phenotype cells, panel cells received and any other Rh variant cells they had available.Results were analysed by expressing the sum of reaction grades for each Mab with each variant cell as a percentage of the sum of reaction grades of that Mab with normal phenotype cells. Reactions with enzyme-treated cells were found to be highly variable between laboratories, and further analysis of these results was not attempted. Anti-D Mabs were sorted into groups which had the same pattern of reactions with different phenotype cells. 24 such patterns were identified, 17 corresponding to previously reported patterns and 7 new ones.     

Coccidioidomycosis: host response and vaccine development

Coccidioidomycosis is caused by the dimorphic fungi in the genus Coccidioides. These fungi live as mycelia in the soil of desert areas of the American Southwest, and when the infectious spores, the arthroconidia, are inhaled, they convert into the parasitic spherule/endospore phase. Most infections are mild, but these organisms are frank pathogens and can cause severe lethal disease in fully immunocompetent individuals. While there is increased risk of disseminated disease in certain racial groups and immunocompromised persons, the fact that there are hosts who contain the initial infection and exhibit long-term immunity to reinfection supports the hypothesis that a vaccine against these pathogens is feasible. Multiple studies have shown that protective immunity against primary disease is associated with T-helper 1 (Th-1)-associated immune responses. The single best vaccine in animal models, formalin-killed spherules (FKS), was tested in a human trial but was not found to be significantly protective. This result has prompted studies to better define immunodominant Coccidioides antigen with the thought that a subunit vaccine would be protective. These efforts have defined multiple candidates, but the single best individual immunogen is the protein termed antigen 2/proline-rich antigen (Ag2/PRA). Studies in multiple laboratories have shown that Ag2/PRA as both protein and genetic vaccines provides significant protection against mice challenged systemically with Coccidioides. Unfortunately, compared to the FKS vaccine, it is significantly less protective as measured by both assays of reduction in fungal CFU and assays of survival. The capacity of Ag2/PRA to induce only partial protection was emphasized when animals were challenged intranasally. Thus, there is a need to define new candidates to create a multivalent vaccine to increase the effectiveness of Ag2/PRA. Efforts of genomic screening using expression library immunization or bioinformatic approaches to identify new candidates have revealed at least two new protective proteins, expression library immunization antigen 1 (ELI-Ag1) and a beta-1,3-glucanosyltransferase (GEL-1). In addition, previously discovered antigens such as Coccidioides-specific antigen (CSA) should be evaluated in assays of protection. While studies have yet to be completed with combinations of the current candidates, the hypothesis is that with increased numbers of candidates in a multivalent vaccine, there will be increased protection. As the genome sequences of the two Coccidioides strains which are under way are completed and annotated, the effort to find new candidates can increase to provide a complete genomic scan for immunodominant proteins. Thus, much progress has been made in the discovery of subunit vaccine candidates against Coccidioides and there are several candidates showing modest levels of protection, but for complete protection against pulmonary challenge we need to continue the search for additional candidates.     

Elimination of False-Positive Results in a Luminex Assay for Pneumococcal Antibodies

In our 14-valent Luminex assay for pneumococcal antibodies, we identified two groups of sera that caused false-positive results. The first group bound nonspecifically to the Luminex microspheres. The second group reacted specifically with bovine serum albumin (BSA). We describe here methods that eliminated the false-positive reactivity of both groups.We previously described a multiplexed pneumococcal antibody assay based on the Luminex xMAP technology (11). Several other laboratories subsequently also described multiplexed Luminex assays for detecting these antibodies (1, 6, 15). In addition, the Luminex xMAP technology has been used for multiplexed assays to measure antibody responses to vaccines for other infectious diseases, including those caused by Neisseria meningitidis, Haemophilus influenza type b, Bordetella pertussis, Clostridium tetani, Corynebacterium diphtheriae, Bacillus anthracis, and papilloma virus (3, 4, 10, 12, 13, 17). Waterboer et al. (18) documented an intrinsic problem with the use of the Luminex technology for serological assays. They found that some human sera bind directly to the carboxylated MicroPlex (formally MultiAnalyte) microspheres, causing a very high level of nonspecific background. These workers also found that SeroMAP microspheres, introduced by Luminex Corporation specifically for use in serological assays, reduced but did not eliminate the nonspecific-binding problem.Using our original protocol (11) for the 14-valent pneumococcal antibody assay with lot B MicroPlex microspheres, we encountered serum samples with very high-level false-positive results that were near or above the analytical measurement range (AMR) of the assay (Table ​(Table1).1). Approximately 15 of every 1,000 sera tested for pneumococcal antibodies exhibited this behavior. We termed these samples “polyspecific,” although they did not react specifically to pneumococcal polysaccharides (PnPs). We tested a panel of 33 of these polyspecific sera and 1 control serum sample not showing polyspecific reactivity against an unconjugated MicroPlex microsphere and an unconjugated SeroMAP microsphere. The serum samples used in this study were submitted to ARUP Laboratories for pneumococcal antibody testing. All samples were deidentified according to protocols approved by the University of Utah Institutional Review Board (no. 7275). Serum samples were diluted 1:25 in phosphate-buffered saline (PBS), pH 7.2, with 5 μg/ml pneumococcal C-polysaccharide (C-Ps) (Staten Serum Institut, Copenhagen, Denmark), 5 μg/ml pneumococcal polysaccharide 22F (American Type Culture Collection, Manassas, VA), and 0.0015% bromcresol purple (BCP) (Sigma-Aldrich, St. Louis, MO). A MicroPlex (region 7) (Luminex Corporation, Austin, TX) microsphere and a SeroMAP (region 8) (Luminex Corporation, Austin, TX) microsphere were pelleted by centrifugation and resuspended in blocking/storage (B/S) buffer consisting of PBS with 0.1% bovine serum albumin (BSA) (Sigma-Aldrich, St. Louis, MO) or in BSA-free StabliGuard immunoassay stabilizer (SG01) (SurModics, Inc., Eden Prairie, MN). Serum dilutions were incubated with the uncoupled microspheres for 20 min at room temperature with shaking, washed once with PBS by filtration, incubated for 20 min at room temperature with shaking with phycoerythrin (PE)-labeled affinity-purified anti-human IgG (γ) (Southern Biotech, Birmingham, AL) in B/S buffer, and washed once with PBS. Microspheres were counted with a Luminex 100 analyzer. The MicroPlex and SeroMAP microspheres were compared in the two diluents in the same assay run, with the same sample dilutions and PE conjugate, to eliminate run-to-run variation. As shown in Fig. ​Fig.1A,1A, all 33 of the polyspecific sera tested reacted strongly to the unconjugated MicroPlex microsphere suspended in B/S buffer, with median fluorescence intensity (MFI) values that ranged from 905 to 18,674. In contrast, the MFI of the control serum sample was 38. Compared to those for the MicroPlex microsphere, the MFI values for the SeroMAP microsphere suspended in B/S buffer were low. All 33 of the polyspecific sera, however, had background MFI values above 110, compared to the control serum sample, which had an MFI of 28. Twenty-four of the 33 sera (72.7%) had MFI values above the cutoff value of 200. A background MFI value of 200 could falsely elevate the pneumococcal antibody assay results by 0.1 μg/ml or more for 5 of the 14 serotypes. If the long-term protective level after pneumococcal vaccine immunization is considered to be 1 μg/ml, a background MFI level of 200 could lead to misinterpretation of protective status. In addition, 10 of the polyspecific sera had background MFI levels above 500 with the SeroMAP microsphere, and 5 of these sera had MFI levels above 1,000. Two of the polyspecific sera, no. 3 and 23, had very high levels of nonspecific reactivity to the SeroMAP microspheres, with MFI values of 4,877 and 2,666, respectively.Open in a separate windowFIG. 1.Nonspecific reactivity of human sera to Luminex microspheres. Shown are median fluorescence intensities for 33 polyspecific sera and a control serum sample reacted against unconjugated MicroPlex (clear bars) and SeroMAP (solid bars) microspheres. (A) Microspheres suspended in B/S buffer. (B) Microspheres suspended in StabliGuard.

TABLE 1.

IgG concentrations in serum before (protocol 1) and after (protocol 2) removal of nonspecific binding to microspheres

Evaluation of Coccidioides Antigen Detection in Dogs with Coccidioidomycosis

Antigen detection has been reported to be a promising method for rapid diagnosis of coccidioidomycosis in humans. Coccidioides antigen detection has not been previously reported in dogs with coccidioidomycosis and was evaluated in 60 cases diagnosed based on detection of anti-Coccidioides antibodies at titers of 1:16 or more in serum. Controls included dogs with presumed histoplasmosis or blastomycosis, other fungal infections, or nonfungal diseases and healthy dogs. Urine and serum specimens were tested using an enzyme immunoassay for Coccidioides galactomannan antigen. Antibody testing was performed at commercial veterinary reference laboratories. Antigen was detected in urine or serum of 12 of 60 (20.0%), urine only in 2 of 57 (3.5%), and serum only in 11 of 58 (19.0%) dogs with coccidioidomycosis. Antigen was detected in the urine of 3 of 43 (7.0%) and serum of 1 of 37 (2.7%) dogs with histoplasmosis or blastomycosis but not in 13 dogs with other fungal infections (serum, 9; urine, 13), 41 dogs with nonfungal diseases (urine, 41; serum, 18), or healthy dogs (serum, 21; urine, 21). Detection of antigen was an insensitive method for diagnosis of coccidioidomycosis in dogs in which the diagnosis was based primarily upon detection of antibodies at titers of 1:16 or higher, and the highest sensitivity was in serum.     

Mumps Class-Specific Immunoglobulins in Radioimmunoassay and Conventional Serology

Antibodies against mumps virus have been studied by using immunoglobulin class-specific indicators labeled with ¹²⁵I in the radioimmunoassay (RIA) procedure. The immunoglobulins in paired acute and convalescent sera were allowed to react with mumps virus in a solid-phase RIA system. Class-specific immunoglobulin indicators (anti-immunoglobulin M [IgM] and anti-immunoglobulin G [IgG]) labeled with ¹²⁵I revealed that immunoglobulins of early antisera were preponderantly IgM, whereas immunoglobulins of late antisera were predominantly IgG. These indicators detected antibodies of the early (IgM) and late (IgG) phases of the immune response. These findings are consistent with the classical temporal order of appearance of 19s (IgM) and 7s (IgG) globulins. Specificity of these indicators for reacting with fractionated 7s and 19s globulins is also presented. Mumps virus RIA obtained with anti-IgG correlated well with conventional serological data obtained by neutralization and hemagglutination inhibition, but most strongly with complement-fixation data. In addition, antibody bound by solid phase was capable of distinguishing between related antigens of the myxovirus group.