Comparison of antibody, antigen, and metabolite assays for hospitalized patients with disseminated or peripheral candidiasis.

Repeat serum samples from 22 patients with proven disseminated candidiasis and 42 with simple peripheral colonization were assayed for Candida antibodies by coelectrosyneresis, immunoprecipitation, and A and B immunofluorescence, for metabolites by D-arabinitol measurement, and for antigens by the mannan immunoassay and Cand-tec latex agglutination (mean number of samples tested, 2.5 per patient). For the antibody and metabolite assays, the results showed no statistical difference between the two groups. By contrast, the results of both antigen assays were positive for a significantly larger number of patients with disseminated candidiasis than of those with simple peripheral colonization. Results were regardless of whether the patients were neutropenic. They were not predictive of death. We calculated that the mannan antigen assay had 29% sensitivity and 97% specificity for the diagnosis of disseminated candidiasis. Likelihood ratios of a positive and a negative result of this test were 9.2 and 0.7, respectively, for this diagnosis. In the latex agglutination test, likelihood ratios were 2.5, 1.5, 1.6, and 0.3 when the test was positive for dilutions of 1/8, 1/4, and 1/2 and was negative, respectively.     

Effects of gastrointestinal candidiasis, antibiotics, dietary arabinitol, and cortisone acetate on levels of the Candida metabolite D-arabinitol in rat serum and urine.

We studied the effects of gastrointestinal (GI) colonization by Candida albicans, dietary arabinitol, intragastric antibiotics, and cortisone on levels of the Candida metabolite D-arabinitol in rat serum and urine. Rats given conventional laboratory chow, intragastric gentamicin and chloramphenicol, and 6.0 x 10(8) live C. albicans B311 blastoconidia by gavage had minimal invasive GI disease and no more DL-arabinitol in the urine than controls given killed C. albicans. However, colonized and uncolonized rats given intragastric antibiotics had transiently higher urine arabinitol levels than the corresponding controls given saline. Rats given conventional laboratory chow (which contained 50 micrograms of arabinitol per g) had higher serum and urine arabinitol levels than rats given no dietary arabinitol, but the differences were less than expected. Moreover, intragastric antibiotics did not cause increased arabinitol excretion in rats given no dietary arabinitol. Rats given intragastric antibiotics and live C. albicans but no dietary arabinitol had no more arabinitol in their serum or urine than controls given antibiotics and killed C. albicans or saline and live or killed C. albicans. Lastly, cortisone acetate (10 mg/kg of body weight per day intramuscularly for 10 days) did not cause increased serum or urine arabinitol levels. We conclude that neither GI colonization by C. albicans nor cortisone should interfere with the usefulness of arabinitol as a marker for invasive candidiasis; antibiotics appear to increase arabinitol excretion by suppressing GI bacteria capable of consuming dietary arabinitol.     

Comparison of two enzyme-linked immunosorbent assays for antigen quantitation: direct competition and antibody inhibition.

Two enzyme-linked immunosorbent assays for antigen quantitation, direct competition and antibody inhibition, were used to measure rabbit immunoglobulin G in polystyrene microtiter plates and were compared for sensitivity and reproducibility. Both procedures repetitively detected this antigen in the 1- to 100-ng/ml range. Both procedures were predictably reproducible, with direct competition procedures having steeper slopes in ranges tested. Antibody inhibition does not require conjugated antigen and can detect sample antigens if available stock antigens can be passively bound to a solid-phase polystyrene plate.     

Diagnosis of systemic candidiasis by latex agglutination for serum antigen.

Three latex agglutination test procedures for detecting Candida antigen in human serum were compared in a retrospective study of 69 patients and 20 normal volunteers. Untreated human serum was reacted with two different latex reagents; one reagent also was reacted with serum treated with protease and heat. The test procedure with treated serum was best, detecting serum antigen in 17 of 21 patients (81%) with disseminated candidiasis. Judging by autopsy-proven cases, there was an increase in positive test results in the last 2 weeks of life. When untreated sera were tested with this reagent, only 3 (14%) of the 21 patients with disseminated candidiasis had detectable antigen in serum. A subset of these same sera was tested by a commercial latex reagent (Candida Detection System lot C001; Ramco Laboratories, Inc., Houston, Tex.) and untreated serum. Of 18 patients with disseminated candidiasis, 5 (28%) had at least one positive serum. Sera from patients with less severe clinical forms of candidiasis were usually negative regardless of the test procedure used. With one exception, sera from control patients were negative or were positive only in sera containing rheumatoid factor. Latex agglutination tests for Candida spp. in treated serum may prove to be a useful procedure for the rapid diagnosis of severe disseminated candidiasis.     

Comparison of assays for antibody to HTLVI.

Indirect immunofluorescence, competitive radioimmunoassay, HTLV I-enzyme linked immunosorbent assay and gelatin particle agglutination Serodia-ATLA were compared in terms of their ability to detect antibody to human T cell leukaemia virus I (HTLV I). The sensitivities were 96.9%, 92%, 97.0%, and 100%, respectively, and the specificities 99.3%, 98.9%, 98.6%, and 96.3%. Particle agglutination was very simple to perform and was the most sensitive, though the least specific test. Antibody titres were 10-100 times higher when measured by particle agglutination than by other tests, and antibody titers were considerably higher in patients with neurological disease related to HTLV I than in those with other conditions. Serodia-ATLA is the method of choice for preliminary screening of specimens for antibody to HTLV I, but positive results must be confirmed by another technique.     

Use of DBA/2N mice in models of systemic candidiasis and pulmonary and systemic aspergillosis.

Mouse models of systemic candidiasis and pulmonary and systemic aspergillosis were established by using DBA/2N mice, which are known to be deficient in the C5 component of complement. In experiments comparing lethality in the respective models in DBA/2N versus outbred CFW mice, results showed that the 50% lethal dose values for the DBA/2N mice were 10- to 1,000-fold lower than those for the outbred mice, depending on the experiment. Additionally, onset of death was somewhat delayed for the DBA/2N mice. In the case of the pulmonary aspergillosis model, administration of cortisone acetate was necessary to ensure lethality after intranasal infection, but only a single dose was necessary.     

Detection of human calicivirus antigen and antibody by enzyme-linked immunosorbent assays.

Enzyme-linked immunosorbent assays (ELISAs) were developed to detect human calicivirus (HCV) antigen and antibody to HCV. The ELISAs were specific for HCV and as sensitive as a previously developed radioimmunoassay. These ELISAs were used to search for evidence of HCV infection in the United States, where HCV gastroenteritis has rarely been reported. One hundred sixty-three stool samples collected from children hospitalized with diarrhea were examined; one sample was positive in the ELISA. Typical calicivirus particles were found in this stool sample, and these particles reacted with a hyperimmune guinea pig anti-HCV serum by immune electron microscopy. The age-related acquisition of antibody to HCV in hospitalized infants and children (from birth to 19 years old) without gastroenteritis and in healthy adults was also evaluated. The pattern of acquisition of antibody to HCV was similar to that for group A rotaviruses, namely, beginning in infancy and becoming 100% by the age of 4 years. These data suggest that HCV is associated with infantile gastroenteritis in the United States, that infections with HCV are common, and that many infections with HCV (Sapporo strain) may not require hospitalization.     

A modified cytoplasmic antigen of Candida albicans for serodiagnosis of systemic candidiasis.

A modified cytoplasmic antigen, prepared by Natamycin degradation of Candida albicans cells is described. The reactivity of this antigen in detecting precipitins to Candida albicans proved tobe very similar qualitatively and quantitatively to a standardised reference antigen, prepared by X-press disruption when both were compared by immunological techniques; the majority of antigenic components in each proving to be identical. When tested against 127 human sera of unknown antibody content the two antigens showed 100% correlation by counterimmunoelectrophoresis and 87.5% correlation by double diffusion. The modified antigen proved to be reproducible and reliable in use and is easily prepared in the routine laboratory.     

Quantitation of antibody isotypes in solid-phase assays. Comparison of myeloma protein and monoisotypic antibody standards

Two methods have been proposed for the standardization of isotype specific antibody assays. In one, myeloma proteins directly attached to plastic surfaces are used as standards, whereas the other method employs antigen coated surfaces followed by monoisotypic antibodies as standards. These standardization methodologies have been investigated by submitting 4 monoisotypic human antibodies to a solid-phase assay standardized by the myeloma method. Specific antibody concentrations of each were determined so that each could serve as a monoisotypic standard. Three purified monoclonal mouse antibodies were also tested which allowed use of the same preparation as a monoisotypic antibody standard or as a 'myeloma protein' standard. Ten times more myeloma protein than specific antibody is needed for the same level of binding of the anti-isotype antibody. Therefore, assays standardized with myeloma proteins give erroneously high concentrations for sample antibodies. The same concentration of antibodies of different specificities (used with different antigen coats) gave very comparable levels of binding of the labeled antibody. This supports the claim that for quantitation of antibodies an antibody standard can be used that is of different specificity to the sample antibody to be measured.     

The effects of surgical removal of Peyer's patches in rat on systemic antibody responses to intestinal antigen.

A method is described for the in vivo surgical removal of all Peyer's patches from the small intestine and caecum of the rat. Over two postoperative months, this procedure had no apparent morphological effects on the small intestine or intraepithelial lymphocyte numbers, nor were the numbers of IgA producing cells in the small intestine or serum immunoglobulin levels permanently influenced. However, circulating specific antibody to human serum albumin was significantly elevated in animals without Peyer's patches 3 weeks after surgery, in comparison with a sham operated group of animals. Subsequent intestinal immunization in animals without Peyer's patches with a lipid-conjugated human serum albumin resulted in a diminished primary but a comparatively normal secondary systemic antibody response to this antigen.     

Mucosal and systemic candidiasis in congenitally immunodeficient mice.

Colony counts and light microscopy were used to assess the capacity of Candida albicans to colonize, infect the alimentary tract, and cause disseminated disease in athymic (nu/nu), euthymic (nu/+), beige (bg/bg), black (bg/+), beige athymic (bg/bg nu/nu), or beige euthymic (bg/bg nu/+) germfree mice. The alimentary tracts of all six genotypes of germfree mice were quickly colonized after exposure to yeast-phase C. albicans. Only bg/bg nu/nu mice showed obvious morbidity and mortality after mucosal colonization with C. albicans. Histopathology of C. albicans-colonized immunocompetent (nu/+, bg/+) and singly immunodeficient (nu/nu, bg/bg, bg/bg nu/+) mice showed minimal to moderate mucosal infections, whereas doubly immunodeficient (bg/bg nu/nu) mice showed extensive yeast and hyphal infection of the palate, tongue, esophagus, and stomach. A progressive systemic infection in C. albicans-colonized mice occurred only in bg/bg nu/nu mice 12 to 16 weeks after colonization and mucosal infection. Thus, it appears that a combination of defective cell-mediated immunity and phagocytic cell defects (polymorphonuclear leukocytes and/or macrophages) predisposed mice to severe mucosal and systemic candidiasis of endogenous origin. This is the first report of a mouse strain that is not only naturally susceptible to mucosal and systemic candidiasis of endogenous origin but also shows lethality at early (1 to 4 weeks) and late (12 to 16 weeks) times after alimentary tract colonization.     

Retrovirus-induced immunodeficiency in mice exacerbates gastrointestinal candidiasis.

Dysfunction of neutrophils in patients infected with human immunodeficiency virus is at least partly responsible for secondary microbial diseases in these individuals, including invasive gastrointestinal (GI) candidiasis. Immunoregulatory disturbances associated with the development of AIDS in human immunodeficiency virus-infected patients exacerbates Candida albicans infection of the upper GI tract and frequently leads to oropharyngeal and esophageal candidiasis. In this article, we present the first report of a murine model of invasive GI candidiasis associated with an AIDS-related murine immunodeficiency syndrome that results from infection of C57BL/6 mice with a previously described retrovirus complex (LP-BM5). Mice of the inbred strain were infected with C. albicans by oral-intragastric inoculation as infants and with the retrovirus by the intraperitoneal route 30 days later. Control mice of the same strain were infected with C. albicans as above and subsequently infected with the avirulent, ecotropic helper virus (MBI-5). Animals were killed 90 days after retroviral challenge. Total and differential blood cell counts, CD4+ T-cell counts in the spleen, and the histopathology of the gastric mucosa of experimental and control animals were determined. The virulent LP-BM5-infected animals developed murine AIDS and showed eruptive and suppurative lesions, with associated C. albicans mainly in regions of the cardial-atrium fold of the stomach. Well-defined abscesses with entrapped C. albicans hyphae were observed in the region of the cardial-atrium fold of control mice. A significant increase in the number of C. albicans CFU in homogenized and plated segments of the GI tract was recognized in mice with murine AIDS versus the control animals. The murine model of GI candidiasis reported here permits examination of the nature of C. albicans interaction with the gastric mucosa both in the immunocompetent host under conditions in which the yeast exists predominantly as a commensal organism and in the immunosuppressed host during progressive stages of AIDS induced by a retroviral infection.     

Comparison of nine commercially available assays for quantification of antibody response to hepatitis B virus surface antigen

Quantitative measurement of anti-HBs is used to evaluate the response to hepatitis B vaccination in health care workers and to optimize postexposure management. The different guidelines for hepatitis B vaccination and booster policy imply that the measurement of anti-HBs levels by different assays is accurate and consistent, yielding comparable quantitative results. We measured anti-HBs levels in 200 serum samples from patients and health care professionals by nine different anti-HBs assays and compared the quantitative results and the performance characteristics of the different test systems. The assay specificity ranged between 96.8 and 100% when sera from individuals without a vaccination history and with negative anti-HBc status were defined as true negatives. Sensitivity ranged between 93.5 and 100%. A high number of sera showed discrepancies between measurements by the different systems. The mean coefficient of variation between the different measurements was 47.1% (range, 15.0 to 201.0%), and the factors of multiplication ranged from 2.8 to 105. Hemolysis or lipemia did not seem to influence the measurement, and there was no difference between anti-HBc-positive and -negative individuals. The classical enzyme immunoassays tend to find lower anti-HBs levels than the automated systems, with higher values by the Abbott AXSYM assay. The serial dilution of the international standard preparation was measured accurately by most of the assays. In conclusion, the quantitative measurement of anti-HBs levels is not reliable, even though an international standard is used for the calibration of the systems. Some systems showed specific problems that should be addressed by the manufacturers.     

Evaluation of five hepatitis delta virus marker assays for detection of antigen and antibody.

Five commercially available assays for hepatitis delta (HD) virus markers were compared for sensitivity, specificity, and reproducibility: three assays for antibody (anti-HD), provided by Diagnostics Pasteur, Organon Teknika, and Abbott Laboratories, and two assays for antigen (HD Ag), from Pasteur and Organon Teknika. The assay from Organon Teknika is the less sensitive assay for anti-HD detection. Although the sensitivities of the Pasteur and Abbott assays for anti-HD detection are similar, the use of Abbott reagents is hampered by the lack of specificity when HD Ag is present. The greater sensitivity for HD Ag detection is obtained with Organon assay.     

Detection of an antibody response in immunocompetent patients with systemic candidiasis orCandida albicans colonisation

Tests to detect circulating antibodies toCandida albicans antigens were performed in sera from 27 immunocompetent patients, 15 of whom had deep-seated candidiasis and 12 of whom were colonised byCandida albicans. For the diagnosis of deep-seated candidiasis in patients with either deep-seated candidiasis orCandida albicans colonisation, counterimmuno-electrophoresis had a sensitivity of 87 % and a specificity of 75 %. Using immunoblotting it could be shown that antibodies to 35K, 47K, 68K and 88K antigens ofCandida albicans occurred more frequently in patients with deep-seated candidiasis than in colonised patients. The presence of dense bands in immunoblots representing antibodies against the 47K and/or 68K antigen served to discriminate significantly between deep-seated and superficial candidiasis (p < 0.05).     

Improvement in the specificity of assays for detection of antibody to hepatitis B core antigen.

Reducing agents dramatically alter the specificity of competitive assays for antibody to hepatitis B core antigen (anti-HBc). A specificity improvement was demonstrated with a new assay which utilizes microparticle membrane capture and chemiluminescence detection as well as a current radioimmunoassay procedure (Corab: Abbott Laboratories, Abbott Park, Ill.). The effect was most noticeable with elevated negative and weakly reactive samples. In both systems, reductants increased separation of a negative population (n = 160) from assay cutoffs. With a selected population (n = 307), inclusion of reductant eliminated apparent anti-HBc activity in 54 of 81 samples in the 30 to 70% inhibition range. Reductant-stable anti-HBc samples were strongly associated with the presence of antibody to hepatitis B surface antigen (21 of 27). The association persisted below the detection limits of current assays to 0.3 to 0.4 Paul Ehrlich Institute units per ml. Only 1 of 54 reduction-sensitive borderline samples was confirmed to be positive for antibody to hepatitis B surface antigen. The modified procedures had unchanged or slightly improved sensitivity for immunoglobulin G (IgG)-associated anti-HBc activity. Although IgM anti-HBc detection was reduced from four- to eightfold in the presence of reductants, sensitivities remained at least twofold greater than tha of an enzyme immunoassay (Corzyme M; Abbott) designed to detect acute-phase levels of IgM anti-HBc. The use of reducing agents should significantly improve the reliability of anti-HBc testing, especially near assay cutoffs.     

Puumala virus antibody and immunoglobulin G avidity assays based on a recombinant nucleocapsid antigen.

Puumala virus is the causative agent of nephropathia epidemica (NE), a hantavirus infection which occurs widely in northern and central Europe and is generally diagnosed by the indirect immunofluorescence (IF) method. We have now expressed the Puumala virus Sotkamo strain nucleocapsid (N) protein-coding S genome segment as a beta-galactosidase fusion protein in Escherichia coli by using the pEX2 expression vector. The recombinant protein was purified by cutting the protein band from an agarose gel, melting the agarose, and removing the protein by freezing, incubation on ice, and centrifugation. The recovery was about 1 to 5 mg/200 ml of bacterial suspension, sufficient for coating 100 to 500 enzyme immunoassay microtiter plates. In a study of 312 IF-positive and 233 IF-negative serum samples from NE patients, the recombinant-N-protein enzyme immunoassay detected immunoglobulin G antibodies to Puumala virus with 97.8% sensitivity and 98.5% specificity compared with the IF test results. In addition, an immunoglobulin G avidity enzyme immunoassay was developed and used successfully to diagnose acute NE from a single serum sample. The results demonstrate that the bioengineered antigen is suitable for use in routine diagnostic assays for Puumala virus immunity and recent infection.     

Use of recombinant hepatitis delta antigen in diagnostic assays for HDV antibody

The gene encoding the hepatitis delta virus (HDV) structural antigen (HD Ag) was inserted into a Rous sarcoma virus expression vector and the recombinant plasmid used to direct the synthesis of recombinant HD Ag (rHD Ag) in a continuous hepatoma cell line. A competitive radioimmunoassay for serum antibody to HDV using rHD Ag was developed and was found to be equally suitable for diagnostic purposes to a radioimmunoassay using infected liver-derived HD Ag. Similarly, rHD Ag was shown to be serologically equivalent to liver-derived HD Ag within the limit of the blocking titrations performed. The rHD Ag-positive cell line was also used in an indirect immunofluorescence assay to detect anti-HD. Similar titres of anti-HD were detected by both radioimmunoassay and immunofluorescence and identical samples were positive for anti-HD by either assay. In a sample of prison inmates with high prevalence of both HBV and HDV, anti-HD was confined almost exclusively to those with persistent HBV infection and not to those in whom HBV infection had cleared. The availability of rHD Ag will permit wider development of diagnostic anti-HD assays, and the use of two such assays is presented in this study.     

Physiological and metabolic alterations accompanying systemic candidiasis in mice.

Mice challenged intravenously with 10(6) viable Candida albicans died between 1 and 16 days after infection. Near the time of death, over 98% of the recoverable fungi came from the kidneys. Physiologically, animals were in renal failure near the time of death as evidenced by elevated blood urea nitrogen (BUN) and blood creatinine levels and a creatinine clearance rate which was about one-half normal. No abnormalities in liver glucogen and blood glucose levels were detectable. When mice were challenged with 4.5 X 10(6) viable C. albicans, they all died within 12 h. Near the time of death they had normal BUN values and were hyperglycemic. In mice receiving 4.5 X 10(6) heat-killed C. albicans, no deaths occurred and liver glycogen, blood glucose, and BUN levels all remained within a normal range and were different from responses to bacterial endotoxin. Cumulatively, the results demonstrate two distinct syndromes for the pathogenesis of experimental C. albicans infections. At the lower dose, mice were in renal failure associated with progressive renal infection. At the higher dose, renal failure was not observed. If a toxin was associated with death from the latter dose, it was not similar to bacterial endotoxin.