Diagnosis Of Systemic Candidiasis Research Articles

P394 Evaluation of diagnostic potential of recombinant secretory aspartyl proteinase 2 (Sap2) protein from Candida parapsilosis for use in systemic candidiasis

Poster session 3, September 23, 2022, 12:30 PM - 1:30 PM ObjectivesSystemic candidiasis is the fourth most common bloodstream infection in ICU patients worldwide. Along with Candida albicans, infections caused by non-albicans Candida (NAC) species are increasingly becoming more prevalent globally along with the emergence of drug resistance. The diagnosis of systemic candidiasis is difficult due to the absence of significant clinical symptoms in patients. Since conventional diagnostics methods for candidiasis show less sensitivity and specificity, novel immunodiagnostic techniques are needed for early diagnosis. We investigated the diagnostic potential of recombinant secreted aspartyl proteinase 2 (rSap2) from C. parapsilosis for the detection of Candida infection.MethodsGenomic DNA was isolated from C. parapsilosis, followed by PCR amplification of Sap2 gene using designed gene-specific primers. Sap2 protein expression and purification was performed using Ni-NTA affinity chromatography under denaturing conditions. The denatured protein was subsequently refolded using a multi-step dialysis procedure. CD and FTIR studies were performed to confirm refolding. Mice were immunized with rSap2 protein and serum ELISA assays were performed for testing immunogenicity. Immunoblotting assays and human serum ELISAs were performed using whole-cell Candida and rSap2 protein in proven systemic candidiasis patient serum and controls, recruited at PGIMER, Chandigarh.ResultsSap2 protein from C. parapsilosis was successfully cloned and expressed using an E. coli-based prokaryotic expression system. Protein refolding was performed in vitro using step-wise dialysis. Structural analysis by CD and FTIR spectroscopy revealed the refolded protein to be in its near native conformation. Immunogenicity analysis demonstrated the rSap2 protein to be highly immunogenic as evident from significantly high titers of Sap2-specific antibodies in antigen immunized BALb/c mice, compared to sham-immunized controls. The diagnostic potential of rSap2 protein was evaluated using immunoblotting and ELISA assays using serum from proven systemic candidiasis patients and controls. Our immunoblotting results demonstrate that the recombinant Sap2 protein was recognized as a single band of approximately 41 kDa by systemic candidiasis patient serum samples and no cross reactivity was observed in healthy controls. On evaluating the diagnostic potential of the rSap2 antigen using an ELISA-based approach, our results show that anti-Sap2 Ig, IgG and IgM antibodies could be detected in the sera of proven candidiasis patients. Of note, the differences in Sap2 antibody titers observed amongst patients and controls were similar to the serological response observed when heat-killed whole-cell Candida was used as a coating antigen.ConclusionIn summary, the rSap2 protein from C. parapsilosis has the potential to be used in the diagnosis of systemic candidiasis, providing a rapid, convenient, accurate, and cost-effective strategy. Our results indicate that the rSap2 protein from C. parapsilosis can be used to detect and diagnose systemic candidiasis infection in human patients and can be used as an alternative/replacement of whole-cell Candida-based ELISA procedures, which are currently in use.

Expression and Purification along with Evaluation of Serological Response and Diagnostic Potential of Recombinant Sap2 Protein from C. parapsilosis for Use in Systemic Candidiasis.

Systemic candidiasis is the fourth most common bloodstream infection in ICU patients worldwide. Although C. albicans is a predominant species causing systemic candidiasis, infections caused by non-albicans Candida (NAC) species are increasingly becoming more prevalent globally along with the emergence of drug resistance. The diagnosis of systemic candidiasis is difficult due to the absence of significant clinical symptoms in patients. We investigated the diagnostic potential of recombinant secreted aspartyl proteinase 2 (rSap2) from C. parapsilosis for the detection of Candida infection. The rSap2 protein was successfully cloned, expressed and purified using Ni-NTA chromatography under denaturing conditions using an E. coli-based prokaryotic expression system, and refolded using a multi-step dialysis procedure. Structural analysis by CD and FTIR spectroscopy revealed the refolded protein to be in its near native conformation. Immunogenicity analysis demonstrated the rSap2 protein to be highly immunogenic as evident from significantly high titers of Sap2-specific antibodies in antigen immunized Balb/c mice, compared to sham-immunized controls. The diagnostic potential of rSap2 protein was evaluated using immunoblotting and ELISA assays using proven candidiasis patient serum and controls. Immunoblotting results indicate that reactivity to rSap2 was specific to candidiasis patient sera with no cross reactivity observed in healthy controls. Increased levels of anti-Sap2-specific Ig, IgG and IgM antibodies were observed in candidiasis patients compared to controls and was similar in sensitivity obtained when whole Candida was used as coating antigen. In summary, the rSap2 protein from C. parapsilosis has the potential to be used in the diagnosis of systemic candidiasis, providing a rapid, convenient, accurate and cost-effective strategy.

Systemic Candidosis in Dogs Associated with Canine Distemper Virus

Background: Candidosis is an opportunistic fungal infection caused by yeasts of the genus Candida, which normally reside on the surfaces of the mucous membranes and in the skin of several animal species and healthy humans; however the fungi can convert into pathogenic microorganisms and result in invasive infections with systemic involvement due to the impairment of the immune system. Systemic candidosis is rare in dogs and few reports of this mycosis are available in literature. Therefore, the objective of this study was to describe three cases of systemic candidiasis associated to canine distemper in dogs, highlighting the main epidemiological, clinical and pathological aspects.Cases: Three cases of systemic infection by Candida sp. were diagnosed in dogs. The animals predominantly presented neurological clinical manifestations, followed by unspecific alterations with an evolution of 5 to 30 days. Macroscopically, the lesions were characterized by white-yellowish multifocal to coalescent areas surrounded by reddish borders in the kidneys and heart (cases 1, 2 and 3); liver and submandibular lymph node (case 1); and lung (case 2). In the brains of the three dogs were observed multifocal, blackened and/or reddish and friable areas. In case 1, was observed an increased volume of the right carpometacarpal joint, that when cut, released a reddish and turbid content. In case 2, there was deposition of a whitish lumpy material on the epicardial surface, aorta artery, pericardial sac and spleen; infarction on the right testicle, hydroureter, pyelonephritis and hydronephrosis. Microscopically, the lesions consisted of necrossupurative inflammation associated to hemorrhage, vasculitis, congestion, thrombosis, infarction and fungal structures with distinct morphological patterns which included blastoconidia, pseudohyphae and hyphae. The fungal structures were strongly positive for the anti-Candida albicans polyclonal antibody in the immunohistochemistry. All 3 dogs were concomitantly infected by the canine distemper virus, and 2 of the animals also presented other comorbidities.Discussion: The diagnosis of systemic candidiasis was made based on the morphotintorial characteristics of the agent and confirmed by immunohistochemistry. The yeasts of the genus Candida live as commensals, but when there is an imbalance of the normal microbiota or the immune system of the host is impaired, the yeasts can convert themselves into opportunistic pathogenic microorganisms. The concomitant infection with the canine distemper virus may have favored the infection and proliferation of the agent in the tissues, as the virus causes immunosuppression. The extensive vascular lesions associated to fungi in the vascular lumen and wall observed in the affected organs suggest hematogenous dissemination causing acute infarctions. The clinical signs of systemic candidosis are extremely variable and reflect the affected organs and hardly ever the infections are considered in the differential diagnoses. The dogs presented neurological clinical signs compatible with canine distemper, however the other clinical alterations were unspecific and insufficient to correlate with a secondary infection and none of the cases diagnosed in the necroscopic examination suggested systemic candidiasis during the clinical investigation. The histochemical methods used in this study contributed effectively for the prior diagnosis of candidosis, making evident the variable morphological characteristics of the fungi. It is concluded that the systemic candidosis in dogs with canine distemper affects young and adult animals with predominantly neurological and unspecific clinical signs resulting from necrossupurative and vascular lesions in several organs, including the articular involvement little described in dogs and testicular lesions only reported in humans.

Clinicopathologic review of 19 patients with systemic candidiasis with skin lesions

A diagnosis of systemic candidiasis is often delayed or missed owing to the absence of sensitive, specific, and timely diagnostic tools. Skin lesions are not common, but they can help to rapidly establish a diagnosis. We report on a 14-year experience of systemic candidiasis with skin lesions in our institution. We report the prevalence, clinical findings, histologic findings, etiologic Candida species, underlying conditions, treatment modalities, and outcomes of the cases and compare them with the previous reports. We reviewed the medical records and laboratory data of patients diagnosed with systemic candidiasis from June 1989 to September 2002 at Asan Medical Center, Seoul, Korea. We thoroughly reviewed the data on those patients with characteristic skin lesions. We included the cases in which Candida organisms were either shown or cultured from the skin. We also included the patients who had developed the characteristic rash at the onset of infection if there was no other possible explanation for the rash. Of 53 documented systemic candidiasis cases, 19 (35.8%) had the characteristic skin lesions. Fifteen patients (78.9%) had hematologic problems and were neutropenic. The skin lesions were a maculopapular or nodular rash and plaques. In addition to the trunk and proximal extremities, the rash also involved the face and distal extremities. The rashes were mostly purpuric, not consistently associated with underlying thrombocytopenia but also associated with underlying vascular damage as a result of Candida organisms. The underlying vascular damage also caused intraepidermal necrotic and vesicular change. One case of transepidermal elimination of organisms was newly found. The most common causative species was Candida tropicalis in the 19 patients with skin lesions, in contrast with Candida albicans in a total of 53 patients. The mortality rate was 84.2%. The prevalence of systemic candidiasis-associated skin lesions may be higher than previously reported. Dermatologists should be familiar with the clinical appearance of skin lesions and suspect this fatal infection when seeing neutropenic patients with a resistant fever and accompanying rash.

Is the persistence of elevated serum C-reactive protein a predictive marker in the diagnosis of systemic candidiasis in premature newborns?

Is the persistence of elevated serum C-reactive protein a predictive marker in the diagnosis of systemic candidiasis in premature newborns?

Acute renal failure caused by fungal bezoar: A late complication of Candida sepsis associated with central catheterization

The authors report a case of acute renal failure caused by fungal bezoar in the renal pelvis. The patient was successfully treated with bilateral percutaneous nephrostomy drainage. He had been admitted because of necrotizing enterocolitis, at the age of 26 days. Eventually, his bowel was reduced to 40 cm of small intestine, including 5 cm of terminal ileum. Candida sepsis developed during central total parenteral nutrition, at the age of 76 days. Five weeks after the diagnosis of systemic candidiasis, sudden anuria developed, and ultrasonography showed echogenic material in both renal pelvises. Bilateral percutaneous nephrostomy catheters were placed in the renal pelvises, and local irrigation with amphotericin B was performed for 3 weeks. The renal function of the baby was completely recovered, without systemic antifungal treatment.

Diagnosis of systemic candidiasis by enzyme immunoassay detection of specific antibodies to mycelial phase cell wall and cytoplasmic candidal antigens.

Diagnosis of systemic Candida infections was attempted by the use of an enzyme-linked immunosorbent assay (EIA) to detect IgG antibodies towards cell wall-bound and cytoplasmic candidal antigens. Cell wall antigens were sequentially solubilized by treatment of germinated blastoconidia of Candida albicans (ATCC 26555 strain) with beta-mercaptoethanol (beta ME extract) and digestion with Zymolyase 20T, a beta-glucanase preparation (Zymolyase extract). Protoplasts obtained after treatment with Zymolyase were osmotically lysed (cytoplasmic antigens). Sera were obtained from patients with systemic (n = 28) and superficial (n = 46) candidiasis. Control sera were obtained from normal healthy individuals (n = 31) and from hospitalized patients at low (n = 36) and at high (n = 13) risk of developing systemic candidiasis yet showing no symptoms of candidal infection. Detection of antibodies in crude sera samples by EIA using all of these antigenic extracts was highly specific (98-100%), but sensitivity of the method was low (3.5-17.8%). However, adsorption of sera with latex microspheres coated with purified Candida mannan in order to selectively remove anti-mannan antibodies prior to EIA improved the diagnostic efficiency of this test. Improvement was particularly noticeable when the beta ME extract was used as antigenic preparation, yielding a sensitivity of 89.2% and a specificity of 98.6%.

Detection of antibodies to Candida albicans germ tube as a possible aid in diagnosing systemic candidiasis in bone marrow transplant patients.

Indirect immunofluorescence assays to detect antibodies to Candida albicans blastospore and germ tube were performed in sera of 29 bone marrow transplant patients. Antibodies to germ tube were present in the sera of six patients, in four of whom a Candida albicans infection was highly probable, while in the other two patients it was not possible to determine the previous course. No healthy blood donors had these antibodies. On the other hand, detection of antibodies to Candida albicans blastospore showed low specificity in the diagnosis of systemic candidiasis. These preliminary findings suggest that the detection of antibodies to Candida albicans germ tube may be an important aid in the diagnosis of systemic candidiasis in bone marrow transplant patients.

The Lack of Utility of the Latex Agglutination Test for Detection of Candida Antigen in the Diagnosis of Systemic Candidiasis

The Ramco latex agglutination test in the diagnosis of systemic candidiasis was utilized for 11 serum samples from 10 patients with systemic candidiasis, 21 serum samples from patients colonized with Candida species, and 20 control serum samples from patients with stable medical problems and no evidence of Candida albicans infection. This study was double-blind and the results of the latex agglutination test did not influence the decision for antifungal therapy. Nine of 10 patients with systemic candidiasis had positive titers (greater than or equal to 1:4); however, these were determined only 1 to 5 days before culture positivity. Nine of 21 (43%) of colonized patients were falsely positive (greater than or equal to 1:4) and all of the control samples were negative. The Ramco latex agglutination test was unreliable and inconsistent in this small sample group to establish an early diagnosis of systemic candidiasis.

Neonatal Systemic Candidiasis Diagnosed by Ultrasound

The diagnosis of systemic candidiasis in ill high-risk neonates is often delayed as there are no specific clinical features and the significance of positive cultures for Candida species may be uncertain. Early diagnosis and treatment is essential to reduce mortality and may be aided by ultrasound examination of the commonly involved organs - the brain and kidneys. In this paper we describe 2 cases where the diagnosis was first raised following ultrasound examination, and review the spectrum of abnormalities seen on renal and cranial ultrasound examination. We emphasise the importance of using this imaging technique in the high-risk neonate in initiating earlier diagnosis and treatment, or in establishing the significance or otherwise of positive cultures.

Mannan and D-arabinitol concentrations in serum from a patient with Candida albicans endocarditis

In an attempt to clarify the comparative values of serological and microbiological examinations for the early diagnosis of systemic candidiasis, antibodies against Candida albicans, serum mannan, and the D-arabinitol creatinine ratio were investigated in a patient with aortic valve endocarditis associated with carcinoma of the bile duct. Candida precipitins and the antibody titer against Candida cell wall mannan were examined by an immunodiffusion technique and hemagglutination test, respectively. Serum mannan was tested by enzyme-linked immunosorbent assay (ELISA) using the biotin-streptavidin procedure. The upper limit of negativity of the assay was determined by adding 0.06 to the absorbance of pooled serum from healthy laboratory workers. This value was about 0.8 ng/ml with ELISA. The D-arabinitol concentration in serum was examined by an enzymatic fluorometric method. Rising antibody titers against C. albicans, mannan antigenemia, and an elevated D-arabinitol creatinine ratio were first observed between the 11th and 12th hospital days. Blood cultures obtained on 8th, 9th, and 11th hospital days grew C. albicans after 3 to 4 days of incubation. Of 11 serum samples, 5 were positive for mannan, whereas D-arabinitol creatinine ratio was positive in 7 of 9 samples. Blood cultures was the earliest evidence of Candida infections in our cases. However, because of saprophytic nature of Candida species, tests for antibodies, antigenemia, and the D-arabinitol creatinine ratio in combination with blood cultures are necessary to confirm systemic candidiasis at an early stage of infection.

Detection of antibodies to Candida albicans germ tube in the diagnosis of systemic candidiasis.

Sera from 109 subjects were tested for the presence of anti-Candida albicans antibodies by an indirect immunofluorescence assay. Aliquots of the sera were adsorbed with heat-killed blastospores to remove the antibodies against the surface of the yeast-phase cell wall and tested for anti-germ tube cell wall antibodies. Unadsorbed sera stained the entire cell wall of yeast and germ-tubes. Immunoglobulin G (IgG) antibodies were found in all patients with systemic candidiasis and in 81.2% of patients with Candida albicans isolated from skin and mucous membranes. IgA and IgG were found in 67.4 and 57.1%, respectively, of controls without evidence of candidiasis. After the adsorption only sera from patients with systemic candidiasis showed antibodies, predominantly IgA, against germ tube cell wall. Adsorption of the sera thus increased the specificity, efficiency, and positive and negative predictive values of the test. The test achieved the highest sensitivity in adsorbed sera for the combination of IgA and IgG.

Diagnosis of systemic candidiasis: development of co-counterimmunoelectrophoresis.

To improve the results of counterimmunoelectrophoresis for serological diagnosis of candidiasis, a new method was devised to characterize human sera precipitins: co-counterimmunoelectrophoresis. Cellulose acetate was chosen as the support in order to observe identity reactions between precipitin lines from serum systems run conjointly (thus, co-counterimmunoelectrophoresis). Different experimental antisera (anti-somatic and anti-cytoplasmic antigens and anti-whole fixed germ tube antisera) were tested for reactions of identity with sera from selected patients with systemic candidiasis caused by Candida albicans. The reaction of patient sera and anti-germ tube antiserum with a selected somatic antigen always resulted in an identity reaction as proven by the continuity of major precipitin lines. The precipitating system, named the co-specific precipitin line, involved polysaccharide antigens; it was not present in human control sera.

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.

Systemic Candidiasis in Very Low-Birth-Weight Infants (<1,500 Grams)

Previous reports in the literature have documented that systemic infection with Candida albicans in very premature infants is frequently fatal (54%) or associated with significant morbidity in survivors (25%). Five patients with a mean birth weight of 829 g had a diagnosis of systemic candidiasis during their stay in a newborn intensive care unit. All infants survived with minimal sequelae following aggressive early treatment with amphotericin B and 5-flucytosine. A review of these five extremely premature infants and 26 previously reported patients suggests the following: (1) disseminated candidiasis is common in the absence of positive findings in blood, CSF, and/or urine cultures; (2) transient candidemia rarely resolves without therapy; (3) meningitis and osteoarthritis occur more frequently than in older patients with disseminated disease; and (4) premature infants tolerate amphotericin B and 5-flucytosine well. Infants who are found to have systemic cultures positive for candidiasis should be treated by (1) removing all factors that predispose to systemic candidiasis (eg, indwelling catheters, broad-spectrum antibiotics); (2) early initiation of systemic antifungal therapy with amphotericin B and 5-flucytosine; and (3) searching for additional foci of disease. After the disease is recognized and treatment is prompt and aggressive, outcome can be substantially improved.

Computerized tomography in the diagnosis of systemic candidiasis in patients with acute leukemia.

Seven patients with acute leukemia and systemic candidiasis presented with a clinical syndrome of fever, abdominal pain, organomegaly, and a cholestatic pattern of hepatic dysfunction with an elevated alkaline phosphatase and normal transaminases. The abdominal CT scan demonstrated diffuse hepatic and splenic abscesses in all seven patients. Culture and histology of liver biopsy specimens was nondiagnostic in four of five cases. The CT-directed percutaneous needle aspirations of these lesions yielded diagnostic material in two of three cases. Culture-negative visceral abscesses in persistently febrile patients with acute leukemia should be recognized as being due to candidiasis. The abdominal CT scan may be useful in identifying this clinical-radiographic syndrome and in facilitating rapid diagnosis. Promptly administered antifungal therapy may lead to successful eradication of this infection.

Rapid diagnosis of systemic candidiasis in children with cancer by pyrolysis gas liquid chromatography.

A pyrolysis gas liquid chromatographic method for detection of Candida antigen in patients with disseminated candidiasis is described. The test was positive in 73% of cases and no false positives or false negatives were noted in controls.

Partially purified antibodies used in a solid-phase radioimmunoassay for detecting candidal antigenemia

The development of a solid-phase radioimmunoassay procedure for the detection of Candida albicans antigens in serum of mice is described. Antibodies against C. albicans that were used in the radioimmunoassay procedure were partially purified from immune serum by a C. albicans antigen-coupled affinity column. Elution of anti-C. albicans antibodies from the column was by glucose and mannose; 4 mg of protein was recovered per ml, which contained 50% of the candidal agglutinin activity of immune serum. Also, 81% of the protein (partially purified antibody) recovered was adsorbed by whole C. albicans cells. Anti-C. albicans antibodies were either coupled to Sepharose 4B for use as the solid phase to bind candidal antigen in serum of infected animals, or radioiodinated (125I) for use as a tracer molecule to bind to the candidal antigen solid-phase complex. Although control experiments indicated that at least 100 ng of candidal antigen should be present in a serum specimen for a positive radioimmunoassay test, candidal antigenemia was detected in 70.4% of infected mice even in cases where blood cultures for C. albicans were negative. With further refinement and adaptability to human serum, the radioimmunoassay test may become a helpful tool for use in the diagnosis of systemic candidiasis.

A radioimmunoassay method for the rapid detection of Candida antibodies in experimental systemic candidiasis.

Rabbits were employed as experimental models to evaluate a solid-phase radioimmunoassay (RIA) method for the diagnosis of systemic candidiasis. Ten rabbits were inoculated subcutaneously to mimic superficial candidiasis and were found to produce no antibodies to Candida as determined by both immunodiffusion and RIA procedures. However, 94 per cent of 18 rabbits systemically infected by intravenous injection of Candida cells were observed to produce antibody as assessed by the RIA technique. These data encourage further tests with human sera and the continued development ofthis RIA procedure as a useful tool in the early serodiagnosis of systemic candidiasis.

Use and value of serologic tests for the diagnosis of systemic candidiasis in cancer patients: A prospective study of 146 patients

Sera from 146 cancer patients at risk for disseminated candidiasis were studied prospectively with immunodiffusion (ID), counterelectrophoresis (CEP), and latex agglutination (LA) tests to determine their diagnostic value in the detection of antibodies to theCandida species. Serial serum samples, cultures, and clinical data were obtained after a malignancy was diagnosed. Patients were classified into three groups (I, II, and III) on the basis of cultural, histological, and clinical evidence for superficial (Group I) versus disseminated (Group III)Candida infection. Thirty-two of 78 patients (41%) in Group I had positive ID, CEP, and LA titers. In Group II, those patients lacking histological confirmation of disseminated infection, 16 of 18 (89%) had positive titers. Thirty-six of 50 (72%) in Group III were positive by all three tests. Heavy colonization of the gastrointestinal tract, without evidence of tissue invasion, produced positive test results. Negative serologic tests were encountered in immunosuppressed patients with rapidly progressive candidiasis.C. krusei infections produced specific antibody titers detected by the homologous antigen but not byC. albicans antigen. Stable or decreasing LA titers were correlated with clinical improvement in patients receiving effective antifungal therapy.