High detection rates of cryptococcal antigen in pulmonary cryptococcosis by Eiken Latex Agglutination test with pronase pretreatment

Two different kits for the detection of serum cryptococcal antigen in patients with pulmonary cryptococcosis were evaluated. The Eiken test (the Eiken Co., Tokyo), which uses pronase for pretreatment of serum, was compared with the Crypto-LA test (International Biological Laboratories, Cranbury, NJ), which did not use pronase prior to testing. Cryptococcal antigen was detected in 21 of 23 patients (91%) with the Eiken test and in only 10 of 23 patients (43%) with the Crypto-LA test (p<0.01 by Mcnemar test). However, the sensitivity of two tests was identical without use of pronase, as both tests could detect as little as 10⁴ cells/ml ofCryptococcus neoformans and 10 ng/ml of capsular polysaccharide ofC. neoformans. In those serum specimens for which both tests were positive, titers were much higher for the Eiken test, but there was a statistically significant correlation between the two tests (coefficient correlation 0.79,p<0.01). Cryptococcal antigen titer levels measured by the Eiken test correlated well with clinical courses. There was one false-positive reaction among 82 sera of non-cryptococcal patients. Pronase enhanced the sensitivity of the Eiken test, which appeared to be useful in patients with pulmonary cryptococcal disease, and its use may prevent unneeded lung biopsies.     

Pulmonary nodules: An unusual onset of HIV infection belatedly diagnosed

BackgroundCryptococcosis is a generally systemic and potentially lethal fungal infection. Although HIV infection is a predisposing condition, especially if the CD₄₊ lymphocyte count is less than 100 cells/mm³, other forms of immunosuppression may be associated with this opportunistic fungal condition, such as prolonged steroid therapy or solid organ transplantation. Pulmonary presentation must be included in the differential diagnosis of pneumonia or pulmonary neoplasia in the immunosuppressed patient.Case reportWe report a case of pulmonary cryptococcosis in a non-diagnosed HIV patient. In a 44 year-old male suffering from dyspnea and chest pain the image of a pulmonary nodule was observed in a radiological finding. In the histopathological study, intracellular structures suggestive of fungal conidia, and morphologically compatible with Cryptococcus, were observed. HIV serology and cryptococcal antigen detection in serum were requested, given the possibility of cryptococcosis. Cryptococcus neoformans var. grubii was isolated from the culture of the pulmonary biopsy. The patient was finally diagnosed with pulmonary cryptococcosis and HIV-1 infection. With a proper antifungal treatment the patient evolved satisfactorily.ConclusionsThe best strategy to avoid opportunistic infections such as cryptococcosis in HIV-infected patients consists of an early diagnosis and a highly active antiretroviral treatment. In our case, the diagnosis of a pulmonary infection by C. neoformans var. grubii allowed a late diagnosis of HIV-1 infection.     

Cryptococcosis in HIV-negative Patients with Renal Dialysis: A Retrospective Analysis of Pooled Cases

Cryptococcosis is a lethal fungal infection mainly caused by Cryptococcus neoformans/C. gattii species. Currently, our understanding of cryptococcosis episodes in HIV-negative patients during renal dialysis remains scarce and fragmented. Here, we performed an analysis of pooled cases to systemically summarize the epidemiology and clinical characteristics of cryptococcosis among HIV-negative patients with renal dialysis. Using pooled data from our hospital and studies identified in four medical databases, 18 cases were identified and analyzed. The median duration time of renal dialysis for peritoneal renal dialysis and hemodialysis cases was 8 months and 36 months, respectively. Several non-neoformans/gattii species were identified among the renal dialysis recipients with cryptococcosis, particularly Cryptococcus laurentii and Cryptococcus albidus, which share similar clinical manifestations as those caused by C. neoformans and C. gattii. Our analyses suggest that physicians should consider the possibility of the occurrence of cryptococcosis among renal dialysis recipients even when cryptococcal antigen test result is negative. The timely removal of the catheter is crucial for peritoneal dialysis patients with cryptococcosis. In addition, there is a need for optimized antifungal treatment strategy in renal dialysis recipients with cryptococcal infections.     

Evidence for Interference by Immune Complexes in the Serodiagnosis of Cryptococcosis

The latex agglutination test was used to compare cryptococcal antigen titers before and after protease treatment in 19 patients with pulmonary cryptococcosis. Antigen was detected by the LA test in 13 of 33 serum samples before protease treatment, and in an additional 13 samples following treatment. Of 26 antigen-positive serum samples, 22 (84.6%) showed an increased antigen titer after protease treatment. Using the cell slide agglutination test, antibody was detected in 3 of 19 cases. In one of these 3, antigen could only be detected after protease treatment. Soluble immune complex was prepared in vitro using anti-C. neoformans rabbit antiserum and polysaccharide antigen of C. neoformans, and the effects of immune complexes on the LA test were examined. In this experimental model, soluble immune complexes seemed to be observed in antibody excess region, because the antigen titers were increased after the protease treatment. We concluded that C. neoformans capsular polysaccharide antigen and anti-C. neoformans antibody formed soluble immune complexes in patients' sera, which interfered with antigen detection by the latex agglutination test without protease treatment.     

Clinical Utility of the Cryptococcal Antigen Lateral Flow Assay in a Diagnostic Mycology Laboratory

Background

Cryptococcus neoformans causes life-threatening meningitis. A recently introduced lateral flow immunoassay (LFA) to detect cryptococcal antigen (CRAG) is reportedly more rapid and convenient than standard latex agglutination (LA), but has not yet been evaluated in a diagnostic laboratory setting.

Methods

One hundred and six serum, 42 cerebrospinal fluid (CSF), and 20 urine samples from 92 patients with known or suspected cryptococcosis were tested by LA and LFA, and titres were compared. Results were correlated with laboratory-confirmed cryptococcosis. Serial samples were tested in nine treated patients.

Results

Twenty-five of 92 patients had confirmed cryptococcosis; all sera (n = 56) from these patients were positive by LFA (sensitivity 100%, 95% confidence interval (CI) 93.6–100%) compared with 51/56 positive by LA (sensitivity 91.1%, 95% CI 80.7–96.1%). Fifty sera from 67 patients without cryptococcosis tested negative in both assays. While LA yielded more false negative results (5/56) this did not reach statistical significance (p = 0.063). Nine CSF samples from patients with cryptococcal meningitis yielded positive results using both assays while 17/18 urine samples from patients with cryptococcosis were positive by the LFA. The LFA detected CRAG in C. gattii infection (n = 4 patients). Agreement between titres obtained by both methods (n = 38 samples) was imperfect; correlation between log-transformed titres (r) was 0.84. Turn-around-time was 20 minutes for the LFA and 2 h for LA. The cost per qualitative sample was 18USD and 91 USD, respectively and per quantitative sample was 38USD and 144USD, respectively.

Conclusions

Qualitative agreement between the LFA and LA assays performed on serum and CSF was good but agreement between titres was imperfect. Ease of performance of the LFA and the capacity for testing urine suggest it has a role in the routine laboratory as a rapid diagnostic test or point-of-care test.     

Cryptococcal Meningitis in Senegal: Epidemiology, Laboratory Findings, Therapeutic and Outcome of Cases Diagnosed from 2004 to 2011

Background

Cryptococcal meningitis is one of the most important opportunistic infection and a major contributor to early mortality. In sub-Saharan Africa, particularly in Senegal, prevalence of cryptococcal meningitis remains high. This study aimed to describe the epidemiology, laboratory profile, therapeutic and outcome of cases diagnosed in Dakar.

Methods

We analyzed the cryptococcosis cases diagnosed at the department of parasitology–mycology in Fann Teaching Hospital in Dakar from 2004 to 2011. The diagnosis was confirmed by culture on Sabouraud’s dextrose agar and/or by India ink preparation and/or by cryptococcal antigen detection. The diagnosis methods were assessed by using culture as reference.

Results

A total of 106 cases of cryptococcal meningitis were diagnosed. The prevalence of cryptococcal meningitis was 7.8 %. The mean age of the patients was 40.17 ± 9.89 years. There were slightly more male (53.8 %) than female (46.2 %) patients; 89.6 % were found to be infected with HIV, and the median CD4+ count was 27/mm³. Approximately 79.5 % of the patients had <100 CD4+ lymphocytes/mm³. India ink staining presented sensitivity at 94.11 % and specificity at 100 %. Sensitivity and specificity of cryptococcal antigen detection in cerebrospinal fluid were, respectively, 96.96 and 15.78 %. The most frequently used antifungal drug was fluconazole (86.7 %), and the mortality rate was 62.2 % (66 deaths).

Conclusion

Early diagnosis is essential to control cryptococcosis, and countries should prioritize widespread and reliable access to rapid diagnostic cryptococcus antigen assays. But it is important to make available conventional methods (India ink and culture) in the maximum of laboratory in regional health facilities.     

Equine Pulmonary Cryptococcosis: A Comparative Literature Review and Evaluation of Fluconazole Monotherapy

Cryptococcus is the most common fungal respiratory pathogen in Australian horses, manifesting primarily as pulmonary granulomas. Disease severity at presentation is dependent on the athletic use of the horse. The diagnosis and estimation of disease severity are centred around clinical findings, cytological evaluation of respiratory tract secretions, diagnostic imaging, and antigen titre testing. Both the lateral flow assay and the latex cryptococcal antigen titre are used, and important similarities and differences between species are discussed. Cryptococcus gattii occurs with greater frequency than Cryptococcus neoformans in equine pulmonic cryptococcosis and can be successfully treated with enteral fluconazole monotherapy, with disease severity determining treatment length.     

Cryptococcal phospholipase B antigen is not detected in serum of patients infected with Cryptococcus neoformans using a sandwich enzyme-linked immunosorbent assay

Extracellular phospholipase B (PLB) is a virulence determinant of Cryptococcus neoformans and Cryptococcus gattii. In this study, we developed a sensitive enzyme-linked immunosorbent assay (ELISA) for PLB antigen with a detection limit of 3.9 ng mL(-1). PLB was detected in culture supernatants of C. neoformans and C. gattii. PLB, however, was not detected in sera of seven human patients and 10 feline patients with active cryptococcosis. Furthermore, none of five rats with extensive pulmonary C. gattii infection had a positive ELISA test result. In conclusion, cryptococcal PLB could not be detected in serum using a PLB antigen-based ELISA. Despite its sensitivity, this ELISA is of limited diagnostic value. Exploration of further extracellular molecules suitable for serodiagnosis of active cryptococcal infection is warranted.     

The changing face of AIDS-related opportunism: Cryptococcosis in the highly active antiretroviral therapy (HAART) era. Case reports and literature review

Only nine cases of AIDS-related cryptococcosis have been reported until now in patients receiving highly active antiretroviral therapy (HAART),all of them with abnormal clinical features. Two HIV-infected patients who experienced an atypical relapse of cryptococcosis shortly after the start of HAART and despite maintenance antifungal treatment, are described. Six different relapses of cryptococcal meningitis were observed in a 28-month period in a patient who obtained a poor immune recovery after HAART (as shown by a CD⁴⁺ lymphocyte count ranging from 78 to 149 cellsL, opposed to a baseline level of 98 cellsL). On the other hand, a patient with favorable immunological response to HAART(as expressed by a CD⁴⁺ count growing from 7 to 186 cellsL),experienced isolated multiple indolent cryptococcal abscesses involving head,neck, the anterior thoracic wall, and regional lymph nodes, with repeatedly negative cultures, and diagnosis obtained by both histopathologic study and positive serum antigen assay. Both our case reports are representative of novel correlations between opportunistic pathogens and immune reactivity, descending from the introduction of HAART. The first episode describes an exceedingly elevated number of disease relapses despite HAART and antifungal maintenance treatment, which may descend from an incomplete immune response to antiretroviral therapy, possibly responsible for failure in obtaining eradication of yeasts, but also for lack of disease dissemination (usually leading to a lethal multivisceral involvement in the pre-HAART era). The abnormal disease course and localization of second reported patient well depicts an immune reconstitution syndrome probably representing a flare-up of a latent fungal infection, caused by a rapidly effective HAART. In patients treated with HAART, AIDS-related cryptococcosis cannot therefore be ruled out by the absence of neurological involvement, and by persistingly negative cultures.This revised version was published online in October 2005 with corrections to the Cover Date.     

55例社区获得性肺隐球菌病病例分析

本文旨在通过研究社区获得性肺隐球菌病的临床特点,提高医务人员对肺隐球菌病的认识,做到早期诊断和治疗.采用回顾性病例分析研究,统计2003年1月~2009年12月入住复旦大学附属中山医院的55例确诊及临床诊断为肺隐球菌病患者的临床资料.55例患者中,男性34例,女性21例;年龄17~75岁,平均(44.8±13.4)岁....     

Performance of Cryptococcal Antigen Lateral Flow Assay Using Saliva in Ugandans with CD4 <100

Background

Cryptococcal meningitis can best be diagnosed by cerebrospinal fluid India ink microscopy, cryptococcal antigen detection, or culture. These require invasive lumbar punctures. The utility of cryptococcal antigen detection in saliva is unknown. We evaluated the diagnostic performance of the point-of-care cryptococcal antigen lateral flow assay (CrAg LFA) in saliva.

Methods

We screened HIV-infected, antiretroviral therapy naïve persons with symptomatic meningitis (n = 130) and asymptomatic persons with CD4+<100 cells/µL entering into HIV care (n = 399) in Kampala, Uganda. The diagnostic performance of testing saliva was compared to serum/plasma cryptococcal antigen as the reference standard.

Results

The saliva lateral flow assay performance was overall more sensitive in symptomatic patients (88%) than in asymptomatic patients (27%). The specificity of saliva lateral flow assay was excellent at 97.8% in the symptomatic patients and 100% in asymptomatic patients. The degree of accuracy of saliva in diagnosing cryptococcosis and the level of agreement between the two sample types was better in symptomatic patients (C-statistic 92.9, κ-0.82) than in asymptomatic patients (C-statistic 63.5, κ-0.41). Persons with false negative salvia CrAg tests had lower levels of peripheral blood CrAg titers (P<0.001).

Conclusion

There was poor diagnostic performance in testing saliva for cryptococcal antigen, particularly among asymptomatic persons screened for preemptive treatment of cryptococcosis.     

Enzyme-linked immunosorbent assay for detection and quantification of Cryptococcus neoformans antigen

An enzyme-linked immunosorbent assay was standardized for the detection of cryptococcal antigen in serum and cerebrospinal fluid. The system was evaluated in clinical samples from patients infected by human immunodeficiency virus with and without previous cryptococcosis diagnosis. The evaluated system is highly sensitive and specific, and when it was compared with latex agglutination there were not significant differences. A standard curve with purified Cryptococcus neoformans antigen was settled down for the antigen quantification in positive samples.     

Genetic control of the humoral response to cryptococcal capsular polysaccharide in mice

Cryptococcus neoformans is responsible for opportunistic infections in patients with cellular immune defects. However, C. neoformans-specific capsular polysaccharide antibodies have been shown to participate in host defenses during cryptococcosis. We investigated the humoral response after primary immunization in various inbred strains of mice and the genetic control. Our data strengthen the arguments for the T-independent type-2 nature of cryptococcal antigen, since CBA/N mice were unable to produce specific antibodies. They show that the influence of the genetic background is predominant for the good response with at least four independent autosomal genes governing this response, including an Igh control as reported for other polysaccharides. Immunization of intra-H-2 recombinant mice on a B10 background allowed us to identify a major histocompatibility complex control located in the subregion E . The genetic control of antibody production following immunization with cryptococcal polysaccharide might explain the high variability of humoral responses during cryptococcosis.     

Value and Interpretation of Serological Tests for the Diagnosis of Cryptococcosis

MAXIMAL SEROLOGICAL DIAGNOSIS OF CRYPTOCOCCOSIS MAY BE ACCOMPLISHED THROUGH THE CONCURRENT USE OF THREE TESTS: the latex agglutination (LA) test for cryptococcal antigen, and the indirect fluorescent antibody (IFA) and tube agglutination (TA) tests for Cryptococcus neoformans antibodies. These tests were applied to 141 serum and cerebral spinal fluid specimens from 66 culturally proven cases of cryptococcosis and to 42 sera from normal subjects and from patients with other systemic mycotic diseases. The LA test was sensitive and completely specific; of the sera from proven cases, 55% were positive. With the TA test, 37% of the specimens were positive and the test was highly specific. With the IFA test, 38% of the specimens were positive and the test appears to be the least specific of the three. Cross-reactions were most evident with blastomycosis and histoplasmosis case sera. When the three tests were used concurrently, 87% of the cryptococcosis case specimens were positive and permitted a presumptive diagnosis of C. neoformans infections in 61 (92%) of the 66 patients whose specimens were examined.     

Field Evaluation of Two Rapid Diagnostic Tests for Neisseria meningitidis Serogroup A during the 2006 Outbreak in Niger

The Pastorex^® (BioRad) rapid agglutination test is one of the main rapid diagnostic tests (RDTs) for meningococcal disease currently in use in the “meningitis belt”. Earlier evaluations, performed after heating and centrifugation of cerebrospinal fluid (CSF) samples, under good laboratory conditions, showed high sensitivity and specificity. However, during an epidemic, the test may be used without prior sample preparation. Recently a new, easy-to-use dipstick RDT for meningococcal disease detection on CSF was developed by the Centre de Recherche Médicale et Sanitaire in Niger and the Pasteur Institute in France. We estimate diagnostic accuracy in the field during the 2006 outbreak of Neisseria meningitidis serogroup A in Maradi, Niger, for the dipstick RDT and Pastorex^® on unprepared CSF, (a) by comparing each test''s sensitivity and specificity with previously reported values; and (b) by comparing results for each test on paired samples, using McNemar''s test. We also (c) estimate diagnostic accuracy of the dipstick RDT on diluted whole blood. We tested unprepared CSF and diluted whole blood from 126 patients with suspected meningococcal disease presenting at four health posts. (a) Pastorex^® sensitivity (69%; 95%CI 57–79) was significantly lower than found previously for prepared CSF samples [87% (81–91); or 88% (85–91)], as was specificity [81% (95%CI 68–91) vs 93% (90–95); or 93% (87–96)]. Sensitivity of the dipstick RDT [89% (95%CI 80–95)] was similar to previously reported values for ideal laboratory conditions [89% (84–93) and 94% (90–96)]. Specificity, at 62% (95%CI 48–75), was significantly lower than found previously [94% (92–96) and 97% (94–99)]. (b) McNemar''s test for the dipstick RDT vs Pastorex^® was statistically significant (p<0.001). (c) The dipstick RDT did not perform satisfactorily on diluted whole blood (sensitivity 73%; specificity 57%).Sensitivity and specificity of Pastorex^® without prior CSF preparation were poorer than previously reported results from prepared samples; therefore we caution against using this test during an epidemic if sample preparation is not possible. For the dipstick RDT, sensitivity was similar to, while specificity was not as high as previously reported during a more stable context. Further studies are needed to evaluate its field performance, especially for different populations and other serogroups.     

Cryptococcus neoformans meningoencephalitis in a patient with polyarteritis nodosa

Case of 59-year-old male with chronic obstructive pulmonary disease and a number of comorbidities, who has developed meningoencephalitis caused by Cryptococcus neoformans var. grubii with polyarteritis nodosa diagnosed during hospitalization, was presented. Before evidence of meningoencephalitis, the patient was being treated with ketoconazole and low doses of fluconazole (200 mg/day) for alleged candidiasis. The dosage was increased (800 mg/day) following laboratory diagnosis of C. neoformans based on positive latex agglutination test and biochemical identification of encapsulated yeast isolated from the blood and CSF. Later, the yeast identification was confirmed by sequencing analysis. Owing to inadequate clinical response, fluconazole therapy was switched to voriconazole (400 mg/day) and later to intravenous amphotericin B (1.0 mg/kg per day). Despite of a temporary stabilization and improvement, which correlated with decline of cryptococcal antigen titers (from 1:1024 to 1:8), after 6 weeks, the patient’s underlying condition deteriorated due to severe pancolitis and serious nosocomial bacterial infections. The patient died of multiorgan failure several days later. Our case demonstrates a possible connection between the development of life-threatening cryptococcosis and an autoimmune vasculitis disease and emphasizes that the outcome of the management of cryptococcal meningoencephalitis is highly dependent on early diagnosis, adequate treatment, including dosage, and last but not least control of underlying disease and risk factors.     

Recovery of Cryptococcus neoformans from the nasopharynx of AIDS patients

Nasopharyngeal swabbings, obtained from AIDS patients, were plated onto Niger seed agar containing antibiotics. Cryptococcus neoformans was isolated from 35 out of 84 patients (41.7%) diagnosed as primary cryptococcal cases before antifungal administration, and 8 out of 86 (9.3%) cryptococcosis patients on antifungal therapy. The fungus could not be isolated from any of 447 samples from 194 AIDS patients not diagnosed with cryptococcosis. These findings are novel in that the presence of C. neoformans in AIDS patients at this site has never been looked at previously. This revised version was published online in June 2006 with corrections to the Cover Date.     

Pulmonary cryptococcosis in late pregnancy and review of published literature

Naturally occurring maternal immunosuppression increases the risk of infection by a variety of pathogens during pregnancy and the postpartum period. Pulmonary cryptococcosis during pregnancy is relatively rare. Here, we report on two cases of pulmonary cryptococcosis during pregnancy and puerperium. Both cases were successfully treated using oral fluconazole after parturition to avoid fetal toxicity. For the two patients, the placentas were checked and found to be pathologically normal, and the cryptococcal serum antigen in both infants was negative. Pulmonary cryptococcosis should be considered during differential diagnosis as a possible cause of abnormal chest shadow in pregnant patients.     

Characterization of<Emphasis Type="Italic">Cryptococcus neoformans</Emphasis> var.<Emphasis Type="Italic">neoformans</Emphasis> serotype A and A/D in samples from Egypt

The cryptococcal polysaccharide antigen was detected in 10 cerebrospinal fluid (CSF) and 23 serum samples from cryptococcal meningitis and intestinal cryptococcosis by the cryptococcal antigen latex agglutination system (CALAS). CALAS titers in CSF and serum samples of cryptococcal meningitis ranged over 8-2048 and 32-2048, respectively, while in cases of intestinal cryptococcosis, serum titers ranged over 8-2048. The isolates of yeast Cryptococcus neoformans were determined to be of serotype A or of the A/D pair. The total leukocyte count and biochemical parameters in CSF were significantly increased as indicators of microbial infection. Furthermore, the in vitro change of the teleomorph (sexual state) to the anamorph (asexual state) was also detected and the teleomorph state changed in vivo to the encapsulated anamoph state which is more virulent during infection in vivo than the yeast-like noncapsulated form. Two primers for internal transcribed spacer (ITS) regions of ribosomal DNA were used for molecular detection of C. neoformans. After PCR amplification, a DNA band of 415 bp, visualized on agarose gel, indicated the presence of C. neoformans cells in the tested CSF and serum samples. The primer sensitivity was also characterized using purified yeast chromosomal DNA as template; it was about 20 pg or more chromosomal DNA which represents about 10 cells of C. neoformans. The primers were also specific for ITS regions of C. neoformans and gave negative results with Candida albicans and E. coli chromosomal DNA templates.     

31例非获得性免疫缺陷综合征肺隐球菌病临床分析

目的通过研究非获得性免疫缺陷综合征肺隐球菌病的临床特点,提高医务人员对肺隐球菌病的认识,做到早期诊断和治疗。方法回顾性分析2001年1月至2011年12月在南京医科大学附属杭州市第一人民医院确诊的31例非获得性免疫缺陷综合征肺隐球菌病患者的性别、年龄分布、职业情况、伴随的基础疾病、临床症状、实验室检查、影像学特征、治疗方案及预后等各方面特点。结果肺隐球菌病发病年龄有年轻化趋势,临床特点及影像学表现缺乏特异性,极易误诊及漏诊。治疗方法及药物少,病程长。结论肺隐球菌病虽然临床表现缺乏特异性,但从病例分析结果来看诊治该病仍有一定规律可循,亟需提高对本病的认识,遇到疑似病例应行血乳胶凝集试验及肺活检或手术病理予以明确,药物治疗需要足够疗程。