Recombinant 60-kDa heat shock protein from Paracoccidioides brasiliensis: is it a good antigen for serological diagnosis of paracoccidioidomycosis?

Paracoccidioides brasiliensis and P. lutzii are fungi that cause paracoccidioidomycosis (PCM), the most prevalent systemic mycosis in South America. For serological diagnosis, although 43-kDa glycoprotein (gp43) is regarded as highly specific for PCM, the occurrence of false negative reactions in sera from patients infected with P. lutzii suggests that preparation with only one antigen is not recommended. Heat shock proteins are feasible alternatives as a second antigen because they are often highly immunogenic. In this study, we evaluated the usefulness of recombinant 60-kDa heat shock protein from P. brasiliensis (rPbHsp60) for the serological diagnosis of PCM. Using western blotting assay, we observed that 77.3% of the sera from PCM patients were positive to rPbHsp60, with 90.9% positivity to recombinant gp43 (rgp43). More importantly, sera from healthy subjects had 27% positivity to rPbHsp60 and none to rgp43. When rPbHsp60 was used in ELISA, we did not observe significant differences between the reactions with sera from PCM patients and healthy subjects, while the difference was clearly evident when the antigen was rgp43. Furthermore, rPbHsp60 was recognized by sera from patients with histoplasmosis, aspergillosis, sporotrichosis or tuberculosis in an ELISA test. These results show that rPbHsp60 is not a good antigen for PCM diagnosis.

Lateral flow asaay for cryptococcal antigen: animportant advance to improve thhe continuum of hiv care and reduce cryptococcal meningitis-related mottality / Ensaio de Fluxo Lateral para antígeno criptocócico: um importante avanço para melhorar o continuum de cuidados com HIV e reduzir a mortalidade relacionada à meningite criptocóccica

SUMMARYAIDS-related cryptococcal meningitis continues to cause a substantial burden of death in low and middle income countries. The diagnostic use for detection of cryptococcal capsular polysaccharide antigen (CrAg) in serum and cerebrospinal fluid by latex agglutination test (CrAg-latex) or enzyme-linked immunoassay (EIA) has been available for over decades. Better diagnostics in asymptomatic and symptomatic phases of cryptococcosis are key components to reduce mortality. Recently, the cryptococcal antigen lateral flow assay (CrAg LFA) was included in the armamentarium for diagnosis. Unlike the other tests, the CrAg LFA is a dipstick immunochromatographic assay, in a format similar to the home pregnancy test, and requires little or no lab infrastructure. This test meets all of the World Health Organization ASSURED criteria (Affordable, Sensitive, Specific, User friendly, Rapid/robust, Equipment-free, and Delivered). CrAg LFA in serum, plasma, whole blood, or cerebrospinal fluid is useful for the diagnosis of disease caused by Cryptococcusspecies. The CrAg LFA has better analytical sensitivity for C. gattii than CrAg-latex or EIA. Prevention of cryptococcal disease is new application of CrAg LFA via screening of blood for subclinical infection in asymptomatic HIV-infected persons with CD4 counts < 100 cells/mL who are not receiving effective antiretroviral therapy. CrAg screening of leftover plasma specimens after CD4 testing can identify persons with asymptomatic infection who urgently require pre-emptive fluconazole, who will otherwise progress to symptomatic infection and/or die.

RESUMOA meningite criptocócica continua causando um substancial índice de óbitos em pacientes infectados por HIV em países de baixa e média renda. Ferramentas diagnósticas para detecção do antígeno capsular polissacarídico criptocócico (CrAg) em soro e líquor tais como o teste de aglutinação de látex (latex-CrAg) ou o imunoensaio (EIE) têm sido utilizadas por muitos anos. Técnicas diagnósticas mais aprimoradas seriam cruciais nas fases assintomática e sintomática da criptococose para reduzir a mortalidade. Recentemente, o ensaio de fluxo lateral para detecção do antígeno criptocócico (LFA CrAg) foi incluído no arsenal diagnóstico. Contrariamente aos outros testes, LFA CrAg é um ensaio imunocromatográfico em formato similar ao teste de gravidez, e requer pouca ou nenhuma infraestrutura laboratorial. Este teste preenche os critérios ASSURED (Affordable, Sensitive,Specific, User friendly,Rapid/ robust,Equipment-free,Delivered) da Organização Mundial da Saúde e pode ser utilizado em soro, plasma, sangue total ou líquor para o diagnóstico da criptococose. LFA CrAg tem melhor sensibilidade analítica para o C. gattii que o teste de látex-CrAg ou EIE. A prevenção da doença criptocócica constituiria uma nova aplicação do LFA CrAg, mediante a triagem de amostras de sangue para a identificação de infecção sub-clínica em pacientes infectados pelo HIV que não apresentam sintomas, possuem contagem de CD4 < 100 células/mL e não recebem terapia antirretroviral eficaz. A triagem de CrgA em amostras de plasma remanescente da contagem de CD4 pode identificar pacientes com infecção assintomática que precisam urgentemente de tratamento preemptivo com fluconazol, evitando assim a progressão para doença sintomática e/ou óbito.

Cryptococcosis in Colombia: Results of the national surveillance program for the years 2006-2010 / Criptococosis en Colombia: resultados de la encuesta nacional, 2006-2010

Introduction: A survey on cryptococcosis is being conducted regularly in Colombia since 1997. We present hereby the results corresponding to patients diagnosed from 2006 to 2010. Objective: To analyze the data obtained during this period. Materials and methods: Retrospective analysis of the corresponding surveys. Results: A total of 526 surveys originating from 72% of the Colombian political divisions were received during the 5-year period. Most patients (76.6%) were males and 74.9% were 21-50 years old. The most prevalent risk factor was HIV infection (83.5%) with cryptococcosis defining AIDS in 23% of the cases. In the general population the estimated mean annual incidence rate for cryptococcosis was 2.4 x 106 inhabitants while in AIDS patients this rate rose to 3.3 x 103. In 474 surveys stating clinical features, most frequent complaints were headache 84.5%, fever 63.4%, nausea and vomiting 57.5%, mental alterations 46.3%, meningeal signs 33.0%, cough 26.4% and visual alterations 24.5%. Neurocryptococcosis was recorded in 81.8% of the cases. Laboratory diagnosis was based on direct examination, culture and latex in 29.3% cases. From 413 Cryptococcus isolates analyzed, 95.6% were identified as C. neoformans var. grubii, 1% C. neoformans var. neoformans, and 3.4% C. gattii. Treatment was reported for 71.6% of the cases with amphotericin B alone or in combination with fluconazole prescribed in 28%. Conclusions: Surveys done through passive surveillance continue to be sentinel markers for HIV infection and represent a systematic approach to the study of opportunistic problems regularly afflicting AIDS patients since cryptococcosis requires no compulsory notification in Colombia.

Introducción. Desde 1997 se viene realizando un programa nacional de vigilancia sobre la criptococosis en Colombia. Se presentan los resultados correspondientes a los pacientes diagnosticados entre el 2006 y el 2010. Objetivo. Analizar los datos obtenidos durante este periodo. Materiales y métodos. Análisis retrospectivo de las encuestas. Resultados. Durante los cinco años mencionados se recibieron 526 encuestas representativas del 72 % de la división política colombiana. La mayoría de pacientes (76,6 %) eran hombres y 74,9 % estaban entre los 21 y los 50 años. El factor de riesgo prevalente fue la infección por VIH (83,5 %), y la criptococosis definió el sida en 23 % de los casos. La incidencia anual promedio en la población general fue de 2,4 por un millón de habitantes mientras que, en pacientes con sida, aumentó a 3,3 por 1.000. En 474 encuestas se informaron manifestaciones clínicas; las más frecuentes fueron: cefalea (84,5 %), fiebre (63,4 %), náuseas y vómito (57,5 %), alteraciones mentales (46,3 %), signos meníngeos (33 %), tos (26,4 %) y alteraciones visuales (24,5 %). La neurocriptococosis se reportó en 81,8 % de los casos. El diagnóstico se hizo por examen directo, cultivo y antigenemia en 29,3 % de los casos. De 413 aislamientos recuperados, 95,6 % fueron C. neoformans var. grubii, 1 % C. neoformans var. neoformans, y 3,4 % C. gattii. En 71,6 % de los casos para el tratamiento se administró anfotericina B y en 28 % se combinó con fluconazol. Conclusiones. La vigilancia pasiva continúa siendo un marcador centinela para la infección por VIH, y constituye una aproximación sistemática al estudio de infecciones oportunistas en pacientes con sida, debido a que la criptococosis no es de notificación obligatoria en Colombia.

Screening for cryptococcal antigenemia in anti-retroviral naive AIDS patients in Benin City, Nigeria

Cryptococcus neoformans is the most incriminated fungal pathogen causing meningitis in acquired immune deficiency syndrome [AIDS] patients, and is known to constitute a major cause of deaths in AIDS patients. This study thus aimed to determine the baseline sero-prevalence of Cryptococcus neoformans infection in anti-retroviral naive [ART-naive] AIDS patients using the serum Cryptococcal antigen [crag] detection method. Baseline effect of variation in CD4 counts, as well as sex and age with sero-positivity for crag were also determined. This descriptive cross-sectional study included 150 [61 males and 89 females] ART-naive AIDS patients attending the Human Immunodeficiency Virus clinic [HIV] at the University of Benin Teaching hospital, Benin City, Nigeria, within the period from February 2011- July 2011. Forty [18 males and 22 females] HIV positive outpatients with CD4 counts >200 cells/microl who were ART-naive were recruited and used as controls. The sero-prevalence of crag in the patients and the control group was measured using the cryptococcal antigen latex agglutination system [CALAS] [Meridian Bioscience, Europe] and CD4 counts were measured using flow cytometry [Partec flow cytometer, Germany]. Of the 150 ART-naive AIDS patients with CD4 counts

Métodos diagnósticos en candidemia: una revisión sistemática de la literatura con meta-análisis / Diagnostic methods in candidemia: a systematic review of literature with meta-analysis

Candidemia is a disease with high morbidity and mortality especially in critical care patients. Early diagnosis enables early treatment. Objectives: To conduct a systematic review of the literature in order to establish the best laboratory tests for the diagnosis of candidemia in critical patients. Materials and Methods: We conducted a systematic review of available literature in PubMed. Serological studies were subjected to meta-analysis in metadisk-Beta 1.1.1. Results: 4 studies of 1286 reviewed were included. Three were about serological tests and one about molecular testing (RT-PCR). The sensitivity and specificity for RT-PCR, antibody testing and antigen and antibody tests were 87 percent and 100 percent, 47.5 percent and 82.6 percent, 96 percent and 81 percent, respectively. Diagnostic Odds Ratio of antigenemia was 1.51 (95 percent CI = 0,032-70,964, p = 0.001). Conclusions: RT-PCR has better diagnostic performance, measuring antigenemia plus antibodies improves sensitivity, specificity, LR + and LR-- . There is insufficient evidence to support this.

La candidemia es una patología con alta morbilidad y mortalidad, especialmente en los pacientes sometidos a servicios de cuidado crítico. El diagnóstico precoz permite realizar tratamiento temprano. Objetivos: Realizar una revisión sistemática de la literatura para establecer cuáles son las pruebas de laboratorio con mejor rendimiento diagnóstico y operativo para el diagnóstico de candidemia en cuidado intensivo. Materiales y Métodos: Se realizó una revisión sistemática de la literatura disponible en PubMed, se sometieron a meta-análisis estudios de pruebas serológicas en MetaDisc-Beta 1.1.1. Resultados: Se incluyeron 4 estudios de 1.286 revisados, 3 de pruebas serológicas y 1 de RPC-RT. La sensibilidad y especificidad fue de 87 y 100 por ciento para RPC-RT, 47,5 y 82,6 por ciento para pruebas de anticuerpos, 96 y 81 por ciento para pruebas de antígeno y anticuerpo. La ORD de antigenemia 1,51(IC95 por ciento = 0,03270,964; p = 0,001). Conclusiones: RPC-RT tiene mejor rendimiento diagnóstico, la medición de antigenemia más anticuerpos mejora la sensibilidad, especificidad, LR+ y LR-. No hay suficiente evidencia que soporte esto.

Human serum antibody reactivity towards Paracoccidioides brasiliensis antigens treated with sodium metaperiodate / Reatividade de anticorpos de soros humanos a antígenos de Paracoccidioides brasiliensis tratados com metaperiodato sódico

In this study, we evaluated the profile of anti-Paracoccidioides brasiliensis immunoglobulin isotypes in serum from patients with the acute and chronic forms of paracoccidioidomycosis, using the whole Paracoccidioides brasiliensis antigen and the antigen treated with sodium metaperiodate. All the immunoglobulin isotypes present in the serum from patients with the acute and chronic forms of paracoccidioidomycosis presented higher reactivity towards the whole antigen than to the antigen treated with metaperiodate (P < 0.05). The reactivity of IgG and IgM to the antigen treated with metaperiodate was greater in serum from patients with the acute form of the disease (P < 0.05), while IgA was more reactive in serum from patients with the chronic form (P < 0.05). There was greater reactivity of IgG1 and IgG2 to the whole antigen and the antigen treated with metaperiodate in the serum from patients with paracoccidioidomycosis than there was in serum from patients with other parasitic infections (P < 0.05). Furthermore, IgG1 from patients with the acute form recognized the 19kDa, 27kDa and 31kDa antigens in the western blot test. Thus, the results suggest that modifications to the epitopes of Paracoccidioides brasiliensis antigens may help to improve the immunodiagnosis of paracoccidioidomycosis.

Neste trabalho, foi avaliado o perfil de isotipos de imunoglobulinas anti-Paracoccidioides brasiliensis em soros de pacientes com formas crônica e aguda de paracoccidiodomicoses usando antígeno total e tratado com meta-periodato. Todos os tipos de imunoglobulinas presentes nos soros de pacientes com formas aguda e crônica apresentaram alta reatividade ao antígeno total quando comparado ao tratado com meta-periodato (P < 0,05). Houve maior reatividade de IgG e IgM anti-antígeno tratado com meta-periodato em soros de pacientes com forma aguda da doença (P < 0,05), enquanto IgA foi mais reativa em soros da forma crônica (P < 0,05). Houve maior reatividade de IgG1 e IgG2 com antígeno total e tratado com meta-periodato em soros de pacientes comparados aos com outras parasitoses (P < 0,05). Além disso, IgG1 de pacientes com a forma aguda reconhecem antígenos de 19kDa, 27kDa e 31kDa por western blot. Assim, os resultados sugerem que alterações nos epitopos de antígenos de Paracoccidioides brasiliensis podem auxiliar no aprimoramento do imunodiagnóstico da paracoccidioidomicose.

Galactomannan antigenemia for the diagnosis of invasive aspergillosis in neutropenic patients with hematological disorders.

OBJECTIVES: Invasive aspergillosis (IA) is among the most common invasive fungal infections in neutropenic patients with hematological disorders in the authors' institution, King Chulalongkorn Memorial Hospital (KCMH), Bangkok, Thailand Previous studies have reported the Aspergillus galactomannan enzyme immunosorbent assay (GMEIA) may be a useful diagnostic tool for IA. The authors evaluated the performance of the GM EIA for the diagnosis and monitoring of the course of IA in KCMH. MATERIAL AND METHOD: The authors prospectively performed the study from June 2002 to January 2004 in a consecutive series of adult neutropenic patients with hematological disorders who were at risk for developing IA. During hospitalization, serum galactomannan levels were measured once or twice weekly using the Platellia Aspergillus EIA test kit. The sensitivity and specificity of the GM EIA were calculated according to the proportion of patients with true and false positive and negative tests. RESULTS: There were 50 treatment episodes in 44 patients with 5 proven, 12 probable, and 33 possible or no IA. The cutoff GM index of > 0.75 was determined with a sensitivity of 94.1% and a specificity of 78.8%. There was a close relationship between clinical outcome and the kinetics of GM indices. CONCLUSION: The GM EIA is a useful diagnostic toolfor the diagnosis and monitoring of the course oflA in the presented institute.

Criptococosis diseminada en pacientes con SIDA. Análisis clÝnico, microbiológico e inmunológico de 51 pacientes / Disseminated cryptococcosis in patients with AIDS. Clinical, microbiological, and immunological analysis of 51 patients

In the context of HIV infection, cryptococcal meningitis is the most common mycosis threatening the patient's life. We conducted a retrospective evaluation to determine the epidemiological, microbiological, immunological and clinical characteristics of disseminated cryptococcosis in 51 hospitalised HIV seropositive patients. All the individuals (n = 51) presented reactive serology for HIV (ELISA and/or Western blot) and none fulfilled strict HAART treatment, previous to the opportunistic infection. CD4+ lymphocyte T counts showed levels between 361 and 0 cells/microliter (mean = 45). All patients but one had counts lower than 100 cells/microliter. Cryptococcosis presented as unique episode in 35 patients (68.6) and in 16 as relapse (31.3). In all of them we detected central nervous system involvement. The induction treatment was carried out with amphotericin B (AMB), continued with maintenance therapy with fluconazole. Lethality rate was 36.7, slightly superior among patients in relapse (40) compared to those who presented a first episode of the mycosis (35.2). In those individuals for whom data were available, 65.2 of blood cultures, 94.1 of CSF cultures and 79.06 of microscopic CSF examination with India ink were positive. Titers of Cryptococcus neoformans capsular antigen in CSF > or = 1/1000 were found in 36.1 and > or = 1/1000 in 73.6 of serum samples. In conclusion, manifestations and severity of disseminated cryptococcosis continue maintaining the characteristics of half a decade behind, in those patients who are not treated with HAART. Neurological involvement existed in all patients of this cohort. Treatment is not able to modify the parameters of mortality seen in previous communications. Diagnostic methods applied in this study are in accordance with those in the bibliography.

Successful medical treatment of multiple cryptococcomas: report of a case and literature review.

We report a 35-year-old man diagnosed as having CNS cryptococcosis with multiple cryptococcomas, presenting with headache, papilloedema and impaired mental function in a previously healthy man. Cerebrospinal fluid (CSF) examination revealed lymphocytic pleocytosis with low glucose level. Gram's stain, acid fast bacilli stain and Indian ink examination were all negative. CSF cryptococcal antigen was positive, however, several fungal cultures were negative. Early cranial CT scan showed focal cerebritis over the right temporal lobe while subsequent imaging studies showed multiple contrast-enhancing masses with severe surrounding brain oedema over bilateral frontoparietal areas. Brain biopsy showed cryptococcal granulomatous lesions. Treatment was successful with antifungal agents and steroids without surgical removal.

Serum cyrptococcal antigen: diagnostic value in the diagnosis of AIDS-related cryptococcal meningitis.

RATIONALE: The incidences of HIV-AIDS patients with opportunistic infections of the central nervous system are increasing. Of these, cryptococcal meningitis is the most important and serious. A simple method for the diagnosis of cryptococcal meningitis is needed despite its variable clinical features and the lack of a capacity in most health facilities in Thailand to exclude it from other diseases especially mass lesions in the brain. OBJECTIVE: To identify the capability and cut off point of serum cryptococcal antigen for diagnosis and screening of cryptococcal meningitis in HIV-AIDS patients. METHODS: One hundred consecutive cases of HIV-AIDS patients suspected of having central nervous system infections were prospectively recruited for the study. The serum of all patients were examined for cryptococcal antigen by latex agglutination test, the Pastorex Cryptococcus manufactured by Sanofi Diagnostic Pasteur, France. If a test was positive, the serum dilution was carried out using 10-fold serial dilution. Every patient went through pre-defined standard investigations to derive at a definite diagnosis. The gold standard for diagnosis of cryptococcal meningitis was the presence of encapsulated yeast forms in the cerebrospinal fluid or a positive culture for cryptococcal neoformans from the cerebrospinal fluid. RESULTS: Of 100 patients enrolled in this study, 58 patients had cryptococcal meningitis and serum cryptococcal antigen was detectable in 60 patients. If the cut-off point for a positive test was when the serum cryptococcal antigen titer was more than zero, then, the sensitivity of the test was 91.4 per cent, the specificity was 83.3 per cent, likelihood ratio if test positive (LR+) was 5.47, likelihood ratio if test negative (LR-) was 0.1, false positive was 16.7 per cent, false negative was 8.6 per cent. CONCLUSION: We conclude that serum cryptococcal antigen is a simple and rapid screening method for diagnosis of cryptococcal meningitis.

Meningite criptocócica em pacientes imunocompetentes: relato de dois casos e revisäo da literatura / Cryptococcal meningitis in immunocompetent patients: report of two cases and literature review

A criptococose é uma doença sistêmica causada pelo fungo Cryptococcus neoformans, que acomete com mais freqüência os pulmões e o sistema nervoso central e, menos freqüentemente, a pele, o sistema esquelético e a próstata. Até a década de 80, antes do surgimento da AIDS, era relativamente rara. Apesar dela ocorrer com freqüência no hospedeiro imunodeprimido, cerca de um terço dos pacientes com a doença näo apresenta uma condiçäo de base, nem fatores predisponentes evidentes. Relatamos dois casos de pacientes imunocompetentes que apresentaram meningite criptocócica, enfatizando epidemiologia, etiopatogenia, clínica, diagnóstico e conduta terapêutica

Influencia del tratamiento con zidovudina (AZT) sobre los niveles de antígeno polisacárido capsular de Cryptococcus neoformans en los fluídos biológicos / Influence of zidovudine (AZT) on levels of capsular antigen of Cryptococcus neoformans in body fluids

Fueron investigados los títulos de antígeno polisacárido capsular de Cryptococcus neoformans en el momento del diagnóstico en 25 pacientes con criptococosis asociada al SIDA. Siete pacientes recibirían en el momento del diagnóstico de la micosis 500-600 mg/día de zidovudina (AZT) y otros 18 no recobirían ninguna medicación antirretroviral. Todos ellos recibieron tratamiento antifúngico específico inmediatamente después de realizado el diagnóstico micológico. Los títulos de los pacientes tratados con AZT fueron más bajos que aquellos no tratados con ningún esquema antirretroviral, aunque la diferencia observada careció de significación estadística (p>0,05). El promedio del tiempo de sobrevida (tomando desde el momento del diagnóstico de la micosis hasta la muerte) fue significativamente más largo (504,43 ñ 160 días) (p = 0,002). Tampoco se observaron diferencias significativas entre los recuentos de linfocitos CD4+ y la prevalencia de diferentes infecciones intercurrentes en ambos grupos. Las diferencias observadas entre ambas poblaciones estudiadas puede demostrar indirectamente la eficacia de la terapéutica antirretroviral para retardar el daño inmunológico provocado por el VIH sobre el sistema inmune de los pacientes