Comparative analysis of diagnostic methods for the detection of Cryptococcus neoformans meningitis.

BACKGROUND: Cryptococcosis is a devastating opportunistic infection in immunocompromised individuals, primarily in people living with HIV/AIDS. This study evaluated a protocol for the early diagnosis of meningitis due to C. neoformans, utilizing established molecular techniques from serum and CSF samples. METHODS: The 18S and 5.8S (rDNA-ITS) sequence-specific nested PCR assays were compared with direct India ink staining and the latex agglutination test for detection of C. neoformans in serum and cerebrospinal fluid (CSF) from 49 Brazilian suspected meningitis patients. Results were validated with samples obtained from 10 patients negative for cryptococcosis and HIV, and by analysis of standard C. neoformans strains. PRINCIPAL FINDINGS: The 5.8S DNA-ITS PCR was more sensitive (89-100%) and specific (100%) than the 18S rDNA PCR and conventional tests (India ink staining and latex agglutination) for identification of C. neoformans. While the 18S PCR exhibited a sensitivity (72%) similar to that of the latex agglutination assay in serum samples, it was superior to the latex agglutination assay when testing CSF, with a sensitivity of 84%. However, the latex agglutination was superior to the 18SrDNA PCR in specificity in CSF (92%). The 5.8S DNA-ITS PCR yielded the highest levels of accuracy (96-100%) of any test for detection (serological and mycological) of C. neoformans in both serum and CSF. CONCLUSION: Use of the nested 5.8S PCR was superior to other techniques for the diagnosis of cryptococcosis. The possibility of using serum, a non-invasively collected material, in a targeted 5.8S PCR analysis to identify Cryptococcus spp. is recommended, especially in immunosuppressed patients. Our results indicate that nested 5.8S PCR can increase the diagnostic capability of cryptococcosis, and we suggest its use to monitor patients in the future.

Anti-GM-CSF Neutralizing Autoantibodies in Colombian Patients with Disseminated Cryptococcosis.

BACKGROUND: Cryptococcosis is a potentially life-threatening fungal disease caused by encapsulated yeasts of the genus Cryptococcus, mostly C. neoformans or C. gattii. Cryptococcal meningitis is the most frequent clinical manifestation in humans. Neutralizing autoantibodies (auto-Abs) against granulocyte-macrophage colony-stimulating factor (GM-CSF) have recently been discovered in otherwise healthy adult patients with cryptococcal meningitis, mostly caused by C. gattii. We hypothesized that three Colombian patients with cryptococcal meningitis caused by C. neoformans in two of them would carry high plasma levels of neutralizing auto-Abs against GM-CSF. METHODS: We reviewed medical and laboratory records, performed immunological evaluations, and tested for anti-cytokine auto-Abs three previously healthy HIV-negative adults with disseminated cryptococcosis. RESULTS: Peripheral blood leukocyte subset levels and serum immunoglobulin concentrations were within the normal ranges. We detected high levels of neutralizing auto-Abs against GM-CSF in the plasma of all three patients. CONCLUSIONS: We report three Colombian patients with disseminated cryptococcosis associated with neutralizing auto-Abs against GM-CSF. Further studies should evaluate the genetic contribution to anti-GM-CSF autoantibody production and the role of the GM-CSF signaling pathway in the immune response to Cryptococcus spp.

Cryptococcal meningitis due to Cryptococcus gattii in an immunocompetent patient: uncommon presentation of a thunderclap headache.

Thunderclap headache is a medical emergency presented as the worst headache ever, is characterised by an abrupt onset and maximal intensity within seconds to minutes. However, cerebrovascular causes are among the most common causes of thunderclap headache, and other non-vascular life-threatening aetiologies should be considered in evaluating a patient. We describe a 23-year-old previously healthy Latino woman who presented to our hospital after a month of repetitive severe, abrupt-onset headaches. Her prior medical history was unremarkable. After a normal brain MRI with angio-MRI, a lumbar puncture was performed with normal opening pressure, hypoglycorrhachia, increased proteins and a leucocyte; India ink staining was positive for encapsulated yeast, cultures were positive for Cryptococcus gattii The patient received appropriate antifungal treatment with a good response. This case highlights the particular presentation of cryptococcal meningitis due to C. gattii among immunocompetent patients.

Cost-Effectiveness Analysis of the Implementation of Cryptococcal Antigen Lateral Flow Assay for the Diagnosis of Cryptococcal Meningitis in Symptomatic People Living With Human Immunodeficiency Virus in Brazil.

OBJECTIVES: Cryptococcal meningitis constitutes a significant source of mortality in the developing world. Annually, approximately 625 000 deaths occur worldwide among patients with human immunodeficiency virus (HIV) infection. This study aims to assess the cost-effectiveness of implementing cryptococcal antigen lateral flow assay (CRAG-LFA) screening in Brazil compared with the current practice. METHODS: An economic evaluation using a Monte Carlo microsimulation was conducted, considering the perspective of the Brazilian Public Health System, to calculate the cost-effectiveness of 4 diagnosis tests: (1) CRAG-LFA, (2) the cryptococcal antigen latex agglutination (CRAG-LA) test, (3) India ink, and (4) nontracking as a baseline. The time horizon comprised 1 year for the intervention and 5 years for the budgetary impact analysis. Two primary effectiveness outcomes were considered: years of life and quality-adjusted life-years. RESULTS: CRAG-LFA has extended dominance vis à vis CRAG-LA and India ink. CRAG-LFA would cost $418.46 more than CRAG-LA for the treatment of each symptomatic patient living with HIV, with an incremental cost effectiveness ratio of $2478.75/quality-adjusted life year. The budgetary impact analysis estimated that the incorporation of CRAG-LFA would have an additional cost of $1 959 236.50 in 5 years. CONCLUSIONS: These findings suggest that, for patients living with HIV in the Brazilian Public Health System, the adoption of CRAG-LFA screening is cost-effective compared with the use of CRAG-LA and India ink. It represents an opportunity to prevent cryptococcal meningitis and its mortality in Brazil.

Comparison of two commercial tests (Immy vs. Dynamiker) for cryptococcal capsular antigen.

INTRODUCTION: Lateral flow assay is an advanced method useful in the early diagnosis of cryptococcal meningitis. We aimed to compare two commercial tests for cryptococcal capsular antigen in the sera of asymptomatic patients with human immunodeficiency virus in Barranquilla, Colombia. METHODS: Thawed (n=162) previously collected serums (2016-2019) were processed using IMMY and Dynamiker cryptococcal antigen lateral flow assay. RESULTS: Compared to IMMY's results, Dynamiker's sensitivity, specificity, positive predictive value, negative predictive value, and kappa index were 100%, 89.9%, 48.3%, 100.0%, and 0.61, respectively. CONCLUSIONS: The Dynamiker test had excellent sensitivity, acceptable specificity, and a low detection threshold for cryptococcal antigen in the tested samples.

Teste diagnóstico, point of care, de Cryptococcal Antigen Lateral Flow Assay (CRAG-LFA) para detecção de infecção por Cryptococcus e diagnóstico de meningite criptocócica em pessoas vivendo com o vírus da imunodeficiência humana (PVHIV) / Cryptococcal diagnostic test, point of care Lateral Antigen Flow Assay (CRAG-LFA) for detection of Cryptococcus infection and diagnosis of cryptococcal meningitis in people living as human immunodeficiency virus (PVHIV)

CONTEXTO: A criptococose é uma micose sistêmica causada por duas espécies do basidiomiceto encapsulado do gênero Cryptococcus. A infecção acomete principalmente o sistema nervoso central (SNC) e o trato respiratório e sua apresentação mais comum é a meningite criptocócica (MC) ou meningoencefalite. Mundialmente, a MC é resultado de cerca de 15% de todas as mortes relacionadas ao HIV. Estima-se que os 223.100 casos de MC, em 2014, resultaram em 181.100 óbitos, sendo 75% (135.900) na África Subsaariana, seguida pelo Sudeste Asiático e América Latina. Após o surgimento da terapia antirretroviral (TARV) para o HIV, a incidência da criptococose diminuiu significativamente em países desenvolvidos (39%), entretanto, a incidência e a mortalidade pela infecção criptocócica ainda é extremamente alta em países com epidemia de HIV incontrolada e acesso limitado aos medicamentos e aos cuidados de saúde. TECNOLOGIA: Teste diagnóstico, point of care, de Cryptococcal Antigen Lateral Flow Assay (CRAG-LFA). PERGUNTAS DE PESQUISA: 1) O teste CRAG-LFA é sensível e específico na detecção da infecção por Cryptococcus em PVHIV com células CD4+ ≤ 200 cél/mm³ comparado ao teste de aglutinação do látex? 2) O teste CRAG-LFA é sensível e específico no diagnóstico da meningite criptocócica em PVHIV sintomáticos, independente da contagem de células CD4+, se comparado ao teste de aglutinação do látex e tinta da China, em amostra de líquor? EVIDÊNCIAS CLÍNICAS: Para a Pergunta 1 de pesquisa, a estimativa de sensibilidade de CRAG-LFA na detecção da infecção criptocócica foi de 100% e as estimativas de especificidade variaram de 99% a 100%, em relação ao teste de aglutinação por látex (CRAG-LA). Já a sensibilidade combinada dos dois estudos primários incluídos foi 100% (IC95%; 96 - 100) e a especificidade combinada de 99% (IC95%; 99 - 100). Para a Pergunta 2 de pesquisa, a estimativa de sensibilidade de CRAGLFA no diagnóstico de meningite criptocócica em PVHIV foi de 100% e as estimativas de especificidade variaram de 98% a 99%. A sensibilidade combinada dos três estudos primários meta-analisados foi de 100% (IC95%; 92 - 100) e a especificidade combinada de 99% (IC95%; 97 - 100), em relação ao teste de tinta da China. Como evidências analisadas para responder à Pergunta 2 de pesquisa, também foram incluídas duas revisões sistemáticas com meta-análise, a primeira reportou que os valores de sensibilidade e especificidade combinados de CRAG-LFA no líquor foram 98,9% (IC 95%, 97,9% a 99,5%) e 98,9% (IC 95%, 98,0% a 99,5%), respectivamente, já a segunda revisão descreveu que, no líquor, CRAG-LA (10 coortes diagnósticas, 1810 participantes) teve uma sensibilidade resumida de 97,1% (91,9 - 99) e uma especificidade de 99,1% (93,8 - 99,9) enquanto o teste CRAG-LFA (6 coortes diagnósticas, 3.099 participantes) teve uma sensibilidade resumida de 99,5% (97,2 - 99,9) e especificidade de 99,5% (94,2 - 99,9). Embora houvesse alguma evidência estatística fraca de que o CRAG-LFA pode ter melhor sensibilidade no líquor (p = 0,07) do que CRAG-LA, suas especificidades foram comparáveis (p = 0,54). EVIDÊNCIAS ECONÔMICAS: Foi conduzida avaliação econômica do tipo Microssimulação de Monte Carlo, com horizonte temporal de um ano. Considerou-se dois desfechos primários de efetividade: anos de vida e anos de vida ajustados por qualidade (QALY). A análise de custo-efetividade mostrou que os dois testes, CRAG-LA e CRAG-LFA, são custo-efetivos, dominando a alternativa tinta da China e o cenário de não realização da detecção. Para o diagnóstico de meningite criptocócica em PVHIV sintomáticas o uso do teste diagnóstico CRAG-LFA revelou-se custo-efetivo comparado ao teste CRAG-LA, e ao teste tinta da China. A análise de sensibilidade determinística e probabilística indica um favorecimento, na maioria das vezes, do teste CRAG-LFA. ANÁLISE DE IMPACTO ORÇAMENTÁRIO: A análise de impacto orçamentário (AIO) foi realizada para um horizonte temporal de cinco anos, assumindo-se um market share inicial de 20% para o LFA, com incrementos anuais no mesmo valor, chegando a 100% no quinto ano. Para a pergunta 1 de pesquisa a incorporação de LFA teria um custo adicional de aproximadamente 52,7 milhões de reais. Estimou-se também o impacto orçamentário de 100% de adoção da detecção em PVHIV CD4≤ 200 cél/mm³ assintomáticos. Nesse contexto, ocorreria uma economia da ordem de 55 milhões de reais em cinco anos, considerando detecção precoce e tratamento preemptivo, em relação ao custo de tratamento da meningite. Para a pergunta 2, para cada 1.000 PVHIV por ano, em cinco anos teríamos 809 indivíduos tratados a um custo adicional de R$ 10.697.431,27. RECOMENDAÇÕES INTERNACIONAIS: Foi realizada busca por recomendações de uso do teste CRAG-LFA por outras instituições e agências de ATS, a recomendação identificada é proveniente da Organização Mundial da Saúde (OMS). De acordo com a OMS, o diagnóstico e o tratamento precoce da meningite criptocócica são essenciais para reduzir a mortalidade por doença criptocócica em pacientes com infecção pelo vírus da imunodeficiência humana (HIV). A recomendação do órgão é para que os países deem prioridade a ensaios de antígeno criptocócico de diagnóstico rápido, de preferência ensaios de fluxo lateral como o CRAG-LFA, para uso no líquor, soro, plasma ou sangue total. Para adultos, adolescentes e crianças vivendo com HIV com suspeita de um primeiro episódio de meningite criptocócica, a punção lombar imediata com medição da pressão de abertura do líquor e aplicação de um teste diagnóstico rápido para o antígeno criptocócico é recomendado como o preferido abordagem diagnóstica. MONITORAMENTO DO HORIZONTE TECNOLÓGICO: Foram identificados dois outros testes registrados no segundo semestre de 2020 na Anvisa: CRAG Lateral Flow Assay e Biosynex® CryptoPS como testes rápidos CRAG-LFA. Na técnica de LA foram localizados 4 (quatro) registros sanitários na ANVISA. Já no FDA foram encontrados 3 (três) registros sanitários, sendo 1 (um) utilizando a técnica de sonda de DNA. Também foram consultadas as bases de dados de Propriedade Intelectual da WIPO e Espacenet Patent para buscar depósitos de testes de diagnósticos para meningite criptocócica. A consulta buscou os dados recentes de depósitos concedidos entre 2017 e 2020. Foram localizadas 05 patentes concedidas, sendo 04 (quatro) para metodologias em biologia molecular e um (1) para espectroscopia de Raman. CONSIDERAÇÕES FINAIS: Dadas as evidências apresentadas, o teste CRAG-LFA tem potencial custo-efetivo para a detecção da infecção criptocócica e prevenção da mortalidade relacionada à meningite criptocócica em PVHIV. RECOMENDAÇÃO PRELIMINAR: Diante do exposto, a Conitec, em sua 95ª reunião ordinária, realizada no dia 03 de março de 2021, deliberou que a matéria fosse disponibilizada em consulta pública com recomendação preliminar favorável à incorporação no SUS do teste point of care de Cryptococcal Antigen Lateral Flow Assay (CRAG-LFA) para detecção de infecção por Cryptococcus em pessoas vivendo com o vírus da imunodeficiência humana (PVHIV) com CD4+ ≥200 células/mm³ e diagnóstico de meningite criptocócica em PVHIV independente da contagem de células CD4+. Os membros do plenário concordaram, a partir das evidências apresentadas que o teste diagnóstico é custo-efetivo, tem baixo impacto orçamentário e, além disso, foi considerado um teste de fácil aplicação na prática clínica, superando os testes já disponíveis, utilizados como comparadores. A matéria foi disponibilizada em consulta pública. CONSULTA PÚBLICA: A Consulta Pública nº 18/2021 foi realizada entre os dias 18/03/2021 e 06/04/2021. Foram recebidas 82 contribuições, sendo 45 pelo formulário para contribuições técnico-científicas e 38 pelo formulário para contribuições sobre experiência ou opinião de pacientes, familiares, amigos ou cuidadores de pacientes, profissionais de saúde ou pessoas interessadas no tema. Foram consideradas apenas as contribuições encaminhadas no período estipulado e por meio do site da Conitec, em formulário próprio. As contribuições recebidas destacaram as vantagens clínicas do uso do teste CRAG-LFA, considerando economia de recursos, na identificação precoce da condição e tratamento preemptivo. Vantagens relacionadas ao manuseio do teste, sua aplicabilidade e facilidade na interpretação também foram citadas. Não FORAM ADICIONADAS REFERÊNCIAS QUE ALTERASSEM A ANÁLISE DA EVIDÊNCIA APRESENTADA NO RELATÓRIO. RECOMENDAÇÃO FINAL: Os membros do plenário presentes na 97ª reunião ordinária da Conitec, no dia 05 de maio de 2021, deliberaram, por unanimidade, recomendar a incorporação, no SUS, do teste diagnóstico, point of care, de Cryptococcal Antigen Lateral Flow Assay (CRAG-LFA) para detecção da infecção por Cryptococcus em pessoas vivendo com o vírus da imunodeficiência humana (PVHIV) com células CD4+ ≤ 200 cél/mm³ e diagnóstico de meningite criptocócica em PVHIV sintomáticos, independente da contagem de células CD4+, conforme estabelecido pelo Ministério da Saúde. Não foram adicionadas referências que alterassem a análise da evidência apresentada. Foi assinado o Registro de Deliberação nº 610/2021. DECISÃO: Incorporar o teste diagnóstico, point of care, de Cryptococcal Antigen Lateral Flow Assay (CRAG-LFA) para detecção de infecção por Cryptococcus em pessoas vivendo com o vírus da imunodeficiência humana (PVHIV) com CD4+ ³200 células/mm³ e diagnóstico de meningite criptocócica em PVHIV independente da contagem de células CD4+, no âmbito do Sistema Único de Saúde ­ SUS, conforme Portaria nº 28, publicada no Diário Oficial da União nº 108, Seção 1, página 177, em 11 de junho de 2021.(AU)

Comparison of two commercial tests (Immy vs. Dynamiker) for cryptococcal capsular antigen

Abstract INTRODUCTION Lateral flow assay is an advanced method useful in the early diagnosis of cryptococcal meningitis. We aimed to compare two commercial tests for cryptococcal capsular antigen in the sera of asymptomatic patients with human immunodeficiency virus in Barranquilla, Colombia. METHODS Thawed (n=162) previously collected serums (2016-2019) were processed using IMMY and Dynamiker cryptococcal antigen lateral flow assay. RESULTS Compared to IMMY's results, Dynamiker's sensitivity, specificity, positive predictive value, negative predictive value, and kappa index were 100%, 89.9%, 48.3%, 100.0%, and 0.61, respectively. CONCLUSIONS The Dynamiker test had excellent sensitivity, acceptable specificity, and a low detection threshold for cryptococcal antigen in the tested samples.

Visible DNA microarray and loop-mediated isothermal amplification (LAMP) for the identification of Cryptococcus species recovered from culture medium and cerebrospinal fluid of patients with meningitis.

Cryptococcal meningitis affects normal hosts and immunocompromised patients exhibiting high mortality rates. The objective of this study was to design two molecular assays, visible microarray platforms and loop-mediated isothermal amplification (LAMP), to identify Cryptococcus spp. and the species neoformans and gattii from the cerebral spinal fluid (CSF). To identify Cryptococcus and the two species, we designed two microarrays DNA platforms based on the internal transcribed spacer (ITS) region and CAP59 gene and LAMP assays specific for Cryptococcus species. The assays were tested using CSF from patients with cryptococcal meningitis. CSF from patients with cryptococcal meningitis was cultured in Sabouraud culture medium, and the Cryptococcus spp. grown in the culture medium were also tested for LAMP and microarray platforms. The results were compared to DNA sequencing of the same genetic regions. A total of 133 CSF samples were studied. Eleven CSFs were positive for Cryptococcus (9 C. neoformans and 2 C. gattii), 15 were positive for bacteria, and 107 were negative. The CAP59 platform correctly identified 73% of the CSF samples, while the ITS platform identified 45.5%. CAP59 platform correctly identified 100% of the Cryptococcus isolates, and ITS platform identified 70%. The two sets of LAMP primers correctly identified 100% of the Cryptococcus isolates. However, for CSF samples, the amplification occurred only in 55.5% of C. neoformans. The methodologies were reliable in the identification of Cryptococcus species, mainly for isolates from culture medium, and they might be applied as adjunctive tests to identify Cryptococcus species.

Production of a latex agglutination reagent for the rapid diagnosis of cryptococcal meningitis / Producción de un reactivo de látex para el diagnóstico rápido de criptococosismeníngea

Abstract Cryptococcosis is a fungal disease affecting more than one million people per yearworldwide. Its main etiological agents are Cryptococcus neoformans species complex and Cryp-tococcus gattii species complex. Cryptococcal meningitis (CM) is considered an AIDS-definingcondition. Rapid diagnosis by cryptococcal antigen assays, either the latex agglutination test(LA) or the lateral flow assay, is key to decreasing mortality due to cryptococcal disease. Theaim of the study was to develop a latex agglutination reagent (LA-ANLIS) for the rapid and reliable diagnosis of cryptococcosis in Argentina. This reagent will be produced in order to supplythe NMLN (National Mycology Laboratory Network). The evaluation of LA-ANLIS performanceand its comparison with the Cryptococcus Antigen Latex Agglutination Test System (LA-IMMY)(Immuno-Mycologics, Inc., USA) were conducted in 94 samples of cerebrospinal fluid. LA-ANLISand LA-IMMY compared exhibited 100% positive agreement and 97% negative agreement. LA-ANLIS showed 94% sensitivity and 97% specificity with the positive and negative predictivevalues of 94% and 97%, respectively. The LA-ANLIS is a reliable, reproducible and cost-effectivereagent, especially useful in countries where the commercial kit is not generally available andmust be obtained at a high cost. National production of reagents is the best choice for a reliableaccess to the rapid diagnosis of CM in Argentina.

Resumen La criptococosis es una enfermedad fúngica que afecta a más de un millón de personas por año en todo el mundo. Los principales agentes etiológicos pertenecen a los complejos de especies Cryptococcus neoformans y Cryptococcus gattii. La criptococosis meníngea (CM) se considera una enfermedad marcadora de sida. El diagnóstico rápido de esta enfermedad a través de la detección del antígeno de Cryptococcus, ya sea por aglutinación en partículas de látex o por inmunocromatografía, es clave para disminuir la mortalidad. El objetivo del presente estudio fue desarrollar un reactivo de aglutinación en partículas de látex para el diagnóstico rápido y certero de la CM en Argentina. Este reactivo (denominado en adelante LA-ANLIS) será producido para abastecer a la Red Nacional de Laboratorios de Micología. Se evaluó el desempeno del reactivo LA-ANLIS, y se realizó una comparación con el reactivo comercial Immuno-Mycologics, Inc. (en adelante, LA-IMMY) utilizando 94 muestras de líquido cefalorraquídeo. Hubo un 100% de acuerdo positivo y un 97% de acuerdo negativo entre los resultados obtenidos con los reactivos LA-ANLIS y LA-IMMY. El reactivo LA-ANLIS mostró una sensibilidad del 94% y una especificidad del 97%; los valores predictivos positivo y negativo fueron del 94 y del 97%, respectivamente. Se concluye que el LA-ANLIS es un reactivo confiable y rentable, que arroja resultados reproducibles, por lo que es especialmente útil en países donde los reactivos comerciales generalmente no están disponibles o sus costos son elevados. La producción nacional de reactivos es la mejor opción para asegurar el acceso de todos los hospitales al diagnóstico rápido de la CM en Argentina.

Production of a latex agglutination reagent for the rapid diagnosis of cryptococcal meningitis.

Cryptococcosis is a fungal disease affecting more than one million people per year worldwide. Its main etiological agents are Cryptococcus neoformans species complex and Cryptococcus gattii species complex. Cryptococcal meningitis (CM) is considered an AIDS-defining condition. Rapid diagnosis by cryptococcal antigen assays, either the latex agglutination test (LA) or the lateral flow assay, is key to decreasing mortality due to cryptococcal disease. The aim of the study was to develop a latex agglutination reagent (LA-ANLIS) for the rapid and reliable diagnosis of cryptococcosis in Argentina. This reagent will be produced in order to supply the NMLN (National Mycology Laboratory Network). The evaluation of LA-ANLIS performance and its comparison with the Cryptococcus Antigen Latex Agglutination Test System (LA-IMMY) (Immuno-Mycologics, Inc., USA) were conducted in 94 samples of cerebrospinal fluid. LA-ANLIS and LA-IMMY compared exhibited 100% positive agreement and 97% negative agreement. LA-ANLIS showed 94% sensitivity and 97% specificity with the positive and negative predictive values of 94% and 97%, respectively. The LA-ANLIS is a reliable, reproducible and cost-effective reagent, especially useful in countries where the commercial kit is not generally available and must be obtained at a high cost. National production of reagents is the best choice for a reliable access to the rapid diagnosis of CM in Argentina.

Cryptococcal meningitis presenting as acute flaccid paralysis: A case report.

Cryptococcus is a cosmopolitan fungus with tropism for the nervous system and a higher prevalence of infection in immunosuppressed patients. Neurological compromise caused by this microorganism mainly debuts as a meningeal syndrome (headache, fever, neck stiffness) with predominant encephalic involvement. In this report we present the rare case of a non-HIV patient with flaccid paralysis and peripheral nerve involvement due to crytpococcal meningitis. This is a 53-years-old woman, with a past-medical history of diabetes, who presented with dysarthria, unilateral peripheral facial paralysis, asymmetric ascending quadriparesis, generalized hyporeflexia and urinary retention. Neuroimaging was initially reported as negative for vascular or demyelinating diseases. Electrophysiological studies were performed, and acute flaccid paralysis of undetermined etiology was defined as a temporal clinical diagnosis. Cerebrospinal fluid molecular analysis confirmed the presence of Cryptococcus neoformans var. gatti; posteriorly, antifungal treatment with amphotericin B and fluconazole was started. Polyneuroradiculopathy symptoms significantly improved over the in-hospital stay. In conclusion, spinal cord and peripheral nerve involvement by Cryptococcus is an infrequent cause of acute flaccid paralysis that should be considered in the differential diagnosis even in HIV-negative patients.

Visible DNA microarray and loop-mediated isothermal amplification (LAMP) for the identification of Cryptococcus species recovered from culture medium and cerebrospinal fluid of patients with meningitis

Cryptococcal meningitis affects normal hosts and immunocompromised patients exhibiting high mortality rates. The objective of this study was to design two molecular assays, visible microarray platforms and loop-mediated isothermal amplification (LAMP), to identify Cryptococcus spp. and the species neoformans and gattii from the cerebral spinal fluid (CSF). To identify Cryptococcus and the two species, we designed two microarrays DNA platforms based on the internal transcribed spacer (ITS) region and CAP59 gene and LAMP assays specific for Cryptococcus species. The assays were tested using CSF from patients with cryptococcal meningitis. CSF from patients with cryptococcal meningitis was cultured in Sabouraud culture medium, and the Cryptococcus spp. grown in the culture medium were also tested for LAMP and microarray platforms. The results were compared to DNA sequencing of the same genetic regions. A total of 133 CSF samples were studied. Eleven CSFs were positive for Cryptococcus (9 C. neoformans and 2 C. gattii), 15 were positive for bacteria, and 107 were negative. The CAP59 platform correctly identified 73% of the CSF samples, while the ITS platform identified 45.5%. CAP59 platform correctly identified 100% of the Cryptococcus isolates, and ITS platform identified 70%. The two sets of LAMP primers correctly identified 100% of the Cryptococcus isolates. However, for CSF samples, the amplification occurred only in 55.5% of C. neoformans. The methodologies were reliable in the identification of Cryptococcus species, mainly for isolates from culture medium, and they might be applied as adjunctive tests to identify Cryptococcus species.

Cryptococcal Meningitis Presenting as New-Onset Seizures in an Immunocompetent Patient.

This report describes a 30-year-old immunocompetent male with new-onset seizures, later found on imaging to have 2 enhancing lesions in the brain. The patient underwent a left parietal craniectomy with resection of one of the masses, which demonstrated focal areas of necrosis and many small cystic structures positive for periodic acid-Schiff and Gomori's methenamine silver special stain. Numerous laboratory examinations, including HIV test, rapid plasma reagin, toxoplasma immunoglobulin G and immunoglobulin M, Lyme, cytomegalovirus, tuberculosis, cysticercosis, and Echinococcus serology, were all negative. Despite negative cerebrospinal fluid (CSF) culture and several negative CSF antigen tests, continued investigation, and follow-up, CSF antigen testing ultimately revealed Cryptococcus as the causative agent. In light of the mysterious and unusual presentation, the authors discuss potential infectious differential diagnoses in patients with atypical clinical presentation, laboratory tests, and surgical pathology.

Prospective cohort of AIDS patients screened for cryptococcal antigenaemia, pre-emptively treated and followed in Brazil.

BACKGROUND: Cryptococcal meningitis has a high morbidity and mortality among AIDS population. Cryptococcal antigen (CrAg) detection is considered an independent predictor for meningitis and death. Since 2011, the World Health Organization recommends CrAg screening for people living with HIV/AIDS (PLHAs) with CD4 counts <100-200 cells/µl. Its implementation is still limited in low-middle-income countries. We aimed to estimate the prevalence and predictors of CrAg positivity in PLHAs. We also evaluated outcomes among those who were CrAg-positive. METHODS: Prospective cohort conducted at an infectious diseases hospital, in Brazil. Adults with CD4 <200 cells/µl, without previous cryptococcal disease and regardless of symptoms, were enrolled from 2015 to 2018. CrAg tests were performed by LFA. Lumbar puncture was done in CrAg+ individuals and pre-emptive therapy was offered for those without meningitis. RESULTS: Of 214 individuals recruited, 88% were antiretroviral experienced, of which only 11.6% with viral suppression. Overall, CrAg prevalence was 7.9% (95% CI, 4.7-12.4). In CD4 ≤100 cells/µl group it was 7.5% (95% CI, 4.1-12.6) and 9.1% (95% CI, 3.4-19.0) in the group with CD4 101 to 199 cells/µl (p = 0.17). Prevalence in asymptomatic subjects was 5.3% (95% CI, 1.4-13.1). One among 17 CrAg+ participants had documented meningoencephalitis and no subclinical meningitis was detected. Adherence to pre-emptive treatment was 68.7% (11/16). There were no statistically significant differences in sociodemographic, clinical or laboratory characteristics to predict CrAg positivity. No case of cryptococcal disease was diagnosed among CrAg + subjects, followed by a median of 12 months. CONCLUSIONS: CrAg screening for severely immunosuppressed PLHAs in Brazil yielded a prevalence of 7.9%. No difference was found in the prevalence of CrAg stratified by CD4 values (CD4 <100 versus CD4 101-199 cells/µl). No clinical nor laboratory factors predicted CrAg positivity, corroborating the need for the implementation of universal CrAg screening for PLHAs with CD4 <200 cells/µl in similar settings.

Cryptococcus deuterogattii VGIIa Infection Associated with Travel to the Pacific Northwest Outbreak Region in an Anti-Granulocyte-Macrophage Colony-Stimulating Factor Autoantibody-Positive Patient in the United States.

The region encompassing the Pacific Northwest (PNW), Vancouver Island, Oregon, and Washington has been the location of an ongoing Cryptococcus gattii outbreak since the 1990s, and there is evidence that the outbreak is expanding along the West Coast into California. Here we report a clinical case of a 69-year-old, HIV-negative man from North Carolina who was diagnosed with a fungal brain mass by magnetic resonance imaging (MRI) and pathology. He had traveled to Seattle and Vancouver 3 years earlier and to Costa Rica 4 months prior to presentation. Phenotypic evidence showed that the fungal mass isolated from the patient's brain represented C. gattii In agreement with the phenotypic results, multilocus sequence typing (MLST) provided genotypic evidence that assigned the infecting organism within the C. gattii species complex and to the C. deuterogattii VGIIa clade. Whole-genome sequencing revealed >99.99% identity with the C. deuterogattii reference strain R265, indicating that the infecting strain is derived from the highly clonal outbreak strains in the PNW. We conclude that the patient acquired the C. gattii infection during his travel to the region 3 years prior and that the infection was dormant for an extended period of time before causing disease. The patient tested positive for anti-granulocyte-macrophage colony-stimulating factor (GM-CSF) autoantibodies, supporting earlier reports that implicate these autoantibodies as a risk factor associated with C. gattii infection.IMPORTANCE Mortality rates associated with C. gattii infections are estimated to be between 13% and 33%, depending on an individual's predisposition, and C. gattii has caused at least 39 deaths in the PNW region. There have been four other international travel cases reported in patients from Europe and Asia with travel history to the PNW, but this report describes the first North American traveler who acquired C. deuterogattii infection presenting within the United States and the first case of a C. deuterogattii outbreak infection associated with anti-GM-CSF autoantibodies. Early and accurate diagnoses are important for disease prevention and treatment and for control of infectious diseases. Continual reporting of C. deuterogattii infections is necessary to raise awareness of the ongoing outbreak in the PNW and to alert travelers and physicians to the areas of endemicity with potential risks.

Cryptococcal meningitis: an unusual presentation of primary HIV infection.

Acute HIV infection presenting as severe opportunistic disease is a very uncommon event associated with more rapid HIV progression and higher mortality. We describe the case of a patient with cryptococcal meningitis in the setting of primary HIV infection.