Genotype and mating type distribution within clinical Cryptococcus neoformans and Cryptococcus gattii isolates from patients with cryptococcal meningitis in Uberaba, Minas Gerais, Brazil

We molecularly characterized 81 cryptococcal isolates recovered from cerebrospinal fluid samples of 77 patients diagnosed between 1998 and 2007 as having cryptococcal meningitis in Uberaba Minas Gerais, Brazil. Fifty-seven (74%) were male with a mean age 35.6 years. Seventy-two (88.9%) of the isolates were from 68 AIDS patients and cryptococcosis was the first AIDS-defining condition in 38 (55.9%) patients. Cryptococcosis and AIDS were simultaneously diagnosed in 25 (65.8%) of these 38 patients. Genotypes were characterized through the use of URA5 restriction fragment length polymorphisms analysis, the genetic variability was determined using PCR-fingerprinting with the minisatellite-specific primer M13, and the mating type and serotypes were established by PCR. Seventy-six of the 81 isolates were Cryptococcus neoformans (93.8%), while the remaining five were C. gattii (6.1%), but all were mating type a. C. neoformans isolates were genotype VNI (serotype A), while C. gattii isolates were VGII. Four of the latter isolates were identical, but only two were from AIDS patients. Six of the nine isolates from non-AIDS patients were VNI. PCR fingerprints of the isolates from two of the three AIDS patients with clinical relapse were 100% identical. The predominance of VNI and mating type a is in accordance with data from other parts of the world. The occurrence of VGII in Minas Gerais indicates a geographical expansion within Brazil.

Cryptococcal meningitis in HIV negative pregnant women: case report and review of literature

INTRODUCTION: Cryptococcosis has become an important entity due to the epidemic of AIDS and therefore it is a significant opportunistic infection. However, there are case reports of cryptococcal meningitis in immune competent pregnant women. Since pregnancy is considered a period of relative immunosuppression, which likely prevents fetal rejection, this could explain the occurrence of opportunistic infections. OBJECTIVE: To report a case of cryptococcosis, and review all cases involving pregnancy and neurocryptococcal infection in immune competent pregnant patients. METHODS: Case report and systematic review of the literature using the MEDLINE and SciELO databases. DISCUSSION: A total of 27 patients were analyzed from 19 studies. The mean age at diagnosis was 26.4 years. There were six patients in their first trimester of pregnancy, 10 in the second, eight in the third and three post-partum. The most prevalent symptoms were headache (85.2%), altered vision (44.4%), altered mental status (44.4%), nausea (40.7%) and fever (33.3%). There were nine deaths (33.3%). Most of the patients received intravenous amphotericin B as treatment (77.8%). The majority (66.6%) of the patients accomplished a term delivery with healthy infants. CONCLUSION: Cryptococcal meningitis should be considered during pregnancy in cases of unexplained headache, altered vision, altered mental status, nausea and fever. Patients with a confirmed diagnosis should be admitted and treated with amphotericin B.

LATERAL FLOW ASSAY FOR CRYPTOCOCCAL ANTIGEN: AN IMPORTANT ADVANCE TO IMPROVE THE CONTINUUM OF HIV CARE AND REDUCE CRYPTOCOCCAL MENINGITIS-RELATED MORTALITY

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.

Clinical and microbiological features of cryptococcal meningitis

Introduction In this study, the clinical features, underlying diseases and clinical outcomes of patients with cryptococcosis were investigated. In addition, a molecular analysis of the Cryptococcus neoformans species complex isolated from these patients was performed. Methods A prospective study of 62 cases of patients with cryptococcal infection was conducted at the Hospital de Doenças Tropicais de Goiás Dr. Anuar Auad from 2009-2010. Cryptococcal meningitis cases were diagnosed by direct examination and cerebrospinal fluid (CSF) sample culture. The profiling of these patients was assessed. The CSF samples were submitted to India ink preparation and cultured on Sabouraud dextrose agar, and C. neoformans was identified by the production of urease, a positive phenoloxidase test and assimilation of carbohydrates. C. neoformans and C. gattii isolates were distinguished by growth on L-canavanine-glycine-bromothymol blue medium, and molecular analysis was conducted via PCR fingerprinting reactions using M13 and (GACA)4 primers. Results From the 62 patients with cryptococcosis, 71 isolates of CSF were obtained; 67 (94.4%) isolates were identified as C. neoformans var. grubii/VNI, and 4 (5.6%) were identified as C. gattii/VGII. Of these patients, 53 had an HIV diagnosis. The incidence of cryptococcosis was higher among patients 20-40 years of age, with 74.2% of the cases reported in males. Cryptococcus-related mortality was noted in 48.4% of the patients, and the symptoms were altered sensorium, headache, fever and stiff neck. Conclusions The high morbidity and mortality observed among patients with cryptococcosis demonstrate the importance of obtaining information regarding the epidemiological profile and clinical course of the disease in the State of Goiás, Brazil.

Ventriculoperitoneal shunts for treating increased intracranial pressure in cryptococcal meningitis with or without ventriculomegaly

Introduction Cryptococcosis is an opportunistic mycosis, especially in patients that are human immunodeficiency virus (HIV)-positive, and frequently involves the central nervous system. Methods We assessed the potential of ventriculoperitoneal shunting (VPS) in preventing mortality due to uncontrollable intracranial hypertension (ICH) in 15 patients with acquired immunodeficiency syndrome (AIDS)-related cryptococcal meningitis. Results After 2 weeks of antifungal therapy consisting of amphotericin B deoxycholate with or without fluconazole, patients with persistent ICH underwent VPS, despite having persistent Cryptococcus neoformans infection. In 12 patients, the uncontrollable ICH was resolved by VPS. Conclusions Patients with cryptococcal meningoencephalitis who have ICH must be considered for VPS even with positive cerebrospinal fluid cultures.

Treatment of experimental cryptococcal meningitis with fluconazole: impact of dose and addition of flucytosine on mycologic and pathophysiologic outcome

Fluconazole is effective in the therapy of cryptococcal meningitis in patients with AIDS. The optimal dosage of fluconazole and the impact of combination with flucytosine are not known. In this study, rabbits with experimental cryptococcal meningitis were given fluconazole at low, intermediate, or high dose or in combination with a low or intermediate dose of flucytosine. Serial cerebrospinal fluid (CSF) examinations showed that all three doses of fluconazole and low-dose fluconazole in combination with intermediate-dose flucytosine were effective in reducing CSF cryptococcal titer, lactate, white blood cell count, and cryptococcal antigen (CRAG) titers. The intermediate and high doses of fluconazole reduced CSF fungal (P < .05) and CRAG (P < .001) titers earlier than low-dose fluconazole alone or in combination with flucytosine. Only the highest dose of fluconazole reduced brain edema after 7 days. In this model of cryptococcal meningitis, there was evidence of a dose response with fluconazole but no in vivo synergism with flucytosine.

Oral SCH 39304 as primary, salvage, and maintenance therapy for cryptococcal meningitis in AIDS

To determine the efficacy and toxicity of SCH 39304 in the treatment and suppression of cryptococcal meningitis, we conducted a prospective, noncomparative study in three groups of patients: patients with acute cryptococcal meningitis, patients with acute cryptococcal meningitis in whom other therapies have failed (salvage), and patients who required maintenance therapy. As primary therapy, the patients received up to 14 days or 1 g of amphotericin B followed by SCH 39304 200 mg once daily for 12 weeks. As maintenance therapy, the patients received SCH 39304 600 mg once weekly for 12 months. Of five salvage patients, none completed the study. Two patients died, two patients clinically deteriorated, and one patient was noncompliant. Two of three patients with acute cryptococcal meningitis completed the 12-week primary therapy, and one patient was discontinued from therapy because of a skin rash (95% confidence interval, 14-100%). All four patients who were receiving weekly maintenance therapy followed up to 27 weeks were clinically stable with no change in their serum cryptococcal antigen titer from baseline when the study was prematurely terminated. Elevation of liver function test results developed in three patients and skin rash developed in one patient. The unique pharmacologic and pharmacokinetic properties of SCH 39304 (low incidence of toxicity, long serum half-life, and good penetration into the cerebrospinal fluid) lend promise to pursue other triazole antifungals at higher doses as primary therapy and less frequent dosing for maintenance therapy.

Cryptococcus neoformans meningitis with negative cryptococcal antigen: Evaluation of a new immunochromatographic detection assay.

Detection of cryptococcal antigen in serum or cerebrospinal fluid allows cryptococcal meningitis diagnosis within few hours with >90% sensitivity. In an HIV-positive patient with Cryptococcus neoformans meningitis, initial antigen detection by immunoagglutination was negative. We thus evaluated a new immunochromatographic detection assay that exhibited a higher sensitivity.

Cryptococcus gattii molecular type VGII as agent of meningitis in a healthy child in Rio de Janeiro, Brazil: report of an autochthonous case

Cryptococcus gattii causes meningoencephalitis in immunocompetent hosts, occurring endemically in some tropical and subtropical regions. Recently, this fungus was involved in an outbreak in Vancouver Island and British Columbia (Canada). In this temperate region, the VGII type is predominant. The paper describes an autochthonous case of meningoencephalitis by C. gattii VGII in a previously health child in Rio de Janeiro, considered nonendemic region of Brazil. The fungus was identified by biochemical tests and the molecular type was determined by URA5-RFLP. The present report highlights the need for clinical vigilance for primary cryptococcal meningitis in nonendemic areas.

Epidemiology of infectious meningitis in the State of Amazonas, Brazil

INTRODUCTION: In the State of Amazonas, particularly in the capital Manaus, meningitis has affected populations of different cultures and social strata over the years. Bacterial meningitis is caused by several different species and represents a major issue of public health importance. The present study reports the meningitis case numbers with different etiologies in Amazonas from January 1976 to December 2012. METHODS: Since the 1970s, the (currently named) Tropical Medicine Foundation of Doutor Heitor Vieira Dourado [Fundação de Medicina Tropical Doutor Heitor Vieira Dourado (FMT-HVD)] has remained a reference center in Amazonas for the treatment of meningitis through the diagnosis and notification of cases and the confirmation of such cases using specific laboratory tests. RESULTS: The foundation has achieved coverage of over 90% of the state medical records for many years. Between 1990 and 2012, meningitis cases caused by Haemophilus influenzae decreased with the introduction of the H. influenzae vaccine. Meningococcal disease previously had a higher frequency of serogroup B disease, but starting in 2008, the detection of serogroup C increased gradually and has outpaced the detection of serogroup B. Recently, surveillance has improved the etiological definition of viral meningitis at FMT-HVD, with enteroviruses, Epstein-Barr virus (EBV) and varicella zoster virus (VZV) prevailing in this group of pathogens. With the advent of acquired immunodeficiency syndrome (AIDS), cryptococcal meningitis has become an important disease in Amazonas. Additionally, infectious meningitis is an important burden in the State of Amazonas. CONCLUSIONS: Changes in the epidemiological profile for the different etiology-defined cases are the result of continuous epidemiological surveillance and laboratory capacity improvements and control measures, such as Haemophilus influenzae vaccination.

Cryptococcal Antigenemia in Immunocompromised Human Immunodeficiency Virus Patients in Rural Tanzania: A Preventable Cause of Early Mortality

Background.  Cryptococcal meningitis is a leading cause of death in people living with human immunodeficiency virus (HIV)/acquired immune deficiency syndrome. The World Health Organizations recommends pre-antiretroviral treatment (ART) cryptococcal antigen (CRAG) screening in persons with CD4 below 100 cells/µL. We assessed the prevalence and outcome of cryptococcal antigenemia in rural southern Tanzania. Methods.  We conducted a retrospective study including all ART-naive adults with CD4 <150 cells/µL prospectively enrolled in the Kilombero and Ulanga Antiretroviral Cohort between 2008 and 2012. Cryptococcal antigen was assessed in cryopreserved pre-ART plasma. Cox regression estimated the composite outcome of death or loss to follow-up (LFU) by CRAG status and fluconazole use. Results.  Of 750 ART-naive adults, 28 (3.7%) were CRAG-positive, corresponding to a prevalence of 4.4% (23 of 520) in CD4 <100 and 2.2% (5 of 230) in CD4 100-150 cells/µL. Within 1 year, 75% (21 of 28) of CRAG-positive and 42% (302 of 722) of CRAG-negative patients were dead or LFU (P<.001), with no differences across CD4 strata. Cryptococcal antigen positivity was an independent predictor of death or LFU after adjusting for relevant confounders (hazard ratio [HR], 2.50; 95% confidence interval [CI], 1.29-4.83; P = .006). Cryptococcal meningitis occurred in 39% (11 of 28) of CRAG-positive patients, with similar retention-in-care regardless of meningitis diagnosis (P = .8). Cryptococcal antigen titer >1:160 was associated with meningitis development (odds ratio, 4.83; 95% CI, 1.24-8.41; P = .008). Fluconazole receipt decreased death or LFU in CRAG-positive patients (HR, 0.18; 95% CI, .04-.78; P = .022). Conclusions.  Cryptococcal antigenemia predicted mortality or LFU among ART-naive HIV-infected persons with CD4 <150 cells/µL, and fluconazole increased survival or retention-in-care, suggesting that targeted pre-ART CRAG screening may decrease early mortality or LFU. A CRAG screening threshold of CD4 <100 cells/µL missed 18% of CRAG-positive patients, suggesting guidelines should consider a higher threshold.

Cerebrospinal fluid levels of chemokines in HIV infected patients with and without opportunistic infection of the central nervous system

Chemokines are chemoattractant cytokines involved in the immune response of a wide variety of diseases. There are few studies assessing their role in opportunistic infections in HIV-infected patients. In this study, we measured CC and CXC chemokines in cerebrospinal fluid (CSF) samples obtained from 40 HIV-infected patients with or without opportunistic infections of the central nervous system (CNS). CSF samples were also analyzed for quantification of total protein, cell count and HIV-1 RNA. HIV+ patients with cryptococcal meningitis had higher levels of CCL2, CCL3, CCL5, CXCL9 and CXCL10 when compared to patients without opportunistic neurological infections. Furthermore, HIV+ patients with associated cryptococcal meningitis had higher levels of CCL3, CXCL9 and CXCL10 when compared to HIV+ patients with associated toxoplasmic encephalitis. CCL3 and CXCL9 levels were positively correlated with CSF HIV-1 RNA levels, CSF protein concentration, and CSF cell count. CXCL10 level was correlated with the CSF viral load and the CSF cell count and CCL5 level was correlated with the CSF cell count. In conclusion, the profile of chemokines in CSF of HIV patients may differ according to the modality of the presented opportunistic infection and according to other biological markers, such as viral load in CSF. These differences are probably related to different patterns of neuroinflammatory responses displayed by patients with different opportunistic neurological infections. (C) 2009 Elsevier B.V. All rights reserved.

HIV infection and AIDS in a small municipality in Southeast Brazil

OBJECTIVE: Studies on the aspects of HIV infection in small Brazilian municipalities are invaluable to appropriately design control strategies, better allocate resources, and improve health care services. The objective of the study was to assess the clinical and epidemiological aspects of HIV infection in a small municipality. METHODS: A descriptive study was carried out in Miracema, a small municipality in the northwestern area of the state of Rio de Janeiro, Brazil, between July 1999 and December 2003. All HIV-infected adult patients followed up at the local HIV/AIDS Program were included. Clinical and epidemiologic characteristics were prospectively assessed through standardized questionnaires. RESULTS: A total of 65 adult patients who attended the local HIV/AIDS Program were analyzed. Most (34) were women (male to female ratio: 0.9). An absolute predominance of patients who were born in Miracema or neighboring municipalities (94%), lived in Miracema (90.7%), were single (70.8%), attributed the acquisition of HIV infection to unprotected heterosexual intercourse (72.3%) and had a past history of snorting cocaine (27.7) was found Central nervous system disorders (including five cases of cryptococcal meningitis) and acute pulmonary pneumocystosis-like respiratory failure were major causes of morbidity. Most patients (56.9%) were at presented in advanced stages of HIV infection. CONCLUSIONS: The predominance of patients on advanced stages of HIV infection suggest the existence of a large pool of undiagnosed cases in the community. A major feature of the cohort was an inverted male to female ratio. Further investigations over a broader geographic area are urgently needed for better understanding the clinical and epidemiological characteristics of HIV infection in small Brazilian municipalities and rural areas.

FEVER OF UNDETERMINED ORIGIN IN PATIENTS WITH THE ACQUIRED IMMUNODEFICIENCY SYNDROME IN BRAZIL: REPORT ON 55 CASES

The medical records of patients with AIDS admitted to a general hospital in Brazil from 1989 to 1997 were reviewed retrospectively with the aim at defining the frequency and etiology of fever of undetermined origin (FUO) in HIV-infected patients of a tropical country and to evaluate the usefulness of the main diagnostic procedures. 188 (58.4%) out of 322 patients reported fever at admission to hospital and 55 (17.1%) had FUO. Those with FUO had a mean CD4+ cell count of 98/ml. A cause of fever was identified for 45 patients (81.8%). Tuberculosis (32.7%), Pneumocystis carinii pneumonia (10.9%), and Mycobacterium avium complex (9.1%) were the most frequent diagnoses. Other infectious diseases are also of note, such as cryptococcal meningitis (5.5%), sinusitis (3.6%), Salmonella-S. mansoni association (3.6%), disseminated histoplasmosis (3.6%), neurosyphilis (1.8%), and isosporiasis (1.8%). Four patients had non-Hodgkin's lymphoma (7.3%). We conclude that an initial aggressive diagnostic approach should be always considered because biopsies (lymph node, liver and bone marrow) produced the highest yield in the diagnosis of FUO and the majority of the diagnosed diseases are treatable. The association of diseases is common and have contributed to delay the final diagnosis of FUO in most cases. In our study area the routine request of hemocultures for Salmonella infection and the investigation of cryptococcal antigen in the serum should be considered.

Neurocryptococcosis: diagnosis by PCR method

Cryptococcus neoformans detection was optimized using PCR technique with the objective of application in the clinical laboratory diagnosis. The amplification area was ITS and 5,6S which encodes the ribosomal RNA (rRNA). A total of 72 cerebrospinal fluid (CSF) samples were used, obtained from cases with and without AIDS. The patients had cryptococcal meningitis (n = 56) and meningitis caused by other agents (n = 16). The results demonstrated that PCR test had the highest sensitivity rates, superior to culture (85.7%) and to India ink test (76.8%). PCR was found to be sensitive in detecting 1 cell/mL and highly specific since it did not amplify other fungal DNA. The comparative analysis of the methods showed that PCR is more sensitive and specific and is applicable as an important laboratorial resource for neurocryptococcosis diagnosis.