Genetic influence of Toll-like receptors on non-HIV cryptococcal meningitis: An observational cohort study.

BACKGROUND: Cryptococcal meningitis (CM) is a significant source of mortality, the pathogenesis of which has not been fully understood, especially in non-HIV infected populations. We aimed to explore the potential genetic influence of Toll-like receptor (TLR) on non-HIV CM. METHODS: This observational cohort study was done in two stages: a discovery stage and a validation stage. A case-control genetic association study was conducted between 159 non-HIV CM patients and 468 healthy controls. TLR SNPs significantly related to susceptibility went further validation in a second cohort of 583 subjects from a certain district. Associations among TLR SNPs, cerebrospinal fluid (CSF) cytokine concentrations, and clinical severity were explored in a third cohort of 99 previously untreated non-HIV CM patients. Logistic regression model was used to determine the independent predictors for disease severity. FINDINGS: In the discovery stage, eight TLR SNPs exhibited significant genetic susceptibility to non-HIV CM, one of which was validated in a population validation of HIV-infected cases while none survived in non-HIV cases. CSF cytokine detections showed that 18 cytokines were significantly over-expressed in severely ill patients. Two of the 8 SNPs (rs5743604 and rs3804099) were also significantly associated with disease severity. Specifically, the rs3804099 C/T genotype was further found to be correlated to 12 of the 18 up-regulated cytokines in severe patients. In addition, high levels of interleukin (IL)-10 in CSF (OR 2·97, 95% CI 1·49-5·90; p = 0·002) was suggested as an independent predictor for severity after adjusted for possible confounders. INTERPRETATION: TLR participates in both the occurrence and the pathogenesis of non-HIV CM. The in situ immune responses of CM were under genetic influence of TLR and contributed to disease severity. FUND: National Natural Science Foundation of China and National Key Basic Research Program of China (973 Program).

Cryptococcosis in patients with hematological diseases: a 14-year retrospective clinical analysis in a Chinese tertiary hospital.

BACKGROUND: Cryptococcal infection has become a public health challenge globally. However, information about cryptococcal infection in patients with hematological diseases remains relatively rare. METHODS: HIV-uninfected cryptococcosis cases with hematological diseases admitted to Huashan Hospital from January 2001 to December 2014 were reviewed. RESULTS: In total, 33 cryptococcosis patients were enrolled, including 12 malignant and 21 non-malignant hematological cases. Twenty-six patients had central nervous system (CNS) involvement, which was observed more often in patients with non-malignancies than with malignancies (20/21 vs. 6/12, P = 0.001) Most patients (25/26) with CNS infection were confirmed by cerebrospinal fluid (CSF) culture or smear, and 100% (20/20) of them tested positive for the CSF cryptococcal antigen test. Eighteen out of 26 cryptococcal meningitis patients were treated with amphotericin B (AmB)-based therapy, 16 of them with AmB deoxycholate (d-AmB) and 2 patients with liposomal AmB. The clinical success rate was 55.6%. D-AmB was well-tolerated at 0.35-0.59 mg/kg/d (median 0.43 mg/kg/d) and only 12 patients had mild adverse events. CONCLUSIONS: CNS cryptococcal infection was more frequent in patients with hematological non-malignancies, and cryptococcal antigen test as well as the CSF fungal culture or smear are suggested for early diagnosis. D-AmB could be used as an alternative therapy for CNS-infected patients with hematological diseases.

Dectin-2 polymorphism associated with pulmonary cryptococcosis in HIV-uninfected Chinese patients.

Dectin-2 is a C-type lectin receptor that can recognize critical structures of fungi and involve in the host immune response after pulmonary fungal infections. We aimed to investigate the association between Dectin-2 genetic polymorphisms and cryptococcosis among a series of human immunodeficiency virus (HIV)-uninfected Chinese patients. In this case control study, a total of 251 patients with cryptococcosis and 464 healthy controls were included. One tag-single nucleotide polymorphism (SNP) (rs11045418) located at 5'-flanking region of Dectin-2 gene was selected and genotyped in this study. Among 251 patients, there were 108 (43%) meningitis patients including 73 (67.7%) healthy ones, 74 (29.5%) pulmonary infected patients including 49 (66.2%) healthy ones, and 69 (27.5%) patients with both neural and pulmonary infection including 38 (55.1%) immunocompetent ones. One hundred and forty-three (74 plus 69) patients with pulmonary cryptococcosis and 177 (108 plus 69) patients with cryptococcal meningitis were compared with controls, respectively. Three samples from 143 pulmonary infected patients failed in genotyping. There was a significant difference between 86 immunocompetent pulmonary infected patients and controls in the overdominant model (C/T vs. T/T + C/C; OR, 0.59; 95%CI, 0.37-0.94; P, .026). Similar but not significant difference was found between the overall pulmonary infected patients and the controls in the overdominant model (OR, 0.77; 95%CI, 0.52-1.12; P, .17). No such difference was found between controls and patients with cryptococcal meningitis. Our study firstly showed a genetic association between Dectin-2 and pulmonary cryptococcosis.

In vitro and in vivo evidence for amphotericin B as a P-glycoprotein substrate on the blood-brain barrier.

Amphotericin B (AMB) has been a mainstay therapy for fungal infections of the central nervous system, but its use has been limited by its poor penetration into the brain, the mechanism of which remains unclear. In this study, we aimed to investigate the role of P-glycoprotein (P-gp) in AMB crossing the blood-brain barrier (BBB). The uptake of AMB by primary brain capillary endothelial cells in vitro was significantly enhanced after inhibition of P-gp by verapamil. The impact of two model P-gp inhibitors, verapamil and itraconazole, on brain/plasma ratios of AMB was examined in both uninfected CD-1 mice and those intracerebrally infected with Cryptococcus neoformans. In uninfected mice, the brain/plasma ratios of AMB were increased 15 min (3.5 versus 2.0; P < 0.05) and 30 min (5.2 versus 2.8; P < 0.05) after administration of verapamil or 45 min (6.0 versus 3.9; P < 0.05) and 60 min (5.4 versus 3.8; P < 0.05) after itraconazole administration. The increases in brain/plasma ratios were also observed in infected mice treated with AMB and P-gp inhibitors. The brain tissue fungal CFU in infected mice were significantly lower in AMB-plus-itraconazole or verapamil groups than in the untreated group (P < 0.005), but none of the treatments protected the mice from succumbing to the infection. In conclusion, we demonstrated that P-gp inhibitors can enhance the uptake of AMB through the BBB, suggesting that AMB is a P-gp substrate.

Identification of clinically relevant fungi and prototheca species by rRNA gene sequencing and multilocus PCR coupled with electrospray ionization mass spectrometry.

BACKGROUND: Multilocus PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS) is a new strategy for pathogen identification, but information about its application in fungal identification remains sparse. METHODS: One-hundred and twelve strains and isolates of clinically important fungi and Prototheca species were subjected to both rRNA gene sequencing and PCR/ESI-MS. Three regions of the rRNA gene were used as targets for sequencing: the 5' end of the large subunit rRNA gene (D1/D2 region), and the internal transcribed spacers 1 and 2 (ITS1 and ITS2 regions). Microbial identification (Micro ID), acquired by combining results of phenotypic methods and rRNA gene sequencing, was used to evaluate the results of PCR/ESI-MS. RESULTS: For identification of yeasts and filamentous fungi, combined sequencing of the three regions had the best performance (species-level identification rate of 93.8% and 81.8% respectively). The highest species-level identification rate was achieved by sequencing of D1/D2 for yeasts (92.2%) and ITS2 for filamentous fungi (75.8%). The two Prototheca species could be identified to species level by D1/D2 sequencing but not by ITS1 or ITS2. For the 102 strains and isolates within the coverage of PCR/ESI-MS identification, 87.3% (89/102) achieved species-level identification, 100% (89/89) of which were concordant to Micro ID on species/complex level. The species-level identification rates for yeasts and filamentous fungi were 93.9% (62/66) and 75% (27/36) respectively. CONCLUSIONS: rRNA gene sequencing provides accurate identification information, with the best results obtained by a combination of ITS1, ITS2 and D1/D2 sequencing. Our preliminary data indicated that PCR/ESI-MS method also provides a rapid and accurate identification for many clinical relevant fungi.

Association of Fcγ receptor IIB polymorphism with cryptococcal meningitis in HIV-uninfected Chinese patients.

BACKGROUND: As important regulators of the immune system, the human Fcγ receptors (FcγRs) have been demonstrated to play important roles in the pathogenesis of various infectious diseases. The aim of the present study was to identify the association between FCGR polymorphisms and cryptococcal meningitis. METHODOLOGY/PRINCIPAL FINDINGS: In this case control genetic association study, we genotyped four functional polymorphisms in low-affinity FcγRs, including FCGR2A 131H/R, FCGR3A 158F/V, FCGR3B NA1/NA2, and FCGR2B 232I/T, in 117 patients with cryptococcal meningitis and 190 healthy controls by multiplex SNaPshot technology. Among the 117 patients with cryptococcal meningitis, 59 had predisposing factors. In patients with cryptococcal meningitis, the FCGR2B 232I/I genotype was over-presented (OR = 1.652, 95% CI [1.02-2.67]; P = 0.039) and the FCGR2B 232I/T genotype was under-presented (OR = 0.542, 95% CI [0.33-0.90]; P = 0.016) in comparison with control group. In cryptococcal meningitis patients without predisposing factors, FCGR2B 232I/I genotype was also more frequently detected (OR = 1.958, 95% CI [1.05-3.66]; P = 0.033), and the FCGR2B 232I/T genotype was also less frequently detected (OR = 0.467, 95% CI [0.24-0.91]; P = 0.023) than in controls. No significant difference was found among FCGR2A 131H/R, FCGR3A 158F/V, and FCGR3B NA1/NA2 genotype frequencies between patients and controls. CONCLUSION/SIGNIFICANCE: We found for the first time associations between cryptococcal meningitis and FCGR2B 232I/T genotypes, which suggested that FcγRIIB might play an important role in the central nervous system infection by Cryptococcus in HIV-uninfected individuals.

Genotypes coding for mannose-binding lectin deficiency correlated with cryptococcal meningitis in HIV-uninfected Chinese patients.

BACKGROUND: There is increasing evidence that mannose-binding lectin (MBL) has a complex role in many diseases, particularly in infectious diseases. However, the relationship between MBL deficiency and cryptococcal meningitis has not been clarified. The purpose of this study was to investigate the correlation between MBL polymorphism and non-HIV cryptococcal meningitis. METHODS: A case-controlled genetic association study was conducted. Patients with cryptococcal meningitis and control subjects were genotyped for 6 alleles of MBL2 gene (H/L, Y/X, P/Q, A/D, A/B, and A/C). The distributions in allele frequency, genotypes, haplotypes, and genotype groups were compared between patients and control subjects. RESULTS: Study participants included 103 HIV-uninfected patients with cryptococcal meningitis and 208 healthy control subjects, all of Chinese Han ethnicity. The homozygous mutative genotypes (O/O) of the coding region were associated with cryptococcal meningitis (P = .023; odds ratio [OR], 4.29; 95% confidence interval [CI], 1.11-19.88), the correlation more overt in immunocompetent patients (P = .005; OR, 6.65; 95% CI, 1.49-33.05). MBL-deficient participant group was associated with cryptococcal meningitis (P = .039; OR, 2.09; 95% CI, .96-4.51), particularly in immunocompetent patients (P = .028; OR, 2.51; 95% CI, .96-6.22). CONCLUSIONS: This is the first to show genotypes coding for MBL deficiency are associated with cryptococcal meningitis in nonimmunocompromised hosts.

Epidemiology and risk factors for non-Candida albicans candidemia in non-neutropenic patients at a Chinese teaching hospital.

The purpose of this study was to describe the epidemiology of nosocomial candidemia and identify risk factors involved in infections caused by non-C. albicans Candida species in a Chinese tertiary care center over a 10-year period. A total of 102 cases of nosocomial candidemia in non-neutropenic patients admitted from 1998 through 2007 were included in the study. Candida albicans remained the most common causative agent, accounting for 57.8% of all cases, followed by C. tropicalis (12.8%), C. parapsilosis (10.8%) and C. glabrata (10.8%). Comparison of C. albicans and non-C. albicans candidemia by multivariate logistic regression showed that factors independently associated with non-C. albicans candidemia included head trauma (OR, 5.34; 95% CI, 1.18-24.17; P = 0.029) and bacterial sepsis (OR, 3.58; 95% CI, 1.17-10.98; P = 0.026). Factors independently associated with C. albicans candidemia included tracheal intubation (OR, 0.26; 95% CI, 0.08-0.92; P = 0.037), and increased peripheral WBC count (OR, 0.84; 95% CI, 0.74-0.95; P = 0.006).

[The polymorphism of cytochrome P(450) 2C19 gene in patients with invasive fungal infections.].

OBJECTIVE: To investigate the polymorphism profile of cytochrome P(450) 2C19 (CYP2C19) in Chinese patients with invasive fungal infections. METHODS: Two major single nucleotide polymorphism loci of the CYP2C19 gene (CYP2C19*2 and CYP2C19*3) were genotyped with PCR and restriction fragment length polymorphism (PCR-RFLP) in 134 patients with invasive fungal infections and 134 healthy volunteers. Allele frequencies and the proportions of metabolizer phenotypes were compared. RESULTS: In patients with invasive fungal infections, CYP2C19*1, CYP2C19*2 and CYP2C19*3 alleles showed frequencies of 58.2%, 36.6% and 5.2%. In healthy volunteers, the frequencies of CYP2C19*1, CYP2C19*2 and CYP2C19*3 were 63.4%, 34.3% and 2.2%. There was no significant difference in allele frequencies between the two groups. Of the patients with invasive fungal infections, 33.6% were homozygous extensive metabolizers, 50.0% heterozygous extensive metabolizers and 16.4% poor metabolizers. Of the healthy volunteers, 40.3% were homozygous extensive metabolizers, 48.5% heterozygous extensive metabolizers and 11.2% poor metabolizers. The proportions of metabolizer phenotypes were similar between the two groups. CONCLUSIONS: Significant CYP2C19 polymorphism was detected in both groups. Approximately two thirds of the Chinese patients were either heterozygous extensive metabolizers or poor metabolizers. The genetic polymorphism may have important effect on drug metabolism in these patients.

[Factors associated with mortality in cryptococcal meningitis].

OBJECTIVE: To investigate factors associated with mortality in non-AIDS patients with cryptococcal meningitis. METHODS: We retrospectively reviewed 154 cases of non-HIV cryptococcal meningitis in a tertiary care hospital in China, from 1997 through 2007. RESULTS: The 1-year attributable mortality was 19.6% (28/143), and overall mortality was 28.7% (41/143). Advanced age (> or = 60 years), delay in diagnosis (> 4 months), hematologic malignancy, solid malignancy, altered mental status (coma, seizure, herniation), and CSF drainage or shunting were factors associated with increased death; factors associated with increased survival were amphotericin B based initial therapy and flucytosine containing therapy. In multivariate analysis, age > or = 60 years, the time from symptom onset to diagnosis > 4 months, coma, cerebral herniation, and non-amphotericin B based initial therapy were independently associated with increased overall mortality; factors independently associated with cause-specific mortality were time from symptom onset to diagnosis > 4 months, cerebral herniation and non-amphotericin B based initial therapy. CONCLUSION: A variety of factors were associated with mortality in non-AIDS cryptococcal meningitis. Amphotericin B based initial treatment was independently correlated to improved 1-year survival.

[Evaluation of low-dose amphotericin B and flucytosine in the treatment of cryptococcal meningitis].

OBJECTIVE: To evaluate the efficacy and safety of low-dose amphotericin B (< 0.7 mg x kg(-1) x d(-1)) and flucytosine in patients with non-AIDS-associated cryptococcal meningitis. METHODS: In non-AIDS patients with cryptococcal meningitis admitted to Huashan Hospital, Fudan University in Shanghai from January 1998 to December 2007, 31 were initially treated with low-dose amphotericin B and flucytosine for more than 1 week. The clinical characteristics, clinical responses, drug-related adverse reactions and outcome of these patients were retrospectively evaluated. RESULTS: Among the 31 patients enrolled in this study, 8 patients had one or more predisposing factors. Headache, fever, meningeal irritation and vomiting were common clinical symptoms and signs when cryptococcal meningitis was diagnosed. The result of cranial CT scan and/or MRI showed abnormality in 22 cases (78.6%). When the therapy of low-dose amphotericin B and flucytosine ended, the complete response rate was 19.4% (6/31), partial response rate was 54.8% (17/31), and total effective rate was 74.2%. Except for 1 patient lost to follow-up, the 1-year attributable and all-cause mortality among the remaining 30 patients were 0 (0/30) and 10.0% (3/30) respectively. On the other hand, 26 (83.9%) patients had amphotericin B-related adverse reactions, including renal impairment, liver injury, arrhythmia, and anemia, etc. However, most of these reactions were tolerable. CONCLUSION: Low-dose amphotericin B and flucytosine can be used in non-AIDS-associated cryptococcal meningitis with both acceptable efficacy and safety.

Cryptococcal meningitis in non-HIV-infected patients in a Chinese tertiary care hospital, 1997-2007.

Information remains sparse about non-HIV patients with cryptococcal meningitis in the era of triazole therapy. Particularly of interest are the clinical manifestations and prognosis of the infection in these previously healthy patients. We retrospectively reviewed 154 non-HIV-infected patients with cryptococcal meningitis who presented in our hospital from 1997 to 2007. We compared the clinical features and outcomes between predisposed and otherwise healthy hosts. The number of cases per year showed a steady increase over time. The majority of patients were otherwise apparently healthy (103 patients, 66.9%) and predisposing factors were identified in only 51 (33.1%) patients. Corticosteroid medication accounted for the most common underlying factor in these cases (n = 21). Morbidity was appallingly high, with seizures in 28.6%, cranial nerves palsies in 51.5% and cerebral herniation in 19.5%. Despite these complications, overall mortality during 1 year was 28.7% (41/143), close to that reported from other centers with non-HIV patients. Death attributed to cryptococcosis occurred in 19.6% (28/143) patients with most receiving amphotericin B as a component of their initial therapy. Among surviving patients who had lumbar punctures at weeks 2 and 10, those given amphotericin B for initial therapy achieved higher rates of overall response than those receiving initial fluconazole therapy at either week 2 (84.4% of 96 patients vs. 33.3% of 24 patients, P <0.001) or week 10 (85.0% of 93 patients vs. 66.7% of 24 patients, P = 0.041). In multivariate analysis, coma, cerebral herniation, and initial antifungal therapy without amphotericin B were independently correlated with both increased overall and attributable mortality, while advanced age (>/= 60 years) was correlated with increased overall mortality only. Patients with apparently normal immune status were overall younger than those who were immunocompromised. In addition, previously healthy patients for whom diagnosis was delayed had more severe disease, experiencing more brain herniation, coma, seizures, hydrocephalus and more surgical shunt procedures. On the other hand, immunocompromised patients were more commonly found to have high fever and brain parenchymal involvement. However, both groups had a similar treatment response and 1-year survival.