The delayed clearance of Talaromyces marneffei in blood culture may be associated with higher MIC of voriconazole after antifungal therapy among AIDS patients with talaromycosis.

OBJECTIVES: This study aimed to investigate the influencing factors of delayed clearance of Talaromyces marneffei (T. marneffei) in blood culture of patients with acquired immune deficiency syndrome (AIDS) complicated with talaromycosis after antifungal therapy. METHODS: The patients with AIDS complicated with talaromycosis were retrospectively enrolled, and divided into two groups according to the blood T. marneffei culture results in two weeks after antifungal therapy. The baseline clinical data were collected and the antifungal susceptibility of T. marneffei was tested. RESULTS: A total of 190 patients with AIDS and talaromycosis were enrolled, of whom 101 cases remained positive for T. marneffei (Pos-group) while the other 89 cases were negative in blood culture (Neg-group) after two weeks' antifungal treatment. The Pos-group had a higher baseline Aspartate aminotransferase (AST, 78.5 vs. 105 U/L; P = 0.073) and lower CD4+ T cells level (11 vs. 7 cells/µl; P = 0.061). The percentage of isolates with higher MICs of voriconazole (VOR) and fluconazole (FLU) in the Pos-group were significantly higher than those in the Neg-group (χ2 = 12.623, P < 0.001 and χ2 = 9.356, P = 0.002, respectively). By multivariate logistic regression, the MIC value for VOR was identified as the prognostic variable that may influence the clearance of T. marneffei in blood culture after antifungal therapy among AIDS patients with talaromycosis. CONCLUSIONS: The delayed negative conversion of blood T. marneffei-culture may be associated with some factors especially higher MIC of VOR, indicating the possibility of drug resistance of T. marneffei.

HIV-infected patients rarely develop invasive fungal diseases under good immune reconstitution after ART regardless high prevalence of pathogenic filamentous fungi carriage in nasopharynx/oropharynx.

Purpose: We aimed to investigate the prevalence and risk factors of filamentous fungi (FF) carriage in human immunodeficiency virus (HIV)-infected patients in Guangdong province, along with its subsequent incidence of invasive fungal disease (IFD). Methods: Seven hundred and sixteen HIV-infected individuals from the outpatient clinic and 293 sex-matched healthy controls were recruited prospectively from May 1 to August 31, 2017. Fungi were isolated from oropharyngeal and nasopharyngeal swabs, then identified by morphological and molecular biological techniques. Logistic regression analysis was used to identify risk factors of pathogenic FF carriage. Pathogenic FF carriers were followed up through the end of 2019. Results: Of the 716 included HIV-infected patients, 602 (84.1%) were male, the median age was 34 (27-42) years, and the median CD4+ count was 385 (254-542) cells/µl. Pathogenic FF were isolated in 119 (16.6%) cases with HIV infection and 40 (13.7%) healthy controls. Mucorales were found in 3 HIV-infected individuals and Talaromyces marneffei in 2 HIV-infected individuals, but not in healthy controls. History of cured opportunistic infections (OIs; OR, 1.97; 95% CI, 1.23-3.13, p = 0.004), and smoking (OR, 1.55; 95%CI, 1.03-2.32, p = 0.035) were independent risk factors of pathogenic FF carriage in HIV-infected individuals. A total of 119 pathogenic FF carriers with HIV infection were followed. During follow-up, 119 (100%) cases received antiretroviral therapy (ART) for at least 28 months, 107 (90%) cases had CD4+ counts>200 cells/µl, and none developed IFD. Discussion: Pathogenic FF carriage is common in HIV-infected individuals but may not develop IFD in those who achieved immune reconstitution. Smoking and cured OIs history increase the risk of pathogenic FF carriage. Smoking abstinence and ART adherence are especially important for these patients.

The Association of TLR2, TLR3, and TLR9 Gene Polymorphisms With Susceptibility to Talaromycosis Among Han Chinese AIDS Patients in Guangdong.

Background: Talaromycosis (TM) caused by Talaromyces marneffei (T. marneffei) is a growing public health concern. Although Toll-like receptor (TLR) genes play a critical role in the host defense against fungal infection, the influence of polymorphisms in these genes on the susceptibility of acquired immune deficiency syndrome (AIDS) patients to TM remains unknown. This study aims to uncover the associations of single nucleotide polymorphisms (SNPs) in TLR genes with TM susceptibility among patients with AIDS. Methods: Altogether 200 AIDS patients complicated with TM, 200 matched AIDS patients without TM, and 76 healthy controls (HCs) were enrolled in this case-control study. In total, 23 SNPs in the TLR2, TLR4, and TLR9 genes, which may influence the susceptibility of AIDS patients to TM, were checked by the time of flight mass spectrometry (TOF/MS) method among these Han Chinese subjects. Results: No significant differences in genotype or allele frequencies of selected SNPs were found among the TM group, Non-TM group, and HC group. Haplotype analysis also demonstrated no correlation of these SNPs with TM. However, subgroup analysis showed that the genotype TT and the T allele in TLR2 SNP rs1339 were more frequent in typical TM cases than controls (50.0 vs. 35.8%, 70.5 vs. 59.7%); the frequency of the GT genotype in TLR2 SNP rs7656411 was markedly higher in severe TM cases compared to controls (57.8 vs. 34.4%). Conclusion: Our results demonstrate a genetic connection of TLR2 SNPs rs1339 and rs7656411 with an increased susceptibility and severity of TM among Han Chinese populations.

Development of peptide-based chemiluminescence enzyme immunoassay (CLEIA) for diagnosis of dengue virus infection in human.

Dengue is the most prevalent mosquito-borne viral disease in tropical and subtropical regions worldwide. Since its clinical symptoms are non-specific and easily mistaken as other kinds of infection, laboratory diagnosis is required to confirm dengue infections. In this study, ten peptides (E1-E10) from the envelope protein of dengue virus (DENV) were first identified using bioinformatic tool. The screened peptides were then synthesized for the peptide-based chemiluminescence enzyme immunoassay (CLEIA). Two peptides, E1 and E7, were found as the best candidate antigen and therefore used as downstream application in the development of low-cost peptide-based anti-DENV immunoglobulin M antibodies (IgM) indirect CLEIA. 176 serum samples were used to study the presence of anti-DENV IgM antibodies to evaluate the diagnostic ability of IgM-CLEIA. Receiver operating characteristic curve (ROC) was used to estimate the diagnostic cut-off value. The sensitivity and the specificity reached 82.5% and 94.6% respectively when peptide E1 was used, but declined to 79.2% and 92.9% respectively when peptide E7 was used. Therefore, the combination of E1 and E7 was used to improve the sensitivity and the specificity to 85.0% and 96.4% respectively in 1.5 h assay time, providing a potentially practical use for the diagnosis of DENV infections in patients' serum.

Expression and characterization of a Talaromyces marneffei active phospholipase B expressed in a Pichia pastoris expression system.

Phospholipase B is a virulence factor for several clinically important pathogenic fungi, including Candida albicans, Cryptococcus neoformans and Aspergillus fumigatus, but its role in the thermally dimorphic fungus Talaromyces marneffei remains unclear. Here, we provide the first report of the expression of a novel phospholipase gene, designated TmPlb1, from T. marneffei in the eukaryotic expression system of Pichia pastoris GS115. Sensitive real-time quantitative reverse-transcription PCR (qRT-PCR) demonstrated that the expression of TmPlb1 increased 1.85-fold in the yeast phase compared with the mycelial phase. TmPlb1 contains an open reading frame (ORF) of 732 bp that encodes a protein of 243 amino acids. The conserved serine, aspartate and histidine catalytic triad and the G-X-S-X-G domain of TmPLB1 provide the structural basis for its molecular activity. The ORF of TmPlb1 was successfully cloned into a pPIC9K vector containing an α-mating factor secretion signal that allowed the secretory expression of TmPLB1 in P. pastoris. The heterologous protein expression began 12 h after methanol induction and peaked at 96 h. Through analysis with SDS-polyacrylamide gel electrophoresis (SDS-PAGE), western blotting and mass spectrometry, we confirmed that TmPLB1 was successfully expressed. Through Ni-affinity chromatography, TmPLB1 was highly purified, and its concentration reached 240.4 mg/L of culture medium. With specific substrates, the phospholipase A1 and phospholipase A2 activities of TmPLB1 were calculated to be 5.96 and 1.59 U/mg, respectively. The high purity and activity of the TmPLB1 obtained here lay a solid foundation for further investigation.

Mutational analysis of Pneumocystis jirovecii dihydropteroate synthase and dihydrofolate reductase genes in HIV-infected patients in China.

We investigated Pneumocystis jirovecii dihydropteroate synthase (DHPS) and dihydrofolate reductase (DHFR) genes for mutations in 25 Chinese HIV-infected patients with P. jirovecii pneumonia. We identified DHPS mutations in 3 (12%) patients and DHFR mutations in 1 (4%) patient. The prevalence of DHPS and DHFR mutations in China remains low, as it does in other developing countries.

Genetic diversity analysis of Penicillium marneffei isolated from AIDS patients in Guangdong, China using randomly amplified polymorphic DNA.

BACKGROUND: Penicillium marneffei (P. marneffei) is an emerging pathogenic fungus that can cause invasive mycosis in patients with AIDS. The epidemiological features of P. marneffei infection in AIDS patients in Guangdong province remain unclear so far. This study aimed to investigate the genetic diversity within a population of 163 P. marneffei isolates obtained from AIDS patients and search for the dominant clinical strains in Guangdong province. METHODS: One hundred and sixty-three P. marneffei isolates obtained from AIDS patients in Guangdong province during January 2004 and December 2009 were studied by randomly amplified polymorphic DNA (RAPD) using two random primers (H2 and H22). The degree of similarity between samples was calculated through similarity coefficients from RAPD fragment data and the dendrogram was assessed using the unweighted pair group method with arithmetic mean (UPGMA). RESULTS: Two primers showed a high degree of discrimination and good stability. Primer H2 yielded eight different patterns (H2-1 to H2-8) among 163 isolates with the discriminatory power being 0.413. Primer H22 identified seven types (H22-1 to H22-7) among 163 isolates with the discriminatory power being 0.467. Genetic similarity coefficients based on RAPD data among 163 P. marneffei isolates ranged from 0.681 to 0.957, 61.96% of which were no less than 0.83. The discriminatory power of the two primers was 0.524. One hundred and sixty-three P. marneffei isolates were clustered into nine distinct groups (groups I to IX) at the similarity coefficient value of 0.83 and group I was the most common, including 101 strains (61.96%). CONCLUSION: The RAPD analyses could provide important information as to the degree of genetic diversity and the relationship among clinical P. marneffei isolates, revealing genetic polymorphism and dominant strains.

Microsatellite analysis of clinical isolates of the opportunistic fungal pathogen Penicillium marneffei from AIDS patients in China.

BACKGROUND: Penicillium marneffei is an opportunistic fungus that may cause fatal disease, and usually infects acquired immune deficiency syndrome (AIDS) patients. The molecular epidemiology of this fungus remains enigmatic. METHODS: A multilocus microsatellite typing (MLMT) system based on 11 microsatellite loci was applied to 169 unrelated isolates of P. marneffei obtained from AIDS patients, in order to identify their genetic diversity. These patients came from the provinces of Guangdong and Guangxi, areas endemic for P. marneffei in China. RESULTS: For the overall population, the average number of alleles per locus ranged from 3 to 8 (mean 5.5), while the discriminatory power (DP) of each locus ranged from 0.235 to 0.651 (mean 0.512). By combining the information generated for 11 loci, MLMT detected 159 different multilocus genotypes (MTs), resulting in a high degree of discrimination (DP = 0.999). One hundred and sixty-nine isolates were further clustered into 9 types (from A to I) at the similarity coefficient of 0.80, with type A (80 isolates) and type B (60 isolates) being the most common types. Within 5 subpopulations from different regions of China, the distribution of MTs of P. marneffei isolates was diverse. Although 169 isolates shared a high genetic similarity (range 0.71-0.933), isolates from Guangxi and Guangdong provinces could be differentiated from each other and clustered into 2 categories by unweighted pair-group method with arithmetic mean (UPGMA) cluster analysis. CONCLUSIONS: By MLMT, the genetic diversity of clinical P. marneffei isolates could be discriminated, the dominant strain of P. marneffei cultured from AIDS patients in China could be identified, and clinical isolates of P. marneffei from Guangxi Province could be differentiated from those from Guangdong Province.

Cloning, characterization, and expression of a novel secretory lipase-like gene from Clonorchis sinensis.

A secretory lipase-like gene was isolated from total cDNA of adult Clonorchis sinensis. The gene has an open reading frame of 1,218 bp long and encodes for a protein of 406 amino acids including a putative signal peptide of 20 amino acids. The deduced amino acid sequence including signal peptide has 42-45% identity with lipase of other species and two typical enzymic active sites that contain consensus sequence (Gly-X-Ser-X-Gly) of lipase. The cDNA encoding this protein was subcloned into pET-28a (+) expression vector and expressed in Escherichia coli. The expressed fusion protein has a molecular mass of about 45 kDa. Prediction of signal peptide and Western blot analysis indicated that the secretory lipase-like protein is an excretory-secretory product of C. sinensis. Immunostaining revealed that the secretory lipase-like protein was localized in the tegument of the adult worm and metacercaria. These results provide basis for further studies on the nutrition taking and invasion of C. sinensis mediated by the secretory lipase-like protein.

[Clinical value of the detection of hepatitis C virus core antigen].

AIM: To explore the value of detecting hepatitis C virus core antigen in HCV infected diseases. METHODS: The HCV-RNA was tested by fluorescent quantitative polymerase chain reaction (FQ-PCR). The core antigens of HCV and anti-HCV were tested by enzyme-linked immunosorbent assay (ELISA). RESULTS: The positive rate of HCV-RNA in 191 patients was 71.2%, about 136 in 191. The positive rate of anti-HCV was 97.4%, about 186 in 191. The positive rate of HCV core antigen was 33.0%, about 63 in 191. After antiviral therapy for 2 patients,their HCV-RNA and anti-HCV were detected negative but their HCV core antigen was detected positive. In another patient, his anti-HCV was negative but his HCV core antigen was positive. CONCLUSION: As a supplemental test for anti-HCV examination the detection of HCV core antigen is of important clinical value in HCV infection diagnosis.

A clinical, epidemiology and virological study of a dengue fever outbreak in Guangzhou, China-2002-2006.

We analysed the clinical and epidemiological characteristics of dengue fever (DF) during the dengue virus (DENV)-1 outbreak in Guangzhou, China. Clinical and epidemiological data of 1342 patients with DF from May 2002 to November 2006 were analyzed retrospectively. The average age was 34.7±13.2 years. The ratio of male to female was 1.05:1. The peak time of the epidemic lasted from August to October. The most common manifestations included fever (100%), headache (85.9%), myalgia (64.5%), bone soreness (46.6%), fatigue (78.2%), skin rash (65.9%) and positive tourniquet test (51.3%). Leukopenia, thrombocytopenia, elevated alanine aminotransferase (ALT), elevated aspartate aminotransferase (AST) and hypopotassemia were found in 66%, 61.3%, 69%, 85.7% and 28.4% of the patients respectively. Only 2(0.15%) patients fulfilled the WHO case definition criteria for dengue haemorrhagic fever (DHF), the others were all diagnosed as classic DF. However, 64(4.8%) patients had severe clinical manifestations (internal haemorrhage, shock, marked thrombocytopenia, myocarditis, sepsis, pneumonia and encephalopathy). Anti-dengue IgM was detected in 90% of patients. Dengue viruses were isolated using the Aedes albopictus C6/36 cell line and identified as DENV-1 by RT-PCR. A 346bp fragment from RT-PCR product of every isolate was sequenced to compare with published sequences of other DENV-1 viruses. The nucleotide homology were 97%, 97% and 98% compared with those of DENV-1 strains of dengue fever outbreak in Cambodia, in 1997 and 1999 in China, respectively. In conclusion, the epidemic of dengue fever was caused by DENV-1 infection from 2002 to 2006 in Guangzhou. Patients with severe clinical manifestations are few, but in some of them, the diagnosis of DHF may be missed if the WHO classification is strictly applied, especially in adults.

[Analysis on clinical and epidemiological characteristics of 1032 patients with Dengue fever in Guangzhou].

OBJECTIVE: To analyze the clinical and epidemiological characteristics of Dengue fever (DF) during the Dengue-1 epidemic in Guangzhou. METHODS: Clinical and epidemiological data of 1032 patients with DF from May 2002 to November 2003 were retrospectively analyzed. Dengue virus were isolated by cell culture and typed by reverse transcriptase polymerase chain reaction (RT-PCR). RESULTS: Age of the patients ranged from 55 days to 91 years old (average 34.7 +/- 13.2 years) with sex ratio 1.03:1. Incubation period ranged from 2 to 12 days with mean periods of 5.3 +/- 2.4 days. Most (45.0%) cases appeared in September and the epidemic last from July to November. Dengue outbreak had involved 675 cases in 26 common places. The common manifestations were seen as fever (100%), headache (90.9%), myalgia (68.4%), bone soreness (48.8%), fatigue (79.3%), skin rash (60.1%), positive tourniquet test (45.3%), leukopenia (63.3%) and thrombocytopenia (60.8%), respectively. Dengue virus was isolated from serum of 19 out of 54 patients' and identified as Dengue virus type 1. DNA sequence analyzes on rates of nucleotide homology were 97%, 97% and 98% compared with those of Dengue virus type 1 strain of DF outbreak in Cambodia, in 1997 and 1999 in China. CONCLUSION: The epidemic of DF in Guangzhou in 2002/2003 was caused by Dengue virus type-1 with most patients showing classic type of the disease. Date suggested that change can happen from non-endemic to hypoendemic regions in Guangdong province.