Usefulness of a microimmunodiffusion test for the detection of Penicillium marneffei antigenemia,antibodies, and exoantigens

Eight sera from culturally-proven cases of penicilliosis marneffei and their corresponding isolates were examined for circulating antibody(ies) and antigen, and exoantigens, respectively, using a microimmunodiffusion (MID) test. Two of the 8 sera produced strong precipitins (1-2) when reacted against control Penicillium marneffei antigen (5-week-old shaken cultures at 25 C) in the presence of control rabbit anti-P. marneffei serum. Five of the 8 sera produced a strong precipitin line when reacted against control hyperimmune serum to P. marneffei. These five sera, and one additional serum, which tested negative for antibody to P. marneffei, demonstrated the presence of antigen by reacting only against the anti-P. marneffei serum. Serological evaluations of the sera revealed that the MID test is capable of detecting antibody and antigen in AIDS patients having penicilliosis marneffei infections. Exoantigen analysis of the 8 P. marneffei isolates, which were previously identified using this conventional and time-consuming macro- and micro-morphological characteristics, showed the presence of 1 to 4 specific exoantigens in MID. With the exoantigen analysis, the identity of all of the isolates was confirmed as P. marneffei. Our studies indicated that the serological tests are useful for detecting circulating antibody and/or antigen in patients' sera, and that the exoantigen test is reliable for confirming the identity of P. marneffei cultures. This revised version was published online in June 2006 with corrections to the Cover Date.     

中国栽培白灵侧耳的RAPD和IGS分析

以RAPD和IGS为分子标记,研究了中国栽培白灵侧耳Pleurotus nebrodensis菌株的遗传多样性,RAPD结果表明,供试菌株间存在多态性;对供试菌株的IGS分析表明,白灵侧耳P. nebrodensis的IGS1区域较保守,菌株间没有多态性;IGS2区域进化较快,菌株间具丰富的多态性。IGS2-RFLP可应用于白灵侧耳的菌株鉴定鉴别,较RAPD更稳定,更具可操作性。     

Sequence analysis of rDNA intergenic spacer (IGS) of <Emphasis Type="Italic">Porphyra haitanensis</Emphasis>

The intergenic spacer region (IGS) has been used for the first time to analyze the genetic variability of Porphyra haitanensis from different areas. In order to determine that whether the IGS sequences could be used for classification and identification in intraspecies of Porphyra, the partial IGS sequences of cultivated strains of P. haitanensis (isolated from Putian-Fujian Province, Shantou-Guangdong Province and Ningbo-Zhejiang Province), were amplified, sequenced and analyzed. The sequence analysis indicated that the partial IGS sequences from the three stains were the external transcribed spacers (ETS) of 3′ end of the IGS gene. In the three stains, the length of IGS sequences ranged from 1,085 to 1,100 bp and the G + C content varied from 50.88% to 51.27%. There were 55 variable sites which occupied approximately 5% of the ETS sequences. Similarity analysis and multisequencing alignment of sequences indicated that the partial IGS sequences of the three stains of P. haitanensis had notable variabilities. Therefore, the IGS sequence could be used as the critical genetic marker in intraspecies of P. haitanensis. Furthermore, IGS sequence analysis will be a powerful tool for genetic diversity and classification in intraspecies of other Porphyra species.     

Insights into the pathogenicity of Penicillium marneffei.

Penicillium marneffei is a significant pathogen of AIDS patients in Southeast Asia. This fungus is unique in that it is the only dimorphic member of the genus. Pathogenesis of P. marneffei requires the saprobic mold form to undergo a morphological change upon tissue invasion. The in vivo form of this fungus reproduces as a fission yeast that capably evades the host immune system. The processes that control these morphological changes, better termed as phase transition, can be replicated in vitro by incubation of the mold form at 37 degrees C. The unidentified molecular mechanisms regulating phase transition in this fungus are now being uncovered using modern methodologies and novel strategies. A better comprehension of these underlying regulatory pathways will provide insight into eukaryotic cellular development as well as the potential factors responsible for infections caused by P. marneffei and other fungi. Such knowledge may lead to better chemotherapeutic interventions of fungal diseases.     

Detection and genotyping of Giardia intestinalis isolates using intergenic spacers(IGS)-based PCR

Giardia intestinalis infections arise primarily from contaminated food or water. Zoonotic transmission is possible, and at least 7 major assemblages including 2 assemblages recovered from humans have been identified. The determination of the genotype of G. intestinalis is useful not only for assessing the correlation of clinical symptoms and genotypes, but also for finding the infection route and its causative agent in epidemiological studies. In this study, methods to identify the genotypes more specifically than the known 2 genotypes recovered from humans have been developed using the intergenic spacer (IGS) region of rDNA. The IGS region contains varying sequences and is thus suitable for comparing isolates once they are classified as the same strain. Genomic DNA was extracted from cysts isolated from the feces of 5 Chinese, 2 Laotians and 2 Koreans infected with G. intestinalis and the trophozoites of WB, K1, and GS strains cultured in the laboratory, respectively. The rDNA containing the IGS region was amplified by PCR and cloned. The nucleotide sequence of the 3' end of IGS region was determined and examined by multiple alignment and phylogenetic analysis. Based on the nucleotide sequence of the IGS region, 13 G. intestinalis isolates were classified to assemblages A and B, and assemblage A was subdivided into A1 and A2. Then, the primers specific to each assemblage were designed, and PCR was performed using those primers. It detected as little as 10 pg of DNA, and the PCR amplified products with the specific length to each assemblage (A1, 176 bp; A2, 261 bp; B, 319 bp) were found. The PCR specific to 3 assemblages of G. intestinalis did not react with other bacteria or protozoans, and it did not react with G. intestinalis isolates obtained from dogs and rats. It was thus confirmed that by applying this PCR method amplifying the IGS region, the detection of G. intestinalis and its genotyping can be determined simultaneously.     

AreA controls nitrogen source utilisation during both growth programs of the dimorphic fungus Penicillium marneffei

The opportunistic pathogen Penicillium marneffei displays a temperature-dependent dimorphic switching program with saprophytic hyphal growth at 25 °C and yeast growth at 37 °C. The areA gene of P. marneffei has been isolated and found to be required for the utilisation of nonpreferred nitrogen sources during both growth programs of P. marneffei, albeit to differing degrees. Based on this functional characterisation and high degree of sequence conservation with other fungal GATA factors, P. marneffei areA represents an orthologue of Aspergillus nidulans areA and Neurospora crassa NIT2. Based on this study it is proposed that AreA is likely to contribute to the pathogenicity of P. marneffei by facilitating growth in the host environment and regulating the expression of potential virulence factors such as extracellular proteases.     

Penicillium marneffei-stimulated dendritic cells enhance HIV-1 trans-infection and promote viral infection by activating primary CD4+ T cells

Penicillium marneffei (P. marneffei) is considered an indicator pathogen of AIDS, and the endemicity and clinical features of P. marneffei have been described. While, how the co-infection of P. marneffei exacerbate deterioration of the immune response remains poorly understood. Here we isolated P. marneffei from the cutaneous lesions of AIDS patients and analyzed its effects on HIV-1-dendritic cells (DCs) interaction. We demonstrated that the monocyte-derived dendritic cells (MDDCs) could be activated by both thermally dimorphic forms of P. marneffei for significantly promoting HIV-1 trans-infection of CD4(+) T cells, while these activated MDDCs were refractory to HIV-1 infection. Mechanistically, P. marneffei-activated MDDCs endocytosed large amounts of HIV-1 and sequestrated the internalized viruses into tetrapasnin CD81(+) compartments potentially for proteolysis escaping. The activated MDDCs increased expression of intercellular adhesion molecule 1 and facilitated the formation of DC-T-cell conjunctions, where much more viruses were recruited. Moreover, we found that P. marneffei-stimulated MDDCs efficiently activated resting CD4(+) T cells and induced more susceptible targets for viral infection. Our findings demonstrate that DC function and its interaction with HIV-1 have been modulated by opportunistic pathogens such as P. marneffei for viral dissemination and infection amplification, highlighting the importance of understanding DC-HIV-1 interaction for viral immunopathogenesis elucidation.     

Exploring the<Emphasis Type="Italic"> Penicillium marneffei</Emphasis> genome

Penicillium marneffei is a dimorphic fungus that intracellularly infects the reticuloendothelial system of humans and bamboo rats. Endemic in Southeast Asia, it infects 10% of AIDS patients in this region. The absence of a sexual stage and the highly infectious nature of the mould-phase conidia have impaired studies on thermal dimorphic switching and host-microbe interactions. Genomic analysis, therefore, could provide crucial information. Pulsed-field gel electrophoresis of genomic DNA of P. marneffei revealed three or more chromosomes (5.0, 4.0, and 2.2 Mb). Telomeric fingerprinting revealed 6-12 bands, suggesting that there were chromosomes of similar sizes. The genome size of P. marneffei was hence about 17.8-26.2 Mb. G+C content of the genome is 48.8 mol%. Random exploration of the genome of P. marneffei yielded 2303 random sequence tags (RSTs), corresponding to 9% of the genome, with 11.7, 6.3, and 17.4% of the RSTs having sequence similarity to yeast-specific sequences, non-yeast fungus sequences, and both (common sequences), respectively. Analysis of the RSTs revealed genes for information transfer (ribosomal protein genes, tRNA synthetase subunits, translation initiation, and elongation factors), metabolism, and compartmentalization, including several multi-drug-resistance protein genes and homologues of fluconazole-resistance gene. Furthermore, the presence of genes encoding pheromone homologues and ankyrin repeat-containing proteins of other fungi and algae strongly suggests the presence of a sexual stage that presumably exists in the environment.     

Separation of strains of the yeasts Xanthophyllomyces dendrorhousand Phaffia rhodozyma based on rDNA IGS and ITS sequence analysis

The type strains of the anamorph Phaffia rhodozyma (CBS 5905) and the teleomorph Xanthophyllomyces dendrorhous (VKM Y-2786) were analyzed by nucleotide sequence analysis and compared to the sequences found in three additional strains (ATCC 24228, ATCC 24230 and CBS 6938). The results of ribosomal DNA Internal transcribed spacer (ITS) and Intergenic spacer (IGS) region analyses indicate that P. rhodozyma, which was isolated from a beech tree, is a distinct species from the other four strains. The latter that were collected from birch trees are considered to be strains of X. dendrorhous. These individual strains of X. dendrorhous, which have geographically distinct isolation sources, can be distinguished by nucleotide substitutions and deletion/insertion gaps in sub-repeat regions of the Intergenic spacer. The conclusions demonstrate that differences in the IGS region provide molecular markers for denoting strains that may differ in their biochemical and physiological capabilities. The hypothesis is presented that strain differences in the IGS region may be useful to demonstrate geographic and host specificity. Received 28 January 1999/ Accepted in revised form 17 April 1999     

A novel approach for screening immunogenic proteins in Penicillium marneffei using the DeltaAFMP1DeltaAFMP2 deletion mutant of Aspergillus fumigatus

Using serum from guinea-pigs immunized with a DeltaAFMP1DeltaAFMP2 deletion mutant of Aspergillus fumigatus to screen a cDNA library of A. fumigatus, we cloned a novel immunogenic 57-kDa protein in A. fumigatus. We also cloned its 55-kDa homologue in Penicillium marneffei, which was possibly related to amino acid biosynthesis and metabolism, with homologues present only in the subphylum Pezizomycotina of Ascomycota. The recombinant 55-kDa protein of P. marneffei reacted strongly with guinea-pig serum immunized with P. marneffei and with the sera of patients with P. marneffei infection. A similar approach could be applied to immunogenic protein screening in other microorganisms for serological diagnosis, epidemiological studies and the study of vaccines.     

Genetic polymorphism of ferula mushroom growing on Ferula sinkiangensis

Mating tests, internal transcribed spacer (ITS) sequence analysis, intergenic spacer 1–restriction fragment length polymorphism (IGS1-RFLP), IGS1 sequence analysis, and IGS2-RFLP analysis were carried out on isolates of 17 morphologically different Pleurotus mushrooms collected on Ferula sinkiangensis. The isolates were divided, based on mating tests and ITS sequence analysis, into two groups identical to P. eryngii var. ferulae and P. nebrodensis, respectively. Single spores from these two groups were incompatible, but those from P. eryngii var. ferulae and P. eryngii were compatible and combined to produce 56.25% dikaryon mycelia with clamp connections. The ITS of P. eryngii var. ferulae and P. nebrodensis (GenBank accession no. AY311408) were both 638 bp in size but differed by 3% in sequence. P. eryngii var. ferulae and P. eryngii (GenBank accession no. AY368658) were identical in ITS size and sequence. P. nebrodensis was the dominant population of Pleurotus mushroom growing on F. sinkiangensis. It exhibited genetic diversity. The two species could also be distinguished by IGSI-RFLP, similar to identification by mating tests and ITS sequence analysis. Difference in IGS1-RFLP existed between P. eryngii var. ferulae and P. nebrodensis. The sequence difference reached 2.28%. Both IGS1 size and IGS1-RFLP were similar among the different samples of P. nebrodensis. The 17 isolates were separated into five types based on IGS2 size and IGS2-RFLP, with both interspecies and extraspecies differences. P. nebrodensis exhibited polymorphism and was divided into four types. These results agreed with macroscopic differences. IGS2 might be the effective domain of genetically polymorphic ribosomal DNA in P. nebrodensis mushrooms found in Xinjiang, China.     

马尔尼菲青霉病8例临床分析

目的分析总结马尔尼菲青霉病的临床及实验室特征。方法以回顾性分析的方法 ,对我科2003～2009年期间诊治的8名明确诊断为马尔尼菲青霉感染患者的临床特征、皮损组织病理学特点及其皮损组织、骨髓或咽拭子等进行真菌培养的菌落及真菌形态等实验室检查结果进行分析。结果①马尔尼菲青霉感染多发生于HIV感染患者或AIDS患者。②皮损可与全身症状同时或先后出现,皮损表现为淡红色丘疱疹、坏死性丘疹、传染性软疣样丘疹、皮肤溃疡及血痂。③多伴有多系统损害。④37℃培养呈酵母相,25℃呈菌丝相,皮损组织病理可以看到典型的"桑葚样"改变。结论马尔尼菲青霉感染好发于HIV感染患者或其他免疫功能低下的患者,常表现为特征性皮疹,皮损组织、骨髓或分泌物于25℃、37℃真菌培养结合皮肤病理是明确诊断的关键。     

Comparative analysis of the intergenic spacer regions and population structure of the species complex of the pathogenic yeast Cryptococcus neoformans

Cryptococcus neoformans is an opportunistic basidiomycete responsible for the high incidence of cryptococcosis in patients with AIDS and in other immune-compromised individuals. This study, which focused on the molecular structure and genetic variability of the two varieties in the C. neoformans and Cryptococcus gattii species complex, employed sequence analysis of the intergenic spacer regions, IGSI and IGSII. The IGS region is the most rapidly evolving region of the rDNA families. The IGSI displayed the most genetic variability represented by nucleotide base substitutions and the presence of long insertions/deletions (indels). In contrast, the IGSII region exhibited less heterogeneity and the indels were not as extensive as those displayed in the IGSI region. Both intergenic spacers contained short, interspersed repeat motifs, which can be related to length polymorphisms observed between sequences. Phylogenetic analysis undertaken in the IGSI, IGSII and IGSI +5S rRNA + IGSII regions revealed the presence of six major phylogenetic lineages, some of which segregated into subgroups. The major lineages are represented by genotypes 1 (C. neoformans var. grubii), genotype 2 (C. neoformans var. neoformans), and genotypes 3, 4, 5 and 6 represented by C. gattii. Genotype 6 is a newly described IGS genotypic group within the C. neoformans species complex. With the inclusion of IGS subgenotypic groups, our sequence analysis distinguished 12 different lineages. Sequencing of clones, which was performed to determine the presence of multiple alleles at the IGS locus in several hybrid strains, yielded a single IGS sequence type per isolate, thus suggesting that the selected group of cloned strains was mono-allelic at this locus. IGS sequence analyses proved to be a powerful technique for the delineation of the varieties of C. neoformans and C. gattii at genotypic and subgenotypic levels.     

Genomic and experimental evidence for a potential sexual cycle in the pathogenic thermal dimorphic fungus Penicillium marneffei

All meiotic genes (except HOP1) and genes encoding putative pheromone processing enzymes, pheromone receptors and pheromone response pathways proteins in Aspergillus fumigatus and Aspergillus nidulans and a putative MAT-1 alpha box mating-type gene were present in the Penicillium marneffei genome. A putative MAT-2 high-mobility group mating-type gene was amplified from a MAT-1 alpha box mating-type gene-negative P. marneffei strain. Among 37 P. marneffei patient strains, MAT-1 alpha box and MAT-2 high-mobility group mating-type genes were present in 23 and 14 isolates, respectively. We speculate that P. marneffei can potentially be a heterothallic fungus that does not switch mating type.     

Imported penicilliosis marneffei in the United States: report of a second human infection

The first natural infection due to Penicillium marneffei in a human was reported in the United States in 1973. We describe a second case of penicilliosis marneffei that was diagnosed in Florida. In both instances, the patients had a history of travel in Southeast Asia where P. marneffei is endemic. The Florida patient had recurrent episodes of hemoptysis attributed to bronchitis and bronchiectasis. In spite of therapy with various antibacterial antibiotics for tuberculosis, the granulomatous lesions in the left upper lobe of the lungs persisted. The diagnosis of penicilliosis marneffei was established by isolating and identifying the dimorphic species of Penicillium, P. marneffei. The histopathologic features of the lung tissue included granulomata with central areas of necrosis and neutrophilic infiltration with many yeast-like, tissue-form cells of P. marneffei, which multiplied by a fission rather than a budding process.     

An STE12 homolog from the asexual, dimorphic fungus Penicillium marneffei complements the defect in sexual development of an Aspergillus nidulans steA mutant

Penicillium marneffei is an opportunistic fungal pathogen of humans and the only dimorphic species identified in its genus. At 25 degrees P. marneffei exhibits true filamentous growth, while at 37 degrees P. marneffei undergoes a dimorphic transition to produce uninucleate yeast cells that divide by fission. Members of the STE12 family of regulators are involved in controlling mating and yeast-hyphal transitions in a number of fungi. We have cloned a homolog of the S. cerevisiae STE12 gene from P. marneffei, stlA, which is highly conserved. The stlA gene, along with the A. nidulans steA and Cryptococcus neoformans STE12alpha genes, form a distinct subclass of STE12 homologs that have a C2H2 zinc-finger motif in addition to the homeobox domain that defines STE12 genes. To examine the function of stlA in P. marneffei, we isolated a number of mutants in the P. marneffei-type strain and, in combination with selectable markers, developed a highly efficient DNA-mediated transformation procedure and gene deletion strategy. Deletion of the stlA gene had no detectable effect on vegetative growth, asexual development, or dimorphic switching in P. marneffei. Despite the lack of a detectable function, the P. marneffei stlA gene complemented the sexual defect of an A. nidulans steA mutant. In addition, substitution rate estimates indicate that there is a significant bias against nonsynonymous substitutions. These data suggest that P. marneffei may have a previously unidentified cryptic sexual cycle.     

IGS基因及RAPD标记分析在加特隐球酵母基因分类中的应用

根据ITS1-5.8S-ITS2区域的特异核酸序列变化,加特隐球酵母Cryptococcus gattii(≡新型隐球酵母加特变种Cryptococcu neoformans var.gattii)可分为6种基因型。本研究通过检测加特隐球酵母的IGS基因,发现其IGS序列有着更丰富的变异和信息位点。通过结合加特隐球酵母RAPD(随机扩增的多态性DNA)图谱比较研究,与IGS和ITS的序列分析结果大体一致,说明新近发现的加特隐球酵母ITS8型确实有别于以前报道过的其他加特隐球酵母ITS基因型。研究证明IGS1及IGS2基因片段分析可以作为加特隐球酵母基因分类鉴定中有效的辅助鉴别的分子生物学方法,联合多种基因分类鉴定的方法可以更有效地揭示新型隐球酵母加特变种种内不同基因亚型间的遗传进化关系。     

Detection of Talaromyces marneffei from Fresh Tissue of an Inhalational Murine Pulmonary Model Using Nested PCR

Penicilliosis marneffei, often consecutive to the aspiration of Talaromyces marneffei (Penicillium marneffei), continues to be one of the significant causes of morbidity and mortality in immunocompromised patients in endemic regions such as Southeast Asia. Improving the accuracy of diagnosing this disease would aid in reducing the mortality of associated infections. In this study, we developed a stable and reproducible murine pulmonary model that mimics human penicilliosis marneffei using a nebulizer to deliver Talaromyces marneffei (SUMS0152) conidia to the lungs of BALB/c nude mice housed in exposure chamber. Using this model, we further revealed that nested PCR was sensitive and specific for detecting Talaromyces marneffei in bronchoalveolar lavage fluid and fresh tissues. This inhalation model may provide a more representative analysis tool for studying the development of penicilliosis marneffei, in addition to revealing that nested PCR has a predictive value in reflecting pulmonary infection.