Clinical and Microbiological Characteristics of Proven Invasive Aspergillosis Due to Rare/Cryptic Species in Allogeneic Hematopoietic Stem Cell Transplant Recipients.

There are few reports on the clinical course of proven invasive aspergillosis (IA) due to rare/cryptic species in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients. We retrospectively reviewed the electronic medical records of patients who underwent allo-HSCT between January 2012 and December 2018. Of 934 allo-HSCT recipients, 10 were diagnosed with proven IA and 61 were diagnosed with probable IA. DNA sequencing was performed in cases of proven IA, and Aspergillus could be identified to the species level in 8 of the 10 cases. Three were due to A. fumigatus, and 5 were due to rare/cryptic Aspergillus species, namely, A. turcosus, A. felis, A. viridinutans, A. nidulans, and A. calidoustus. In these 8 patients, no patients with IA due to A. fumigatus died, whereas 3 of the 5 with IA due to rare/cryptic species died within 12 weeks. The 2 surviving cases of IA due to rare/cryptic species were treated with surgical resection and antifungal treatment. Susceptibility testing for cryptic species in 4 cases showed an amphotericin B MIC > 1 mg/L in 3 cases, itraconazole MIC > 1 mg/L in 2 cases, and voriconazole MIC > 1 mg/L in 2 cases. In conclusion, more than half of the causative pathogens of proven IA were rare/cryptic species, so it is important to accurately identify the Aspergillus species. In addition, surgical treatment might be an important option in cases of proven IA, given the possibility that the causative organisms are azole-resistant A. fumigatus or rare/cryptic species.

Factors Associated with Breakthrough Fungemia Caused by Candida, Trichosporon, or Fusarium Species in Patients with Hematological Disorders.

Limited data are available on breakthrough fungemia, defined as fungemia that develops on administration of antifungal agents, in patients with hematological disorders. We reviewed the medical and microbiological records of adult patients with hematological diseases who had breakthrough fungemia between January 2008 and July 2019 at Toranomon Hospital and Toranomon Hospital Kajigaya in Japan. A total of 121 cases of breakthrough fungemia were identified. Of the 121 involved patients, 83, 11, 5, and 22 were receiving micafungin, voriconazole, itraconazole, and liposomal amphotericin B, respectively, when the breakthrough occurred. Of the 121 causative breakthrough fungal strains, 96 were Candida species, and the rest were 13 cases of Trichosporon species, 7 of Fusarium species, 2 of Rhodotorula mucilaginosa, and 1 each of Cryptococcus neoformans, Exophiala dermatitidis, and Magnusiomyces capitatus. The crude 14-day mortality rate of breakthrough fungemia was 36%. Significant independent factors associated with the crude 14-day mortality rate were age of ≥60 years (P = 0.011), chronic renal failure (P = 0.0087), septic shock (P < 0.0001), steroid administration (P = 0.0085), and liposomal amphotericin B breakthrough fungemia (P = 0.0011). An absolute neutrophil count of >500/µL was significantly more common in candidemia in the multivariate analysis (P = 0.0065), neutropenia and nonallogeneic hematopoietic stem cell transplants were significantly more common in Trichosporon fungemia (P = 0.036 and P = 0.033, respectively), and voriconazole breakthrough fungemia and neutropenia were significantly more common in Fusarium fungemia (P = 0.016 and P = 0.016, respectively). The epidemiological and clinical characteristics of breakthrough fungemia of patients with hematological disorders were demonstrated. Some useful factors to predict candidemia, Trichosporon fungemia, and Fusarium fungemia were identified.

Aureobasidium melanigenum catheter-related bloodstream infection: a case report.

BACKGROUND: Aureobasidium melanigenum is a ubiquitous dematiaceous fungus that rarely causes invasive human infections. Here, we present a case of Aureobasidium melanigenum bloodstream infection in a 20-year-old man with long-term catheter use. CASE PRESENTATION: A 20-year-old man receiving home care with severe disabilities due to cerebral palsy and short bowel syndrome, resulting in long-term central venous catheter use, was referred to our hospital with a fever. After the detection of yeast-like cells in blood cultures on day 3, antifungal therapy was initiated. Two identification tests performed at a clinical microbiological laboratory showed different identification results: Aureobasidium pullulans from matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and Cryptococcus albidus from a VITEK2 system. Therefore, we changed the antifungal drug to liposomal amphotericin B. The fungus was identified as A. melanigenum by DNA sequence-based analysis. The patient recovered with antifungal therapy and long-term catheter removal. CONCLUSION: It is difficult to correctly identify A. melanigenum by routine microbiological testing. Clinicians must pay attention to the process of identification of yeast-like cells and retain A. melanigenum in cases of refractory fungal infection.

Surfeit 4 Contributes to the Replication of Hepatitis C Virus Using Double-Membrane Vesicles.

A number of positive-strand RNA viruses, such as hepatitis C virus (HCV) and poliovirus, use double-membrane vesicles (DMVs) as replication sites. However, the role of cellular proteins in DMV formation during virus replication is poorly understood. HCV NS4B protein induces the formation of a "membranous web" structure that provides a platform for the assembly of viral replication complexes. Our previous screen of NS4B-associated host membrane proteins by dual-affinity purification, liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS), and small interfering RNA (siRNA) methods revealed that the Surfeit 4 (Surf4) gene, which encodes an integral membrane protein, is involved in the replication of the JFH1 subgenomic replicon. Here, we investigated in detail the effect of Surf4 on HCV replication. Surf4 affects HCV replication in a genotype-independent manner, whereas HCV replication does not alter Surf4 expression. The influence of Surf4 on HCV replication indicates that while Surf4 regulates replication, it has no effect on entry, translation, assembly, or release. Analysis of the underlying mechanism showed that Surf4 is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs and the structural integrity of RNA replication complexes. Surf4 also participates in the replication of poliovirus, which uses DMVs as replication sites, but it has no effect on the replication of dengue virus, which uses invaginated/sphere-type vesicles as replication sites. These findings clearly show that Surf4 is a novel cofactor that is involved in the replication of positive-strand RNA viruses using DMVs as RNA replication sites, which provides valuable clues for DMV formation during positive-strand RNA virus replication.IMPORTANCE Hepatitis C virus (HCV) NS4B protein induces the formation of a membranous web (MW) structure that provides a platform for the assembly of viral replication complexes. The main constituents of the MW are double-membrane vesicles (DMVs). Here, we found that the cellular protein Surf4, which maintains endoplasmic reticulum (ER)-Golgi intermediate compartments and the Golgi compartment, is recruited into HCV RNA replication complexes by NS4B and is involved in the formation of DMVs. Moreover, Surf4 participates in the replication of poliovirus, which uses DMVs as replication sites, but has no effect on the replication of dengue virus, which uses invaginated vesicles as replication sites. These results indicate that the cellular protein Surf4 is involved in the replication of positive-strand RNA viruses that use DMVs as RNA replication sites, providing new insights into DMV formation during virus replication and potential targets for the diagnosis and treatment of positive-strand RNA viruses.

Lect2 Controls Inflammatory Monocytes to Constrain the Growth and Progression of Hepatocellular Carcinoma.

Leukocyte cell-derived chemotaxin-2 (LECT2) was originally identified as a hepatocyte-secreted chemokine-like factor and a positive target of ß-catenin signaling. Here, we dissected out the mechanisms by which LECT2 modulates hepatocellular carcinoma (HCC) development using both HCC mouse models and human HCC samples. We have demonstrated that LECT2 exhibits dual abilities as it has profound repercussions on the tumor phenotype itself and the immune microenvironment. Its absence confers Ctnnb-1-mutated tumor hepatocytes a stronger ability to undergo epithelial to mesenchymal transition and fosters the accumulation of pejorative inflammatory monocytes harboring immunosuppressive properties and strong tumor-promoting potential. Consistent with our HCC mouse model, a low level of LECT2 in human HCC is strongly associated with high tumor grade and the presence of inflammatory infiltrates, emphasizing the clinical value of LECT2 in human liver tumorigenesis. Conclusion: Our findings have demonstrated that LECT2 is a key player in liver tumorigenesis because its absence reshapes the tumor microenvironment and the tumor phenotype, revealing LECT2 as a promising immunotherapeutic option for HCC.

IL-10 promoter transactivation by the viral K-RTA protein involves the host-cell transcription factors, specificity proteins 1 and 3.

Kaposi's sarcoma-associated herpesvirus (KSHV)/human herpesvirus-8 (HHV-8) causes a persistent infection, presenting latent and lytic replication phases during its life cycle. KSHV-related diseases are associated with deregulated expression of inflammatory cytokines, including IL-6 and IL-10, but the mechanisms underlying this dysregulation are unclear. Herein, we report a molecular mechanism for KSHV-induced IL-10 gene expression. KSHV replication and transcription activator (K-RTA) is a molecular switch for the initiation of expression of viral lytic genes, and we describe, for the first time, that K-RTA significantly activates the promoter of the human IL-10 gene. Of note, mutations involving a basic region of K-RTA reduced the association of K-RTA with the IL-10 promoter. Moreover, the host-cell transcription factors, specificity proteins (SP) 1 and 3, play a pivotal cooperative role in K-RTA-mediated transactivation of the IL-10 promoter. K-RTA can interact with SP1 and SP3 directly in vitro, and electrophoresis mobility shift assays (EMSAs) revealed co-operative interaction involving K-RTA, SP1, and SP3 in binding to the IL-10 promoter. As DNase I footprinting assays indicated that K-RTA did not affect SP3 binding to the IL-10 promoter, SP3 can function to recruit K-RTA to the IL-10 promoter. These findings indicate that K-RTA can directly contribute to IL-10 up-regulation via a functional interplay with the cellular transcription factors SP1 and SP3.

CRISPR/Cas9 Genome Editing To Demonstrate the Contribution of Cyp51A Gly138Ser to Azole Resistance in Aspergillus fumigatus.

A pan-azole-resistant Aspergillus fumigatus strain with the cyp51A mutations Gly138Ser and Asn248Lys was isolated from a patient receiving long-term voriconazole treatment. PCR fragments containing cyp51A with the mutations were introduced along with the Cas9 protein and single guide RNA into the azole-resistant/susceptible strains. Recombinant strains showed increased susceptibility via the replacement of Ser138 by glycine. Genetic recombination, which has been hampered thus far in clinical isolates, can now be achieved using CRISPR/Cas9 genome editing.

Micafungin Breakthrough Fungemia in Patients with Hematological Disorders.

Limited data are available on micafungin breakthrough fungemia (MBF), fungemia that develops on administration of micafungin, in patients with hematological disorders. We reviewed medical and microbiological records of patients with hematological disorders who developed MBF between January 2008 and June 2015. A total of 39 patients with MBF were identified, and Candida (30 strains) and non-Candida (9 strains) fungal species were recognized as causative strains. Among 35 stored strains, Candida parapsilosis (14 strains), Trichosporon asahii (7 strains), Candida glabrata (5 strains), and other fungal species (9 strains) were identified by sequencing. Neutropenia was identified as an independent predictor of non-Candida fungemia (P = 0.023). T. asahii was the most common causative strain (7/19) during neutropenia. The 14-day crude mortality rate of patients treated with early micafungin change (EMC) to other antifungal agents was lower than that of the patients not treated with EMC (14% versus 43%, P = 0.044). Most of the stored causative Candida strains were susceptible (80%) or showed wild-type susceptibility (72%) to micafungin. The MICs of voriconazole for T. asahii were low (range, 0.015 to 0.12 µg/ml), whereas the MICs of amphotericin B for T. asahii were high (range, 2 to 4 µg/ml). MBF caused by non-Candida fungus should be considered, especially in patients with neutropenia. EMC could improve early mortality. Based on epidemiology and drug susceptibility profiling, empirical voriconazole-containing therapy might be suitable for treating MBF during neutropenia to cover for T. asahii.

Intraspecies variation in the efficacy of adjunctive recombinant interferon-γ therapy against cryptococcal meningoencephalitis in mice.

The efficacy of recombinant interferon γ (rIFN-γ) for cryptococcal meningoencephalitis has been poorly understood. Compared to Cryptococcus gattii, rIFN-γ significantly improved the survival in experimental meningoencephalitis due to Cryptococcus neoformans. The number of phagocytic macrophages and the levels of inflammatory cytokines production for ex vivo co-incubation with C. neoformans were increased after rIFN-γ stimulation but not C. gattii. Intraspecies differences of phagocytosis by the rIFN-γ-activated macrophages might be associated to the severity of cryptococcal infection.

Crystal Structure of Human Leukocyte Cell-derived Chemotaxin 2 (LECT2) Reveals a Mechanistic Basis of Functional Evolution in a Mammalian Protein with an M23 Metalloendopeptidase Fold.

Human leukocyte cell-derived chemotaxin 2 (LECT2), which is predominantly expressed in the liver, is a multifunctional protein. LECT2 is becoming a potential therapeutic target for several diseases of worldwide concern such as rheumatoid arthritis, hepatocellular carcinoma, and obesity. Here, we present the crystal structure of LECT2, the first mammalian protein whose structure contains an M23 metalloendopeptidase fold. The LECT2 structure adopts a conserved Zn(II) coordination configuration but lacks a proposed catalytic histidine residue, and its potential substrate-binding groove is blocked in the vicinity of the Zn(II)-binding site by an additional intrachain loop at the N terminus. Consistent with these structural features, LECT2 was found to be catalytically inactive as a metalloendopeptidase against various types of peptide sequences, including pentaglycine. In addition, a surface plasmon resonance analysis demonstrated that LECT2 bound to the c-Met receptor with micromolar affinity. These results indicate that LECT2 likely plays its critical roles by acting as a ligand for the corresponding protein receptors rather than as an enzymatically active peptidase. The intrachain loop together with the pseudo-active site groove in LECT2 structure may be specific for interactions between LECT2 and receptors. Our study reveals a mechanistic basis for the functional evolution of a mammalian protein with an M23 metalloendopeptidase fold and potentially broadens the implications for the biological importance of noncatalytic peptidases in the M23 family.

Clinical and Microbiological Characteristics of Breakthrough Candidemia in Allogeneic Hematopoietic Stem Cell Transplant Recipients in a Japanese Hospital.

Few data on breakthrough candidemia (BC), defined as candidemia that develops on administration of antifungal agents (AFAs), in allogeneic hematopoietic stem cell transplant (allo-HSCT) recipients are available. The medical and microbiological records of recipients of an allo-HSCT obtained between December 2008 and December 2014 were reviewed. Of 768 allo-HSCT cases, 26 developed BC. Among the 26 causative strains, 22 strains were stored and identified by sequencing. The following species were isolated: Candida parapsilosis (9 strains), C. glabrata (4 strains), C. guilliermondii (3 strains), and other Candida species (6 strains). The AFAs being used when BC developed were micafungin (17 cases), liposomal amphotericin B (5 cases), itraconazole (2 cases), and voriconazole (2 cases). All 17 cases who developed BC during micafungin administration were administered 150 mg/day of micafungin. The susceptibilities of the causative Candida species to the administered AFAs when breakthrough occurred ranged from susceptible to resistant. Especially, 85% of the Candida species that caused BC during micafungin administration were susceptible to micafungin. Additionally, 75% of the strains were wild type for susceptibility to the administered AFAs when breakthrough occurred. Systemic steroid administration and a longer severe neutropenic phase (≥5 days) were independent risk factors for BC (P = 0.016 and P = 0.015, respectively). BC developed in allo-HSCT recipients even when they received a sufficient dose of AFA, including micafungin, to which the causative Candida species were susceptible and/or had wild-type susceptibility in vitro Systemic steroid administration and a longer severe neutropenic phase were host-based factors associated with BC.

Prolactin Regulatory Element Binding Protein Is Involved in Hepatitis C Virus Replication by Interaction with NS4B.

UNLABELLED: It has been proposed that the hepatitis C virus (HCV) NS4B protein triggers the membranous HCV replication compartment, but the underlying molecular mechanism is not fully understood. Here, we screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis and identified 202 host proteins. Subsequent screening of replicon cells with small interfering RNA identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. The interaction between PREB and NS4B was confirmed by immunoprecipitation, immunofluorescence, and proximity ligation assays. PREB colocalized with double-stranded RNA and the newly synthesized HCV RNA labeled with bromouridine triphosphate in HCV replicon cells. Furthermore, PREB shifted to detergent-resistant membranes (DRMs), where HCV replication complexes reside, in the presence of NS4B expression in Huh7 cells. However, a PREB mutant lacking the NS4B-binding region (PREBd3) could not colocalize with double-stranded RNA and did not shift to the DRM in the presence of NS4B. These results indicate that PREB locates at the HCV replication complex by interacting with NS4B. PREB silencing inhibited the formation of the membranous HCV replication compartment and increased the protease and nuclease sensitivity of HCV replicase proteins and RNA in DRMs, respectively. Collectively, these data indicate that PREB promotes HCV RNA replication by participating in the formation of the membranous replication compartment and by maintaining its proper structure by interacting with NS4B. Furthermore, PREB was induced by HCV infection in vitro and in vivo. Our findings provide new insights into HCV host cofactors. IMPORTANCE: The hepatitis C virus (HCV) protein NS4B can induce alteration of the endoplasmic reticulum and the formation of a membranous web structure, which provides a platform for the HCV replication complex. The molecular mechanism by which NS4B induces the membranous HCV replication compartment is not understood. We screened for NS4B-associated membrane proteins by tandem affinity purification and proteome analysis, followed by screening with small interfering RNA. We identified prolactin regulatory element binding (PREB) to be a novel HCV host cofactor. PREB is induced by HCV infection and recruited into the replication complex by interaction with NS4B. Recruited PREB promotes HCV RNA replication by participating in the formation of the membranous HCV replication compartment. To our knowledge, the effect of NS4B-binding protein on the formation of the membranous HCV replication compartment is newly described in this report. Our findings are expected to provide new insights into HCV host cofactors.

Identification of a Novel Rhizopus-specific Antigen by Screening with a Signal Sequence Trap and Evaluation as a Possible Diagnostic Marker of Mucormycosis.

Mucormycosis is the second most common mould infection, often indistinguishable from other invasive mould infections such as aspergillosis. Although an appropriate antifungal therapy is effective at an early stage of the infection, there is no reliable diagnostic method for decision making. Thus, it is necessary to develop an efficient method that can detect mucormycosis rapidly and accurately. We searched for secreted or membrane-bound proteins of Rhizopus oryzae, which is the most common pathogen of mucormycosis, using the method of a signal sequence trap by retrovirus-mediated expression (SST-REX). Among the identified proteins, a Rhizopus-specific antigen was selected as a candidate, and efficacy of this specific antigen was evaluated using R. oryzae-infected mice. Of 302 clones obtained from the SST-REX library, a hypothetical protein (23 kDa, named "protein RSA") was selected as a candidate because of its highest prevalence of clones. Protein RSA was detected at significantly higher concentrations in serum and in lung homogenates of the infected mice as compared to those of uninfected mice. Our study indicates that protein RSA may be a promising biomarker of R. oryzae infection. SST-REX may be useful for comprehensive screening of prospective eukaryotic biomarkers of intractable mould infections.

Interferon-γ promotes phagocytosis of Cryptococcus neoformans but not Cryptococcus gattii by murine macrophages.

Among invasive fungal infections, cryptococcosis caused by inhalation of Cryptococcus neoformans or Cryptococcus gattii is particularly dangerous because it can disseminate to the central nervous system and cause life-threatening meningitis or meningoencephalitis. Previous reports described significant differences in the histopathological features of C. neoformans and C. gattii infection, such as greater pathogen proliferation and a limited macrophage response in mouse lung infected by C. gattii. To elucidate the difference in pathogenicity of these two Cryptococcus species, we investigated the interaction of C. neoformans and C. gattii with murine macrophages, the first line of host defense, by confocal laser microscopy. Only thin-capsulated, and not thick-capsulated C. neoformans and C. gattii were phagocytosed by macrophages. Preactivation with interferon-γ increased the phagocytic rate of thin-capsulated C. neoformans up to two-fold, but did not promote phagocytosis of thin-capsulated C. gattii. Lipopolysaccharide preactivation or Aspergillus fumigatus conidia co-incubation had no effect on internalization of thin-capsulated C. neoformans or C. gattii by macrophages. Phagocytosis of live thin-capsulated C. neoformans, but not that of live thin-capsulated C. gattii, induced interleukin-12 release from macrophages. However, phagocytosis of heat-killed or paraformaldehyde-fixed thin-capsulated C. neoformans did not increase IL-12 release, showing that the internalization of live yeast is important for initiating the immune response during C. neoformans-macrophage interactions. Our data suggest that macrophage response to C. gattii is limited compared with that to C. neoformans and that these results may partially explain the limited immune response and the greater pathogenicity of C. gattii.

The Candida glabrata sterol scavenging mechanism, mediated by the ATP-binding cassette transporter Aus1p, is regulated by iron limitation.

During disseminated infection by the opportunistic pathogen Candida glabrata, uptake of sterols such as serum cholesterol may play a significant role during pathogenesis. The ATP-binding cassette transporter Aus1p is thought to function as a sterol importer and in this study, we show that uptake of exogenous sterols occurred under anaerobic conditions in wild-type cells of C. glabrata but not in AUS1-deleted mutant (aus1Δ) cells. In aerobic cultures, growth inhibition by fluconazole was prevented in the presence of serum, and AUS1 expression was upregulated. Uptake of sterol by azole treated cells required the presence of serum, and sterol alone did not reverse FLC inhibition of growth. However, if iron availability in the growth medium was limited by addition of the iron chelators ferrozine or apo-transferrin, growth of wild-type cells, but not aus1Δ cells, was rescued. In a mouse model of disseminated infection, the C. glabrata aus1Δ strain caused a significantly decreased kidney fungal burden than the wild-type strain or a strain in which AUS1 was restored. We conclude that sterol uptake in C. glabrata can occur in iron poor environment of host tissues and thus may contribute to C. glabrata pathogenesis.

Expression, high-pressure refolding and purification of human leukocyte cell-derived chemotaxin 2 (LECT2).

Human leukocyte cell-derived chemotaxin 2 (LECT2) is a chemotactic factor for neutrophils and a 16-kDa secreted protein consisting of 133 amino acids with three intramolecular disulfide bonds. Here, we propose an efficient method for the preparation of human LECT2 using a high hydrostatic pressure (HHP, 200 MPa) refolding technique. When LECT2 was over-expressed in Escherichia coli cells, most of LECT2 molecules became insoluble inclusion bodies (IBs). HHP was applied to the refolding of LECT2 from insoluble IBs, which dramatically improved the yield of the active LECT2. CD and NMR measurements demonstrated that the refolded LECT2 had a tertiary structure indistinguishable from the solubly expressed LECT2. In addition, both the refolded and solubly expressed LECT2 showed the same level of chemotactic activity.

The mannan of Candida albicans lacking ß-1,2-linked oligomannosides increases the production of inflammatory cytokines by dendritic cells.

Mannans are mannose polymers attached to cell wall proteins in all Candida species, including the pathogenic fungus Candida albicans. Mannans are sensed by pattern recognition receptors expressed on innate immune cells. However, the detailed structural patterns affecting immune sensing are not fully understood because mannans have a complex structure that includes α- and ß-mannosyl linkages. In this study, we focused on the ß-1,2-mannosides of N-linked mannan in C. albicans because this moiety is not present in the non-pathogenic yeast Saccharomyces cerevisiae. To investigate the impact of ß-1,2-mannosides on immune sensing, we constructed a C. albicans ∆mnn4/∆bmt1 double deletant. Thin-layer chromatography and nuclear magnetic resonance analyses revealed that the deletant lacked ß-1,2-mannosides in N-linked mannan. Mannans lacking the ß-1,2-mannosides induced the production of higher levels of inflammatory cytokines, including IL-6, IL-12p40 and TNF-α, in mice dendritic cells compared to wild-type mannan. Our data show that ß-1,2-mannosides in N-linked mannan reduce the production of inflammatory cytokines by dendritic cells.

Crystallization and preliminary X-ray analysis of human leukocyte cell-derived chemotaxin 2 (LECT2).

Human leukocyte cell-derived chemotaxin 2 (LECT2) is a chemotactic factor for neutrophils that plays multifunctional roles in liver regeneration, regulation of neuritic development and proliferation of chondrocytes and osteoblasts. In addition, the C-terminal region of LECT2 belongs to the zinc metalloendopeptidase M23 (PF01551) family. Purified LECT2 was crystallized using the sitting-drop vapour-diffusion method at 293 K. Crystals of selenomethionine-substituted LECT2 that diffracted X-rays to 1.94 Šresolution were obtained using a reservoir solution consisting of 0.2 M ammonium sulfate, 0.1 M HEPES pH 7.5, 25%(w/v) PEG 8000. The crystal belonged to space group P212121, with unit-cell parameters a=59.4, b=63.5, c=64.0 Å. The calculated Matthews coefficient (VM=2.10 Å3 Da(-1), solvent content 40%) indicates that the crystal consists of two molecules per asymmetric unit.

Application of nested PCR for diagnosis of histoplasmosis.

Histoplasmosis is a fungal infection that, although not endemic in Japan, has seen a rise in the number of Japanese cases since the mid-1980s. Diagnosis of the disease is not straightforward, and the main method of detection, fungal culture (which has biosafety-related issues), is of low sensitivity in general. Alternative methods that depend on antibody or antigen detection have had limited use. We have developed a histoplasmosis detection method based on PCR amplification of the Histoplasma capsulatum M antigen gene. We compared this method with fungal culture and serological diagnostic techniques. Among five cases that were finally diagnosed as histoplasmosis, the fungal culture method was only successful in identifying one such case. Although the presence of anti-H. capsulatum antibodies was confirmed in three cases, our PCR method identified four of five cases of histoplasmosis. The performance of our PCR method could not be compared with the antigen detection method, which is used in the United States but is not routinely used in Japan. However, the PCR method was shown to have high sensitivity and specificity for H. capsulatum. Although the number of histoplasmosis cases examined in this study was small, our data suggest that the molecular diagnosis technique has potential for increasing the reliability of histoplasmosis diagnosis when used in combination with established methods.