Sporothrix brasiliensis, S. globosa, and S. mexicana, three new Sporothrix species of clinical interest

Sporothrix schenckii is the species responsible for sporotrichosis, a fungal infection caused by the traumatic implantation of this dimorphic fungus. Recent molecular studies have demonstrated that this species constitutes a complex of numerous phylogenetic species. Since the delineation of such species could be of extreme importance from a clinical point of view, we have studied a total of 127 isolates, most of which were received as S. schenckii, including the available type strains of species currently considered synonyms, and also some close morphological species. We have phenotypically characterized all these isolates using different culture media, growth rates at different temperatures, and numerous nutritional tests and compared their calmodulin gene sequences. The molecular analysis revealed that Sporothrix albicans, S. inflata, and S. schenckii var. luriei are species that are clearly different from S. schenckii. The combination of these phenetic and genetic approaches allowed us to propose the new species Sporothrix brasiliensis, S. globosa, and S. mexicana. The key phenotypic features for recognizing these species are the morphology of the sessile pigmented conidia, growth at 30, 35, and 37 degrees C, and the assimilation of sucrose, raffinose, and ribitol.     

Rapid identification of Sporothrix species by T3B fingerprinting

This article describes PCR fingerprinting using the universal primer T3B to distinguish among species of the Sporothrix complex, S. brasiliensis, S. globosa, S. mexicana, and S. schenckii. This methodology generated distinct banding patterns, allowing the correct identification of all 35 clinical isolates at the species level, confirmed by partial calmodulin (CAL) gene sequence analyses. This methodology is simple, reliable, rapid, and cheap, making it an ideal routine identification system for clinical mycology laboratories.     

Antifungal susceptibilities of Sporothrix albicans, S. brasiliensis, and S. luriei of the S. schenckii complex identified in Brazil

We studied 40 strains of the species complex formerly classified as the single species Sporothrix schenckii to identify new species within this complex and evaluate their antifungal susceptibility profiles. Based on phenotypic tests (ability to grow at 37°C, colony diameters, and pigmentation of the colonies, as well as assimilation of sucrose and raffinose) and molecular assays (amplification of a fragment of the calmodulin gene), here we report the identification of S. albicans, S. brasiliensis, S. luriei, and S. schenckii; two isolates of these species were detected as itraconazole-resistant strains.     

Passive immunization with monoclonal antibody against a 70-kDa putative adhesin of Sporothrix schenckii induces protection in murine sporotrichosis

Cell-mediated and innate immunity are considered the most important mechanisms of host defense against fungus infections. However, recent studies demonstrated that specific antibodies show different degrees of protection against mycosis. In a previous study, antigens secreted by Sporothrix schenckii induced a specific humoral response in infected animals, mainly against the 70-kDa molecule, indicating a possible participation of antibodies to this antigen in infection control. In the present study, an IgG1 mAb was produced against a 70-kDa glycoprotein of S. schenckii in order to better understand the effect of passive immunization of mice infected with S. schenckii. Results showed a significant reduction in the number of CFU in organs of mice when the mAb was injected before and during S. schenckii infection. Similar results were observed when T-cell-deficient mice were used. Moreover, in a second schedule treatment, the mAb was injected after infection was established, and again we observed a significant reduction in CFU associated with an increase of IFN-gammaproduction. Also, the 70-kDa antigen is shown to be a putative adhesin present on the surface of this fungus. In conclusion, we report for the first time the protective effect of a specific antibody against S. schenckii.     

Receptor for α1-Microglobulin on T Lymphocytes: Inhibition of Antigen-Induced Interleukin-2 Production

The human plasma protein α₁-microglobulin (α₁m) was found to inhibit the antigen-induced interleukin-2 (IL-2) production of two different mouse T-helper cell hybridomas. α₁m isolated from human plasma and recombinant α₁m isolated from baculovirus-infected insect cell cultures had similar inhibitory effects. Flow cytometric analysis showed a binding of plasma and recombinant α₁m to the T-cell hybridomas as well as to a human T-cell line. Radiolabelled plasma and recombinant α₁m bound to the T-cell hybridomas in a saturable manner and the binding could be eliminated by trypsination of the cells. The affinity constants for the cell binding were calculated to be 0.4–1 × 10⁵  M ⁻¹ using Scatchard plotting, and the number of binding sites per cell was estimated to be 5 × 10⁵–1 × 10⁶. The cell-surface proteins of one of the T-cell hybridomas were radiolabelled, the cells lysed and α₁m-binding proteins isolated by affinity chromatography. SDS–PAGE and autoradiography analysis of the eluate revealed major bands with M _r-values around 70, 35 and 15 kDa. The results thus suggest that α₁m binds to a specific receptor on T cells and that the binding leads to inhibition of antigen-stimulated IL-2 production by T-helper cells.     

Experimental acute respiratory Burkholderia pseudomallei infection in BALB/c mice

Burkholderia pseudomallei is the causative agent of melioidosis, which is considered a potential deliberate release agent. The objective of this study was to establish and characterise a relevant, acute respiratory Burkholderia pseudomallei infection in BALB/c mice. Mice were infected with 100 B. pseudomallei strain BRI bacteria by the aerosol route (approximately 20 median lethal doses). Bacterial counts within lung, liver, spleen, brain, kidney and blood over 5 days were determined and histopathological and immunocytochemical profiles were assessed. Bacterial numbers in the lungs reached approximately 10⁸ cfu/ml at day 5 post-infection. Bacterial numbers in other tissues were lower, reaching between 10³ and 10⁵ cfu/ml at day 4. Blood counts remained relatively constant at approximately 1.0 × 10² cfu/ml. Foci of acute inflammation and necrosis were seen within lungs, liver and spleen. These results suggest that the BALB/c mouse is highly susceptible to B. pseudomallei by the aerosol route and represents a relevant model system of acute human melioidosis.     

New, special stain for histopathological diagnosis of cryptococcosis.

The Masson-Fontana silver stain for melanin was employed for the differentiation of pathogenic fungal species in human or mouse tissues. The fungi studied were Candida albicans, Candida tropicalis, Candida glabrata (Torulopsis glabrata), Cryptococcus neoformans, Cryptococcus bacillisporus, Coccidioides immitis, Blastomyces dermatitidis, Histoplasma capsulatum, Paracoccidioides brasiliensis, Sporothrix schenckii, Rhizopus rhizopodiformis, and Aspergillus fumigatus. The tissue sections stained with Masson-Fontana silver stain showed a dark brown to black color in the wall of cryptococci, whereas the walls of remaining fungal species were hyaline, except for those of S. schenckii. The yeastlike cells of S. schenckii showed faint brown pigment on the wall. Cultures of these fungi showed staining characteristics identical to those of the in vivo results. Cultures of four nonpathogenic Cryptococcus species, Cryptococcus uniguttulatus, Cryptococcus laurentii, Cryptococcus terreus, and Cryptococcus luteolus, were also tested for staining by the Masson-Fontana procedure. Of these, only C. laurentii stained positively, and the pigment on the cell wall was as intense as that of the cells of C. neoformans. These results indicate that the Masson-Fontana silver stain can be used as a specific stain in the histological diagnosis of cryptococcosis.     

Molecular Identification and Phenotypic Characterisation of Sporothrix globosa from Clinical Cases of Eastern Assam,North-east India

Sporotrichosis is known to be endemic in the state of Assam, North-east India, which is situated in the Sub-Himalayan region. This disease is an acute or chronic infection caused by Sporothrix schenckii species complex which currently includes several species of clinical relevance such as Sporothrix brasiliensis, Sporothrix globosa, Sporothrix schenckii sensu stricto, Sporothrix albicans, Sporothrix mexicana, Sporothrix pallida and Sporothrix luriei. S. globosa is the prevalent species in India. Eight culture-positive patients were diagnosed from suspected consecutive cases of two lymphocutaneous and six fixed cutaneous forms over a period of 4 years in a clinical mycology laboratory of a tertiary care centre in Eastern Assam. Phenotypic speciation was inconclusive using the criteria of Marimon et al. because of atypical growth pattern shown by the isolates. Our isolates showed good growth at 37°C ranging from 6 to 27 mm; four of the isolates showed growth of 11–27 mm unlike S. globosa strains reported earlier. Molecular identification was done by sequencing both the internal transcribed spacer (ITS) region and the calmodulin (CAL) protein encoding gene (partial). All the isolates were identified as S. globosa. Molecular confirmation of species using ITS region and CAL protein encoding gene (partial) is necessary for isolates of S. globosa showing atypical biopatterns.     

Brucella detection in blood: comparison of the BacT/Alert standard aerobic bottle, BacT/Alert FAN aerobic bottle and BacT/Alert enhanced FAN aerobic bottle in simulated blood culture

The objective of this study was to compare the performances of the standard aerobic bottle (StAe), FAN aerobic (FANAe) and enhanced FAN aerobic (E-FANAe) (the charcoal component of the FANAe was revised recently to improve the feasibility of Gram smear interpretation) blood culture bottles for BacT/Alert system for the detection of Brucella melitensis in simulated blood culture. Triplicate strains of eight clinical isolates of B. melitensis were studied. Each bottle was inoculated with 5 mL of freshly collected human blood at three different targeted bacterial inocula (10¹, 10² and 10³ CFU/bottle). All bottles were monitored for up to 21 days or until they became positive. The results of time to detection (TTD) on the eight B. melitensis samples were as follows: at 10¹ CFU/bottle, the E-FANAe had a mean TTD significantly shorter than the StAe (48 h vs. 56.2 h, P  < 0.05); and at 10³ CFU/bottle, the FANAe and E-FANAe had a mean TTD significantly shorter than the StAe (41.2 h and 40 h vs. 45.6 h, P  < 0.05). The reproducibilities (no.of positive signals/no.of all bottles) of three bottle systems were as follows: at 10¹ CFU/bottle, the reproducibilities of StAe, FANAe and E-FANAe were 96, 83 and 58%, respectively. At 10³ CFU/bottle, the reproducibilities of StAe, FANAe and E-FANAe were 95, 95 and 91%, respectively. Positive results for the presence of bacteria in Gram smears were confirmed in 68% of StAe, 54% of FANAe and 90% of E-FANAe. In case of suspected brucellosis, the combination of one StAe bottle and one E-FANAe bottle seems to provide the highest and fastest recovery of the organism.     

Detection of Sporothrix schenckii in clinical samples by a nested PCR assay

Cutaneous sporotrichosis is a chronic granulomatous fungal infection caused by Sporothrix schenckii with worldwide distribution. Its traditional diagnosis is time-consuming and difficult to differentiate from that of a clinical sporotrichoid lesion caused by various pathogens. In this study, a nested PCR assay for the detection of S. schenckii was evaluated by using a sequence of 18S rRNA gene as a target. For the examination of specificity and sensitivity, five clinical isolates with 1 ATCC 10213 strain of S. schenckii, 10 strains of clinical common fungi, 3 strains of Mycobacterium spp., Staphylococcus aureus, and normal human skin tissue were used. The expected fragment was amplified from six S. schenckii isolates in the first round and nested PCR but not from other microorganisms and human DNA. Their sequences were 100% identical to the S. schenckii 18S rRNA gene sequence deposited in GenBank. A detection limit of 40 fg of S. schenckii DNA extract was determined with ethidium bromide staining. Serial dilution studies demonstrated that the nested PCR could detect a DNA amount of 1 CFU of S. schenckii in tissue samples. We further investigated the nested PCR assay for the detection of S. schenckii from the tail tissues of 5 experimentally infected mice and from the clinical biopsy specimens of 12 patients with sporotrichosis confirmed by culture or histochemical staining. The nested PCR assay was positive in all 5 infected mice and in 11 of the 12 clinical specimens. The high sensitivity and specificity of this nested PCR indicate that the assay can provide rapid diagnosis with sufficient accuracy to be clinically useful for patients with sporotrichosis.     

Isolation and characterization of Sporothrix schenckii from clinical and environmental sources associated with the largest U.S. epidemic of sporotrichosis.

The largest recorded epidemic of sporotrichosis in the United States occurred in 1988 and involved a total of 84 cases in 15 states. All cases were associated with Wisconsin-grown sphagnum moss. Twenty-one clinical isolates of Sporothrix schenckii and 69 environmental isolates of Sporothrix spp. from the epidemic were characterized and compared. The environmental isolates were recovered from 102 samples of sphagnum moss and other material by using direct plating techniques. Characteristics examined included macroscopic and microscopic morphology, conversion to a yeast phase, exoantigen reactions, and virulence in mice. On the basis of these studies, eight environmental isolates were identified as S. schenckii, five were identified as Ophiostoma stenoceras, and the remainder were identified as Sporothrix species. The environmental isolates of S. schenckii were recovered from moss samples from one Pennsylvania nursery and from three New York State Soil and Water Conservation districts, but none were recovered from moss directly from the bogs in Wisconsin.     

An unbiased stereological study on subpopulations of rat liver macrophages and on their numerical relation with the hepatocytes and stellate cells

Studies on liver macrophages have elucidated their key roles in immunological, fibrotic and regenerative responses, and shown that macrophages are not a homogeneous population. In the rat, two sets of liver macrophages coexist, identified by ED1 and ED2 antibodies. Those sets have different quantitative responses in liver injuries and may have different tasks throughout the injury and recovery phases. Nevertheless, the total number (N), number per gram (N g⁻¹) and proportion of those macrophages in relation to other liver cells has never been quantified using design-based stereology. Thus, we combined immunocytochemistry with those tools to produce an unbiased estimate of the N of ED1⁺ and of ED2⁺ cells. A smooth fractionator sampling scheme was applied to the liver of five male Wistar rats (3 months old), to obtain systematic uniform random sections (30 µm thick); these were immunostained with the monoclonal antibodies: ED1, a pan-macrophagic marker; and ED2, which identifies the completely differentiated macrophages, i.e. Kupffer cells. The N of ED1⁺ cells was 340 × 10⁶, estimated with a coefficient of error (CE) of 0.04, and that of ED2⁺ cells was 283 × 10⁶, with a CE of 0.05. These figures correspond to 10.7% and 8.9%, respectively, of the total liver cells. The new data constitute reference values for correlative inferences. Also, the methodological strategy, by its accuracy and precision, is valuable for future investigations on the liver cell composition in various models of disease, and especially for studying the more subtle variations that occur during the injury and recovery phases.     

Effect of clindamycin in a model of acute murine toxoplasmosis

Objective: To characterize the antitoxoplasma activity of clindamycin in a murine model of acute toxoplasmosis.
Method: Rates of survival and mean survival times of Swiss Webster mice infected intraperitoneally with 10⁶-10² tachyzoites of the RH strain of Toxoplasma gondii treated with clindamycin or sulfamethoxazole (positive control) or untreated (negative control) were compared. Survivors were submitted to examination of untreated brain tissue preparations, intraperitoneal and peroral subinoculations of brain tissue homogenates into fresh mice, and to patho-histology, including immunohistochemistry, of brain and lungs.
Results: The effect of clindamycin treatment (400 mg/kg/day) on infected Swiss Webster mice was inoculum size dependent, ranging from no survivals in animals infected with 10⁶ parasites, to 100% survivals with an inoculum of 10². Treatment initiated 24 h before and at time of infection prolonged mean survival times comparably to sulfamethoxazole, and significantly when compared to untreated controls. In contrast, treatment initiated 48 h postinfection with an inoculum of 10⁶ did not postpone death. In the clindamycin-treated survivors, there was no biological or histologic evidence for the persistence of toxoplasma.
Conclusions: The results obtained show that at an appropriate parasite dose/drug dose ratio, clindamycin is strongly toxoplasmacidal in a murine model of acute toxoplasmosis.     

The impact of substance P signalling on the development of experimental staphylococcal sepsis and arthritis

Substance P (SP), acting on the neurokinin-1 receptor (NK-1R), is a neuropeptide, involved in the inflammatory processes. It promotes vasodilatation and increases vasopermeability, thus ensuing extravasation and accumulation of leucocytes at sites of injury. The aim of this study was to assess the impact of SP signalling on the responses during staphylococcal infection and the accompanying arthritis. Three experiments were performed where NK-1R−/− mice and controls were intravenously infected with different doses of Staphylococcus aureus. Clinical assessment of arthritis was performed as well as histological analysis of bone and cartilage destruction in the joints. In addition, the impact of NK-1R mutation on bacterial load in the kidneys as well as the phagocytic capacity of blood leucocytes were studied. Mice lacking the NK-1R displayed significantly higher bacterial load in the kidneys and significantly more severe synovitis and cartilage/bone destruction than the controls when inoculated with 1.4 × 10⁷ staphylococci. Infection with 3.5 × 10⁸ CFU/mouse induced sepsis. Thus, 11 days after bacterial inoculation 15 of 19 mice in the NK-1R−/− group had died versus 8 of 15 in the control group. Phagocytosis test revealed that significantly fewer macrophages from NK-1R−/− mice were able to phagocytose S. aureus when compared with macrophages from congenic control mice. Blocking the biological responses to substance P via its receptor NK-1R results in a less efficient clearance of bacteria leading to more severe arthritic lesions in mice.     

Antibiotic-resistant obligate anaerobes during exacerbations of cystic fibrosis patients

Pseudomonas aeruginosa and Staphylococcus aureus are thought to cause the majority of lung infections in patients with cystic fibrosis (CF). However, other bacterial pathogens may contribute to the pathophysiology of lung disease. Here, obligate anaerobes were identified in a cross-sectional study, and cell numbers and antibiotic susceptibilities of facultative and obligate anaerobes from 114 sputum samples from nine children and 36 adults with CF were determined. Furthermore, in 12 CF patients, we investigated whether conventional intravenous antibiotic therapy, administered during acute exacerbations, would affect the numbers of obligate anaerobes. Fifteen genera of obligate anaerobes were identified in 91% of the CF patients. Cell numbers (mean: 2.2 × 10⁷ ± standard deviation 6.9 × 10⁷ CFU/mL of sputum sample) were comparable to those of P. aeruginosa and S. aureus . Staphylococcus saccharolyticus and Peptostreptococcus prevotii were most prevalent. Infection with P. aeruginosa did not increase the likelihood that obligate anaerobes are present in sputum specimens. Single obligate anaerobic species persisted for up to 11 months in sputum plugs in vivo . Patients with and without obligate anaerobes in sputum specimens did not differ in lung function. Intravenous therapy directed against P. aeruginosa during acute exacerbations increased lung function, but did not reduce the numbers of obligate anaerobes. Obligate anaerobic species differed widely in their patterns of resistance against meropenem, piperacillin–tazobactam, clindamycin, metronidazole and ceftazidime. In 58% of patients with acute exacerbations, obligate anaerobes were detected that were resistant to the antibiotics used for treatment. Antibiotic therapy, optimized to target anaerobes in addition to P. aeruginosa , may improve the management of CF lung disease.     

Synthesis of melanin-like pigments by Sporothrix schenckii in vitro and during mammalian infection

Melanin has been implicated in the pathogenesis of several important human fungal pathogens. Existing data suggest that the conidia of the dimorphic fungal pathogen Sporothrix schenckii produce melanin or melanin-like compounds; in this study we aimed to confirm this suggestion and to demonstrate in vitro and in vivo production of melanin by yeast cells. S. schenckii grown on Mycosel agar produced visibly pigmented conidia, although yeast cells grown in brain heart infusion and minimal medium broth appeared to be nonpigmented macroscopically. However, treatment of both conidia and yeast cells with proteolytic enzymes, denaturant, and concentrated hot acid yielded dark particles similar in shape and size to the corresponding propagules, which were stable free radicals consistent with identification as melanins. Melanin particles extracted from S. schenckii yeast cells were used to produce a panel of murine monoclonal antibodies (MAbs) which labeled pigmented conidia, yeast cells, and the isolated particles. Tissue from hamster testicles infected with S. schenckii contained fungal cells that were labeled by melanin-binding MAbs, and digestion of infected hamster tissue yielded dark particles that were also reactive. Additionally, sera from humans with sporotrichosis contained antibodies that bound melanin particles. These findings indicate that S. schenckii conidia and yeast cells can produce melanin or melanin-like compounds in vitro and that yeast cells can synthesize pigment in vivo. Since melanin is an important virulence factor in other pathogenic fungi, this pigment may have a similar role in the pathogenesis of sporotrichosis.     

Deoxyribonucleic Acid Base Composition and Hybridization Studies on the Human Pathogen Sporothrix schenckii and Ceratocystis Species

Deoxyribonucleic acid (DNA) base composition, as measured by guanine plus cytosine (G + C) content, was determined in 17 strains of Sporothrix schenckii and Ceratocystis species. The average G + C content for S. schenckii was 54.7 +/- 0.2 mol%. No strain of Ceratocystis, with the exception of C. minor, gave values for G + C content similar to those obtained for S. schenckii. All DNA samples from strains of S. schenckii cross-hybridized with high percentages of relative binding. DNA samples from C. stenoceras and C. pilifera showed low degrees of homology with S. schenckii DNA. The DNA from C. minor hybridized with 75% binding with that from a strain of S. schenckii, as compared with the homologous reaction.     

Differential induction of Th1-prone immunity by human dendritic cells activated with Sporothrix schenckii of cutaneous and visceral origins to determine their different virulence

Sporotrichosis is caused by a thermo-dependent dimorphic fungus, Sporothrix schenckii. The major clinical manifestations occur in the skin; however, cases of visceral manifestations have also been increasingly reported with some being observed in immune compromised patients. Different virulence of individual S. schenckii strain as well as immune status of the host could contribute to form such different clinical manifestations. Thus, the purpose of the study was to investigate whether different virulence of individual S. schenckii could be a factor for such clinical difference. We investigated the interactions between human monocyte-derived dendritic cells (MoDCs) and S. schenckii, assessed by (i) morphological features, (ii) surface marker expressions, cytokine productions, (iii) signaling pathways and (iv) allostimulatory activity of the activated MoDCs. Immature MoDCs, obtained from peripheral blood monocytes supplemented with granulocyte macrophage colony-stimulating factor and IL-4, were stimulated with S. schenckii strains of both yeasts and conidia forms of different origins (cutaneous isolates: KMU4649, IFM5906 and IFM46010; visceral isolates: KMU4648, IFM41598 and ATCC26331) to be used for various assays. Through the analysis, we found that the cutaneous S. shenckii of cutaneous origins were more potent to activate MoDCs to induce strong T(h)1 response, as evidenced by abundant IFN-gamma production, while the S. shenckii of visceral origins induced only minimal dendritic cell activation and T(h)1 induction. The p38 mitogen-activated protein kinase and c-Jun N-terminal kinase signaling pathways appeared to be associated with the differential activation of the MoDCs by S. schenckii of cutaneous and the visceral origins. Overall, we concluded that the differential activation of MoDCs by S. schenckii of cutaneous and visceral origins to induce T(h)1 response, other than immune status or the host, may be a factor for their different clinical manifestations.     

Experimental sporotrichosis in Syrian hamsters.

Syrian hamsters were infected with Sporothrix schenckii by subcutaneous footpad inoculation. Two types of infection could be uniformly induced: a self-limited, lymphatic infection resembling the classical disease in humans, and a generalized nonfatal infection. An infecting dose of approximately 5,300 yeast cells produced the localized subcutaneous-lymphatic disease which was limited to a single limb. In contrast, a 1,000-fold increase in the inoculum temporarily overwhelmed the animals' defense mechanisms, producing a systemic infection involving the liver and spleen. These models were used to demonstrate the development of increased resistance to subsequent infection following either infection or active immunization with ribosomal fractions or trypsinized cell wall antigens of S. schenckii incorporated in Freund complete adjuvant. Agglutination titers were detectable in all animals that were either infected or immunized. In one group of infected animals, the titers persisted for at least 1 year after three booster doses of Formalin-killed S. schenckii. The ability to produce an infection in hamsters which closely resembles the disease seen in humans makes the animal a good model with which to study experimental sporotrichosis.     

Kinetics of the Reaction between a Monoclonal IgM Anti-I and Rabbit Red Cells

Rabbit red cells were found to bind a maximum of 1.8 × 10⁵ IgM anti-I-molecules. A K-value of 5.0 ± 0.8 × 10⁸ 1/M was determined for the reaction between the cells and intact anti-I at 5°C; the K-value was little affected by changes in temperature. Hybrid 17S and 8S molecules composed of varying amounts of cold agglutinin and inactive IgM were prepared by reduction with 2-mercaptoethanol and reassociation. The results obtained with the reassociated products indicate random reassociation at the half subunit level. It is concluded that a 17S fraction with four or more active half subunits shows binding similar to intact IgM, while 8S fractions with two active half subunits show binding similar to the cysteine-reduced 8S IgM.