Efficacy of Liposomal Nystatin in a Rabbit Model of Cryptococcal Meningitis.

Cryptococcal meningitis (CM) causes significant global morbidity and mortality. Current therapeutic strategies rely on deoxycholated or liposomal forms of the polyene amphotericin B. Nystatin is also a polyene with broad-spectrum antimicrobial activity. Treatment with systemic nystatin has been limited by toxicity, which is a consistent challenge with polyene therapeutics. One mechanism to improve the toxicity is usage of a liposomal form of the active agent. Previous data from a murine candidemia model indicated that liposomal nystatin may be an effective antifungal drug formulation. Since the rabbit model of CM is a highly predictive preclinical system for evaluating antifungal therapeutics, we tested the effectiveness of two doses of daily liposomal nystatin, 3 and 8 mg/kg in the rabbit model of CM. Treatment with liposomal nystatin in this model did not reduce the fungal burden in the cerebrospinal fluid. A subsequent clinical trial also did not find activity in a human population. These data indicate that liposomal nystatin in the current form and at the tested dosages is not an effective therapy for CM. The data provide further evidence for the predictive power of the rabbit model of CM as a vital preclinical system for testing novel antifungal therapeutics for CM.

A ketogenic diet enhances fluconazole efficacy in murine models of systemic fungal infection.

Invasive fungal infections are a significant public health concern, with mortality rates ranging from 20% to 85% despite current treatments. Therefore, we examined whether a ketogenic diet could serve as a successful treatment intervention in murine models of Cryptococcus neoformans and Candida albicans infection in combination with fluconazole-a low-cost, readily available antifungal therapy. The ketogenic diet is a high-fat, low-carbohydrate diet that promotes fatty acid oxidation as an alternative to glycolysis through the production of ketone bodies. In this series of experiments, mice fed a ketogenic diet prior to infection with C. neoformans and treated with fluconazole had a significant decrease in fungal burden in both the brain (mean 2.66 ± 0.289 log10 reduction) and lung (mean 1.72 ± 0.399 log10 reduction) compared to fluconazole treatment on a conventional diet. During C. albicans infection, kidney fungal burden of mice in the keto-fluconazole combination group was significantly decreased compared to fluconazole alone (2.37 ± 0.770 log10-reduction). Along with higher concentrations of fluconazole in the plasma and brain tissue, fluconazole efficacy was maximized at a significantly lower concentration on a keto diet compared to a conventional diet, indicating a dramatic effect on fluconazole pharmacodynamics. Our findings indicate that a ketogenic diet potentiates the effect of fluconazole at multiple body sites during both C. neoformans and C. albicans infection and could have practical and promising treatment implications.IMPORTANCEInvasive fungal infections cause over 2.5 million deaths per year around the world. Treatments for fungal infections are limited, and there is a significant need to develop strategies to enhance antifungal efficacy, combat antifungal resistance, and mitigate treatment side effects. We determined that a high-fat, low-carbohydrate ketogenic diet significantly potentiated the therapeutic effect of fluconazole, which resulted in a substantial decrease in tissue fungal burden of both C. neoformans and C. albicans in experimental animal models. We believe this work is the first of its kind to demonstrate that diet can dramatically influence the treatment of fungal infections. These results highlight a novel strategy of antifungal drug enhancement and emphasize the need for future investigation into dietary effects on antifungal drug activity.

Susceptibility to Cryptococcus neoformans Infection with Bruton's Tyrosine Kinase Inhibition.

Patients receiving the Bruton's tyrosine kinase (BTK) inhibitor ibrutinib have an increased likelihood of fungal infections. The objectives of this study were to determine if Cryptococcus neoformans infection severity was isolate dependent with BTK inhibition and whether blocking BTK impacted infection severity in a mouse model. We compared four clinical isolates from patients on ibrutinib to virulent (H99) and avirulent (A1-35-8) reference strains. BTK knockout (KO) and wild-type (WT) C57 mice and WT CD1 mice were infected by intranasal (i.n.), oropharyngeal aspiration (OPA), and intravenous (i.v.) routes. Infection severity was assessed by survival and fungal burden (CFU per gram of tissue). Ibrutinib (25 mg/kg) or vehicle was administered daily through intraperitoneal injections. In the BTK KO model, no isolate-dependent effect on fungal burden was observed, and infection severity was not significantly different from that of the WT with i.n., OPA, and i.v. routes. Ibrutinib treatment did not impact infection severity. However, when the four clinical isolates were compared to H99, two of these isolates were less virulent, with significantly longer survival and reduced rates of brain infection. In conclusion, C. neoformans infection severity in the BTK KO model does not appear to be isolate dependent. BTK KO and ibrutinib treatment did not result in significantly different infection severities. However, based on repeated clinical observations of increased susceptibility to fungal infections with BTK inhibitor therapy, further work is needed to optimize a mouse model with BTK inhibition to better understand the role that this pathway plays in susceptibility to C. neoformans infection.

Efficacy of APX2039 in a Rabbit Model of Cryptococcal Meningitis.

Cryptococcal Meningitis (CM) is uniformly fatal if not treated, and treatment options are limited. We previously reported on the activity of APX2096, the prodrug of the novel Gwt1 inhibitor APX2039, in a mouse model of CM. Here, we investigated the efficacy of APX2039 in mouse and rabbit models of CM. In the mouse model, the controls had a mean lung fungal burden of 5.95 log10 CFU/g, whereas those in the fluconazole-, amphotericin B-, and APX2039-treated mice were 3.56, 4.59, and 1.50 log10 CFU/g, respectively. In the brain, the control mean fungal burden was 7.97 log10 CFU/g, while the burdens were 4.64, 7.16, and 1.44 log10 CFU/g for treatment with fluconazole, amphotericin B, and APX2039, respectively. In the rabbit model of CM, the oral administration of APX2039 at 50 mg/kg of body weight twice a day (BID) resulted in a rapid decrease in the cerebrospinal fluid (CSF) fungal burden, and the burden was below the limit of detection by day 10 postinfection. The effective fungicidal activity (EFA) was -0.66 log10 CFU/mL/day, decreasing from an average of 4.75 log10 CFU/mL to 0 CFU/mL, over 8 days of therapy, comparing favorably with good clinical outcomes in humans associated with reductions of the CSF fungal burden of -0.4 log10 CFU/mL/day, and, remarkably, 2-fold the EFA of amphotericin B deoxycholate in this model (-0.33 log10 CFU/mL/day). A total drug exposure of the area under the concentration-time curve from 0 to 24 h (AUC0-24) of 25 to 50 mg · h/L of APX2039 resulted in near-maximal antifungal activity. These data support the further preclinical and clinical evaluation of APX2039 as a new oral fungicidal monotherapy for the treatment of CM. IMPORTANCE Cryptococcal meningitis (CM) is a fungal disease with significant global morbidity and mortality. The gepix Gwt1 inhibitors are a new class of antifungal drugs. Here, we demonstrated the efficacy of APX2039, the second member of the gepix class, in rabbit and mouse models of cryptococcal meningitis. We also analyzed the drug levels in the blood and cerebrospinal fluid in the highly predictive rabbit model and built a mathematical model to describe the behavior of the drug with respect to the elimination of the fungal pathogen. We demonstrated that the oral administration of APX2039 resulted in a rapid decrease in the CSF fungal burden, with an effective fungicidal activity of -0.66 log10 CFU/mL/day, comparing favorably with good clinical outcomes in humans associated with reductions of -0.4 log10 CFU/mL/day. The drug APX2039 had good penetration of the central nervous system and is an excellent candidate for future clinical testing in humans for the treatment of CM.

Curvularia alcornii Aortic Pseudoaneurysm Following Aortic Valve Replacement: Case Report and Review of the Literature.

We report the first case of Curvularia alcornii aortic pseudoaneurysm following bioprosthetic aortic valve replacement in an immunocompetent host. Infection was complicated by septic emboli to multiple organs. Despite aggressive surgical intervention and antifungal therapy, infection progressed. We review the literature on invasive Curvularia infection to inform diagnosis and management.

Black mold takes hold and story told.

We present a case of an invasive Curvularia infection in a patient who developed following bilateral orthotopic lung transplantation despite receiving post-transplant antifungal prophylaxis. This infection presented as mold colonies studding the interior surface of his chest tubes. Despite surgical washout of his bilateral pleural cavities and antifungal treatment with liposomal amphotericin B, micafungin, and isavuconazonium sulfate, the patient died.

Effect of fluconazole prophylaxis on Candida fluconazole susceptibility in premature infants.

Objectives: Extremely premature infants are at high risk of developing invasive candidiasis; fluconazole prophylaxis is safe and effective for reducing invasive candidiasis in this population but further study is needed. We sought to better understand the effect of prophylactic fluconazole on a selection of fluconazole-resistant Candida species. Methods: We evaluated the susceptibility to fluconazole of Candida isolates from premature infants (<750 g birth weight) enrolled in a multicentre, randomized, placebo-controlled trial of fluconazole prophylaxis. Candida species were isolated through surveillance cultures at baseline (study day 0-7), period 1 (study day 8-28) and period 2 (study day 29-49). Fluconazole MICs were determined for all Candida isolates. Results: Three hundred and sixty-one infants received fluconazole (n = 188) or placebo (n = 173). After the baseline period, Candida colonization was significantly lower in the fluconazole group compared with placebo during periods 1 (5% versus 27%; P < 0.001) and 2 (3% versus 27%; P < 0.001). After the baseline period, two infants (1%) were colonized with at least one fluconazole-resistant Candida in each group. Median fluconazole MIC was similar in both treatment groups at baseline and period 1. However, in period 2, median MIC was higher in the fluconazole group compared with placebo (1.00 versus 0.50 mg/L, P = 0.01). There was no emergence of resistance observed and no patients developed invasive candidiasis with a resistant Candida isolate. Conclusions: Fluconazole prophylaxis decreased Candida albicans and 'non-albicans' Candida colonization and was associated with a slightly higher fluconazole MIC for colonizing Candida isolates.

Pulmonary blastomycosis presenting as primary lung cancer.

BACKGROUND: Blastomycosis is an endemic mycosis in North America that is caused by the dimorphic fungus Blastomyces dermatitidis. The illness is a systemic disease with a wide variety of pulmonary and extra-pulmonary manifestations. The initial presentation of blastomycosis may easily be mistaken for other infectious or non-infectious etiologies. CASE PRESENTATION: We present the case of a 52-year-old African-American male and former smoker that presented to his primary care provider with a 2-week history of non-productive cough, night sweats and weight loss. Initially diagnosed with primary lung malignancy, the patient was subsequently found to have pulmonary blastomycosis mimicking lung cancer. The patient underwent a successful course of treatment with posaconazole. CONCLUSIONS: Chronic blastomycosis can present with clinical and radiographic features indistinguishable from thoracic malignancies. There is no clinical syndrome specific for blastomycosis, thus a high degree of suspicion is required for early diagnosis. In this case report, we review recent evidence in radiographic features, diagnostic considerations and treatment of the disease.

Genomic characterization of recurrent mold infections in thoracic transplant recipients.

Invasive mold disease in thoracic organ transplant recipients is a well-recognized complication, but the long-term persistence of molds within the human body and evasion of host defenses has not been well-described. We present 2 cases of invasive mold disease (Verruconis gallopava and Aspergillus fumigatus) in thoracic transplant recipients who had the same mold cultured years prior to the invasive disease presentation. The paired isolates from the index and recurrent infections in both patients were compared using whole-genome sequencing to determine if the same strain of mold caused both the index and recurrent infections. In Case 1, the isolates were found to be of the same strain indicating that the initial colonizing isolate identified pre-transplant eventually caused invasive mold disease post-transplant while in Case 2, the 2 isolates were not of the same strain. These results demonstrate the distinct possibility of molds both persisting within the human body for years prior to invasive mold disease or the long-term risk of recurrent, persistent infection with more than one strain. Further studies of long-term molecular epidemiology of IMD and risk factors for mold persistence in transplant recipients are encouraged.

In Vitro and In Vivo Evaluation of APX001A/APX001 and Other Gwt1 Inhibitors against Cryptococcus.

Cryptococcal meningitis (CM), caused primarily by Cryptococcus neoformans, is uniformly fatal if not treated. Treatment options are limited, especially in resource-poor geographical regions, and mortality rates remain high despite current therapies. Here we evaluated the in vitro and in vivo activity of several compounds, including APX001A and its prodrug, APX001, currently in clinical development for the treatment of invasive fungal infections. These compounds target the conserved Gwt1 enzyme that is required for the localization of glycosylphosphatidylinositol (GPI)-anchored cell wall mannoproteins in fungi. The Gwt1 inhibitors had low MIC values, ranging from 0.004 µg/ml to 0.5 µg/ml, against both C. neoformans and C. gattii APX001A and APX2020 demonstrated in vitro synergy with fluconazole (fractional inhibitory concentration index, 0.37 for both). In a CM model, APX001 and fluconazole each alone reduced the fungal burden in brain tissue (0.78 and 1.04 log10 CFU/g, respectively), whereas the combination resulted in a reduction of 3.52 log10 CFU/g brain tissue. Efficacy, as measured by a reduction in the brain and lung tissue fungal burden, was also observed for another Gwt1 inhibitor prodrug, APX2096, where dose-dependent reductions in the fungal burden ranged from 5.91 to 1.79 log10 CFU/g lung tissue and from 7.00 and 0.92 log10 CFU/g brain tissue, representing the nearly complete or complete sterilization of lung and brain tissue at the higher doses. These data support the further clinical evaluation of this new class of antifungal agents for the treatment of CM.

Antifungal Activity of SCY-078 and Standard Antifungal Agents against 178 Clinical Isolates of Resistant and Susceptible Candida Species.

SCY-078 in vitro activity was determined for 178 isolates of resistant or susceptible Candida albicans, Candida dubliniensis, Candida glabrata, Candida krusei, Candida lusitaniae, and Candida parapsilosis, including 44 Candida isolates with known genotypic (FKS1 or FKS2 mutations), phenotypic, or clinical resistance to echinocandins. Results were compared to those for anidulafungin, caspofungin, micafungin, fluconazole, and voriconazole. SCY-078 was shown to have excellent activity against both wild-type isolates and echinocandin- and azole-resistant isolates of Candida species.

Simple Strategy for Taming Membrane-Disrupting Antibiotics.

A strategy has been devised for increasing the cellular selectivity of membrane-disrupting antibiotics based on the attachment of a facially amphiphilic sterol. Using Amphotericin B (AmB) as a prototype, covalent attachment of cholic acid bound to a series of α,ω-diamines has led to a dramatic reduction in hemolytic activity, a significant reduction in toxicity toward HEK293T cells, and significant retention of antifungal activity.

Diagnostic difficulty identifying Apophysomyces trapeziformis septic arthritis in a patient with multiple myeloma.

INTRODUCTION: Mucormycosis is a rare fungal infection, but can cause substantial morbidity and mortality in both immunocompromised and immunocompetent patients. Apophysomyces is a mucormycetes species ubiquitous in nature, particularly in soil, decaying wood and other organic matter. Apophysomyces is known to cause cutaneous fungal infections, particularly after penetrating trauma. Septic arthritis is a rare clinical manifestation. CASE PRESENTATION: We describe a case of Apophysomyces trapeziformis causing septic arthritis of the knee of a patient with multiple myeloma. He was treated multiple times for bacterial septic arthritis with minimal improvement. Surgical tissue specimens finally grew mucoraceous mould, and DNA sequencing and morphological assessment of spores identified the mould as A. trapeziformis. The patient was treated with amphotericin B and posaconazole, but ultimately required an above-the-knee amputation for definitive treatment. CONCLUSION: This case illustrates the need to evaluate for fungal infection in a persistent septic arthritis that is culture negative and refractory to empiric antibiotics, particularly in an immunocompromised individual. It also shows the importance of a thorough social history and adequate tissue specimens for culture.

Taming Amphotericin B.

A strategy is introduced for enhancing the cellular selectivity of Amphotericin B (AmB) and other classes of membrane-disrupting agents. This strategy involves attaching the agent to a molecular umbrella to minimize the disruptive power of aggregated forms. Based on this approach, AmB has been coupled to a molecular umbrella derived from one spermidine and two cholic acid molecules and found to have antifungal activities approaching that of the native drug. However, in sharp contrast to AmB, the hemolytic activity and the cytotoxcity of this conjugate toward HEK293 T cells have been dramatically reduced.

Characterization of alpha-toxin hla gene variants, alpha-toxin expression levels, and levels of antibody to alpha-toxin in hemodialysis and postsurgical patients with Staphylococcus aureus bacteremia.

Alpha-toxin is a major Staphylococcus aureus virulence factor. This study evaluated potential relationships between in vitro alpha-toxin expression of S. aureus bloodstream isolates, anti-alpha-toxin antibody in serum of patients with S. aureus bacteremia (SAB), and clinical outcomes in 100 hemodialysis and 100 postsurgical SAB patients. Isolates underwent spa typing and hla sequencing. Serum anti-alpha-toxin IgG and neutralizing antibody levels were measured by using an enzyme-linked immunosorbent assay and a red blood cell (RBC)-based hemolysis neutralization assay. Neutralization of alpha-toxin by an anti-alpha-toxin monoclonal antibody (MAb MEDI4893) was tested in an RBC-based lysis assay. Most isolates encoded hla (197/200; 98.5%) and expressed alpha-toxin (173/200; 86.5%). In vitro alpha-toxin levels were inversely associated with survival (cure, 2.19 µg/ml, versus failure, 1.09 µg/ml; P < 0.01). Both neutralizing (hemodialysis, 1.26 IU/ml, versus postsurgical, 0.95; P < 0.05) and IgG (hemodialysis, 1.94 IU/ml, versus postsurgical, 1.27; P < 0.05) antibody levels were higher in the hemodialysis population. Antibody levels were also significantly higher in patients infected with alpha-toxin-expressing S. aureus isolates (P < 0.05). Levels of both neutralizing antibodies and IgG were similar among patients who were cured and those not cured (failures). Sequence analysis of hla revealed 12 distinct hla genotypes, and all genotypic variants were susceptible to a neutralizing monoclonal antibody in clinical development (MEDI4893). These data demonstrate that alpha-toxin is highly conserved in clinical S. aureus isolates. Higher in vitro alpha-toxin levels were associated with a positive clinical outcome. Although patients infected with alpha-toxin-producing S. aureus exhibited higher anti-alpha-toxin antibody levels, these levels were not associated with a better clinical outcome in this study.

Outbreak of bacteremia due to Burkholderia contaminans linked to intravenous fentanyl from an institutional compounding pharmacy.

IMPORTANCE Many health care facilities compound medications on site to fulfill local demands when customized formulations are needed, national supply is critically low, or costs for manufactured pharmaceuticals are excessive. Small, institutional compounding facilities may perform the same high-risk procedures as large distributors of compounded medications. OBJECTIVES To investigate an outbreak related to contamination of compounded sterile preparations and to determine processes to prevent future outbreaks. DESIGN, SETTING, AND PARTICIPANTS We performed an outbreak investigation of inpatients at Duke University Hospital from August 31 through September 6, 2012. The investigation included a case-control study, compounding facility inspection and environmental sampling, observation of a mock compounding demonstration, and microbiologic and molecular testing of sequestered medication. EXPOSURES Intravenous fentanyl prepared by an institutional compounding pharmacy. MAIN OUTCOMES AND MEASURES Microbiologic and molecular evidence of contamination of a compounded sterile preparation and failure of routine sterility testing. RESULTS Blood cultures of 7 patients during a 7-day period at Duke University Hospital yielded pan-susceptible Burkholderia cepacia complex bacteria. The risk factor common to all patients was receipt of continuous fentanyl infusion prepared by our institutional compounding pharmacy (odds ratio, 11.22; 95% CI, 2.09-∞; P = .01). The outbreak was terminated after sequestration of compounded fentanyl. An intensive evaluation of the compounding facility, its practice, and its procedures was completed. Investigators evaluated the clean room, collected targeted microbiologic samples within the compounding pharmacy environment, and observed a mock demonstration of compounding practice. The B cepacia complex was found in the anteroom sink drain and pH probe calibration fluid from the compounding clean room. Multiple microbiologic analyses of sequestered fentanyl initially failed. Ultimately, a batched, vacuum-assisted filtration method produced B cepacia complex from a single lot. Molecular analyses using repetitive element polymerase chain reaction and pulsed-field gel electrophoresis confirmed a clonal Burkholderia contaminans strain from patients, fentanyl, and environmental samples. CONCLUSIONS AND RELEVANCE An outbreak of B contaminans bacteremia was linked to contamination of locally compounded intravenous fentanyl. Health care facilities that house institutional compounding facilities must be vigilant in efforts to prevent, recognize, and terminate medication-related outbreaks.

Impact of surfactant protein D, interleukin-5, and eosinophilia on Cryptococcosis.

Cryptococcus neoformans is an opportunistic fungal pathogen that initiates infection following inhalation. As a result, the pulmonary immune response provides a first line of defense against C. neoformans. Surfactant protein D (SP-D) is an important regulator of pulmonary immune responses and is typically host protective against bacterial and viral respiratory infections. However, SP-D is not protective against C. neoformans. This is evidenced by previous work from our laboratory demonstrating that SP-D-deficient mice infected with C. neoformans have a lower fungal burden and live longer than wild-type (WT) control animals. We hypothesized that SP-D alters susceptibility to C. neoformans by dysregulating the innate pulmonary immune response following infection. Thus, inflammatory cells and cytokines were compared in the bronchoalveolar lavage fluid from WT and SP-D(-/-) mice after C. neoformans infection. Postinfection, mice lacking SP-D have reduced eosinophil infiltration and interleukin-5 (IL-5) in lung lavage fluid. To further explore the interplay of SP-D, eosinophils, and IL-5, mice expressing altered levels of eosinophils and/or IL-5 were infected with C. neoformans to assess the role of these innate immune mediators. IL-5-overexpressing mice have increased pulmonary eosinophilia and are more susceptible to C. neoformans infection than WT mice. Furthermore, susceptibility of SP-D(-/-) mice to C. neoformans infection could be restored to the level of WT mice by increasing IL-5 and eosinophils by crossing the IL-5-overexpressing mice with SP-D(-/-) mice. Together, these studies support the conclusion that SP-D increases susceptibility to C. neoformans infection by promoting C. neoformans-driven pulmonary IL-5 and eosinophil infiltration.

Quantitative models of the dose-response and time course of inhalational anthrax in humans.

Anthrax poses a community health risk due to accidental or intentional aerosol release. Reliable quantitative dose-response analyses are required to estimate the magnitude and timeline of potential consequences and the effect of public health intervention strategies under specific scenarios. Analyses of available data from exposures and infections of humans and non-human primates are often contradictory. We review existing quantitative inhalational anthrax dose-response models in light of criteria we propose for a model to be useful and defensible. To satisfy these criteria, we extend an existing mechanistic competing-risks model to create a novel Exposure-Infection-Symptomatic illness-Death (EISD) model and use experimental non-human primate data and human epidemiological data to optimize parameter values. The best fit to these data leads to estimates of a dose leading to infection in 50% of susceptible humans (ID50) of 11,000 spores (95% confidence interval 7,200-17,000), ID10 of 1,700 (1,100-2,600), and ID1 of 160 (100-250). These estimates suggest that use of a threshold to human infection of 600 spores (as suggested in the literature) underestimates the infectivity of low doses, while an existing estimate of a 1% infection rate for a single spore overestimates low dose infectivity. We estimate the median time from exposure to onset of symptoms (incubation period) among untreated cases to be 9.9 days (7.7-13.1) for exposure to ID50, 11.8 days (9.5-15.0) for ID10, and 12.1 days (9.9-15.3) for ID1. Our model is the first to provide incubation period estimates that are independently consistent with data from the largest known human outbreak. This model refines previous estimates of the distribution of early onset cases after a release and provides support for the recommended 60-day course of prophylactic antibiotic treatment for individuals exposed to low doses.

Dematiaceous fungi mimicking a conjunctival melanoma.

An 85-year-old, immunocompetent man was referred to the authors due to the presence of an enlarging, pigmented mass of the conjunctiva concerning for a conjunctival melanoma. Wide excision of the mass revealed a pigmented or "dematiaceous" fungus. He was treated with topical natamycin, and the lesion healed well without any evidence of recurrence. Dematiaceous fungi should be considered in the differential for pigmented conjunctival lesions.

A new lineage in Pucciniomycotina: class Tritirachiomycetes, order Tritirachiales, family Tritirachiaceae.

Based on multiple gene analyses (nuclear large subunit, nuclear small subunit, internal transcribed spacer region including the 5.8 s subunit rDNA, and translation elongation factor 1α) and septal pore ultrastructure we describe a new lineage of Pucciniomycotina consisting of a new class, Tritirachiomycetes, new order, Tritirachiales, and new family, Tritirachiaceae. Tritirachium dependens, T. oryzae, T. roseum (reintroduced), T. cinnamomeum and two unidentified species are recognized. Phylogenetic analyses do not support existing morphological circumscription of some species, and the available evidence suggests that morphological evaluation alone is not adequate for species identification.