Epidemiology of Mucormycosis in Greece; Results from a Nationwide Prospective Survey and Published Case Reports.

Mucormycosis has emerged as a group of severe infections mainly in immunocompromised patients. We analysed the epidemiology of mucormycosis in Greece in a multicentre, nationwide prospective survey of patients of all ages, during 2005-2022. A total of 108 cases were recorded. The annual incidence declined after 2009 and appeared stable thereafter, at 0.54 cases/million population. The most common forms were rhinocerebral (51.8%), cutaneous (32.4%), and pulmonary (11.1%). Main underlying conditions were haematologic malignancy/neutropenia (29.9%), haematopoietic stem cell transplantation (4.7%), diabetes mellitus (DM) (15.9%), other immunodeficiencies (23.4%), while 22.4% of cases involved immunocompetent individuals with cutaneous/soft-tissue infections after motor vehicle accident, surgical/iatrogenic trauma, burns, and injuries associated with natural disasters. Additionally, DM or steroid-induced DM was reported as a comorbidity in 21.5% of cases with various main conditions. Rhizopus (mostly R. arrhizus) predominated (67.1%), followed by Lichtheimia (8.5%) and Mucor (6.1%). Antifungal treatment consisted mainly of liposomal amphotericin B (86.3%), median dose 7 mg/kg/day, range 3-10 mg/kg/day, with or without posaconazole. Crude mortality was 62.8% during 2005-2008 but decreased significantly after 2009, at 34.9% (p = 0.02), with four times fewer haematological cases, fewer iatrogenic infections, and fewer cases with advanced rhinocerebral form. The increased DM prevalence should alert clinicians for timely diagnosis of mucormycosis in this patient population.

Filamentous Fungal Keratitis in Greece: A 16-Year Nationwide Multicenter Survey.

In a multicenter, prospective study of filamentous fungal keratitis in Greece, predisposing factors, etiology, treatment practices, and outcome, were determined. Corneal scrapings were collected from patients with clinical suspicion of fungal keratitis, and demographic and clinical data were recorded. Fungal identification was based on morphology, molecular methods, and matrix assisted laser desorption ionization time-of-flight mass-spectrometry. A total of 35 cases were identified in a 16-year study period. Female to male ratio was 1:1.7 and median age 48 years. Corneal injury by plant material, and soft contact lens use were the main risk factors (42.8% and 31.4%, respectively). Trauma was the leading risk factor for men (68.1%), contact lens use (61.5%) for women. Fusarium species were isolated more frequently (n = 21, 61.8%). F. solani was mostly associated with trauma, F. verticillioides and F. proliferatum with soft contact lens use. Other fungi were: Purpureocillium lilacinum (14.7%), Alternaria (11.8%), Aspergillus (8.8%), and Phoma foliaceiphila, Beauveria bassiana and Curvularia spicifera, one case each. Amphotericin B and voriconazole MIC50s against Fusarium were 2 mg/L and 4 mg/L respectively. Antifungal therapy consisted mainly of voriconazole locally or both locally and systemically, alone or in combination with liposomal AmB. Cure/improvement rate with antifungal therapy alone was 52%, keratoplasty was required in 40% of cases, and enucleation in 8%. In conclusion, filamentous fungal keratitis in Greece is rare, but with considerable morbidity. A large proportion of cases resulted in keratoplasty despite appropriate antifungal treatment.

Global Cutaneous Mucormycosis: A Systematic Review.

Cutaneous mucormycosis is the third most common clinical type of mucormycosis. The signs and symptoms vary widely, and it is important to make the diagnosis as early as possible in order to achieve a better outcome. We present a systematic review of its epidemiology, clinical presentation, diagnosis, and treatment, analyzing cases published from 1958 until 2021. The review was conducted according to the PRISMA guidelines and included 693 cases from 485 articles from 46 countries. Most publications were from North America (256 cases, 36.9%) and Asia (216 cases, 31.2%). The most common risk factors were diabetes mellitus (20%) and hematological malignancies (15.7%). However, a large proportion of published cases (275, 39.6%) had no identified underlying disease. The most common mode of transmission was trauma (54%), and 108 (15.6%) cases were healthcare-associated. In this review, 291 (42.5%) patients had localized infection, and 90 (13%) had disseminated mucormycosis. In Europe, N. America and S. America, the most common genus was Rhizopus spp., while in Asia it was Apophysomyces spp. (34.7%). Treatment was performed with antifungals, mainly amphotericin B, and/or surgery. Mortality was significantly lower when both antifungals and surgery were applied (29.6%).

Induction of innate immune responses by KPC-producing Klebsiella pneumoniae of the pandemic sequence type 258-clade I.

Klebsiella pneumoniae -carbapenemase-producing K. pneumoniae (KPC) sequence-type 258 (ST258) has emerged as an important human pathogen throughout the world. Although lacking known virulence factors, it is associated with significant morbidity and high mortality rates. The pathogenicity of KPC K. pneumoniae ST258 strains has not been fully elucidated yet. We sought to investigate the interactions of the KPC K. pneumoniae ST258-clade I with different components of innate immunity. Human serum was used to evaluate the serum bactericidal activity and the J774A.1 murine (BALB/c mice) macrophage cell-line was used to examine phagocytosis, mRNA expression and production of the pro-inflammatory cytokines IL-1ß, TNF-α and IL-6. L-78, a KPC-producing K. pneumoniae ST258-clade I strain was used as representative of the strains circulating in Greek hospitals. K. pneumoniae ATCC 43816, a virulent K2 strain, was used for comparison. Strain L-78 was susceptible to human serum and rapidly phagocytosed by J774A.1 cells, in contrast to the virulent K2 strain, which was serum-resistant and slowly phagocytosed. Stimulation of the J774A.1 cells with the L-78 strain induced production of IL-1ß at concentration levels significantly higher compared to K2, whereas production of TNF-α and IL-6 levels were comparable by the two strains. L-78 was able to induce IL-1ß mRNA and NLRP3 mRNA expression. Our findings indicate that K. pneumoniae ST258-clade I is serum sensitive, rapidly phagocytosed and is capable of eliciting adequate innate immune response in terms of production of pro-inflammatory cytokines.

Epidemiology and Diagnosis of Mucormycosis: An Update.

Mucormycosis is an angioinvasive fungal infection, due to fungi of the order Mucorales. Its incidence cannot be measured exactly, since there are few population-based studies, but multiple studies have shown that it is increasing. The prevalence of mucormycosis in India is about 80 times the prevalence in developed countries, being approximately 0.14 cases per 1000 population. Diabetes mellitus is the main underlying disease globally, especially in low and middle-income countries. In developed countries the most common underlying diseases are hematological malignancies and transplantation. Τhe epidemiology of mucormycosis is evolving as new immunomodulating agents are used in the treatment of cancer and autoimmune diseases, and as the modern diagnostic tools lead to the identification of previously uncommon genera/species such as Apophysomyces or Saksenaea complex. In addition, new risk factors are reported from Asia, including post-pulmonary tuberculosis and chronic kidney disease. New emerging species include Rhizopus homothallicus, Thamnostylum lucknowense, Mucor irregularis and Saksenaea erythrospora. Diagnosis of mucormycosis remains challenging. Clinical approach to diagnosis has a low sensitivity and specificity, it helps however in raising suspicion and prompting the initiation of laboratory testing. Histopathology, direct examination and culture remain essential tools, although the molecular methods are improving. The internal transcribed spacer (ITS) region is the most widely sequenced DNA region for fungi and it is recommended as a first-line method for species identification of Mucorales. New molecular platforms are being investigated and new fungal genetic targets are being explored. Molecular-based methods have gained acceptance for confirmation of the infection when applied on tissues. Methods on the detection of Mucorales DNA in blood have shown promising results for earlier and rapid diagnosis and could be used as screening tests in high-risk patients, but have to be validated in clinical studies. More, much needed, rapid methods that do not require invasive procedures, such as serology-based point-of-care, or metabolomics-based breath tests, are being developed and hopefully will be evaluated in the near future.

Rare fungal infectious agents: a lurking enemy.

In the expanding population of immunocompromised patients and those treated in intensive care units, rare fungal infectious agents have emerged as important pathogens, causing invasive infections associated with high morbidity and mortality. These infections may present either as de novo or as breakthrough invasive infections in high-risk patients with hematologic malignancies receiving prophylactic or empirical antifungal therapy or in patients with central venous catheters. Diagnosis and treatment are challenging. Physicians should have a high index of suspicion because early diagnosis is of paramount importance. Conventional diagnostic methods such as cultures and histopathology are still essential, but rapid and more specific molecular techniques for both detection and identification of the infecting pathogens are being developed and hopefully will lead to early targeted treatment. The management of invasive fungal infections is multimodal. Reversal of risk factors, if feasible, should be attempted. Surgical debridement is recommended in localized mold infections. The efficacy of various antifungal drugs is not uniform. Amphotericin B is active against most yeasts, except Trichosporon, as well as against Mucorales, Fusarium, and some species of Paecilomyces and dimorphic fungi. The use of voriconazole is suggested for the treatment of trichosporonosis and scedosporiosis. Combination treatment, though recommended as salvage therapy in some infections, is controversial in most cases. Despite the use of available antifungals, mortality remains high. The optimization of molecular-based techniques, with expansion of reference libraries and the possibility for direct detection of resistance mechanisms, is awaited with great interest in the near future. Further research is necessary, however, in order to find the best ways to confront and destroy these lurking enemies.

Association Between Candiduria and Candidemia: A Clinical and Molecular Analysis of Cases.

The risk of developing candidemia after candiduria is reportedly very low, but it has not been adequately investigated. The aim of this study was to examine the molecular relatedness between Candida strains isolated from adult patients with candidemia and concomitant candiduria in association with the clinical characteristics of the cases. All episodes of candidemia occurring in a tertiary care academic hospital during a 5-year period were recorded prospectively. Patients with episodes of candiduria occurring two weeks preceding to or one week following a positive for Candida blood culture were included in the study. The genotypic relatedness of Candida strains isolated from blood and urine was investigated by pulsed-field gel electrophoresis after digestion with the BssHII restriction endonuclease. We recorded 141 candidemia episodes, occurring in 134 patients. Twelve episodes of candidemia with concomitant candiduria occurred in 11 patients (8% of all candidemias). In six of these episodes, the strains in the blood-urine pairs belonged to different species. In two episodes, the isolates belonged to the same species but were not genetically related, and only in four (2.8% of all candidemias), the strains were related. All four patients were severely ill and had multiple risk factors for candidemia. These findings indicate that in hospitalized patients with candidemia, concomitant candiduria is rare and usually an independent event, confirming previous reports. In the critically ill, however, the existence of genetically related strains in blood and urine appears to be more frequent, with more probable the hematogenous dissemination.

Reduction of Environmental Contamination With Multidrug-Resistant Bacteria by Copper-Alloy Coating of Surfaces in a Highly Endemic Setting.

OBJECTIVE To evaluate the efficacy of copper-coating in reducing environmental colonization in an intensive-care unit (ICU) with multidrug-resistant-organism (MDRO) endemicity DESIGN Interventional, comparative crossover trial SETTING The general ICU of Attikon University hospital in Athens, Greece PATIENTS Those admitted to ICU compartments A and B during the study period METHODS Before any intervention (phase 1), the optimum sampling method using 2 nylon swabs was validated. In phase 2, 6 copper-coated beds (ie, with coated upper, lower, and side rails) and accessories (ie, coated side table, intravenous [i.v.] pole stands, side-cart handles, and manual antiseptic dispenser cover) were introduced as follows: During phase 2a (September 2011 to February 2012), coated items were placed next to noncoated ones (controls) in both compartments A and B; during phase 2b (May 2012 to January 2013), all copper-coated items were placed in compartment A, and all noncoated ones (controls) in compartment B. Patients were randomly assigned to available beds. Environmental samples were cultured quantitatively for clinically important bacteria. Clinical and demographic data were collected from medical records. RESULTS Copper coating significantly reduced the percentage of colonized surfaces (55.6% vs 72.5%; P<.0001), the percentage of surfaces colonized by MDR gram-negative bacteria (13.8% vs 22.7%; P=.003) or by enterococci (4% vs 17%; P=.014), the total bioburden (2,858 vs 7,631 cfu/100 cm2; P=.008), and the bioburden of gram-negative isolates, specifically (261 vs 1,266 cfu/100 cm2; P=.049). This effect was more pronounced when the ratio of coated surfaces around the patient was increased (phase 2b). CONCLUSIONS Copper-coated items in an ICU setting with endemic high antimicrobial resistance reduced environmental colonization by MDROs. Infect Control Hosp Epidemiol 2017;38:765-771.

Diagnostic value of a semi-nested PCR for the diagnosis of mucormycosis and aspergillosis from paraffin-embedded tissue: A single center experience.

OBJECTIVE: The main aim of our study was to investigate the diagnostic value of a molecular method for the diagnosis of mucormycosis and aspergillosis from formalin-fixed and paraffin-embedded (FFPE) tissues. METHODS: A retrospective chart review identified all cases with histology reports mentioning the presence of fungi with morphological characteristics of either Aspergillus or mucormycetes, for the period 2005-2012. Paraffin blocks were retrieved from the archives of the Department of Pathology. A semi-nested PCR specific for the detection of mucormycetes and Aspergillus species was applied in FFPE tissue from the above blocks. Results were compared with those of histological (gold standard) and microbiological methods. RESULTS: Twenty cases with fungal hyphae in tissue were revealed. Mucormycetes were detected in 9 cases (45%) by PCR, in only 4 of which culture was available. Species of Aspergillus were detected in 8 cases (40%) by PCR, two of which were co-infection with mucormycetes. Five patients had other fungi, non-detectable with this specific PCR. At least one sample per patient was positive by PCR. Seven out of 30 samples tested overall were false negative. The calculated sensitivity of this method in our setting was 79.3% (95% CI: 60.3-91.9%); specificity was 100%. CONCLUSIONS: The specific PCR used appears to be an easy and useful tool for the prompt and accurate diagnosis of mucormycosis and aspergillosis, in combination with histology and direct examination. Mucormycosis was more frequent than aspergillosis during the study period, highlighting the importance of continuous epidemiological surveillance of these serious infections.

The effect of maternal flora on Candida colonisation in the neonate.

Colonisation may be the first step for the development of Candida infection. The source of neonatal colonisation is thought to be the hospital environment or the maternal vaginal tract. This study investigated to what extend Candida isolates in neonates are similar to isolates from their mother's vaginal tract. Vaginal samples were collected from 347 pregnant women within 48 h before delivery. Samples from oral and rectal mucosa of their neonates were collected within 24-72 h after delivery, were cultured and yeast species were identified. Antifungal susceptibility tests against six antifungal agents were performed. All paired isolates from mother and infant were genotyped by pulse field gel electrophoresis. A total of 82 mothers and of 16 infants were found colonised by Candida spp. C. albicans was the most common species in pregnant women (n = 68) followed by C. glabrata (n = 11). Only C. albicans was isolated from infants, mainly (14/16) from rectal site. All colonised neonates were born to mothers colonised by C. albicans. Candida genotyping revealed identical strains in all investigated neonate-mother pairs. All isolates were susceptible to amphotericin B. Our findings strongly suggest that vertical transmission has the principal role in the neonatal colonisation by C. albicans in the very first days of life.

In vitro antifungal susceptibility of filamentous fungi causing rare infections: synergy testing of amphotericin B, posaconazole and anidulafungin in pairs.

OBJECTIVES: Mucormycetes (formerly known as zygomycetes of the order Mucorales) and hyaline moulds such as those of the genus Fusarium or Paecilomyces are emerging as significant human pathogens. The aim of the study was to determine the in vitro antifungal susceptibility of these fungi to older and newer antifungals and to investigate the antifungal activity of amphotericin B, posaconazole and anidulafungin in dual combinations. METHODS: Twenty-one clinical isolates of mucormycetes and 16 of rare hyaline moulds were tested. MICs were determined by EUCAST methodology for conidia-forming moulds and Etesting. For antifungal combinations a chequerboard method based on EUCAST methodology was used. RESULTS: Against mucormycetes, amphotericin B exhibited the lowest MICs, followed by posaconazole. Ravuconazole was active against eight of the Rhizopus isolates (MIC 1 mg/L). Resistance to amphotericin B (MIC ≥ 2 mg/L) and posaconazole (MICs ≥ 4 mg/L) was observed in five and three Rhizopus isolates, respectively. Among Fusarium species variable susceptibility patterns were detected. Amphotericin B exhibited the lowest MICs, followed by voriconazole. Etesting for amphotericin B and posaconazole had excellent agreement with EUCAST methodology (78.6%-100%). Synergy between amphotericin B and anidulafungin was observed against two isolates (one Mucor circinelloides and one Fusarium proliferatum). Synergy or antagonism was not detected in any other combination. CONCLUSIONS: The study showed that mucormycetes and other rare hyaline moulds exhibit variable susceptibilities to antifungals, and hence antifungal testing is valuable. The fact that the combination of amphotericin B with anidulafungin was found synergistic in some cases merits further investigation.