Emergence of zoonotic sporotrichosis in Brazil: a genomic epidemiology study.

BACKGROUND: Zoonotic sporotrichosis is a neglected fungal disease, whereby outbreaks are primarily driven by Sporothrix brasiliensis and linked to cat-to-human transmission. To understand the emergence and spread of sporotrichosis in Brazil, the epicentre of the current epidemic in South America, we aimed to conduct whole-genome sequencing (WGS) to describe the genomic epidemiology. METHODS: In this genomic epidemiology study, we included Sporothrix spp isolates from sporotrichosis cases from Brazil, Colombia, and the USA. We conducted WGS using Illumina NovaSeq on isolates collected by three laboratories in Brazil from humans and cats with sporotrichosis between 2013 and 2022. All isolates that were confirmed to be Sporothrix genus by internal transcribed spacer or beta-tubulin PCR sequencing were included in this study. We downloaded eight Sporothrix genome sequences from the National Center for Biotechnology Information (six from Brazil, two from Colombia). Three Sporothrix spp genome sequences from the USA were generated by the US Centers for Disease Control and Prevention as part of this study. We did phylogenetic analyses and correlated geographical and temporal case distribution with genotypic features of Sporothrix spp isolates. FINDINGS: 72 Sporothrix spp isolates from 55 human and 17 animal sporotrichosis cases were included: 67 (93%) were from Brazil, two (3%) from Colombia, and three (4%) from the USA. Cases spanned from 1999 to 2022. Most (61 [85%]) isolates were S brasiliensis, and all were reported from Brazil. Ten (14%) were Sporothrix schenckii and were reported from Brazil, USA, and Colombia. For S schenckii isolates, two distinct clades were observed wherein isolates clustered by geography. For S brasiliensis isolates, five clades separated by more than 100 000 single-nucleotide polymorphisms were observed. Among the five S brasiliensis clades, clades A and C contained isolates from both human and cat cases, and clade A contained isolates from six different states in Brazil. Compared with S brasiliensis isolates, larger genetic diversity was observed among S schenckii isolates from animal and human cases within a clade. INTERPRETATION: Our results suggest that the ongoing epidemic driven by S brasiliensis in Brazil represents several, independent emergence events followed by animal-to-animal and animal-to human transmission within and between Brazilian states. These results describe how S brasiliensis can emerge and spread within a country. FUNDING: Coordenação de Aperfeiçoamento de Pessoal de Nível Superior, Brazil; the São Paulo Research Foundation; Productivity in Research fellowships by the National Council for Scientific and Technological Development, and Ministry of Science and Technology of Brazil.

Bimodal distribution of azole susceptibility in Sporothrix brasiliensis isolates in Brazil.

Sporothrix brasiliensis is an emerging zoonotic fungal pathogen that can be difficult to treat. Antifungal susceptibility testing was performed on the mold phase of a convenience sample of 61 Sporothrix spp. isolates from human and cat sporotrichosis cases in Brazil using the Clinical and Laboratory Standards Institute standard M38. A bimodal distribution of azole susceptibility was observed with 50% (28/56) of S. brasiliensis isolates showing elevated itraconazole minimum inhibitory concentrations ≥16 µg/mL. Phylogenetic analysis found the in vitro resistant isolates were not clonal and were distributed across three different S. brasiliensis clades. Single nucleotide polymorphism (SNP) analysis was performed to identify potential mechanisms of in vitro resistance. Two of the 28 resistant isolates (MIC ≥16 mg/L) had a polymorphism in the cytochrome P450 gene, cyp51, corresponding to the well-known G448S substitution inducing azole resistance in Aspergillus fumigatus. SNPs corresponding to other known mechanisms of azole resistance were not identified in the remaining 26 in vitro resistant isolates.

Carbapenem-resistant Klebsiella pneumoniae bloodstream infections in haematological malignances and hematopoietic stem cell transplantation: Clinical impact of combination therapy in a 10-year Brazilian cohort.

Bacterial bloodstream infections (BSI) are a common threat among patients with haematological malignancies (HM) and hematopoietic stem cell transplant recipients (HSCT). The purpose of this research was to describe clinical and microbiological aspects of BSI caused by carbapenem-resistant Klebsiella pneumoniae (CRKp) and assess risk factors associated with 30-day mortality in a 10-year cohort of haematological patients. A total of 65 CRKp-BSI episodes occurring in HM patients and HSCT recipients and CRKp-BSI between January 2010 and December 2019 were retrospectively studied. Acute leukemias were the most frequently observed underlying disease (87.7%) and 18 patients (27.7%) received HSCT. Mucosal barrier injury in the gastrointestinal tract was the primary cause of bacteremia (86.1%). Also, 14 individuals (21.6%) had an Invasive Fungal Disease (IFD) throughout the episode. Regarding treatment, in 31 patients (47.7%) empirical therapy was deemed appropriate, whereas 33 (50.8%) patients received a combination therapy. Microbiological data revealed that the majority of isolates (53-58%) had the Polymyxin B co-resistance phenotype, while amikacin resistance was less common (16 samples, or 24.7%). The mortality rates at 14 and 30 days were 32.3% and 36.9%, respectively. In a multivariate Cox regression analysis, prompt appropriate antibiotic administration within three days was associated with a better outcome (Adjusted Hazard Ratio [aHR]: 0.33; 95% Confidence Interval [CI]: 0.14-0.76; p = 0.01), whereas hypotension at presentation (aHR: 3.88; 95% CI: 1.40-10.74; p = 0.01) and concurrent IFD (aHR: 2.97; 95% CI: 1.20-7.37; p = 0.02) were independently associated with death within 30 days. Additionally, a favorable correlation between combination therapy and overall survival was found (aHR: 0.18; 95%CI: 0.06-0.56; p = 0.002). In conclusion, 30-day mortality CRKp-BSI was elevated and most of the isolates were polymyxin B resistant. Early appropriate antimicrobial treatment and the use of combination therapy were linked to a better outcome.

A simple disk pre-diffusion test to predict in vitro aztreonam/avibactam activity against NDM-producing Klebsiella pneumoniae complex.

OBJECTIVES: The aim of this study was to describe a simple test to predict in vitro efficacy of aztreonam/avibactam (ATM-AVI) combination based on a pre-diffusion assay involving routinely available ceftazidime/avibactam (CAZ-AVI) and aztreonam (ATM) disks. METHODS: A total of 113 non-repetitive NDM-producing Klebsiella had the species identified by multiplex PCR. Minimum inhibitory concentrations (MICs) for ATM and ATM-AVI were determined by broth microdilution. For the combined disk pre-diffusion method, a disk containing ceftazidime 10 µg and avibactam 4 µg (CAZ-AVI) was applied on the surface of an uninoculated Mueller-Hinton agar plate. Following incubation for 2 h at 36°C, the disk was removed, the bacterial suspension was applied and a 30 µg ATM disk was placed precisely in the same position as the removed CAZ-AVI disk. Following incubation for 16-20 h, inhibition zone diameters were measured and correlated with ATM-AVI MICs. RESULTS: The distribution of species among the 113 isolates was 85 Klebsiella pneumoniae (75.2%), 19 Klebsiella quasipneumoniae (16.8%) and 9 Klebsiella variicola (8.0%). A total of 99 isolates had only blaNDM and 14 had both blaNDM and blaKPC genes. Regarding the isolates positive for blaNDM only, 38.4% were susceptible to ATM and 7.1% were susceptible, increased exposure. All isolates had ATM-AVI MICs of ≤1 mg/L, and the smallest inhibition zone diameter observed was 23 mm. CONCLUSION: Modified disk pre-diffusion can be used as a simple test to screen for ATM-AVI in vitro activity against Klebsiella, since ATM-AVI disks, gradient strips or microdilution panels are not commercially available.

Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry and real-time PCR in a combined protocol for diagnosis of bloodstream infections: a turnaround time approach.

Bloodstream infections (BSIs) are serious infections associated with high rates of morbidity and mortality. Every hour delay in initiation of an effective antibiotic increases mortality due to sepsis by 7%. Turnaround time (TAT) for conventional blood cultures takes 48h, forcing physicians to streamline therapy by exposing patients to broad-spectrum antimicrobials. Our objective was (1) to evaluate the accuracy and TAT of an optimized workflow combining direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and in-house real-time polymerase chain reaction (PCR) for bacterial identification and antimicrobial resistance profiling directly from positive blood bottles for diagnosing bloodstream infections and (2) to verify the effect of reporting results to medical staff. A total of 103 BSI episodes from 91 patients admitted to three hospitals in São Paulo, Brazil were included. TAT from molecular versus conventional methods was measured and compared. Our protocol showed an overall agreement of 93.5% for genus and 78.5% for species identification; 74.2% for methicillin resistance detection, 89.2% for extended-spectrum ß-lactamase profiling, 77.8% for metallo-ß-lactamase profiling, and 100% for carbapenemase profile and vancomycin-resistance detection when compared with conventional testing. TAT of molecular sample processing according to our protocol was 38h shorter than conventional methods. Antimicrobial interventions were possible in 27 BSI episodes. Antimicrobial discontinuation was achieved in 12 BSI episodes while escalation of therapy occurred in 15 episodes. Antimicrobial therapy was inadequate in three (12%) BSI episodes diagnosed using results of molecular testing. Our in-house rapid protocol for identifying both bacteria and antimicrobial resistance provided rapid and accurate results, having good agreement with conventional testing results. These results could contribute to faster antimicrobial therapy interventions in BSI episodes.

Direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry and real-time PCR in a combined protocol for diagnosis of bloodstream infections: a turnaround time approach

ABSTRACT Bloodstream infections (BSIs) are serious infections associated with high rates of morbidity and mortality. Every hour delay in initiation of an effective antibiotic increases mortality due to sepsis by 7%. Turnaround time (TAT) for conventional blood cultures takes 48 h, forcing physicians to streamline therapy by exposing patients to broad-spectrum antimicrobials. Our objective was (1) to evaluate the accuracy and TAT of an optimized workflow combining direct matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) and in-house real-time polymerase chain reaction (PCR) for bacterial identification and antimicrobial resistance profiling directly from positive blood bottles for diagnosing bloodstream infections and (2) to verify the effect of reporting results to medical staff. A total of 103 BSI episodes from 91 patients admitted to three hospitals in São Paulo, Brazil were included. TAT from molecular versus conventional methods was measured and compared. Our protocol showed an overall agreement of 93.5% for genus and 78.5% for species identification; 74.2% for methicillin resistance detection, 89.2% for extended-spectrum β-lactamase profiling, 77.8% for metallo-β-lactamase profiling, and 100% for carbapenemase profile and vancomycin-resistance detection when compared with conventional testing. TAT of molecular sample processing according to our protocol was 38 h shorter than conventional methods. Antimicrobial interventions were possible in 27 BSI episodes. Antimicrobial discontinuation was achieved in 12 BSI episodes while escalation of therapy occurred in 15 episodes. Antimicrobial therapy was inadequate in three (12%) BSI episodes diagnosed using results of molecular testing. Our in-house rapid protocol for identifying both bacteria and antimicrobial resistance provided rapid and accurate results, having good agreement with conventional testing results. These results could contribute to faster antimicrobial therapy interventions in BSI episodes.

Outcomes of patients with invasive fusariosis who undergo further immunosuppressive treatments, is there a role for secondary prophylaxis?

BACKGROUND: Patients treated for invasive aspergillosis may relapse during subsequent periods of immunosuppression and should receive secondary prophylaxis. Little is known about the frequency of relapse and practices of secondary prophylaxis for invasive fusariosis (IF). OBJECTIVES: Evaluate practices of secondary prophylaxis and the frequency of relapse in patients who survived IF and were exposed to subsequent periods of immunosuppression. METHODS: Multicentre retrospective study of patients with haematological malignancies who developed IF, survived the initial fungal disease period, and were exposed to subsequent periods of immunosuppression. RESULTS: Among 40 patients, 35 received additional chemotherapy and developed neutropenia (median, 24 days; range, 4-104), and five received glucocorticoids for the treatment of graft-vs-host disease. Overall, 32 patients received secondary prophylaxis (voriconazole in 24) for a median of 112 days (range, 12-468). IF relapsed in five patients (12.5%): 2/8 (25%) not on prophylaxis and 3/32 (9.4%) receiving prophylaxis. Among 28 patients with disseminated IF, relapse occurred in 2/2 (100%) not on prophylaxis and in 3/26 (11.5%) on prophylaxis (P = 0.03). All patients who relapsed IF died. CONCLUSIONS: Patients with IF who survive the initial disease may relapse if exposed to subsequent episodes of immunosuppressive therapies. Secondary prophylaxis should be considered, especially if IF was disseminated.

Tailoring antimicrobials in febrile neutropenia: using faster diagnostic and communication tools to improve treatment in the era of extensively resistant pathogens

ABSTRACT Febrile Neutropenia represents a medical emergency and the use of appropriate antimicrobial therapy is essential for a better outcome. Although being time-consuming, conventional cultures and antimicrobial susceptibility tests remain the golden standard practices for microbiology identification. Final reports are typically available within several days. Faster diagnostic tools, such as species identification trough Matrix Assisted Laser Desorption Ionization-Time of Flight (MALDI-TOF) and molecular techniques might help to shorten time to diagnostic and also guide definitive therapy in this scenario. Here we present a case in which the use of a diagnostic molecular workflow combining MALDI-TOF and real-time PCR for relevant genes codifying antibiotic resistant integrated with instant communication report, led to a tailored and more appropriate treatment in a patient presenting with febrile neutropenia.

Rapid detection of blaKPC directly from surveillance rectal swabs by EasyQ KPC.

The performance of EasyQ KPC assay was evaluated for the first time for blaKPC detection directly from surveillance rectal swabs without broth enrichment. Using conventional polymerase chain reaction as gold standard method, EasyQ KPC and culture-based molecular tests demonstrated a sensitivity/specificity of 100%/87.3% and 83.3%/98.2%, respectively.

Clinical relevance of molecular identification of microorganisms and detection of antimicrobial resistance genes in bloodstream infections of paediatric cancer patients.

BACKGROUND: Bloodstream infections (BSIs) are the major cause of mortality in cancer patients. Molecular techniques are used for rapid diagnosis of BSI, allowing early therapy and improving survival. We aimed to establish whether real-time quantitative polymerase chain reaction (qPCR) could improve early diagnosis and therapy in paediatric cancer patients, and describe the predominant pathogens of BSI and their antimicrobial susceptibility. METHODS: Blood samples were processed by the BACTEC system and microbial identification and susceptibility tests were performed by the Phoenix system. All samples were screened by multiplex 16 s rDNA qPCR. Seventeen species were evaluated using sex-specific TaqMan probes and resistance genes blaSHV, blaTEM, blaCTX, blaKPC, blaIMP, blaSPM, blaVIM, vanA, vanB and mecA were screened by SYBR Green reactions. Therapeutic efficacy was evaluated at the time of positive blood culture and at final phenotypic identification and antimicrobial susceptibility results. RESULTS: We analyzed 69 episodes of BSI from 64 patients. Gram-positive bacteria were identified in 61 % of the samples, Gram-negative bacteria in 32 % and fungi in 7 %. There was 78.2 % of agreement between the phenotypic and molecular methods in final species identification. The mecA gene was detected in 81.4 % of Staphylococcus spp., and 91.6 % were concordant with the phenotypic method. Detection of vanA gene was 100 % concordant. The concordance for Gram-negative susceptibilities was 71.4 % for Enterobacteriaceae and 50 % for Pseudomonas aeruginosa. Therapy was more frequently inadequate in patients who died, and the molecular test was concordant with the phenotypic susceptibility test in 50 %. CONCLUSIONS: qPCR has potential indication for early identification of pathogens and antimicrobial resistance genes from BSI in paediatric cancer patients and may improve antimicrobial therapy.

Earlier diagnosis of invasive fusariosis with Aspergillus serum galactomannan testing.

Cross-reactivity of Fusarium species with serum galactomannan antigen (GMI) test has been observed. We sought to evaluate if GMI could help to early diagnose invasive fusariosis and to monitor treatment response. We reviewed the records of all patients with invasive fusariosis between 2008 and 2012 in three Brazilian hospitals. We selected patients who had at least 1 GMI test within 2 days before or after the date of the first clinical manifestation of fusariosis, and analyzed the temporal relationship between the first positive GMI test and the date of the diagnosis of invasive fusariosis, and the kinetics of GMI in relation to patients' response to treatment. We also selected 18 controls to determine the sensitivity and specificity of the test. Among 18 patients, 15 (83%) had at least one positive GMI (median 4, range 1-15). The sensitivity and specificity of was 83% and 67%, respectively. GMI was positive before the diagnosis of invasive fusariosis in 11 of the 15 cases (73%), at a median of 10 days (range 3-39), and after the diagnosis in 4 cases. GMI became negative in 8 of the 15 patients; 3 of these 8 patients (37.5%) were alive 90 days after the diagnosis of fusariosis compared with 2 of 7 (29%) who did not normalize GMI (p = 1.0). GMI is frequently positive in invasive fusariosis, and becomes positive before diagnosis in most patients. These findings may have important implications for the choice of antifungal therapy in settings with high prevalence of invasive fusariosis.

Diagnosis by real-time polymerase chain reaction of pathogens and antimicrobial resistance genes in bone marrow transplant patients with bloodstream infections.

BACKGROUND: Early identification of pathogens and antimicrobial resistance in bloodstream infections (BSIs) decreases morbidity and mortality, particularly in immunocompromised patients. The aim of the present study was to compare real-time polymerase chain reaction (PCR) with commercial kits for detection of 17 pathogens from blood culture (BC) and 10 antimicrobial resistance genes. METHODS: A total of 160 BCs were taken from bone marrow transplant patients and screened with Gram-specific probes by multiplex real-time PCR and 17 genus-specific sequences using TaqMan probes and blaSHV, blaTEM, blaCTX, blaKPC, blaIMP, blaSPM, blaVIM, vanA, vanB, and mecA genes by SYBR Green. RESULTS: Twenty-three of 33 samples identified by phenotypic testing were concordantly positive by BC and real-time PCR. Pathogen identification was discordant in 13 cases. In 12 of 15 coagulase-negative staphylococci, the mecA gene was detected and four Enterococcus spp. were positive for vanA. Two blaCTX and three blaSHV genes were found by quantitative PCR. The blaKPC and metallo-ß-lactamase genes were not detected. Five fungal species were identified only by real-time PCR. CONCLUSIONS: Real-time PCR could be a valuable complementary tool in the management of BSI in bone marrow transplants patients, allowing identification of pathogens and antimicrobial resistance genes.

Recomendações no manejo das complicações infecciosas no transplante de células-tronco hematopoéticas / Management of infectious complications after hematopoietic stem cell transplant

A infecção em receptores de transplante de células-tronco hematopoéticas (TCTH) está relacionada a altas taxas de morbidade e mortalidade. O tipo de transplante, a fonte de célula-tronco hematopoética, a utilização de doadores alternativos e outras medidas relacionadas ao procedimento influenciam diretamente no tipo e na intensidade da imunossupressão, modificando o risco de desenvolver uma infecção. Nesta seção são discutidas as estratégias para monitorização, diagnóstico e tratamento das infecções em receptores de TCTH em três fases: na fase pré-transplante, durante a fase de neutropenia, e na fase pós-pega do enxerto.

Infectious complications following stem cell transplantation are frequent and associated with high morbidity and mortality. Several factors related to the transplant procedure, such as type of transplant, the source of stem cells, the utilization of alternative donors are important determinants of the immune status of the host, and impact on the risk of infection. In this section we will discuss the different approaches for monitoring patients at risk and diagnosing and treating infectious complications in three time periods: before transplant, during neutropenia, and after engraftment.

Epidemiology and risk factors for bloodstream infections after allogeneic hematopoietic stem cell transplantion.

A total of 315 patients who underwent allogeneic Hematopoietic Stem Cell Transplantation (HSCT) during a 4-year period were analysed with the aim of collecting information on bloodstream infections (BSI). Eighty-four patients (27%) developed 112 BSI, with a cumulative risk of 20.6% at 30 days and 27.7% at 180 days. Overall, 127 pathogens were isolated, 95 (75%) gram-positive cocci, 27 (21%) gram-negative rods and 5 (4%) fungi. Enterococcus sp. accounted for 46 of 127 (36%) isolates. In a multivariable analysis only including baseline factors, the type of transplant was the only factor significantly associated with the risk of BSI and the risk was higher for patients receiving transplant from mismatched or unrelated donors. In a case-control study aimed at evaluating the predictive role of additional factors during transplant, the risk appeared to be higher in patients with a positive CMV antigenemia (p = 0.03; OR of 4.82; 95% CI, 1.21-19.17), long duration of severe granulocytopenia (p = 0.015; OR 7.53; 95% CI, 1.92 - 29.58) and lower platelet count (p < 0.001; OR 0.14; 95% CI, 0.05 - 0.40). By day 180 post-transplant, 87 (28%) out of 314 patients had died. The cumulative risk of death was significantly higher among patients with BSI than among other patients.

False-positive galactomannan platelia Aspergillus test results for patients receiving piperacillin-tazobactam.

At the bone marrow transplantation center of the San Martino Hospital (Genoa), we observed an increase in the rate of patients with positive Platelia Aspergillus (PA; Bio-Rad) test results, from 10% (38 of 386 patients) in the period from January 1999 through January 2003 to 36% (21 of 59 patients) in the period from February 2003 through May 2003. Positivity was significantly (P<.001) associated with the administration of piperacillin-tazobactam (PT) (17 [74%] of 23 patients who received PT had positive results vs. 4 [11%] of 36 who did not receive PT). Multivariate analysis found administration of PT ( chi 2=34.7; P<.001) and underlying disease ( chi 2=21.14; P<.001) to be associated with PA positivity. Of 15 PT batches tested, 12 had positive PA test results.