Rapid identification of moulds and arthroconidial yeasts from positive blood cultures by MALDI-TOF mass spectrometry.

Moulds and arthroconidial yeasts are potential life-threatening agents of fungemia in immunocompromised patients. Fast and accurate identification (ID) of these pathogens hastens initiation of targeted antifungal therapy, thereby improving the patients' prognosis. We describe a new strategy that enabled the identification of moulds and arthroconidial yeasts directly from positive blood cultures by MALDI-TOF mass spectrometry (MS). Positive blood cultures (BCs) with Gram staining showing hyphae and/or arthroconidia were prospectively selected and submitted to an in-house protein extraction protocol. Mass spectra were obtained by Vitek MS™ system, and identifications were carried out with in the research use only (RUO) mode with an extended database (SARAMIS™ [v.4.12] plus in-house database). Fusarium solani, Fusarium verticillioides, Exophiala dermatitidis, Saprochaete clavata, and Trichosporon asahii had correct species ID by MALDI-TOF MS analysis of positive BCs. All cases were related to critically ill patients with high mortality fungemia and direct ID from positive BCs was helpful for rapid administration of targeted antifungal therapy.

A multiplex nested PCR for the detection and identification of Candida species in blood samples of critically ill paediatric patients.

BACKGROUND: Nosocomial candidaemia is associated with high mortality rates in critically ill paediatric patients; thus, the early detection and identification of the infectious agent is crucial for successful medical intervention. The PCR-based techniques have significantly increased the detection of Candida species in bloodstream infections. In this study, a multiplex nested PCR approach was developed for candidaemia detection in neonatal and paediatric intensive care patients. METHODS: DNA samples from the blood of 54 neonates and children hospitalised in intensive care units with suspected candidaemia were evaluated by multiplex nested PCR with specific primers designed to identify seven Candida species, and the results were compared with those obtained from blood cultures. RESULTS: The multiplex nested PCR had a detection limit of four Candida genomes/mL of blood for all Candida species. Blood cultures were positive in 14.8% of patients, whereas the multiplex nested PCR was positive in 24.0% of patients, including all culture-positive patients. The results obtained with the molecular technique were available within 24 hours, and the assay was able to identify Candida species with 100% of concordance with blood cultures. Additionally, the multiplex nested PCR detected dual candidaemia in three patients. CONCLUSIONS: Our proposed PCR method may represent an effective tool for the detection and identification of Candida species in the context of candidaemia diagnosis in children, showing highly sensitive detection and the ability to identify the major species involved in this infection.

Predictive models-assisted diagnosis of AIDS-associated Pneumocystis jirovecii pneumonia in the emergency room, based on clinical, laboratory, and radiological data.

We assessed predictive models (PMs) for diagnosing Pneumocystis jirovecii pneumonia (PCP) in AIDS patients seen in the emergency room (ER), aiming to guide empirical treatment decisions. Data from suspected PCP cases among AIDS patients were gathered prospectively at a reference hospital's ER, with diagnoses later confirmed through sputum PCR analysis. We compared clinical, laboratory, and radiological data between PCP and non-PCP groups, using the Boruta algorithm to confirm significant differences. We evaluated ten PMs tailored for various ERs resource levels to diagnose PCP. Four scenarios were created, two based on X-ray findings (diffuse interstitial infiltrate) and two on CT scans ("ground-glass"), incorporating mandatory variables: lactate dehydrogenase, O2sat, C-reactive protein, respiratory rate (> 24 bpm), and dry cough. We also assessed HIV viral load and CD4 cell count. Among the 86 patients in the study, each model considered either 6 or 8 parameters, depending on the scenario. Many models performed well, with accuracy, precision, recall, and AUC scores > 0.8. Notably, nearest neighbor and naïve Bayes excelled (scores > 0.9) in specific scenarios. Surprisingly, HIV viral load and CD4 cell count did not improve model performance. In conclusion, ER-based PMs using readily available data can significantly aid PCP treatment decisions in AIDS patients.

First occurrence of feline sporotrichosis in a metropolitan area of Central-West Brazil.

Sporotrichosis is a neglected mycosis that affects human and animal hosts, including domestic cats. In Brazil, its most frequently diagnosed etiological agent is Sporothrix brasiliensis. Zoonotic transmission of S. brasiliensis occurs via direct contact between an infected cat and a susceptible human host. Notification of confirmed cases of feline sporotrichosis is not mandatory in Brazil. The metropolitan area of Goiania city can be considered a silent area for the occurrence of feline sporotrichosis. In this context, voluntary reporting of feline sporotrichosis cases is recommended for all healthcare professionals. This study aimed to report the first occurrence of S. brasiliensis in a cat from the metropolitan area of Goiania city. Cytopathology, mycology, thermal dimorphism and calmodulin gene amplification tests were performed. The mycological and molecular biological diagnoses corresponded to S. brasiliensis. The etiological agent of zoonotic sporotrichosis was detected in the metropolitan area of Goiania city, and therefore there is a risk of the emergence of new cases of cats infected with S. brasiliensis and the occurrence of zoonotic transmission of this fungus.

Performance of a Real Time PCR for Pneumocystis jirovecii Identification in Induced Sputum of AIDS Patients: Differentiation between Pneumonia and Colonization.

Pneumocystis jirovecii pneumonia (PcP) remains an important cause of morbimortality worldwide and a diagnostic challenge. Conventional methods have low accuracy, hardly discriminating colonization from infection, while some new high-cost or broncho-alveolar lavage-based methods have limited usefulness in developing countries. Quantitative PCR (qPCR) tests may overcome these limitations due to their high accuracy, possibility of automation, and decreasing cost. We evaluated an in-house qPCR targeting the fungus mtSSU gene using induced sputum. Sensitivity of the assay (ten target gene copies/assay) was determined using recombinant plasmids. We prospectively studied 86 AIDS patients with subacute respiratory symptoms in whom PcP was suspected. qPCR results were determined as quantification cycles (Cq) and compared with a qualitative PCR performed in the same IS, serum 1,3-ß-D-Glucan assay, and a clinical/laboratory/radiology index for PcP. The qPCR clustered the patients in three groups: 32 with Cq ≤ 31 (qPCR+), 45 with Cq ≥ 33 (qPCR-), and nine with Cq between 31-33 (intermediary), which, combined with the other three analyses, enabled us to classify the groups as having PcP, not P. jirovecii-infected, and P. jirovecii-colonized, respectively. This molecular assay may contribute to improve PcP management, avoiding unnecessary treatments, and our knowledge of the natural history of this infection.

Rhino-orbito-cerebral mucormycosis caused by Rhizopus microsporus var. microsporus in a diabetic patient with COVID-19.

COVID-19 disease caused by the SARS-CoV-2 coronavirus causes a wide range of clinical manifestations, ranging from mild to severe, with the main ones affecting the respiratory tract, such as pneumonia. In patients with greater severity, the high frequency of bacterial and fungal coinfection stands out, a situation related both to the patient's pre-existing comorbidities and due to the hospitalization itself. Cases of mucormycosis associated with COVID-19 were highlighted in the lay and scientific media, with the increase in mycosis cases being directly and indirectly attributed to the viral infection. This report describes a case of rhino-orbito-cerebral mucormycosis in a diabetic patient hospitalized for COVID-19, whose diagnosis was confirmed by identifying the agent Rhizopus microsporus var. microsporus through culture for fungi and PCR examination.

A neglected disease. Human sporotrichosis in a densely populated urban area in São Paulo, Brazil: clinical-epidemiological and therapeutic aspects.

Human sporotrichosis is caused by different Sporothrix species; however, Sporothrix brasiliensis is the main species, usually related to cat transmission in urban areas. A retrospective descriptive study was conducted at the Institute of Infectology Emílio Ribas from 2010 to 2018. Demography, clinical, diagnostic, and therapeutic data were obtained from medical records. Polymerase chain reaction of the calmodulin gene was performed to identify Sporothrix species. In addition, to evaluate the spread of the disease across São Paulo metropolitan region, TerraView version 4.2.2 software was used for geocoding cases according to residence addresses. Kernell's maps using QGIS software version 2.16.3 were constructed to determine the concentration of cases. Results: 260 cases of sporotrichosis were diagnosed between 2010 and 2018. We observed a 700% increment in the number of human cases in the 2016-2018 triennium compared with the 2013-2015 triennium. Female adults with a median age of 46 years old were the predominant infected group associated with cats' exposition at home care, although the age range of all patients was 01 to 86 years old. The main epidemiological risk of acquiring sporotrichosis was contact with cats, reported by 96.5% of the patients. Molecular identification showed that most of the tested isolates were Sporothrix brasiliensis. Lymphocutaneous form was observed in 59.2% and fixed cutaneous form in 37.5% of the patients. Regarding treatment, itraconazole was the main drug used (94.2%) with a cure rate of 98.8%. We observed an important spread of human sporotrichosis involving cat transmission caused by Sporothrix brasiliensis in a densely populated area of São Paulo state. These results are important to alert clinicians and dermatologists about the occurrence and progression of a neglected tropical disease in an urban area and the urgent necessity to include sporotrichosis as a differential diagnosis in the clinical investigation routine.

Lack of efficacy of echinocandins against high metabolic activity biofilms of Candida parapsilosis clinical isolates.

Candida parapsilosis produces biofilm, which colonizes catheters and other invasive medical devices that are manipulated by health care workers. In previous studies, C. parapsilosis in vitro biofilms have exhibited high resistance rates against conventional antifungals, but susceptibility to both echinocandins and lipid formulations of amphotericin B (lipid complex and liposomal). However, a recent study showed good activity of amphotericin B deoxycholate on the biomass of C. parapsilosis biofilms. Although moderate activity of echinocandins has been demonstrated against low metabolic activity biofilms of C. parapsilosis, few studies have analyzed the action of these drugs on high metabolic activity biofilms. Moreover, high biofilm-forming isolates have been associated with central venous catheter-related fungemia outbreaks and higher mortality rates. Therefore, it is relevant to verify the activity of the main antifungal drugs against high metabolic activity biofilms of C. parapsilosis. Our study aimed to evaluate the in vitro activity of amphotericin B deoxycholate, anidulafungin, caspofungin, and micafungin against high biofilm-forming and high metabolic activity clinical isolates of C. parapsilosis. Our results showed good activity of amphotericin B against C. parapsilosis biofilms, but none of the echinocandin drugs was effective. This suggests that amphotericin B deoxycholate may be a better choice than echinocandins for the treatment of biofilm-associated infections by C. parapsilosis, mainly in countries with insufficient health care resources to purchase lipid formulations of amphotericin B. These results warn of the possibility of persistent catheter-related candidemia caused by high biofilm-forming C. parapsilosis strains when treated with echinocandin drugs.

A Real Time PCR strategy for the detection and quantification of Candida albicans in human blood.

Candidemia is a significant cause of bloodstream infections (BSI) in nosocomial settings. The identification of species can potentially improve the quality of care and decrease human mortality. Quantitative PCR (qPCR) was evaluated for Candida albicans detection using culture suspensions containing C. albicans , spiked human blood, the cloned qPCR target fragment (ITS2 region) and the results of these assays were compared. The assays showed a good detection limit: C. albicans DNA extracted from yeast (sensitivity 0.2 CFU/µL), spiked human blood (sensitivity 10 CFU/mL), and cloned fragment of ITS2 region (sensitivity 20 target copies/µL). The efficiency of ITS2 fragment-qPCR ranged from 89.67 to 97.07, and the linearity (R2) of the standard curve ranged from 0.992 to 0.999. The results showed that this ITS2-qPCR has a great potential as a molecular prototype model for the development of a test to be applied in clinical practice, greatly reducing the time of candidemia diagnosis, which is extremely important in this clinical setting.

First occurrence of feline sporotrichosis in a metropolitan area of Central-West Brazil

ABSTRACT Sporotrichosis is a neglected mycosis that affects human and animal hosts, including domestic cats. In Brazil, its most frequently diagnosed etiological agent is Sporothrix brasiliensis. Zoonotic transmission of S. brasiliensis occurs via direct contact between an infected cat and a susceptible human host. Notification of confirmed cases of feline sporotrichosis is not mandatory in Brazil. The metropolitan area of Goiania city can be considered a silent area for the occurrence of feline sporotrichosis. In this context, voluntary reporting of feline sporotrichosis cases is recommended for all healthcare professionals. This study aimed to report the first occurrence of S. brasiliensis in a cat from the metropolitan area of Goiania city. Cytopathology, mycology, thermal dimorphism and calmodulin gene amplification tests were performed. The mycological and molecular biological diagnoses corresponded to S. brasiliensis. The etiological agent of zoonotic sporotrichosis was detected in the metropolitan area of Goiania city, and therefore there is a risk of the emergence of new cases of cats infected with S. brasiliensis and the occurrence of zoonotic transmission of this fungus.

Diagnosis of neonatal group B Streptococcus sepsis by nested-PCR of residual urine samples.

Group B streptococcus (GBS) remains the most common cause of early-onset sepsis in newborns. Laboratory gold-standard, broth culture methods are highly specific, but lack sensitivity. The aim of this study was to validate a nested-PCR and to determine whether residue volumes of urine samples obtained by non invasive, non sterile methods could be used to confirm neonatal GBS sepsis. The nested-PCR was performed with primers of the major GBS surface antigen. Unavailability of biological samples to perform life supporting exams, as well as others to elucidate the etiology of infections is a frequent problem concerning newborn patients. Nevertheless, we decided to include cases according to strict criteria: newborns had to present with signs and symptoms compatible with GBS infection; at least one of the following biological samples had to be sent for culture: blood, urine, or cerebrospinal fluid; availability of residue volumes of the samples sent for cultures, or of others collected on the day of hospitalization, prior to antibiotic therapy prescription, to be analyzed by PCR; favorable outcome after GBS empiric treatment. In only one newborn GBS infection was confirmed by cultures, while infection was only presumptive in the other three patients (they fulfilled inclusion criteria but were GBS-culture negative). From a total of 12 biological samples (5 blood, 3 CSF and 4 urine specimen), eight were tested by culture methods (2/8 were positive), and 8 were tested by PCR (7/8 were positive), and only 4 samples were simultaneously tested by both methods (1 positive by culture and 3 by PCR). In conclusion, although based on a restricted number of neonates and samples, our results suggest that the proposed nested-PCR might be used to diagnose GBS sepsis as it has successfully amplified the three types of biological samples analyzed (blood, urine and cerebrospinal fluid), and was more sensitive than culture methods as PCR in urine confirmed diagnosis in all four patients. Moreover, PCR has enabled us to use residue volumes of urine samples collected by non invasive, non sterile methods, what is technically adequate as GBS is not part of the normal urine flora, thus avoiding invasive procedures such as suprapubic bladder punction or transurethral catheterization. At the same time, the use of urine instead of blood samples could help preventing newborns blood spoliation.

Case Report: Misleading Serological Diagnosis of Paracoccidioidomycosis in a Young Patient with the Acute Form Disease: Paracoccidioides brasiliensis or Paracoccidioides lutzii?

Negative results in serological routine screening of patients with microbiologically proven Paracoccidioidomycosis (PCM) are occasionally reported. Failure in detecting anti-Paracoccidioides antibodies has been ascribed to factors either related to serological techniques or to the status of the host immune reactivity. Recently, this issue has been renewed by the recognition that the Paracoccidioides genera comprises two species, lutzii and brasiliensis, which have distinct antigenic profiles and, therefore, may elicit different host antibody responses. We describe a patient with the acute form PCM due to Paracoccidioides brasiliensis with negative results on two reference centers' routine screening for P. brasiliensis antibodies, but positive results with Paracoccidioides lutzii antigens. The present case report suggests that antibodies elicited during P. brasiliensis infection recognize antigenic fractions shared by both species, highlighting the difficulties in distinguishing the two infections by means of the currently available routine serological assays.

Severe anemia, panserositis, and cryptogenic hepatitis in an HIV patient infected with Bartonella henselae.

Bartonella spp. constitute emerging pathogens of worldwide distribution. Bacillary angiomatosis is the most frequent skin manifestation of bartonelloses; nevertheless, B. henselae infection should always be considered systemic, especially in immunodeficient individuals. The authors report the case of an AIDS patient with bacillary angiomatosis, who had concurrent severe anemia, hepatitis, peritonitis, pleuritis, and pericarditis. Clinical manifestation, electronic microscopic examination of erythrocytes, and histopathology of a papule biopsy suggested a Bartonella sp. infection. Multiple genes were target by PCR and B. henselae DNA was amplified and sequenced (GenBank accession number EF196804) from the angiomatous papule. Treatment with clarithromycin resulted in resolution of the bacillary angiomatosis, fever, anemia, panserosites, and hepatitis.

Bartonella henselae AS A PUTATIVE CAUSE OF CONGENITAL CHOLESTASIS.

Severe anemia and cholestatic hepatitis are associated with bartonella infections. A putative vertical Bartonella henselae infection was defined on the basis of ultrastructural and molecular analyses in a three-year-old child with anemia, jaundice and hepatosplenomegaly since birth. Physicians should consider bartonellosis in patients with anemia and hepatitis of unknown origin.

Detection of EBV-DNA in serum samples of an immunosuppressed child during a three years follow-up: association of clinical and PCR data with active infection.

Twenty-four whole blood and serum samples were drawn from an eight year-old heart transplant child during a 36 months follow-up. EBV serology was positive for VCA-IgM and IgG, and negative for EBNA-IgG at the age of five years old when the child presented with signs and symptoms suggestive of acute infectious mononucleosis. After 14 months, serological parameters were: positive VCA-IgG, EBNA-IgG and negative VCA-IgM. This serological pattern has been maintained since then even during episodes suggestive of EBV reactivation. PCR amplified a specific DNA fragment from the EBV gp220 (detection limit of 100 viral copies). All twenty-four whole blood samples yielded positive results by PCR, while 12 out of 24 serum samples were positive. We aimed at analyzing whether detection of EBV-DNA in serum samples by PCR was associated with overt disease as stated by the need of antiviral treatment and hospitalization. Statistical analysis showed agreement between the two parameters evidenced by the Kappa test (value 0.750; p < 0.001). We concluded that detection of EBV-DNA in serum samples of immunosuppressed patients might be used as a laboratory marker of active EBV disease when a Real-Time PCR or another quantitative method is not available.

Frequency of the deltaF508 mutation in 108 cystic fibrosis patients in Sao Paulo: comparison with reported Brazilian data.

PURPOSE: To analyze the frequency of the delta F508 (deltaF508) deletion mutation in 108 unrelated cystic fibrosis patients and compare the results with the previously reported data for Brazilian patients. Cystic fibrosis is the leading cause of genetic disease in Caucasians, and the deltaF508 deletion is the most common mutation associated with the disease. METHOD: The frequency of the deltaF508 mutation was assessed by means of a polymerase chain reaction (PCR) followed by detection in 8% silver-stained polyacrylamide gels. RESULTS: Twenty-three of 108 patients (21.3%) were homozygous for the deltaF508 deletion, 50 were heterozygous (46.3%), and the remaining 35 (32.4%) were non-carriers. In terms of alleles, there were 96 mutated (96/216 or 44.45%) and 120 wild-type ones (120/216 or 55.5%). CONCLUSION: The 44.45% of affected alleles that were found is higher than the 33% first described in 1993, but slightly lower than the 48% recently reported. Moreover, our data corroborated the idea that the frequency of the deltaF508 mutation is lower in Brazil in comparison to that found in studies carried out in Europe and North American (circa 70.0%), probably due to increased racial miscegenation. These findings must be taken into account before any genetic screening of the population is proposed in Brazil.

Evaluation of VITEK 2 for discriminating Trichosporon species: misidentification of Trichosporon non-T. asahii.

The VITEK 2 system was evaluated for the identification of 74 Trichosporon invasive and non-invasive clinical isolates, comparing its results with the IGS1 sequencing. The system correctly identified Trichosporon asahii but not non-T. asahii isolates, which represented nearly 50% of the invasive infections in our nosocomial setting.

Evaluation of the MALDI-TOF VITEK MS™ system for the identification of Candida parapsilosis, C. orthopsilosis and C. metapsilosis from bloodstream infections.

Twenty-nine Candida parapsilosis, seventeen Candida orthopsilosis and two Candida metapsilosis bloodstream isolates were submitted for identification by VITEK-MS™ mass spectrometer. Four isolates, two C. orthopsilosis and two C. metapsilosis, were not identified. Inclusion of Superspectra of both species in this database is required to improve its discrimination power.

Serological diagnosis of paracoccidioidomycosis: high rate of inter-laboratorial variability among medical mycology reference centers.

BACKGROUND: Serological tests have long been established as rapid, simple and inexpensive tools for the diagnosis and follow-up of PCM. However, different protocols and antigen preparations are used and the few attempts to standardize the routine serological methods have not succeeded. METHODOLOGY/PRINCIPAL FINDINGS: We compared the performance of six Brazilian reference centers for serological diagnosis of PCM. Each center provided 30 sera of PCM patients, with positive high, intermediate and low titers, which were defined as the "reference" titers. Each center then applied its own antigen preparation and serological routine test, either semiquantitative double immunodifusion or counterimmmunoelectrophoresis, in the 150 sera from the other five centers blindly as regard to the "reference" titers. Titers were transformed into scores: 0 (negative), 1 (healing titers), 2 (active disease, low titers) and 3 (active disease, high titers) according to each center's criteria. Major discordances were considered between scores indicating active disease and scores indicating negative or healing titers; such discordance when associated with proper clinical and other laboratorial data, may correspond to different approaches to the patient's treatment. Surprisingly, all centers exhibited a high rate of "major" discordances with a mean of 31 (20%) discordant scores. Alternatively, when the scores given by one center to their own sera were compared with the scores given to their sera by the remaining five other centers, a high rate of major discordances was also found, with a mean number of 14.8 sera in 30 presenting a discordance with at least one other center. The data also suggest that centers that used CIE and pool of isolates for antigen preparation performed better. CONCLUSION: There are inconsistencies among the laboratories that are strong enough to result in conflicting information regarding the patients' treatment. Renewed efforts should be promoted to improve standardization of the serological diagnosis of PCM.

Factors associated with hyperglycemia and low insulin levels in children undergoing cardiac surgery with cardiopulmonary bypass who received a single high dose of methylprednisolone.

OBJECTIVES: Administering steroids before cardiopulmonary bypass in pediatric heart surgery modulates systemic inflammatory response syndrome and improves postoperative recovery. However, the use of steroids aggravates hyperglycemia, which is associated with a poor prognosis. Adult patients with systemic inflammatory response syndrome usually evolve with hyperglycemia and high insulin levels, whereas >90% of pediatric patients exhibit hyperglycemia and low insulin levels. This study aims to determine: A) the metabolic and inflammatory factors that are associated with hyperglycemia and low insulin levels in children who underwent cardiac surgery with cardiopulmonary bypass and who received a single high dose of methylprednisolone and B) the best predictors of insulin variation using a mathematical model. METHODS: This preliminary study recruited 20 children who underwent heart surgery with cardiopulmonary bypass and received methylprednisolone (30 mg/kg) immediately after anesthesia. Among the 20 patients initially recruited, one was excluded because of the absence of hyperglycemia and lower insulin levels after surgery. However, these abnormalities were confirmed in the remaining 19 children. The C-peptide, CRP, IL-6, and adrenomedullin levels were measured before surgery, immediately after cardiopulmonary bypass, and on the first, second, and third days after cardiac surgery. RESULTS: IL-6, CRP, and adrenomedullin increments were observed, whereas the C-peptide levels remained within reference intervals. CONCLUSION: The multiple regression model demonstrated that in addition to age and glycemia (two well-known factors that are directly involved in glucose metabolism), adrenomedullin and IL-6 levels were independent factors associated with lower insulin concentrations. These four parameters were responsible for 64.7% of the observed insulin variances. In addition, the fact that C-peptide levels did not fall together with insulin could have grounded the medical decision not to administer insulin to patients.