A rare case of cutaneous Papiliotrema (Cryptococcus) laurentii infection in a 23-year-old Caucasian woman affected by an autoimmune thyroid disorder with hypothyroidism.

In recent years, the frequency of infections due to saprophytic fungi has increased. Cryptococcus laurentii, recently classified as Papiliotrema laurentii, is responsible for fungemia, meningitis, and superficial infections. Here, we report the first case of cutaneous Papiliotrema (Cryptococcus) laurentii infection in a 23-year-old Caucasian woman affected by an autoimmune thyroiditis with hypothyroidism. Impairments of the immune system are often associated with unusual fungal infections, which cannot be neglected. The isolate strain was susceptible to Amphotericin B while resistant to fluconazole, itraconazole, voriconazole, and terbinafine. The patient was successfully treated with Amphotericin B.

A significant decreased in the frequency of Clostridioides difficile infection at the Italian Hospital of Desio over the last decade.

Clostridiodes difficile infection (CDI) is the most important cause of healthcare-associated diarrhea. The decreasing trend of CDI from 15% to 4% observed at the Italian Hospital of Desio over a 10-year period is due to prevention strategies. Our data highlight the importance of surveillance studies to control CDI.

A rare case of Clostridium paraputrificum bacteremia in a 78-year-old Caucasian man diagnosed with an intestinal neoplasm.

Clostridium like species, particularly Clostridium perfrigens, are the second most common causes of human anaerobic infections, including myonecrosis and bacteremia. Clostridium paraputrificum is an infrequent isolate, which has been identified in only 1% of reported cases of clostridial infections. We herein report a rare case of C. paraputrificum bacteremia in a 78-year-old Caucasian man diagnosed with an intestinal carcinoma and liver neoplastic lesions. The isolate was susceptible to chloramphenicol, meropenem, metronidazole, vancomycin, and resistant to clindamycin and penicillin, and the patient was successfully treated with metronidazole. Malignancy and inflammatory bowel diseases are often associated with clostridial bacteremia, which cannot be neglected.

Multiple Parasitic Infestation in a Nine-month-old Patient: A Case Report.

We are reporting the case of a nine-month-old Pakistani female with complaint of growth retardation who presented multiple intestinal parasitic infections. Probably because of contamination with fecal matter, the initial microscopic examination of the urinary sample revealed the presence of eggs of Enterobius vermicularis, cysts of Entamoeba coli, and an organism similar to mites. Stool samples were obtained after two weeks and microscopic investigation confirmed the presence of Enterobius vermicularis eggs, cysts of Entamoeba coli, and hookworm eggs. The patient was immediately subjected to mebendazole therapy associated with trimethoprim-sulfamethoxazole, to which she responded well. Follow-up stool re-examinations performed 15 and 30 days after the treatment tested negative for all parasitic ova and cysts. This study reflects the importance of considering multiple parasitic infestations in low socio-economic populations and highlights the need of improving poor hygienic conditions to prevent such infections, in particular in children.

Genus-level identification of dermatophytes by MALDI-TOF MS after 2 days of colony growth.

Matrix-assisted laser desorption-ionization time-of-flight mass spectrometry (MALDI-TOF MS) is becoming a popular technology in clinical microbiology. It is a fast and highly specific method for the routine identification of micro-organisms. In this study, we evaluated the suitability of dermatophyte identification after only 2 days of colony growth using MALDI-TOF MS. Two protein extraction protocols were also evaluated consisting of either formic acid alone or of ethanol-formic acid-acetonitrile to achieve a complete protein extraction. Morphology-based techniques were used as the diagnostic standard methods and MALDI-TOF MS results were obtained using the manufacturer's spectral library. Using the formic acid protein extraction protocol after 2 days of colony growth, 70 and 46% of dermatophytes were properly identified at the genus and species-level respectively. The addition of ethanol-formic acid-acetonitrile extraction protocol increased the identification to 90 and 62%. Based on our observations, we propose a two-step workflow for the fast and reliable identification of dermatophytes after only 2 days of colony growth. This flow chart consists of a first direct deposition procedure with the addition of formic acid, followed by a complete protein extraction when dermatophyte identification is not successful. SIGNIFICANCE AND IMPACT OF THE STUDY: In this study, a two-step workflow for the identification of clinical dermatophytes using MALDI-TOF analysis and commercially available spectral library was developed. The workflow consists of an initial direct deposition of the sample on the MALDI plate and formic acid protein extraction at 2 days of growth culture; if dermatophyte identification is not successful, a complete protein extraction using ethanol-formic acid-acetonitrile is subsequently performed. Using this workflow, the correct isolate identifications increase up to 90%; of these, 27% are identified at the genus-level, providing sufficient information to start an antifungal treatment. The method here proposed represents a fast and useful approach to differentiate dermatophytes grown in culture.

Improvement in the detection of enteric protozoa from clinical stool samples using the automated urine sediment analyzer sediMAX® 2 compared to sediMAX® 1.

Detection of intestinal parasites from fecal samples is routinely performed by direct wet mount examination. This method requires skilled personnel, and it is time consuming. The aim of this work is to demonstrate the usefulness of the newer automated urinary sediment analyser sediMAX 2 for a fast detection of intestinal protozoa in stool samples. A total of 700 consecutively preserved samples consisting of 70 positives and 630 negatives were analyzed. SediMAX 2 takes digital images of each sediment sample, and analysis was conducted using a dilution of stool specimens, allowing determination of typical morphology. Compared to manual microscopy, sediMAX 2 showed sensitivity and specificity of 100 % in the detection of intestinal parasites, as also recently demonstrated for sediMAX 1. However, all clinically important human protozoa were detected using only 15 images for each specimen, compared to 30 images required in sediMAX 1 analysis. Moreover, changing manually the focus, it is possible to carry out a discrimination between morphologically identical Entamoeba complex members, including the pathogenic E. histolytica and the non-pathogenic E. dispar, E. moshkovskii and E. Bangladeshi, from the non-pathogenic Entamoeba coli based on the number of nuclei present in the cells. This study presents sediMAX 2 as an automatic aid to traditional microscopy.

Reducing time to identification of aerobic bacteria and fastidious micro-organisms in positive blood cultures.

Rapid and early identification of micro-organisms in blood has a key role in the diagnosis of a febrile patient, in particular, in guiding the clinician to define the correct antibiotic therapy. This study presents a simple and very fast method with high performances for identifying bacteria by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) after only 4 h of incubation. We used early bacterial growth on PolyViteX chocolate agar plates inoculated with five drops of blood-broth medium deposited in the same point and spread with a sterile loop, followed by a direct transfer procedure on MALDI-TOF MS target slides without additional modification. Ninety-nine percentage of aerobic bacteria were correctly identified from 600 monomicrobial-positive blood cultures. This procedure allowed obtaining the correct identification of fastidious pathogens, such as Streptococcus pneumoniae, Neisseria meningitidis and Haemophilus influenzae that need complex nutritional and environmental requirements in order to grow. Compared to the traditional pathogen identification from blood cultures that takes over 24 h, the reliability of results, rapid performance and suitability of this protocol allowed a more rapid administration of optimal antimicrobial treatment in the patients. SIGNIFICANCE AND IMPACT OF THE STUDY: Bloodstream infections are serious conditions with a high mortality and morbidity rate. Rapid identification of pathogens and appropriate antimicrobial therapy have a key role for successful patient outcome. In this work, we developed a rapid, simplified, accurate, and efficient method, reaching 99 % identification of aerobic bacteria from monomicrobial-positive blood cultures by using early growth on enriched medium, direct transfer to target plate without additional procedures, matrix-assisted laser desorption ionization-time of flight mass spectrometry and SARAMIS database. The application of this protocol allows to anticipate appropriate antibiotic therapy.

The clinical utility of D-dimer/platelet count ratio in pregnant women.

OBJECTIVE: We performed a retrospective study to assess the clinical utility of a new index, D-dimer/platelet count (DD/PLT) ratio, in discriminating preeclampsia from normal pregnancy and gestational hypertension during third trimester, compared to the biomarkers currently used, such as D-dimer (DD), platelet (PLT), lymphocyte (LIN) and neutrophil (NEU) counts, fibrinogen (FIB), PLT/NEU, NEU/LIN and PLT/LIN ratios. STUDY DESIGN: We retrospectively included 213 subjects. Of them, 163 and 50 were singleton pregnant and healthy non-pregnant women, respectively. Among pregnant women, 105 had normal pregnancy, 33 had gestational hypertension, and 25 had preeclampsia. RESULTS: Using Receiver Operating Curve (ROC) analysis, DD/PLT ratio showed significant higher area under the curve (AUC) (0.90; 95% confidence interval (CI) 0.84-0.95) in discriminating preeclampsia from normal pregnancy compared to those of DD, NEU, FIB, LIN, PLT/NEU, NEU/LIN and PLT/LIN ratios (p < .03). In discriminating preeclampsia from gestational hypertension, the DD/PLT AUC (0.90; 95% CI 0.79-0.96) was significantly higher than those of DD, NEU, FIB, LIN, NEU/LIN and PLT/LIN ratios (p < .03), and not statistically different from those of PLT (p = .22) and PLT/NEU ratio (p = .46). CONCLUSIONS: This study shows that DD/PLT ratio helps to discriminate preeclampsia from normal pregnancy and gestational hypertension. Large-scale studies are needed to verify its clinical usefulness, and to suggest more appropriate cutoff values for a widespread use.

Monoclonal free light chain detection and quantification: Performances and limits of available laboratory assays.

The detection and quantification of immunoglobulin free light chains in serum and urine is recommended for the diagnosis and monitoring of monoclonal gammopathies according to the guidelines of the International Myeloma Working Group (IMWG). Several tests are currently available in the clinical laboratory to detect and quantify free light chains but although quality, efficiency, and effectiveness have been improved, the results are still variable and poorly harmonized and standardized. The present review article wants to analyze these aspects, with a keen eye on techniques, such as mass spectrometry, that could replace in the practical clinical laboratory the current methods including Bence-Jones protein assay and free light chain immunoassays.

Detection of intestinal parasites by use of the cuvette-based automated microscopy analyser sediMAX(®).

Microscopy is the reference method for intestinal parasite identification. The cuvette-based automated microscopy analyser, sediMAX 1, provides 15 digital images of each sediment sample. In this study, we have evaluated this fully automated instrument for detection of enteric parasites, helminths and protozoa. A total of 700 consecutively preserved samples consisting of 60 positive samples (50 protozoa, ten helminths) and 640 negative samples were analysed. Operators were blinded to each others' results. Samples were randomized and were tested both by manual microscopy and sediMAX 1 for parasite recognition. The sediMAX 1 analysis was conducted using a dilution of faecal samples, allowing determination of morphology. The data obtained using sediMAX 1 showed a specificity of 100% and a sensitivity of 100%. Some species of helminths, such as Enterobius vermicularis, Strongyloides stercolaris, the Ancylostoma duodenale/Necator americanus complex, and schistosomes were not considered in this work, because they are rare in stool specimens, are not easily detectable with microscopy analysis, and require specific recovery techniques. This study demonstrated for the first time that sediMAX 1 can be an aid in enteric parasite identification.

Identification and expression analysis of Drosophila melanogaster genes encoding beta-hexosaminidases of the sperm plasma membrane.

Sperm surface beta-N-acetylhexosaminidases are among the molecules mediating early gamete interactions in invertebrates and vertebrates, including man. The plasma membrane of Drosophila spermatozoa contains two beta-N-acetylhexosaminidases, DmHEXA and DmHEXB, which are required for egg fertilization. Here, we demonstrate that three putative Drosophila melanogaster genes predicted to code for beta-N-acetylhexosaminidases, Hexo1, Hexo2, and fdl, are all expressed in the male germ line. fdl codes for a homolog of the alpha-subunit of the mammalian lysosomal beta-N-acetylhexosaminidase Hex A. Hexo1 and Hexo2 encode two homologs of the beta-subunit of all known beta-N-acetylhexosaminidases, which we have named beta(1) and beta(2), respectively. Immunoblot analysis of sperm proteins indicated that the gene products associate in different heterodimeric combinations forming DmHEXA, with an alphabeta(2) structure, and DmHEXB, with a beta(1)beta(2) structure. Immunofluorescence demonstrated that all the gene products localized to the sperm plasma membrane. Although none of the genes was testis-specific, fdl was highly and preferentially expressed in the testis, whereas Hexo1 and Hexo2 showed broader tissue expression. Enzyme assays carried out on testis and on a variety of somatic tissues corroborated the results of gene expression analysis. These findings for the first time show the in vivo expression in insects of genes encoding beta-N-acetylhexosaminidases, the only molecules so far identified as involved in sperm/egg recognition in this class, whereas in mammals, the organisms where these enzymes have been best studied, only two types of polypeptide chains forming dimeric functional beta-N-acetylhexosaminidases are present in Drosophila three different gene products are available that might generate numerous dimeric isoforms.