Acute spontaneous non-hemorrhagic adrenal infarction in pregnancy: case-report and literature review.

Unilateral non-hemorrhagic adrenal infarction (NHAI) is a very uncommon cause of acute abdomen in pregnancy. Diagnosis is highly challenging due to its rarity, heterogeneity of clinical presentation, and inconclusiveness of the initial workup. Timely recognition is pivotal to ensuring optimal outcomes. Here we describe a case of spontaneous unilateral NHAI diagnosed in a singleton pregnant woman at 32 weeks' gestation at our centre and provide the findings of an extensive literature review on the topic. We identified 22 articles describing 31 NHAI cases in 30 obstetric patients: NHAI occurs more frequently on the right side and in the third trimester, and diagnosis is formulated more than 24 h after clinical presentation in 50% of cases; second-level imaging is always necessary to reach a definitive diagnosis and start appropriate treatment. A high degree of clinical suspicion is needed to promptly recognize NHAI in pregnancy, thus allowing appropriate multidisciplinary management and timely treatment initiation. Promotion of knowledge and awareness of NHAI as a potential cause of acute abdomen in pregnancy is mandatory to improve clinical practice and, ultimately, perinatal outcomes.

Current Pharmacological Treatment of Type 2 Diabetes Mellitus in Undocumented Migrants: Is It Appropriate for the Phenotype of the Disease?

Type 2 diabetes is increasingly recognized as a spectrum of metabolic disorders sharing chronic hyperglycaemia. In Europe, the continually growing number of migrants from developing countries could affect diabetes phenotypes. We evaluated a population of 426 Italians and 412 undocumented migrants. Using 17 variables (with the exclusion of ethnic origin) we performed a multiple component analysis to detect potential clusters, independently from ethnicity. We also compared the two groups to evaluate potential ethnicity associated differences. We found five clusters of patients with different disease phenotypes. Comparing Italians with undocumented migrants, we noted that the first had more often cardiovascular risk factors and neurologic involvement, while the latter had a higher frequency of diabetic ulcers and renal involvement. Metformin was used in a comparable percentage of patients in all clusters, but other antidiabetic treatments showed some differences. Italians were more often on insulin, due to a larger use of long acting insulin, and received a larger number of oral antidiabetics in combination. Pharmacological treatment of comorbidities showed some differences too. We suggest that type 2 diabetes should be considered as a spectrum of diseases with different phenotypes also in heterogeneous populations, and that this is not due only to ethnic differences.

A Rare Diagnosis After the Fall of a 96-Year-Old Woman: Doege-Potter Syndrome.

INTRODUCTION: Doege-Potter Syndrome (DPS) is a rare but life-threatening paraneoplastic syndrome, characterized by Non-Islet Cell Tumor-Induced Hypoglycemia (NICTH) secondary to a Solitary Fibrous Tumor (SFT), which secretes an incompletely processed form of Insulin-like Growth Factor 2 (IGF-2). RESULTS: A 96-year-old woman was admitted with head trauma due to an accidental fall. During her hospital stay she experienced frequent hypoglycemic episodes. Multiple injections of 33% dextrose and continuous infusion with 10% dextrose were required to maintain normal blood glucose levels. Biochemical analyses revealed hypoinsulinemic hypoglycemia, low C-peptide levels, suppressed insulin-like growth factor-1, normal insulin-like growth factor-2, and an elevated IGF-2:IGF-1 ratio, all consistent with IGF-2 secretion by a non-islet cell tumor. A contrast-enhanced chest and abdominal CT scans showed a single large pleural mass in the left lower hemithorax measuring 15x14 cm without secondary lesions. Histological analysis of biopsied specimens suggested a solitary fibrous pleural tumor; accordingly, a diagnosis of Doege-Potter syndrome was considered. Due to extensive tumor burden and the advanced age of the patient, supportive and non-invasive management was chosen. Dexamethasone therapy was started, and while receiving this therapy she was able to discontinue glucose infusion and successfully maintain euglycemia. DISCUSSION: In the elderly, a sudden and unexplained fall can be the expression of severe hypoglycemia, usually as a complication of insulin therapy or of oral hypoglycemic agents administered to patients with diabetes. However, in patients without diabetes, other causes should be investigated, and the hypothesis of neoplastic diseases should be considered. CONCLUSION: In this case report we describe an uncommon cause of paraneoplastic hypoglycemia occurring in the oldest patient with a non-islet cell tumor reported thus far.

Post-prandial anorexigenic gut peptide, appetite and glucometabolic responses at different eating rates in obese patients undergoing laparoscopic sleeve gastrectomy.

Although different hypotheses have been proposed, the underlying mechanism(s) of the weight loss induced by laparoscopic sleeve gastrectomy (LSG) is still unknown. The aim of this study was to determine whether eating the same meal at different rates (fast vs. slow feeding) evokes different post-prandial anorexigenic gut peptide responses in ten obese patients undergoing LSG. Circulating levels of GLP-1, PYY, glucose, insulin and triglycerides were measured before and 3 months after LSG. Visual analogue scales were used to evaluate the subjective feelings of hunger and satiety. Irrespective of the operative state, either fast or slow feeding did not stimulate GLP-1 release (vs. 0 min); plasma levels of PYY were increased (vs. 0 min) by fast and slow feeding only after LSG. There were no differences in post-prandial levels of GLP-1 when comparing fast to slow feeding or pre-to-post-operative state. Plasma levels of PYY after fast or slow feeding were higher in post, rather than pre-operative state, with no differences when comparing PYY release after fast and slow feeding. Hunger and satiety were decreased and increased, respectively, (vs. 0 min) by food intake. Fast feeding evoked a higher satiety than slow feeding in both pre- and post-operative states, with no differences in hunger. In both pre- and post-operative states, there were similar responses for hunger and satiety after food intake. Finally, LSG improved insulin resistance after either fast or slow feeding. These (negative) findings would suggest a negligible contribution of the anorexigenic gut peptide responses in LSG-induced weight loss.

Solid cell nests of the thyroid gland: morphological, immunohistochemical and genetic features.

AIMS: The correct identification of solid cell nests (SCNs) is an important issue in thyroid pathology because of the spectrum of differential diagnoses of this type of lesion. METHODS AND RESULTS: Ten cases of 295 consecutive thyroidectomies showed the presence of SCNs at histological examination. The identification of the exact SCN type required the distinction of the cystic and solid pattern; SCNs were usually composed of a mixture of main cells (MCs) and C-cells (CCs). The immunohistochemical calcitonin stain identified CCs easily, both inside SCNs and dispersed in islets at the periphery. For the characterization of MCs, we added the utility of p40 to p63. The use of thyroid transcription factor-1 (TTF-1) helped in their identification, as MCs did not react with this marker; the combination of TTF-1 and p40 or p63 IHC stains was useful for the characterization of cystic SCNs of both types 3 and 4. The negativity of mouse monoclonal mesothelioma antibody (HMBE-1) and a very low proliferative index (MIB-1) supported the diagnosis. [Correction added on 23 November 2015, after online publication: MIB-1 was incorrectly defined, the expanded form was deleted.] We discourage the use of galectin-3 (Gal-3) and cytokeratin-19 (CK-19), as they have an important overlap with papillary thyroid carcinoma. The complete absence of any B-Raf proto-oncogene, serine/threonine kinase (BRAF) mutations is an additional fundamental finding. CONCLUSIONS: We reviewed the most relevant morphological and immunohistochemical features of SCNs and have provided a genetic analysis of the BRAF gene because of its expanding use in thyroid pathology.

New case of trichorinophalangeal syndrome-like phenotype with a de novo t(2;8)(p16.1;q23.3) translocation which does not disrupt the TRPS1 gene.

BACKGROUND: Trichorhinophalangeal syndrome (TRPS) is a rare autosomal dominant genetic disorder characterised by distinctive craniofacial and skeletal abnormalities. TRPS is generally associated with mutations in the TRPS1 gene at 8q23.3 or microdeletions of the 8q23.3-q24.11 region. However, three deletions affecting the same chromosome region and a familial translocation t(8;13) co-segregating with TRPS, which do not encompass or disrupt the TRPS1 gene, have been reported. A deregulated expression of TRPS1 has been hypothesised as cause of the TRPS phenotype of these patients. CASE PRESENTATION: We report the clinical and molecular characterisation of a 57-year-old Caucasian woman carrying the t(2;8)(p16.1;q23.3) de novo balanced translocation. The proband presented with peculiar clinical features (severe craniofacial dysmorphism, alopecia universalis, severe scoliosis, mitral valve prolapse, mild mental impairment and normal growth parameters) that partially overlap with TRPS I. Mutational and array CGH analyses ruled out any genetic defect affecting TRPS1 or genomic alteration at the translocation breakpoint or elsewhere in the genome. Breakpoint mapping excluded disruption of TRPS1, and revealed that the chromosome 8q23.3 breakpoint was located within the IVS10 of the long intergenic non-coding RNA LINC00536, at approximately 300 kb from the TRPS1 5' end. Conversely, the 2p16.1 breakpoint mapped within a LINE sequence, in a region that lacks transcriptional regulatory elements. As a result of the translocation, nucleotide base pair additions and deletions were detected at both breakpoint junction fragments, and an evolutionarily conserved VISTA enhancer element from 2p16.1 was relocated at approximately 325 kb from the TRPS1 promoter. CONCLUSIONS: We suggest that the disruption of the genomic architecture of cis regulatory elements downstream the TRPS1 5' region, combined with the translocation of a novel enhancer element nearby TRPS1, might be the pathogenetic mechanism underpinning the proband's phenotype. The clinical and genetic characterisation of the present subject allowed us to make a genetic diagnosis in the context of a known syndrome, contributing to a better comprehension of the complex transcriptional regulation of TRPS1 and TRPS ethiopathogenesis.

It is never too late for a genetic disease: a case of a 79-year-old man with persistent hypokalemia.

BACKGROUND: We describe a 79-year-old man with biochemical and radiological features of Gitelman syndrome: hypokalemia, hypomagnesemia, hyperreninemic hyperaldosteronism in absence of secondary hyperaldosteronism causes, and chondrocalcinosis.
 METHODS AND RESULTS: The diagnosis was confirmed by sequence analysis of the SLC12A3 gene showing the compound heterozygous mutation Gly439Ser and Arg1018Term. Aliskiren, a direct renin inhibitor, in combination with potassium and magnesium oral supplements was effective in ameliorating the electrolytic imbalance without any adverse effects. CONCLUSION: This study has shown for the first time that aliskiren may represent a reliable and safe treatment as an alternative to potassium-sparing diuretics for Gitelman syndrome.

Effect of a somatostatin infusion on circulating levels of adipokines in obese women.

OBJECTIVE: Changes in circulating levels of many adipocyte-derived peptides, including adipokines such as adiponectin, leptin and tumor necrosis factor alpha (TNF-α), have been reported in obesity (OB). Somatostatin (SRIF) inhibits circulating levels of adiponectin and leptin in lean (LN) subjects, but the effect of a SRIF infusion on these adipokines, including TNF-α, in OB is to date unknown. METHODS: Ten young women (5 OB and 5 LN) were studied. All subjects underwent an infusion of SRIF (9 µg/kg/h i.v., over 60 min), with blood samples drawn prior to and at different time intervals after SRIF administration. Plasma levels of adiponectin, leptin and TNF-α were measured at each interval. RESULTS: Basal levels of leptin and TNF-α were significantly higher in OB than LN women, whereas levels of adiponectin were significantly lower in OB than LN subjects. SRIF significantly inhibited plasma concentrations of adiponectin (at 60 min) in both OB and LN women, without affecting those of leptin and TNF-α in either group. In LN subjects, the inhibitory effect of SRIF on plasma adiponectin persisted up to 150 min, whereas SRIF infusion withdrawal in OB women resulted in a prompt restoration of basal levels of the adipokine. CONCLUSIONS: Plasma concentrations of leptin and TNF-α, which are higher in OB than LN subjects, are unaffected by a SRIF infusion, which, in contrast, inhibits circulating levels of adiponectin in both groups, with a delayed return to the baseline secretion of the adipokine in LN subjects.

Postprandial triglyceride profile after a standardized oral fat load is altered in growth hormone (GH)-deficient adult patients and is not improved after short-term GH replacement therapy.

OBJECTIVE: Adult growth hormone deficiency (GHD) has detrimental effects on metabolic profile, leading to an increased cardiovascular mortality and morbidity. Above all, disturbance in postprandial triglyceride metabolism is of major concern because of the crucial role of triglyceride-rich lipoproteins in atherogenesis. The majority of previous studies on GH replacement have shown favourable changes in the fasting lipid profile. Aim of this study is to investigate whether this beneficial effect is exerted also on postprandial triglyceride (TG) metabolism. PATIENTS AND METHODS: We challenged nine GHD patients with a standardized fat loading meal at baseline and after 6 months of GH replacement therapy. Nine healthy control subjects were similarly tested under baseline conditions. Blood samples were obtained before and up to 8 h after fat loading for serum lipid analysis. RESULTS: We found that GHD patients with fasting TG level in the normal range (1·29 ± 0·31 mm) had a delayed postprandial TG clearance compared to healthy controls (triglyceride level at 8 h, 3·82 ± 0·83 vs 1 ± 0·06 mm P < 0·01), and the postprandial hypertriglyceridaemia was not corrected by 6 months of GH therapy. CONCLUSIONS: This study has shown for the first time that GHD adult patients have a higher postprandial triglyceridaemia compared to healthy controls when challenged by a standardized fat load and that this atherogenic feature is not normalized by short-term GH treatment despite a decrease in visceral fat mass described during the replacement therapy.

Effect of somatostatin infusion on peptide YY secretion: studies in the acute and recovery phase of anorexia nervosa and in obesity.

OBJECTIVE: Changes in many gastrointestinal peptides, including the anorexigenic peptide YY (PYY), which is produced by L cells, occur in both anorexia nervosa (AN) and obesity (OB). High PYY levels are present in AN, whereas in morbid OB fasting and postprandial PYY secretion is blunted. Somatostatin (somatotropin release-inhibiting factor (SRIF)) reportedly inhibits plasma PYY concentrations in animals and healthy humans, but the effect of a SRIF infusion on spontaneous PYY secretion in AN and OB is unknown. METHODS: A total of 18 young women, seven with acute AN (A-AN), four with AN in the recovery phase (R-AN), and seven with morbid OB, were studied. All subjects underwent an infusion of SRIF (9 µg/kg i.v./h, over 60 min), with blood samples drawn before and at different time intervals after SRIF administration. Plasma PYY levels were measured at each time point. RESULTS: SRIF significantly inhibited plasma PYY concentrations in R-AN and OB, without affecting PYY titers in A-AN. In OB, the inhibitory effect of SRIF also persisted at 90 min. Withdrawal of SRIF infusion in R-AN resulted in a prompt restoration of basal plasma PYY levels, whereas termination of SRIF infusion in OB was followed by a slower increase of PYY titers toward baseline levels. After infusion, PYY Δ area under the curve (ΔAUC) in R-AN was significantly higher than those in A-AN and OB patients. A significant difference in PYY ΔAUC between A-AN and OB was present. CONCLUSIONS: These results suggest the existence of a hypo- and hyper-sensitivity of L cells to the inhibitory effect of SRIF in A-AN and OB respectively.

GH deficiency in adult B-thalassemia major patients and its relationship with IGF-1 production.

Endocrine complications in Β-thalassemia represent a prominent cause of morbidity. Above all, dysfunction of GH-IGF-1 axis is of a major concern because of its pathogenic role on cardiac and bone disease, frequently described in this clinical setting. The aim of this paper is to analyze GH-IGF-1 axis in a cohort of 25 adult patients affected by Β-thalassemia. We found that GH deficiency was present in only 8% of our patients if diagnosis was based on GH peak below 9µg/L to two GH provocative tests instead of only one, and was mainly related to iron overload. On the contrary, IGF-1 production was impaired in a higher percentage of patients (72%), without significant correlation with iron burden. Of note, patients with hepatitis C virus infection showed lower IGF-1 concentrations than uninfected subjects despite a normal GH reserve, suggesting that partial GH insensitivity at the post-receptor level may play a key role in IGF-1 deficiency described in thalassemic patients.

Hepatitis C virus (HCV) infection may elicit neutralizing antibodies targeting epitopes conserved in all viral genotypes.

Anti-hepatitis C virus (HCV) cross-neutralizing human monoclonal antibodies, directed against conserved epitopes on surface E2 glycoprotein, are central tools for understanding virus-host interplay, and for planning strategies for prevention and treatment of this infection. Recently, we developed a research aimed at identifying these antibody specificities. The characteristics of one of these antibodies (Fab e20) were addressed in this study. Firstly, using immunofluorescence and FACS analysis of cells expressing envelope HCV glycoproteins, Fab e20 was able to recognize all HCV genotypes. Secondly, competition assays with a panel of mouse and rat monoclonals, and alanine scanning mutagenesis analyses located the e20 epitope within the CD81 binding site, documenting that three highly conserved HCV/E2 residues (W529, G530 and D535) are critical for e20 binding. Finally, a strong neutralizing activity against HCV pseudoparticles (HCVpp) incorporating envelope glycoproteins of genotypes 1a, 1b, 2a, 2b and 4, and against the cell culture-grown (HCVcc) JFH1 strain, was observed. The data highlight that neutralizing antibodies against HCV epitopes present in all HCV genotypes are elicited during natural infection. Their availability may open new avenues to the understanding of HCV persistence and to the development of strategies for the immune control of this infection.

Antigen-driven evolution of B lymphocytes in coronary atherosclerotic plaques.

Recent data indicated that adaptive immunity is involved in the process of atherogenesis. Oligoclonal recruitment of T lymphocytes has been described in coronary plaques of patients with acute coronary syndrome. However, the nature of immune response remains to be determined. In the present study, we examined the Ab response in six coronary plaques obtained by endoluminal directional atherectomy. The IgG1/kappa-coding gene repertoires of B lymphocytes present in circulating blood and in coronary plaques were cloned and analyzed. In all of the six plaques, we observed 1) a skewed usage of heavy and light IgG1/kappa Ab-coding genes, 2) an oligoclonal distribution of V(K), J(K), and V(H), D(H), and J(H) genes with overrepresentation of some rarely used IgG genes, and 3) the unequivocal signs of Ag-driven clonal expansion and evolution of B cells. The data document for the first time the presence of a local Ag-driven clonal evolution of B cells in human atherosclerotic plaques.

Anti-HIV-1 response elicited in rabbits by anti-idiotype monoclonal antibodies mimicking the CD4-binding site.

Antibodies against conserved epitopes on HIV-1 envelope glycoproteins (Env), such as the gp120 CD4-binding site (CD4bs), could contribute to protection against HIV-1. Env-based immunogens inducing such a response could be a major component of future anti-HIV-1 strategies. In this proof-of-concept study we describe the generation of two anti-idiotype (AI) murine antibodies mimicking the CD4bs epitope. Sera were collected from long-term non-progressor patients to obtain CD4bs-directed IgG, through sequential purification steps. The purified IgG were then used as Fab fragments to immunize mice for hybridoma generation. Two hybridomas (P1 and P2), reacting only against the CD4bs-directed IgG, were identified and characterized. The P1 and P2 antibodies were shown to recognize the idiotype of the broadly neutralizing anti-CD4bs human mAb b12. Both P1 and P2 Fabs were able to induce a strong anti-gp120 response in rabbits. Moreover, the rabbits' sera were shown to neutralize two sensitive tier 1 strains of HIV-1 in an Env-pseudotype neutralization assay. In particular, 3/5 rabbits in the P1 group and 1/5 in the P2 group showed greater than 80% neutralizing activity against the HXB2 pseudovirus. Two rabbits also neutralized the pseudovirus HIV-MN. Overall, these data describe the first anti-idiotypic vaccine approach performed to generate antibodies to the CD4bs of the HIV-1 gp120. Although future studies will be necessary to improve strength and breadth of the elicited neutralizing response, this proof-of-concept study documents that immunogens designed on the idiotype of broadly neutralizing Abs are feasible and could help in the design of future anti-HIV strategies.

Hepatitis C virus (HCV)-driven stimulation of subfamily-restricted natural IgM antibodies in mixed cryoglobulinemia.

Hepatitis C virus (HCV) infection has been closely related to mixed cryoglobulinemia (MC). During HCV infection, cryoglobulins derive from the restricted expression of few germline genes as VH1-69, a subfamily highly represented in anti-HCV humoral response. Little is known about the self-reacting IgM component of the cryoprecipitate. In the present study, the IgM/K repertoire of an HCV-infected cryoglobulinemic patient was dissected by phage-display on well-characterized anti-HCV/E2 VH1-69-derived monoclonal IgG1/Kappa Fab fragments cloned from the same patient. All selected IgM clones were shown to react with the anti-HCV/E2 antibodies belonging to VH1-69 subfamily. More than 60% of selected clones showed a bias in VH gene usage, restricted to two VH subfamilies frequently described in autoimmune manifestations (VH3-23; VH3-21). Moreover, all selected clones showed an high similarity (>98.5%) to germline genes evidencing their natural origin. A possible hypothesis is that clones belonging to some subfamilies are naturally prone to react against other VH gene subfamilies, as VH 1-69. An antigen-driven stimulation of these subfamilies, and their overexpression as in HCV infection, could lead to a breaking of humoral homeostatic balance exposing the patients to the risk of developing autoimmune disorders.

Perspectives for the utilization of neutralizing human monoclonal antibodies as anti-HCV drugs.

A major problem in hepatitis C virus (HCV) immunotherapy or vaccine design is the extreme variability of the virus. We identified human monoclonal antibodies (mAbs) that neutralize genetically diverse HCV isolates and protect against heterologous HCV quasispecies challenge in a human liver-chimeric mouse model. The results provide evidence that broadly neutralizing antibodies to HCV protect against heterologous viral infection and suggest that a prophylactic vaccine against HCV may be achievable.

Molecular diagnosis of sepsis in neutropenic patients with haematological malignancies.

The rapid diagnosis of an infectious cause in the course of fever of unknown origin plays a pivotal role in the correct management of neutropenic patients. In this study, blood samples from febrile oncohaematological patients were tested using a novel commercial real-time PCR assay (LightCycler SeptiFast; Roche Molecular Systems) and blood culture (BacT/Alert 3D; bioMérieux). Twenty-one (20.4 %) and 34 (33 %) of the 103 samples under study tested positive by blood culture and PCR, respectively. The analysis of concordance evidenced a low correlation between the two approaches (83 %), mainly due to samples that tested negative by culture but positive using the molecular approach. Among 14 discordant cases negative by culture but positive by PCR, 12 were observed in sequential samples of patients with initial concordant results on samples drawn before the administration of a specific antimicrobial therapy. Moreover, DNA of a fastidious organism, Aspergillus fumigatus, not easily detectable by the cultural approach was rapidly detected in the two remaining discordant cases. Overall, the characteristics featured by the molecular method could be of interest in the development of new algorithms for the diagnosis of sepsis in critical patients.

Identification of a broadly cross-reacting and neutralizing human monoclonal antibody directed against the hepatitis C virus E2 protein.

Identification of anti-hepatitis C virus (anti-HCV) human antibody clones with broad neutralizing activity is important for a better understanding of the interplay between the virus and host and for the design of an effective passive immunotherapy and an effective vaccine. We report the identification of a human monoclonal Fab (e137) able to bind the HCV E2 glycoprotein of all HCV genotypes but genotype 5. The results of antibody competition assays and testing the reactivity to alanine mutant E2 proteins confirmed that the e137 epitope includes residues (T416, W420, W529, G530, and D535) highly conserved across all HCV genotypes. Fab e137 neutralized HCV pseudoparticles bearing genotype 1a, 1b, and 4 E1-E2 proteins and to a lesser extent, genotype 2b. Fab e137 was also able to inhibit cell culture-grown HCV (genotype 2a). These data indicate that broadly cross-reacting and cross-neutralizing antibodies are generated during HCV infection.

Development and validation of a molecular method for the diagnosis of medically important fungal infections.

The increasing incidence of severe fungal infections highlights the need for rapid and precise identification methods in clinical mycology. The aim of this study was to develop and validate a culture-indipendent molecular approach that could allow the detection of fungal pathogens in clinical samples, with particular attention to the identification of drug-resistant Candida and Aspergillus species. A real-time multiplex PCR assay was developed using TaqMan probes specific for highly discriminating ITS sequences. In its multiplex format the assay showed a high specificity, clearly discriminating among different species, as well as a high sensitivity (20 CFU/1 mL sample), making it a potentially useful starting point for the development of a more complete molecular diagnostic assay.

Rapid molecular identification of fungal pathogens in corneal samples from suspected keratomycosis cases.

An increase in the incidence of fungal infections has highlighted the need for rapid and precise detection and identification methods in clinical mycology. This report describes the data obtained on corneal samples from 24 patients with suspected keratomycosis using a conventional cultural approach in parallel with PCR amplification and sequencing of the internal transcribed spacers (ITSs) of the rDNA regions. Using the cultural approach, seven samples (58.3 % of the 12 samples positive for an infectious pathogen) tested positive for a fungal aetiology, with final identification taking a mean time of more than 5 days. In two cases, diagnosis required 10 days. Using the ITS-based molecular approach, a direct diagnosis was obtained in only five of the seven fungus-positive cases (71.4 %) starting from the clinical samples, but identification was still possible in all seven cases within 24 h (by using 16 h cultures for the two remaining cases). Despite the less-than-optimal sensitivity when working directly on clinical samples, the obtained data indicate that the molecular strategy used in this study is a useful complement to the conventional diagnostic approaches used for keratomycosis and, in particular, allows precise and fast fungal identification, in response to the clinical requirements. Similar studies on larger panels of patients and on different clinical samples are required for further investigation of the clinical potential of ITS-based approaches in the diagnosis of mycotic infections.