Polyphasic identification of three new species in Alternaria section Infectoriae causing human cutaneous infection.

BACKGROUND: Cutaneous phaeohyphomycosis is an emerging disease in immunocompromised patients, being Alternaria one of the most common genera reported as a causative agent. Species identification is not carried out mainly due to the complexity of the genus. Analysis of the ITS barcode has become standard for fungal identification, but in Alternaria it is only able to discriminate among species-groups or sections. METHODS: We present three cases of cutaneous infection caused by Alternaria isolates morphologically identified as belonging to section Infectoriae. They have been morphologically characterised and phylogenetically delineated with five molecular markers (ITS, ATPase, gapdh, rpb2 and tef1). RESULTS: Mycotic infections have been diagnosed by repeated cultures and histopathological examination in two of the cases. The polyphasic approach has allowed to delineate three new species of Alternaria section Infectoriae, that is A anthropophila, A atrobrunnea and A guarroi. ATPase has been the only locus able to discriminate most of the species (29 out of 31) currently sequenced in this section, including A infectoria the commonest reported species causing alternariosis. Susceptibility test showed different antifungal patterns for the three species, although terbinafine was the most active in vitro drug against these fungi. CONCLUSIONS: The ATPase gene is recommended as an alternative barcode locus to identify Alternaria clinical isolates in section Infectoriae. Our results reinforce the relevance of identification of Alternaria isolates at the species level and the necessity to carry out antifungal susceptibility testing to determine the most adequate drug for treatment.

Systematic analysis of transrectal prostate biopsies using an ink method and specific histopathologic protocol: a prospective study.

Background. Transrectal prostate biopsy is the standard protocol for the screening for prostate cancer. It helps to locate prostatic adenocarcinoma and plan treatment. However, the increasing number of prostate biopsies leads to considerably greater costs for the pathology laboratories. In this study, we compare the traditional method with an ink method in combination with a systematic histopathologic protocol. Methods. Two hundred consecutive transrectal prostate biopsy specimens were received from the radiology department. They were separated into two groups: one hundred were processed as six different specimens in the usual manner. The other one hundred were submitted in six containers, the apex, base, and middle section of which were stained different colours. The samples subject to the ink method were embedded in paraffin and placed in two cassettes which were sectioned using a specific protocol. Results. The comparative study of the nonink and ink methods for histopathologic diagnosis showed no statistical differences as far as diagnostic categories were concerned (P value < .005). The number of PIN diagnoses increased when the ink method was used, but no statistical differences were found. The ink method led to a cost reduction of 48.86%. Conclusions. Our ink method combined with a specific histopathologic protocol provided the same diagnostic quality, tumor location information as the traditional method, and lower pathology expenses.

HIV-1 upregulates intercellular adhesion molecule-1 gene expression in lymphoid tissue of patients with chronic HIV-1 infection.

OBJECTIVES: Intercellular adhesion molecule (ICAM)-1 is an adhesion molecule that plays an important role in the transmission of HIV-1 to CD4+ target cells and in the decrease of these cells in lymphoid tissue (LT). Our main objective was to study ICAM-1 expression in LT from HIV-1-infected persons and to correlate this expression with LT viral load and the immunoarchitecture alteration before and after highly active antiretroviral therapy (HAART). METHODS: Tonsillar LT samples from 16 patients with chronic asymptomatic HIV-1 infection were studied before initiating treatment and after 12 months of HAART. ICAM-1 protein expression was studied by immunohistochemistry in all cases, and ICAM-1 messenger RNA (mRNA) was quantified from frozen tissue in 6 patients using quantitative real-time polymerase chain reaction (PCR). LT viral load was determined by PCR. The LT immunoarchitecture, p24 immunoexpression, and CD4+ cell count were assessed from tissue sections. RESULTS: Before initiating HAART, there was high immunohistochemical ICAM-1 expression in follicular dendritic and endothelial cells and high ICAM-1 mRNA quantification. These findings correlated with a high LT viral load, strong p24 expression, and an effacement of LT immunoarchitecture with a low number of CD4+ cells. After HAART, there was a significant decrease of immunohistochemical and gene ICAM-1 expression. These results correlated with a significant decrease of LT viral load and p24 immunoexpression, a recovery of LT architecture, and a significant increase of CD4+ cells. CONCLUSIONS: HIV-1 upregulates ICAM-1 expression in LT. This finding is associated with a marked effacement of LT architecture. HAART produces downregulation of ICAM-1 expression and recovery of LT architecture by reducing LT viral load significantly.

Immunoarchitecture of lymphoid tissue in HIV-infection during antiretroviral therapy correlates with viral persistence.

Plasma viral load and T-cell subset determinations in blood are the markers used for monitoring HIV-1 infection. However, key pathogenesis events, viral replication and most immunologic changes occur in the lymphoid tissues. We have studied the tonsillar biopsies of 30 patients in the early stages of the disease, before initiating treatment and after 12 and 36 months of fully effective highly active antiretroviral therapy. We have investigated the HIV RNA by polymerase chain reaction (lymphoid tissue viral load), the immunohistochemical HIV-p24 antigen expression, as well as the lymphoid tissue architecture and lymphoid cell subsets using morphometry. The lymphoid tissue viral load and the immunoexpression of p24, which was found to be mainly associated with follicular dendritic cells, decreased significantly after treatment, but did not disappear in all cases, even after 36 months of treatment. A significant improvement of the lymphoid tissue architecture was also observed after treatment, with recovery of follicular structures. These histological changes correlated with the lymphoid tissue viral load. Moreover, the counts of CD4+ increased whereas CD8+ and cytotoxic lymphocytes (CD8+ granzyme B+) decreased significantly, the latter in both interfollicular and intrafollicular areas. However, these cellular counts after treatment did not reach those of lymphoid tissue of non-HIV-infected patients used as control cases. Naive (CD45RA+) and memory (CD45RO+) cells also improved significantly after treatment. In conclusion, in HIV-infection the impact of treatment can only be assessed completely in the lymphoid tissue reservoir, where most of the virus is stored and associated with follicular dendritic cells. Highly active antiretroviral therapy produces a significant recovery of lymphoid tissue architecture and lymphoid cell subsets, which are associated with the decrease of lymphoid tissue viral load. However, these parameters studied in lymphoid tissue are not re-established completely, even after 36 months of highly active antiretroviral therapy.

Low thymidylate synthase expression in the primary tumor predicts favorable clinical outcome in resected gastric cancer patients treated with adjuvant tegafur.

OBJECTIVE: To assess the thymidylate synthase protein (TS) in tumor cells of resected gastric cancer patients treated with adjuvant tegafur (TG), we reviewed the outcome of 94 randomized patients treated either with adjuvant TG plus mitomycin C (MMC) or MMC alone. METHODS: TS was determined in 76 out of 94 patients, previously randomized to receive adjuvant TG, 500 mg/m(2)/day p.o. for 6 months plus four courses of MMC, 10- 20 mg/m(2) i.v. every 6 weeks or MMC alone. An immunohistochemical assessment with the monoclonal antibody TS-106 was performed. RESULTS: Low TS was observed in 38 patients (20 treated with TG-MMC and 18 with MMC) and high TS in the other 38 patients (21 treated with TG-MMC and 17 with MMC). After 10 years' median follow-up time, 61% of adjuvant TG-MMC patients and 43% of MMC patients were alive and disease-free. Disease-free survival and overall survival were significantly better for patients treated with TG-MMC compared to MMC adjuvant (p = 0.0277 and p = 0.05), and low-TS compared to high-TS patients (p = 0.0184 and p = 0.0198). In 38 low-TS patients we observed an 83% chance to be disease-free in TG-MMC-treated patients and 55% in MMC-treated patients (p = 0.04). CONCLUSIONS: A low TS level determines a subset of patients that may benefit from adjuvant oral TG when added to MMC showing a 5-year cure rate of more than 80%.

Immunohistochemical expression of CK20, p53, and Ki-67 as objective markers of urothelial dysplasia.

Urothelial dysplasia and carcinoma in situ (CIS) are related to recurrence and progression of urothelial carcinoma. Distinguishing CIS and dysplasia from reactive atypia is often difficult on the basis of histological features alone. Cytokeratin 20 (CK20), p53, and Ki-67 are related either to neoplastic change or prognosis in urothelial proliferations. The objective of the present study was to establish the immunohistochemical pattern of these three antibodies in urothelial dysplasia and CIS. Three groups of patients were evaluated: 40 nonneoplastic urothelial samples, 50 cases with histologically incontrovertible CIS, and 30 samples with nonconclusive atypical changes (atypia of unknown significance). Monoclonal antibodies (MoAb) against CK20, p53, and Ki-67 (MIB-1) were used on paraffin-embedded samples. Nonneoplastic urothelium showed no reactivity to CK20 except for umbrella cells; p53 and Ki-67 were negative or weakly positive in <10% of basal cells. In the CIS group, 42% showed positivity for all three MoAb; 44%, for two; and 14%, only for one. CK20 was positive through the full thickness of the urothelium in 72% of cases, p53 was positive in 80% of cases, and Ki-67, in 94% of cases. In the third group, the suspected dysplastic cells showed strong positivity in scattered cells through the epithelium in 75% of cases. Aberrant CK20 expression in urothelial cells plus overexpression of p53 and Ki-67 are indicators of dysplastic change in urothelial mucosa. Thus, immunohistochemistry is a useful tool to confirm the diagnosis of CIS and could be helpful to distinguish dysplastic changes from reactive atypia.

Intranuclear inclusions in fine needle aspirates of bronchial low grade mucoepidermoid carcinoma with clear cell change: a report of two cases.

BACKGROUND: Mucoepidermoid carcinoma of the bronchus is a rare neoplasm that can be recognized on histology as well as cytology by the presence of three characteristic cell types: mucus secreting, epidermoid and intermediate. We encountered two cases displaying unusual cytologic features, including clear intranuclear inclusions. CASES: Two females, aged 33 and 39, presented with an intrabronchial tumor and pulmonary parenchymatous mass, respectively. Fine needle aspiration of both tumors showed similar cytologic features, with a dominant population of cells with bland nuclei and wide cytoplasm, and frequent intranuclear inclusions. A minor component of mucus-secreting cells was also recognized. Histologically, both tumors corresponded to the clear cell variant of mucoepidermoid carcinoma. CONCLUSION: The cytologic picture in our cases has not been described previously in fine needle aspirates of mucoepidermoid carcinoma, in neither the bronchus nor salivary gland. The differential diagnosis of a monotonous population of epithelial cells with intranuclear inclusions involves bronchioloalveolar carcinoma, but the absence of the characteristic sheet pattern, as well as the clinical and image findings, excludes this possibility. The lack of atypia and intrabronchial location limits the scope to carcinoid and salivary gland-type tumors of the bronchus. Since we were aware of the possibility of unusual cytologic presentations of mucoepidermoid carcinomas, search for different cellular populations suggested the precise diagnosis.