Amplified in situ hybridization with peptide nucleic acid probes for differentiation of Mycobacterium tuberculosis complex and nontuberculous Mycobacterium species on formalin-fixed, paraffin-embedded archival biopsy and autopsy samples.

The aim of this study was to evaluate sensitivity and specificity of in situ hybridization (ISH) using peptide nucleic acid (PNA) probes and tyramide-based amplification for the differentiation between Mycobacterium tuberculosis (MTB) and mycobacteria other than tuberculosis (MOTT) on formalin-fixed, paraffin-embedded tissue samples. We performed ISH simultaneously with both probes on 86 specimens from different organs: 70 obtained at autopsy and 16 by biopsy, all with a histologic evidence of mycobacterial infection confirmed by Ziehl-Neelsen-positive staining. Taking culture as the "gold standard," the sensitivity and the specificity of the MTB probe were 100% (41/41) and 95% (38/40), respectively. In only 2 cases ISH failed to identify mycobacteria. Culture results were not available in 3 cases. We propose ISH as a relatively simple and rapid method to differentiate mycobacteria on formalin-fixed, paraffin-embedded specimens (it is more specific than usual histologic stains) and as an alternative to polymerase chain reaction, allowing the morphologic evaluation of positive bacilli.

Rantes distribution and cellular localization in the brain of HIV-infected patients.

OBJECTIVE: To study the immunochemical distribution ofRantes chemokine and its correlation with HIV-p24 expression, in brains with HIV-related lesions. MATERIAL AND METHODS: 17 HIV-positive cases of HIV-related brain lesions, 7 HIV-positive cases without cerebral HIV-related lesions (5 with opportunistic brain diseases), and 7 HIV-negative cases as controls (4 with brain lesion) were selected. RESULTS: High expression of Rantes was observed in the cases with inflammatory brain lesions (22/24 HIV-positive and 2/7 HIV-negative patients). Positivity was observed in the diffuse and nodular microglial cells and lymphocytes. In the patients with HIV-related lesions, the presence of Rantes-stained microglia did not correlate with that of HIV-p24-positive cells. Positive astrocytes were only found in the HIV-positive patients. Multinucleated giant cells were always Rantes-negative. CONCLUSIONS: Our results seem to demonstrate the role of Rantes chemokine in inducing inflammatory brain perivascular and microglial reactions both in HIV-positive and -negative patients.

Beta amyloid precursor protein and patterns of HIV p24 immunohistochemistry in different brain areas of AIDS patients.

OBJECTIVES: To evaluate the correlation between immunohistochemical positive patterns (globular and filamentous structures) of beta-amyloid precursor protein (beta-APP), used as a marker of axonal damage, and the different distribution of HIV p24 antigens, in three different brain areas of AIDS patients. METHODS: Eighteen AIDS patients with HIV-related brain lesions were included in the study. Forty-nine sections from basal ganglia, frontal cortex and hippocampus were selected. After microwave oven pre-treatment, the sections were incubated with anti-HIV p24 and anti-beta-APP monoclonal antibodies; the reactions were developed with peroxidase/3,3'diaminobenzidine. The positivity was graded by semi-quantitative scores. Double immunohistochemical staining was used to evaluate the co-localization of the antigens. RESULTS: HIV p24 immunohistochemistry was positive in 44 of 49 sections (89%), with a prevalence of interstitial positive cells and positive microglial nodules in 27 and 13 sections respectively. beta-APP-positive structures were demonstrated in 23 of 44 sections (52%) with HIV-related lesions, and were absent from the five sections without viral expression. Globular and filamentous lesions were observed in 21 of 23 sections and 10 of 23 lesions respectively. Moreover, a high grade of globular type lesion was related to an elevated presence of diffuse interstitial HIV p24-positive cells in basal ganglia; double immunohistochemical reactions demonstrated the co-localization of beta-APP globules and HIV p24 antigens. CONCLUSIONS: The data obtained confirm the coexpression of beta-APP and viral antigens in particular areas of the brain with HIV-related lesions; there is a strict correlation between beta-APP globules (indicating chronic cerebral damage) and the interstitial pattern of HIV p24 immunohistochemistry.

Etiology of microglial nodules in brains of patients with acquired immunodeficiency syndrome.

Microglial nodules associated with opportunistic and HIV-related lesions are frequently found in the brains of AIDS patients. However, in many cases, the causative agent is only presumptively suspected. We reviewed 199 brains of AIDS patients with micronodular lesions to clarify their etiology by immunohistochemistry (to Toxoplasma gondii, cytomegalovirus, herpes simplex virus I/II, varicella zoster virus and HIV-p24 core protein), PCR (for herpetic viruses and Mycobacterium tuberculosis) and electron microscopy. Productive HIV infection was observed in 110 cases (55.1%): 30 cases with Toxoplasma gondii encephalitis, 30 with cytomegalovirus encephalitis, eight with multiple cerebral diseases, while in the remaining 42 cases HIV was the only pathogenetic agent. Multinucleated giant cells (hallmark of HIV infection) were found in the MGNs of 85/110 cases with HIV-related lesions; the remaining 25 cases had only p24 positive cells but no multinucleated giant cells. In these latter cases the micronodular lesions had been initially attributed to the main opportunistic agent found in the brain, or defined as subacute encephalitis. Individual microglial nodules positive for an opportunistic pathogen were generally negative for HIV antigens. In 13 cases no opportunistic agent or HIV productive infection was found. In these cases, PCR and electron microscopy examination for HIV and other viral infections were negative. Our data suggest that HIV-immunohistochemistry should be used for the etiological diagnosis of micronodular lesions in AIDS brains, even in the presence of other pathogens. After extensive search, the etiology of the microglial nodules remains unknown in only a small percentage of cases.

Focal gastric inflammatory infiltrates in inflammatory bowel diseases: prevalence, immunohistochemical characteristics, and diagnostic role.

OBJECTIVES: To date, few studies have evaluated gastric histology in patients with inflammatory bowel disease (IBD). The aim of this prospective controlled study was to establish the frequency of focal gastritis in Crohn's disease (CD) and ulcerative colitis (UC) patients, as well as to evaluate its immunohistochemical characteristics and clinicoanatomical determinants. METHODS: We evaluated 141 consecutive patients with known CD of the large and/or small bowel, 79 patients with UC, and 141 CD- and UC-free controls; all underwent upper gastrointestinal (GI) endoscopy and 13C urea-breath test. Biopsy specimens taken from the antrum, angulus, and gastric body were evaluated by histology and immunohistochemistry. A series of variables, including CD activity index, duration, extent and location of disease, intestinal resection, number of recurrences, and previous and current medical therapy, as well as the presence of dyspeptic symptoms and mucosal lesions at endoscopy, were determined in all CD patients and correlated with the presence or absence of focal gastritis. RESULTS: Helicobacter pylori-associated gastritis was found in 47 patients with CD (33%), in 37 patients with UC (47%), and in 60% of CD-/UC-free controls (p < 0.01). In H. pylori-negative CD patients focal gastritis was found in 43% of cases (40/94), compared with 12% (5/42) of UC patients and 19% (11/57) of controls (p < 0.05). Specificity and positive predictive value of focal gastritis in CD were 84% and 71%, respectively. It was characterized by a focal perifoveolar or periglandular lymphomonocytic infiltrate, with CD8+/CD4+ cells predominant both in CD and UC patients. There were no significant correlations between the occurrence of focal gastritis and any clinicoanatomical CD features. CONCLUSIONS: Focal gastritis is relatively common in CD patients although it is not exclusive to this condition. Its recognition could be useful in the diagnostic workup of any patient with suspected or indeterminate inflammatory bowel disease, as it makes a diagnosis of CD more likely.

Polymerase chain reaction for Mycobacterium tuberculosis complex DNA. Use on negative archival Ziehl-Neelsen cytologic samples.

OBJECTIVE: To evaluate the usefulness of a nested polymerase chain reaction (PCR) for Mycobacterium tuberculosis complex on routinely stained cytologic samples from patients with extrapulmonary tuberculosis. STUDY DESIGN: Nested PCR for the detection of a fragment of the IS6110 insertion sequence of M tuberculosis complex was applied to Ziehl-Neelsen-negative archival cytologic slides of serous effusions (pleural [n = 7], peritoneal [n = 1] and pericardial [n = 1]) and a lymph node fine needle aspirate (n = 1) from nine human immunodeficiency virus (HIV)-positive patients with autopsy-proven active extrapulmonary tuberculosis. Malignant effusions and aspirates from nine HIV-positive patients with non-Hodgkin's lymphoma and pleural effusions from seven HIV-negative patients with heart failure were used as controls. DNA was extracted after removing the coverslip and gently scraping the cytologic sample from the slides. RESULTS: In all cases, enough DNA was obtained for PCR without any significant loss of integrity, as demonstrated by PCR positive for HLA-Dq. PCR for M tuberculosis was positive in 8 of the 10 samples (80%) from patients with tuberculosis but also in three samples (30%) from HIV-positive patients in the control group. None of the samples from the HIV-negative patients was positive. CONCLUSION: PCR for M tuberculosis can be reliably performed on archival cytologic slides from extrapulmonary samples, but although it is highly sensitive, it may lead to positive results in immunocompromised patients without any sign of active tubercular disease.

[Temporal and spatial variations of coliforms and Escherichia coli in fluvial recreational waters (Salado River, Santa Fe, Argentina). Relationship with the quality standards]. / Variaciones temporales y espaciales de coliformes y de Escherichia coli en aguas recreativas fluviales (Río Salado, Santa Fe, Argentina). Relación con los estándares de calidad.

There are no standards accepted by all the countries to fix top concentrations of microbiological indicators in recreational waters. Even now there is still a considerable discussion either in USA as in Europe. The universal application of a bacteriological quality criterion is hard due to several environmental factors that affect the relation between the indicator, the exposition and the health risks. Our purpose was to present a case study as an example of the influence of the climatic conditions in the application of the most known standards (Environmental Protection Agency of USA, Council of European Communities, World Organization of Health, and others from Canada, South Africa and Hong Kong). The pluvial rainfall increased the number of E. coli, thermotolerant coliforms (C Te), and total coliforms (CT) 6-10 fold, in comparison to the number registered during the steady-state conditions of the system. However, not all the standards included that factor. In Summer, hourly, daily and weekly variations were proved, therefore the standards that suggest fortnightly sampling frequencies would not be convenient in that system. Although the main source of variation was time, spatial variability was also detected. The percentage of E. coli among the C Te was very variable, but the average resulted low (26%) compared to the levels in temperate regions of other countries (> 90%). According to the directives proposed by the Commission of European Communities, the parameter has been changed (C Te for E. coli), but the standard has remained (2000/100 ml). Thus, the directive would be more permissive.

Variaciones temporales y espaciales de coliformes y de Escherichia coli en aguas recreativas fluviales (RÝo Salado, Santa Fe, Argentina). Relación con los estßndares de calidad / Temporal and spatial variations of coliforms and Escherichia coli in fluvial recreational waters (Salado River, Santa Fe, Argentina). Relationship with the quality standards

There are no standards accepted by all the countries to fix top concentrations of microbiological indicators in recreational waters. Even now there is still a considerable discussion either in USA as in Europe. The universal application of a bacteriological quality criterion is hard due to several environmental factors that affect the relation between the indicator, the exposition and the health risks. Our purpose was to present a case study as an example of the influence of the climatic conditions in the application of the most known standards (Environmental Protection Agency of USA, Council of European Communities, World Organization of Health, and others from Canada, South Africa and Hong Kong). The pluvial rainfall increased the number of E. coli, thermotolerant coliforms (C Te), and total coliforms (CT) 6-10 fold, in comparison to the number registered during the steady-state conditions of the system. However, not all the standards included that factor. In Summer, hourly, daily and weekly variations were proved, therefore the standards that suggest fortnightly sampling frequencies would not be convenient in that system. Although the main source of variation was time, spatial variability was also detected. The percentage of E. coli among the C Te was very variable, but the average resulted low (26) compared to the levels in temperate regions of other countries (> 90). According to the directives proposed by the Commission of European Communities, the parameter has been changed (C Te for E. coli), but the standard has remained (2000/100 ml). Thus, the directive would be more permissive.

BK virus renal infection in a patient with the acquired immunodeficiency syndrome.

BACKGROUND: We describe herein a patient with the acquired immunodeficiency syndrome and renal failure due to biopsy-proven BK virus (BKV) infection. Three months after the diagnosis of the renal viral infection, his condition remained unchanged. Although BKV has previously been shown to be associated with ureteral stenosis and renal damage in renal transplant patients, to our knowledge, the literature contains only 3 cases describing the presence of BKV lesions in the kidneys of immunosuppressed patients who had not undergone transplantation. METHODS: The presence of BKV infection was demonstrated by means of histology, immunohistochemistry with polyclonal anti-SV40 antibody, immunoelectron microscopy, polymerase chain reaction, and enzymatic cleavage with BamHI. RESULTS: Histologic examination revealed interstitial inflammatory infiltrates and tubules with enlarged and eosinophilic nuclei. CONCLUSIONS: The high frequency of latent BKV infection and its reactivation during immunosuppression suggest that the possibility of its involvement in renal damage should be considered in immunocompromised patients.

Hepatic metastases from medullary thyroid carcinoma appearing twelve years after the eradication of primitive tumor: cytological and radiological aspects.

We report on radiological and cytological findings from a case of medullary thyroid carcinoma (MTC) metastatizing to the liver 12 yr after the eradication of the primary neoplasm. This behavior has never before been described in a sporadic form of MTC.

Is routine histological evaluation an accurate test for Helicobacter pylori infection?

AIM: To compare the diagnostic accuracy of routine histology for Helicobacter pylori infection, with histology by an expert pathologist, and to compare histology with the rapid urease test (RUT), 13C-urea breath test, IgG serology and culture of antrum and corpus specimens, in a consecutive series of untreated patients presenting for upper oesophago-gastro-duodenoscopy. MATERIALS AND METHODS: One-hundred and fifteen consecutive patients underwent multiple tests for H. pylori infection: rapid urease test, 13C-urea breath test, IgG serology and histology and culture on antrum and corpus biopsy specimens. Histology was first evaluated by the pathologists in a routine examination, and then blindly reviewed by an expert pathologist with a special interest in gastrointestinal pathology. The patients were considered to be H. pylori-positive if two or more tests were positive. RESULTS: Eighty-one patients (70.4%) were found to be H. pylori positive. 13C-urea breath test and IgG serology showed the best sensitivity and specificity (100%). Both the antral and body cultures, and the rapid urease test had the highest specificity (100%). Histological diagnosis after re-evaluation by an expert pathologist showed a high sensitivity (98. 8%) and specificity (100%), and was better than routine histology (sensitivity 92.6%; specificity 90.3%). The accuracy of the rapid urease test was greater than that of routine histology, and the combination of these two tests improved the sensitivity of H. pylori detection to up to 100%. CONCLUSION: All diagnostic tests usually utilised in clinical practice have a sensitivity higher than 90%. In patients who were not pre-treated with antisecretory agents or antibiotics, the sensitivity of histological diagnosis, however, seems to be influenced by the accuracy of the histological examination. The sensitivity of routine histology, but not of revised histological diagnosis, is improved by an additional rapid urease test.

Mitral valve repair for anterior leaflet papillary fibroelastoma: two case descriptions and a literature review.

Cardiac papillary fibroelastomas are rare cardiac tumors and have been considered a 'benign' incidental finding that may have significant clinical manifestations. In this paper we report two cases of mitral valve fibroelastoma: one was discovered by chance with transthoracic echocardiography in a young healthy man, the other was an intraoperative incidental finding in a middle aged man with a recent history of acute myocardial infarction. The mitral valve was repaired in both cases after excising the tumor. The patients did well and remain asymptomatic. A literature review was compiled which comprises previous case reports of 34 patients with mitral valve papillary fibroelastomas. Most were asymptomatic, but when symptoms occurred, they could be disabling, such as stroke, cardiac heart failure, myocardial infarction, and sudden death. Papillary fibroelastoma is amenable to simple surgical excision or in addition to mitral valve repair or replacement. Recurrence has not been reported.

Changes in peritoneal membrane after continuous ambulatory peritoneal dialysis--a histopathological study.

Peritoneal membrane changes in continuous ambulatory peritoneal dialysis (CAPD) patients have been widely described but poorly classified. Our aim was to identify the morphological changes occurring after CAPD treatment. To this end, 17 biopsies of parietal peritoneum (1 cm in diameter) were withdrawn at least 5 cm from the catheter entry hole and stained with Van Gieson, hematoxylin-eosin, trichrome, and some immunohistochemical stains: keratin, vimentin, CD34, CD20, CD4, CD8, desmin, and collagen IV. The morphology of mesothelium, vessels, and basement membrane (BM) of mesothelium and vessels, the presence of inflammatory cells, fibrin, and calcifications, and the distribution and thickness of submesothelial tissue were evaluated. Patients were divided into three groups according to the thickness of the sclerotic band replacing mesothelium: group 1, band up to 40 microns; group 2, band less than 40 microns; group 3, no sclerotic band. The main histopathological alterations noted were: loss of mesothelium; sclerotic alteration of vessels or duplication of BM; presence of myofibroblasts; and presence of inflammatory cells (sparse, focal, or perivascular), mainly represented by macrophages and CD4+ lymphocytes. No significant qualitative differences were observed between the three groups. In conclusion, the variable histological changes in peritoneal membrane suggest a routine peritoneal biopsy in any surgical procedure to better understand pathological changes in the course of CAPD treatment.

Use of magnetic beads for tissue DNA extraction and IS6110 Mycobacterium tuberculosis PCR.

Polymerase chain reaction (PCR) techniques are used increasingly for the diagnosis of Mycobacterium tuberculosis infection and can be used on the DNA obtained from both frozen and formalin fixed, paraffin wax embedded tissues. However, the extraction of DNA by means of the conventional phenol/chloroform method is time consuming and requires the use of potentially dangerous chemical reagents. This paper describes a method based upon the use of magnetic beads for the extraction of M tuberculosis DNA from both routinely formalin fixed, paraffin wax embedded tissues and frozen tissues. Magnetic bead extracted DNA from brain, lymph node, and lung tissues collected from patients with human immunodeficiency virus and tuberculosis was compared with that extracted using the phenol/chloroform method. The magnetic bead extraction procedure requires less than two hours, including the time necessary to dewax the tissue sections. In all cases, the DNA extracted with both methods was amplified successfully by PCR for the M tuberculosis IS6110 sequence. Magnetic bead DNA extraction can be used on both frozen and archival tissues: the method is reliable, simple, sensitive, and rapid; in addition, it does not use hazardous procedures or specialised laboratory equipment and can be used for routine DNA isolation from various human tissues.

VZV fulminant necrotizing encephalitis with concomitant EBV-related lymphoma and CMV ventriculitis: report of an AIDS case.

A case of AIDS with varicella zoster virus fulminant necrotizing encephalitis associated with cytomegalovirus ependymitis-subependymitis and a periventricular Epstein-Barr virus-related lymphoma is described. The patient had no herpes zoster cutaneous eruptions and died three days after the onset of symptoms. Varicella zoster virus and cytomegalovirus antigens were found by immunohistochemistry in the same area around a necrotic periventricular lesion; a periventricular lymphoma, large B cell type, was also observed. In situ hybridization with Epstein-Barr virus-encoded- RNAs probe was positive in about 40% of the neoplastic cells. The association of herpes-related lesions in the same cerebral region should be consistent in AIDS cases with acute neurological symptoms.

Nested polymerase chain reaction for Mycobacterium tuberculosis IS6110 sequence on formalin-fixed paraffin-embedded tissues with granulomatous diseases for rapid diagnosis of tuberculosis.

We evaluated the sensitivity and specificity of a nested polymerase chain reaction (PCR) to the Mycobacterium tuberculosis IS6110 sequence on formalin-fixed paraffin-embedded tissue samples from patients with tubercular and other granulomatous lesions. Five groups of patients and samples were studied: (1) 28 samples from HIV-positive patients with tuberculosis, (2) 8 samples from HIV-negative patients with histologically suspected tuberculosis (confirmed by culture in 5 cases), (3) lymph nodes from 5 HIV-positive patients with Mycobacterium avium-intracellulare infection, (4) lymph nodes from 30 patients with sarcoidosis, and (5) specimens from 17 patients with other granulomatous diseases. The DNA was extracted from sections with a total thickness of 60 microm, and PCR amplified an internal fragment of 123 base pairs. All of the cases with M. tuberculosis infection were PCR-positive, although this sensitivity was partially related to the initial concentration of the DNA used for amplification. Two of the group 4 samples also were repeatedly positive, thus reducing the specificity of the method. All of the cases with granulomatous diseases other than sarcoidosis were negative. We propose a simplified and highly sensitive nested PCR for the diagnosis of M. tuberculosis infection on archived material in HIV-positive and HIV-negative patients.

[AIDS-related pulmonary Kaposi's sarcoma: role of high-resolution computerized tomography]. / Sarcoma di Kaposi polmonare nell'AIDS: ruolo della Tomografia Computerizzata con alta risoluzione.

INTRODUCTION: HIV-related Kaposi sarcoma (KS) is characterized by lesion multifocality, stronger progression and recurrent involvement of some internal organs. Pulmonary lesions are found in 18-47% of cases and not necessarily associated with skin involvement. Lung infections are potentially life-threatening and their early and prompt demonstration is a crucial step for both treatment planning and the prognosis of this severe disease. As a rapid recognition of a pulmonary condition leads to a complete or partial regression in at least 50% of cases, we investigated the role and the diagnostic yield of HRCT in depicting HIV-related KS. MATERIAL AND METHODS: The findings of thirty-nine patients with HIV-related pulmonary KS were retrospectively reviewed. We excluded the patients with associated diseases and incomplete radiologic findings and included 12 patients who had a chest radiograph and a HRCT scanning at least. HRCT showed parenchymal and subpleural micronodules (< 10 mm) and macronodules (> 10 mm), with the halo sign in some cases; perivascular and peribronchial infiltrates, linear or irregular opacities, pleural effusions and enlarged lymph nodes were also seen. Chemotherapy response was also evaluated. RESULTS: All 12 patients had advanced AIDS. The chest films showed abnormal patterns, such as peribronchial and perivascular infiltrates which were most often in midlower pulmonary lobes (88.9%) and often symmetric. Nodules were depicted in 50% of cases and were often associated with peribronchial and perivascular infiltrates; they were always bilateral and characterized by the presence of macronodules in most cases. Eleven of 12 HRCT examinations were considered sufficiently accurate for evaluation, while a pleural effusion prevented lung assessment in one case. Peribronchial and perivascular infiltrates were the most frequent abnormal findings (83.3%), with bilateral involvement in 80% and mostly in the midlower lobes (90%). Parenchymal and subpleural nodules were depicted in 58.3% of cases and always had irregular borders; the halo sign was seen around the nodules in 2 cases and macronodules were found in 2 cases. Pleural effusions were seen in 3 cases and enlarged lymph nodes in 4. Lung KS diagnosis was always confirmed at pathology. The response to chemotherapy (ABV protocol) was evaluated in 5 patients: transient and definitive regressions were observed in 1 and 2 cases, respectively, and disease progression was seen in 2 cases. CONCLUSIONS: HRCT allows the accurate assessment of pulmonary KS in its different stages detailing the disease and its spread, which makes biopsy easier. It also permits to avoid more invasive diagnostic procedures and it is useful in the follow-up after chemotherapy.

[Detection of mycobacterium tuberculosis DNA using nested polymerase chain reaction in lymph nodes with sarcoidosis, fixed in formalin and embedded in paraffin]. / Ricerca del DNA del Mycobacterium tuberculosis mediante nested Polymerase Chain Reaction in linfonodi con sarcoidosi fissati in formalina e inclusi in paraffina.

An involvement of mycobacteria in the pathogenesis of sarcoidosis has often been hypothesized, but not confirmed reproducibly. In this study we applied a nested Polymerase Chain Reaction (PCR) to the insertion sequence IS6110 for detection of Mycobacterium tuberculosis (MT) DNA in formalin-fixed and paraffin-embedded tissues from patients with sarcoidosis, with tuberculosis and atypical mycobacteriosis confirmed by culture, and from negative control samples. MT-DNA could be detected in 2/30 samples of sarcoidosis, in 10/10 tuberculoses, in 0/5 atypical mycobacterioses and in 0/10 negative controls. Nested PCR confirmed its high sensitivity and specificity in detecting MT-DNA on archival histopathological specimens. From our results we conclude that in the granulomatous lesions of sarcoidosis MT-DNA is only sporadically demonstrable and probably it doesn't play a role in the pathogenesis of this disease.