Clear cell odontogenic carcinoma: case report with immunohistochemical findings adding support to the challenging diagnosis

Clear cell odontogenic carcinoma (CCOC) is a rare odontogenic tumor associated with aggressive clinical behavior, metastasis, and low survival. We report a case of CCOC affecting the mandible of a 39-year-old man. The tumor presented a biphasic pattern composed of clear cell nests intermingled with eosinophilic cells and separated by collagenous stroma. Immunoreactivity to cytokeratin (CK), specifically AE1/AE3 and CK 8, 14, 18, and 19 was found, as well as to epithelial membrane antigen (EMA). The tumor cells were negative for S100 protein, CK 13, vimentin, smooth muscle actin, laminin and type IV collagen. Low labeling indices for the proliferation markers Ki-67 and proliferating cell nuclear antigen and to p53 protein might predict a favorable prognosis for the lesion. A surgical resection was performed, followed by adjuvant radiotherapy. A 2-year follow-up has shown no signs of recurrence. The significance of histochemical and immunohistochemical resources in the correct diagnosis of CCOC is analyzed.

The reliability of immunohistochemistry as a prescreening method for the diagnosis of hereditary nonpolyposis colorectal cancer (HNPCC) - Results of an international collaborative study

Hereditary nonpolyposis colorectal cancer syndrome (HNPCC) is an autosomal dominant condition accounting for 2–5% of all colorectal carcinomas as well as a small subset of endometrial, upper urinary tract and other gastrointestinal cancers. An assay to detect the underlying defect in HNPCC, inactivation of a DNA mismatch repair enzyme, would be useful in identifying HNPCC probands. Monoclonal antibodies against hMLH1 and hMSH2, two DNA mismatch repair proteins which account for most HNPCC cancers, are commercially available. This study sought to investigate the potential utility of these antibodies in determining the expression status of these proteins in paraffin-embedded formalin-fixed tissue and to identify key technical protocol components associated with successful staining. A set of 20 colorectal carcinoma cases of known hMLH1 and hMSH2 mutation and expression status underwent immunoperoxidase staining at multiple institutions, each of which used their own technical protocol. Staining for hMSH2 was successful in most laboratories while staining for hMLH1 proved problematic in multiple labs. However, a significant minority of laboratories demonstrated excellent results including high discriminatory power with both monoclonal antibodies. These laboratories appropriately identified hMLH1 or hMSH2 inactivation with high sensitivity and specificity. The key protocol point associated with successful staining was an antigen retrieval step involving heat treatment and either EDTA or citrate buffer. This study demonstrates the potential utility of immunohistochemistry in detecting HNPCC probands and identifies key technical components for successful staining.

Rapid diagnosis in pediatric infectious diseases: The past, the present and the future

The focus of rapid diagnosis of infectious diseases of children in the last decade has shifted from variations of the conventional laboratory techniques of antigen detection, microscopy and culture to that of molecular diagnosis of infectious agents. Pediatricians will need to be able to interpret the use, limitations and results of molecular diagnostic techniques as they are increasingly integrated into routine clinical microbiology laboratory protocols. PCR is the best known and most successfully implemented diagnostic molecular technology to date. It can detect specific infectious agents and determine their virulence and antimicrobial genotypes with greater speed, sensitivity and specificity than conventional microbiology methods. Inherent technical limitations of PCR are present, although they are reduced in laboratories that follow suitable validation and quality control procedures. Variations of PCR together with advances in nucleic acid amplification technology have broadened its diagnostic capabilities in clinical infectious disease to now rival and even surpass traditional methods in some situations. Automation of all components of PCR is now possible. The completion of the genome sequencing projects for significant microbial pathogens, in combination with PCR and DNA chip technology, will revolutionize the diagnosis and management of infectious diseases.

Expression analysis of delta-catenin and prostate-specific membrane antigen: Their potential as diagnostic markers for prostate cancer

The current approach to prostate cancer diagnosis has major limitations including the inability of prostate-specific antigen (PSA) assays to accurately differentiate between prostate cancer and benign prostate hyperplasia (BPH) and the imprecision of transrectal ultrasound (TRUS) biopsy sampling. We have employed cDNA microarray screening to compare gene expression patterns in BPH and tumour samples to identify expression markers that may be useful in discriminating between these conditions. Screening of 3 individual cDNA arrays identified 8 genes with expression 3-fold greater in 6 tumour tissues than in 1 nontumour sample and I BPH sample. Real-time PCR was used to confirm the overexpression of these 8 genes and 12 genes selected from the literature against a panel of 17 tumours and I 1 BPH samples. Two genes, delta-catenin (delta-catenin; CTNND2) and prostate-specific membrane antigen (PSMA; FOLH1), were significantly overexpressed in prostate cancer compared to BPH. Prostate epithelial cells stained positively for S-catenin and PSMA in our prostate cancer tissues, whereas the majority of our BPH tissues were negative for both markers. Thus we have identified delta-catenin (not previously associated with prostatic adenocarcinoma) and confirmed the potential of PSMA as potential candidates for the diagnosis and management of prostate cancer. (C) 2002 Wiley-Liss. Inc.

Diagnosis of Helicobacter pylori infection in adults with intellectual disability

Helicobacter pylori infection is common among adults with intellectual disability. The acceptabilities and accuracies of different diagnostic tests in this population are unknown. We aimed to determine (i) patient acceptability and (ii) performance characteristics of serology, fecal-antigen, and urea breath tests among adults with intellectual disability. One hundred sixty-eight such adults underwent H. pylori testing with serology and fecal-antigen tests, and a portion underwent treatment. One year later, the participants were retested with fecal-antigen, serology, and urea breath tests. The numbers of specimens obtained and difficulties in collection reported by caregivers were noted. Test performance characteristics were assessed among participants and 65 of their caregivers, using serology as the reference. All participants provided at least one specimen, despite reported collection difficulties for 23% of fecal and 27% of blood specimens. Only 25% of the participants provided breath specimens; failure to perform this test was associated with lower intellectual ability and higher maladaptive behavior. The sensitivity, specificity, and positive and negative predictive values of the fecal test (baseline and 12 months versus caregivers) were 70 and 63 versus 81, 93 and 95 versus 98, 96 and 92 versus 93, and 53 and 74 versus 93%, respectively; those of the urea breath test (12 months versus caregivers) were 86 versus 100, 88 versus 95, 75 versus 89, and 94 versus 100%, respectively. With assistance, fecal or blood specimens for H. pylori assessment can be provided by most patients with intellectual disability regardless of their level of function or behavior. Only those with greater ability can perform the urea breath test. Using serology as the reference test, the limitations of performance characteristics of the fecal-antigen and urea breath tests are similar to those among a control group of caregivers.

Voltammetric immunosensor for the diagnosis of celiac disease based on the quantification of anti-gliadin antibodies

Antibodies against gliadin are used to detect celiac disease (CD) in patients. An electrochemical immunosensor for the voltammetric detection of human anti-gliadin antibodies (AGA) IgA and AGA IgG in real serum samples is proposed. The transducer surface consists of screen-printed carbon electrodes modified with a carbon nanotube/gold nanoparticle hybrid system, which provides a very useful surface for the amplification of the immunological interactions. The immunosensing strategy is based on the immobilization of gliadin, the antigen for the autoantibodies of interest, onto the nanostructured surface. The antigen–antibody interaction is recorded using alkaline phosphatase labeled anti-human antibodies and a mixture of 3-indoxyl phosphate with silver ions (3-IP/Ag+) was used as the substrate. The analytical signal is based on the anodic redissolution of the enzymatically generated silver by cyclic voltammetry. The electrochemical behavior of this immunosensor was carefully evaluated assessing aspects as sensitivity, non-specific binding and matrix effects, and repeatability and reproducibility. The results were supported with a commercial ELISA test.

Immunodiagnostic confirmation of hydatid disease in patients with a presumptive diagnosis of infection

Information obtained from the routine application of hydatid immunodiagnostic techniques in different clinical situations over a seven-year period is presented. The Immunoelectrophoresis test was used until it was replaced by the simpler, more sensitive and equally specific arc 5 double diffusion (DD5) test. Examination of sera from 1,888 patients with signs and/or symptoms compatible with hydatid disease revealed that the presurgical confirmation of Echinococcus granulosus infection is only obtained by detection of anti-antigen 5 antibodies. The latter were not found in 1,539 presumptive hydatidosis patients whose definitive diagnoses corresponded to other disease conditions. However, false positive latex agglutination test results were obtained in two cases. In all patients whose preoperative serum showed three or more uncharacteristic bands in the absence of anti-antigen 5 antibodies, hydatid cysts were found sur gically. DD5 testing of a fluid sample collected by puncture established its hydatid etiology. Post-operative monitoring of hydatidosis patients demonstrated that persistence of DD5-positivity two years after surgery established the presence of other cysts. Further evidence was obtained in patients with hydatid cysts in intrathoracic, abdominal or other locations associating cyst membrane integrity, antigen release and immunodiagnostic test positivity.

The value of the Widal test in the diagnosis of Prolonged Septicemic Salmonellosis

Twenty patients with prolonged septicemic salmonellosis (Group 1) and 20 with schistosomiasis mansoni (Group 2) were selected for this study. In both groups, the Widal test was done using antigens of the sample Ty 901 (S. typhi). The test was also applied in 6 group 1 patients with antigens prepared from salmonellae isolated from these patients (autoantigens). Titres over 1:200 were considered significant. Ten group 1 patients (50%) were positive for antigen "H" and 5 (25%) were positive for antigen "O". Three patients with negative "H" and "O" reactions became positive with high titres when using autoantigens. Two other cases maintained the same positive titres and one case showed a fourfold increase in titres when the test was done 'with antigens of the Salmonella isolated. The Widal test was positive in most patients infected with group D Salmonellae. Considering titres above 1:200, all cases were negative in Group 2. The authors conclude that the Widal test has low positivity in prolonged septicemic salmonellosis. The test may be valuable in the diagnosis of this disease when using S. paratyphi "A" and "B" antigens and a mixture of Salmonella antigens taken from other groups.

Predictive value of serologic tests in the diagnosis and follow-up of patients with paracoccidioidomycosis

A serologic study was undertaken in a group of 43 patients with active paracoccidioidomycosis who were treated in the same form (ketoconazole), for identical periods of time (6 months), and folio wed-up for various periods posttherapy. The tests employed were agar gel immunodiffusion (AGID) and complement fixation (FC). Also studied were 50 sera from patients with proven histoplasmosis and pulmonary aspergilloma, 30 patients with culturaly proven tuberculosis as well as 92 specimens from healthy individuals, residents in the endemic area for paracoccidioidomycosis. A single lot of yeast filtrate antigen was used throughout the study. The value of each test was measured according to GALEN and GAMBINO6. Both tests were highly sensitive, 89 and 93% respectively. Regarding their specificity, the AGID was totally specific while the CF exhibited 96.6% and 97% specificity in front of tuberculosis patients and healthy individuals respectively and 82% in comparison with patients with other mycoses. The concept of predictive value, that is, the certainty one has in accepting a positive test as diagnostic of paracoccidioidomycosis, favored the AGID procedure (100%) over the CF test. The latter could sort out with 93% certainty a patient with paracoccidioidomycosis among a group of healthy individuals and with 97.5% in the case of TB patients; when the group in question was composed by individuals with other deep mycoses, such certainty was lower (81%). The above results indicate that both the AGID and the CF tests furnish results of high confidence; one should not relay, however, in the CF alone as a means to establish the specific diagnosis of paracoccidioidomycosis.

Intestinal cryptosporidiosis. association with Pneumocystis carinii, cytomegalovirus and Candida sp. Infections

This is a case report of intestinal cryptosporidiosis diagnosed in histological specimen collected from autopsy. The patient was a child of 5 months admitted to the hospital with severe acute diarrhea associated with Pneumocystis carinii pneumonia, cytomegalic sialadenitis, oral and dermal candidiasis. The presence of multiple opportunistic infections in this case indicated immunodeficiency state. Cryptosporidium sp is a possible etiology of acute diarrhea in both immunodeficient and immunocompetent patients and has to be searched for at autopsy when diagnosis was not possible "in vivo".

Comparison on the performance of Leishmania major-like and Leishmania braziliensis braziliensis as antigen for new world leishmaniasis IgG-immunofluorescence test

The performance of an antigen of L. major-like promastigotes for the serological diagnosis of mucocutaneous leishmaniasis in the IgG-immunofluorescent test was compared to that of an antigen of L. braziliensis braziliensis. Each antigen was used to test two hundred and twenty-four sera of etiologies such as mucocutaneous leishmaniasis, deep mycoses, toxoplasmosis, malaria, Chagas' disease, visceral leishmaniasis, anti-nuclear factor, schistosomaiasis, rheumatoid factor and normal controls. Agreement between responses to each antigen was high: 77.2% of leishmaniases sera agreed on a positive or a negative result to both antigens and 91.1 % of control sera. Cross reactivity was restricted to Chagas' disease sera, visceral leishmaniasis, anti-nuclear factor and paracoccidiodomycosis. The quantitative response of leishmaniasis and Chagas' disease sera to both antigens was evaluated by a linear regression; although the y-intercept and the slope were different for each antigen, neither was better than the other in the disclosure of anti-Leishmania antibodies. In the case of Chagas' disease sera the L. major-like antigen was better than L. b. braziliensis' to disclose cross-reacting antibodies.

Malaria serology: performance of six Plasmodium falciparum antigen extracts and of three ways of determining serum titers in IgG and IgM-ELISA

The study evaluated six Plasmodium falciparum antigen extracts to be used in the IgG and IgM enzyme-linked immunosorbent assays (ELISA), for malaria diagnosis and epidemiological studies. Results obtained with eighteen positive and nine negative control sera indicated that there were statistically significant differences among these antigen extracts (Multifactor ANOVA, p< 0.0001). Urea, sodium deoxycholate and Zwittergent antigen extracts performed better than did the three others, their features being very similar for the detection of IgG antibodies. Urea, alkaline and sodium deoxycholate antigen extracts proved to be better than the others for the detection of IgM antibodies. A straight line relationship was found between the optical densities (or their respective log 10) and the log 10 of antibody dilutions, with a very constant slope. Thus serum titers could be determined by direct titration and by two different equations, needing only one serum dilution. For IgM antibody detections, log 10 expression gave results that better correlated with direct titration (95% Bonferroni). For IgG antibody detections, the titer differences were not significant. The reproducibility of antibody titers and antigen batches was also evaluated, giving satisfactory results.

Alkaline soluble Trypanosoma cruzi epimastigote antigen (ASEA) applied to dot-ELISA

The alkaline soluble Trypanosoma cruzi epimastigote antigen (ASEA) was assessed in dot-ELISA for the diagnosis of Chagas' disease. Serum samples (355) from chagasic and non-chagasic patients were studied, and IgG antibodies to ASEA were found in all patients with chronic Chagas' disease. In non-chagasic patients 95.6% were negative, except for those with leishmaniasis (visceral and mucocutaneous), and some patients from control group reacted in low titers. The data indicate that dot-ELISA using ASEA is suitable for seroepidemiologic surveys to be employed in endemic areas for Chagas' disease.

Evaluation of the double diffusion, enzyme immunoassay and immunoblotting techniques, for the diagnosis of human hydatid disease in tropical areas

Hydatid disease in tropical areas poses a serious diagnostic problem due to the high frequence of cross-reactivity with other endemic helminthic infections. The enzyme-linked-immunosorbent assay (ELISA) and the double diffusion arc 5 showed respectively a sensitivity of 73% and 57% and a specificity of 84-95% and 100%. However, the specificity of ELISA was greatly increased by using ovine serum and phosphorylcholine in the diluent buffer. The hydatic antigen obtained from ovine cyst fluid showed three main protein bands of 64,58 and 30 KDa using SDS PAGE and immunoblotting. Sera from patients with onchocerciasis, cysticercosis, toxocariasis and Strongyloides infection cross-reacted with the 64 and 58 KDa bands by immunoblotting. However, none of the analyzed sera recognized the 30 KDa band, that seems to be specific in this assay. The immunoblotting showed a sensitivity of 80% and a specificity of 100% when used to recognize the 30 KDa band.

Dot-ELISA-IgM in saliva for the diagnosis of human leptospirosis using polyester fabric-resin as support (Preliminary Report)

In order to improve the diagnosis of human leptospirosis, we standardized the dot-ELISA for the search of specific IgM antibodies in saliva. Saliva and serum samples were collected simultaneously from 20 patients with the icterohemorrhagic form of the disease, from 10 patients with other pathologies and from 5 negative controls. Leptospires of serovars icterohaemorrhagiae, canicola, hebdomadis, brasiliensis and cynopteri grown in EMJH medium and mixed together in equal volumes, were used as antigen at individual protein concentration of 0.2 µg/µl. In the solid phase of the test we used polyester fabric impregnated with N-methylolacrylamide resin. The antigen volume for each test was 1µl, the saliva volume was 8 µl, and the volume of peroxidase-labelled anti-human IgM conjugate was 30 µl. A visual reading was taken after development in freshly prepared chromogen solution. In contrast to the classic nitrocellulose membrane support, the fabric support is easy to obtain and to handle. Saliva can be collected directly onto the support, a fact that facilitates the method and reduces the expenses and risks related to blood processing.