Nested-PCR for the detection of Mycoplasma hyopneumoniae in bronchial alveolar swabs, frozen tissues and formalin-fixed paraffin-embedded swine lung samples: comparative evaluation with immunohistochemical findings and histological features

The diagnosis of Mycoplasma hyopneumoniae infection is often performed through histopathology, immunohistochemistry (IHC) and polymerase chain reaction (PCR) or a combination of these techniques. PCR can be performed on samples using several conservation methods, including swabs, frozen tissue or formalin-fixed and paraffin-embedded (FFPE) tissue. However, the formalin fixation process often inhibits DNA amplification. To evaluate whether M. hyopneumoniae DNA could be recovered from FFPE tissues, 15 lungs with cranioventral consolidation lesions were collected in a slaughterhouse from swine bred in herds with respiratory disease. Bronchial swabs and fresh lung tissue were collected, and a fragment of the corresponding lung section was placed in neutral buffered formalin for 48 hours. A PCR assay was performed to compare FFPE tissue samples with samples that were only refrigerated (bronchial swabs) or frozen (tissue pieces). M. hyopneumoniae was detected by PCR in all 15 samples of the swab and frozen tissue, while it was detected in only 11 of the 15 FFPE samples. Histological features of M. hyopneumoniae infection were presented in 11 cases and 7 of these samples stained positive in IHC. Concordance between the histological features and detection results was observed in 13 of the FFPE tissue samples. PCR was the most sensitive technique. Comparison of different sample conservation methods indicated that it is possible to detect M. hyopneumoniae from FFPE tissue. It is important to conduct further research using archived material because the efficiency of PCR could be compromised under these conditions.

Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples

Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories.

Immunohistochemical detection of Clostridia species in paraffin-embedded tissues of experimentally inoculated guinea pigs

Blackleg is caused by Clostridium chauvoei, whereas malignant oedema is caused by C. chauvoei, C. septicum, C. sordellii, C. perfringens type A, and/or C. novyi type A. Anti-C. chauvoei, anti-C. septicum, anti-C. sordellii and anti-C. novyi type A polyclonal antibodies were produced in rabbits and purified in a column of DEAE-cellulose. Aliquots of the antisera were conjugated with fluorescein isothiocyanate and the remaining was used for the streptavidin biotin peroxidase technique (SBPT). SBPT was standardized to detect C. chauvoei, C. septicum, C. sordellii and C. novyi type A in formalin-fixed, paraffin-embedded tissues of guinea pigs. SBPT was compared to a fluorescent antibody technique (FAT). Sections and smears of muscle from inoculation area (MIA), heart, liver, spleen and kidney, were obtained for both SBPT and FAT. Cross-reactions between the different Clostridial species were not observed. C. chauvoei and C. septicum were detected in all specimens from the animals inoculated with these microorganisms, while only sections of muscle obtained from all the animals inoculated with C. sordellii and C. novyi type A were positive. The same results observed by the SBPT, were obtained on tissue smears of these microorganisms stained by the FAT. The results indicate that SBPT is suitable for detection of C. chauvoei, C. septicum, C. sordellii and C. novyi type A in formalin-fixed, paraffin-embedded tissues of guinea pigs.

Detection of parvovirus B19 infection in formalin-fixed and paraffin-embedded placenta and fetal tissues

Parvovirus B19 infection was first discovered in 1975 and it is implicated in fetal death from hydrops fetalis the world over. Diagnosis is usually made through histological identification of the intranuclear inclusion in placenta and fetal organs. However, these cells may be scarce or uncharacteristic, making definitive diagnosis difficult. We analyzed histologically placentas and fetal organs from 34 cases of non-immune hydrops fetalis, stained with Hematoxylin and Eosin (HE) and submitted to immunohistochemistry and polymerase chain reaction (PCR). Of 34 tissue samples, two (5.9%) presented typical intranuclear inclusion in circulating normoblasts seen in Hematoxylin and Eosin stained sections, confirmed by immunohistochemistry and PCR. However, PCR of fetal organs was negative in one case in which the placenta PCR was positive. We concluded that parvovirus B19 infection frequency is similar to the literature and that immunohistochemistry was the best detection method. It is highly specific and sensitive, preserves the morphology and reveals a larger number of positive cells than does HE with the advantage of showing cytoplasmic and nuclear positivity, making it more reliable. Although PCR is more specific and sensitive in fresh or ideally fixed material it is not so in formalin-fixed paraffin-embedded tissues, frequently the only one available in such cases.

Comparison of two PCR methods for detection of Leptospira interrogans in formalin-fixed and paraffin-embedded tissues

In this study we compared two polymerase chain reaction (PCR) methods using either 16S ribosomal RNA (rRNA) or 23S rRNA gene primers for the detection of different Leptospira interrogans serovars. The performance of these two methods was assessed using DNA extracted from bovine tissues previously inoculated with several bacterial suspensions. PCR was performed on the same tissues before and after the formalin-fixed, paraffin-embedding procedure (FFPE tissues). The 23S rDNA PCR detected all fresh and FFPE positive tissues while the 16S rDNA-based protocol detected primarily the positive fresh tissues. Both methods are specific for pathogenic L. interrogans. The 23S-based PCR method successfully detected Leptospira in four dubious cases of human leptospirosis from archival tissue specimens and one leptospirosis-positive canine specimen. A sensitive method for leptospirosis identification in FFPE tissues would be a useful tool to screen histological specimen archives and gain a better assessment of human leptospirosis prevalence, especially in tropical countries, where large outbreaks can occur following the rainy season.

HIGH SENSITIVE PCR METHOD FOR DETECTION OF PATHOGENIC Leptospira spp. IN PARAFFIN-EMBEDDED TISSUES

This study describes the development and application of a new PCR assay for the specific detection of pathogenic leptospires and its comparison with a previously reported PCR protocol. New primers were designed for PCR optimization and evaluation in artificially-infected paraffin-embedded tissues. PCR was then applied to post-mortem, paraffin-embedded samples, followed by amplicon sequencing. The PCR was more efficient than the reported protocol, allowing the amplification of expected DNA fragment from the artificially infected samples and from 44% of the post-mortem samples. The sequences of PCR amplicons from different patients showed >99% homology with pathogenic leptospires DNA sequences. The applicability of a highly sensitive and specific tool to screen histological specimens for the detection of pathogenic Leptospira spp. would facilitate a better assessment of the prevalence and epidemiology of leptospirosis, which constitutes a health problem in many countries.

Detection of Leishmania braziliensis in human paraffin-embedded tissues from Tucumán, Argentina by polymerase chain reaction

American cutaneous leishmaniasis (ACL) is an endemic disease in Northern Argentina. We applied the polymerase chain reaction (PCR) followed by a hybridization labelled probe to 21 paraffin embedded human skin biopsies, already analyzed histologically, from leishmaniasis endemic areas in the province of Tucumán, Argentina. We used primers previously designed to detect a Leishmania-specific 120-base-pair fragment of kinetoplast DNA minicircle, other two primer pairs that amplify kDNA minicircles belonging to the L. braziliensis and L. mexicana complexes respectively, and specific oligonucleotide primers to detect L. (V.) braziliensis which amplify the sequence of the ribosomal protein L-14 of this species. The PCR-hybridization showed a sensitivity of 90.5% when compared to the histopathology test which was 61.9%. Five of the total samples analyzed were positive for the L. braziliensis complex whilst none was positive for the L. mexicana complex. The specific primers for L. (V.) braziliensis detected the parasite in four samples. These results are consistent with those reported for close endemic areas and demonstrate that the causative agent of human leishmaniasis in the analyzed cases was L. (V.) braziliensis. PCR should be used as a diagnostic tool for tegumentary leishmaniasis, especially in the mucosal form, and as a valuable technique for the identification of the Leishmania species that causes the disease in certain areas.

Echinococcus granulosus sensu lato GENOTYPES IN DOMESTIC LIVESTOCK AND HUMANS IN GOLESTAN PROVINCE, IRAN

Cystic echinococcosis (CE) is a globally parasitic zoonosis caused by larval stages of Echinococcus granulosus. This study investigated E. granulosus genotypes isolated from livestock and humans in the Golestan province, northern Iran, southeast of the Caspian sea, using partial sequencing data of the cytochrome c oxidase subunit 1 (cox1) and NADH dehydrogenase 1 (nad1) mitochondrial genes. Seventy E. granulosus isolates were collected from animals in slaughterhouses: 18 isolates from sheep, 40 from cattle, nine from camels, two from buffaloes and one from a goat, along with four human isolates (formalin-fixed, paraffin-embedded tissues) from CE patients of provincial hospitals. All isolates were successfully analysed by PCR amplification and sequencing. The sequence analysis found four E. granulosus genotypes among the 74 CE isolates: G1 (78.3%), G2 (2.7%), G3 (15%) and G6 (4%). The G1-G3 complex genotype was found in all of the sheep, goat, cattle and buffalo isolates. Among the nine camel isolates, the frequency of G1-G3 and G6 genotypes were 66.7% and 33.3%, respectively. All four human CE isolates belonged to E. granulosus sensu stricto. This study reports the first occurrence of the G2 genotype in cattle from Iran and confirms the previously reported G3 genotype in camels in the same country.

Production and characterization of monoclonal antibodies to Brazilian isolates of bovine viral diarrhea virus

Three Brazilian isolates of bovine viral diarrhea virus (BVDV), antigenically distinct from the standard North American isolates, were selected to immunize BALB/c mice in order to obtain hybridoma cells secreting anti-BVDV monoclonal antibodies (mAbs). Two hybridoma clones secreting mAbs, reacting specifically with BVDV-infected cells (mAbs 3.1C4 and 6.F11), were selected after five fusions and screening of 1001 hypoxanthine-aminopterin-thymidine-resistant clones. These mAbs reacted in an indirect fluorescent antibody (IFA) assay with all 39 South and North American BVDV field isolates and reference strains available in our laboratory, yet failed to recognize other pestiviruses, namely the hog cholera virus. The mAbs reacted at dilutions up to 1:25,600 (ascitic fluid) and 1:100 (hybridoma culture supernatant) in IFA and immunoperoxidase (IPX) staining of BVDV-infected cells but only mAb 3.1C4 neutralized virus infectivity. Furthermore, both mAbs failed to recognize BVDV proteins by IPX in formalin-fixed paraffin-embedded tissues and following SDS-PAGE and immunoblot analysis of virus-infected cells, suggesting they are probably directed to conformational-type epitopes. The protein specificity of these mAbs was then determined by IFA staining of CV-1 cells transiently expressing each of the BVDV proteins: mAb 3.1C4 reacted with the structural protein E2/gp53 and mAb 6.F11 reacted with the structural protein E1/gp25. Both mAbs were shown to be of the IgG2a isotype. To our knowledge, these are the first mAbs produced against South American BVDV isolates and will certainly be useful for research and diagnostic purposes.

Trypanosoma cruzi: parasite persistence in tissues in chronic chagasic Brazilian patients

Chagas disease in the chronic phase may develop into cardiac and/or digestive forms. The pathogenesis of the disease is not yet clear and studies have been carried out to elucidate the role of parasite persistence in affected organs. The aim of this study was to detect and quantify Trypanosoma cruzi in paraffin-embedded tissue samples from chronic patients using NPCR (nested polymerase chain reaction) and QPCR (quantitative polymerase chain reaction) methods. These results were correlated to anatomopathological alterations in the heart and gastrointestinal tract (GIT). Of the 23 patients studied, 18 presented the cardiac form and five presented the cardiodigestive form of Chagas disease. DNA samples were randomly isolated from formalin-fixed paraffin-embedded sections of heart and GIT tissue of 23 necropsies and were analyzed through NPCR amplification. T. cruzi DNA was detected by NPCR in 48/56 (85.7%) heart and 35/42 (83.3%) GIT samples from patients with the cardiac form. For patients with the cardiodigestive form, NPCR was positive in 12/14 (85.7%) heart and in 14/14 (100%) GIT samples. QPCR, with an efficiency of 97.6%, was performed in 13 samples (11 from cardiac and 2 from cardiodigestive form) identified previously as positive by NPCR. The number of T. cruzi copies was compared to heart weight and no statistical significance was observed. Additionally, we compared the number of copies in different tissues (both heart and GIT) in six samples from the cardiac form and two samples from the cardiodigestive form. The parasite load observed was proportionally higher in heart tissues from patients with the cardiac form. These results show that the presence of the parasite in tissues is essential to Chagas disease pathogenesis.

Histomorphological and immunohistochemical characterization of 172 cutaneous round cell tumours in dogs

This paper describes the use of a panel of antibodies (CD117, CD3, CD79a, CD45, cytokeratin, vimentin and E-cadherin) on formalin-fixed, paraffin-embedded sections of canine cutaneous round cell tumours. Neoplastic tumours were diagnosed by histology and histochemical stains and included 107 mast cell tumours, 31 cutaneous histiocytomas, two localized histiocytic sarcomas, 21 cutaneous lymphomas, three plasma cell tumours, one transmissible venereal tumour and seven unclassified round cell tumours. The histologic diagnosis was modified in 39.5% of the total 172 neoplasms. The staining for CD45 and Ecadherin were variable, and therefore, the final diagnoses of cutaneous histiocytoma and localized histiocytic sarcoma were made based on histology in association with negative results for CD3, CD79a, CD117 and cytokeratin. The cellular origin of unclassified round cell tumours was defined in all cases. Cutaneous B-cell lymphoma and plasma cell tumours were CD79a-positive and could be distinguished from each other by the morphological characteristics. Mast cell tumours and T cell lymphoma were CD117 and CD3 positive, respectively. The positive staining for vimentin and the negative staining for CD3, CD79a, CD117 and cytokeratin favoured the diagnosis of transmissible venereal tumours. Thus, the final diagnosis of cutaneous round cell tumours should be based on the interpretation of immunohistochemical results together with the cellular morphology observed by histology. Therefore, more studies to optimize the specific markers in formalin-fixed, paraffinembedded tissues (especially for histiocytes) are required for definitive diagnosis of round cell tumours in dogs.

An immunohistochemic assay to localize leptospires in tissue specimens

An Immunoperoxidase technique for identification of leptospires in formalin fixed, paraffin embedded kidney sections is presented, using peroxidase-antiperoxidase complex. The anti-leptospiral antibody was raised in rabbit. Possible applications of this technique are discussed.

Detection of codon 12 mutation in the k-ras oncogene in pancreatic tumors

Mutations at codons 12, 13, or 61 of the H-ras, K-ras, and N-ras have been detected in human neoplasias by a variety of techniques. Some of these techniques are very sensitive and can detect K-ras mutation in 90% of the cases of pancreatic adenocarcinomas. We analyzed 11 samples of pancreatic adenocarcinoma, three samples of pancreatic mucinous cystadenoma, and two samples without tumors in formalin-fixed paraffin embedded tissue sections. K-ras mutations at codon 12 were detected by a two-step PCR-enriched technique in all the samples of pancreatic adenocarcinoma, but not in cystadenoma or control samples. This technique may be useful for early detection of pancreatic cancer.

Human parvovirus B19 infection and hydrops fetalis in Rio de Janeiro, Brazil

Formalin-fixed paraffin embedded lung and liver tissue from 23 cases of non immune hydrops fetalis and five control cases, in which hydrops were due to syphilis (3) and genetic causes (2), were examined for the presence of human parvovirus B19 by DNA hybridisation. Using in situ hybridisation with a biotynilated probe one positive case was detected. Using 32P-labelled probes in a dot blot assay format, five further positives were obtained. These were all confirmed as positive by a nested polymerase chain reaction assay. Electron microscopy revealed virus in all these five positive cases. The six B19 DNA positive cases of hydrops fetalis were from 1974, 1980, 1982, 1987 and 1988, four of which occurred during the second half of the year, confirming the seasonality of the disease.

Immunohistochemical expression of HER-2/NEU-CERBB-2 in patients with adenocarcinoma of the stomach

OBJECTIVES: To determine the prevalence of Her-2/Neu-cerbb-2 in the gastric mucosa of patients with gastric adenocarcinoma in a brazilian patient group. METHODS: The immunohistochemical expression of Her-2/Neu was studied in 37 formalin-fixed paraffin-embedded tissue sections. RESULTS: The immunohistochemical reaction produced by the anti-HER-2/Neu antibody was positive in two cases (5.4%). CONCLUSION: The low prevalence of Her-2/Neu observed in these southern brazilian cases is probably due to the great number of poorly differentiated cancers in this serie.