Human papillomavirus detection in cervical dysplasias or neoplasias and in condylomata acuminaata by in situ hybridization with biotinylated DNA probes

Specimens from cervical dysplasias or carcinomas and genital condylomata acuminata were retrospectively analysed by in situ hybridization (ISH) with bioti-nylated DNA probes for human papillomavirus (HPV) types 6, 11, 16 and 18. In the control group no case was positive for HPV DNA. In mild/moderate dysplasias, 4 cases (14%) were positive for HPV 6 or 11 and 2 cases (7%), for HPV 16. In the severe dysplasia/in situ carcinoma group, 9 cases (31%) showed presence of DNA of HPV types 16 or 18. Six invasive carcinomas (20%) were positive for HPV type 16 or 18. Among condylomata acuminata, 22 cases (73%) were positive for HPV types 6 or 11. In all ISH-positive cases only one viral type was detected. No correlation between HPV DNA positivity and histological findings of HPV infection was observed. Although less sensitive than some other molecular biology techniques, in situ hybridization with biotinylated DNA probes proved to be simple and useful for detecting and typing HPV in samples routinely received for histopathological analysis.

Cross-Reactions between Toxocara canis and Ascaris suum in the diagnosis of visceral larva migrans by western blotting technique

Visceral larva migrans (VLM) is a clinical syndrome caused by infection of man by Toxocara spp, the common roundworm of dogs and cats. Tissue migration of larval stages causes illness specially in children. Because larvae are difficult to detect in tissues, diagnosis is mostly based on serology. After the introduction of the enzyme-linked immunosorbent assay (ELISA) using the larval excretory-secretory antigen of T. canis (TES), the diagnosis specificity was greatly improved although cross-reactivity with other helminths are still being reported. In Brazil, diagnosis is routinely made after absorption of serum samples with Ascaris suum antigens, a nematode antigenicaly related with Ascaris lumbricoides which is a common intestinal nematode of children. In order to identify T. canis antigens that cross react to A. suum antigens we analyzed TES antigen by SDS-PAGE and Western blotting techniques. When we used serum samples from patients suspected of VLM and positive result by ELISA as well as a reference serum sample numerous bands were seen (molecular weight of 210-200 kDa, 116-97 kDa, 55-50 kDa and 35-29 kDa). Among these there is at least one band with molecular weight around 55-66 kDa that seem to be responsible for the cross-reactivity between T. canis e A. suum once it disappears when previous absorption of serum samples with A. suum antigens is performed

New Strategies on Molecular Biology Applied to Microbial Systematics

Systematics is the study of diversity of the organisms and their relationships comprising classification, nomenclature and identification. The term classification or taxonomy means the arrangement of the organisms in groups (rate) and the nomenclature is the attribution of correct international scientific names to organisms and identification is the inclusion of unknown strains in groups derived from classification. Therefore, classification for a stable nomenclature and a perfect identification are required previously. The beginning of the new bacterial systematics era can be remembered by the introduction and application of new taxonomic concepts and techniques, from the 50’s and 60’s. Important progress were achieved using numerical taxonomy and molecular taxonomy. Molecular taxonomy, brought into effect after the emergence of the Molecular Biology resources, provided knowledge that comprises systematics of bacteria, in which occurs great evolutionary interest, or where is observed the necessity of eliminating any environmental interference. When you study the composition and disposition of nucleotides in certain portions of the genetic material, you study searching their genome, much less susceptible to environmental alterations than proteins, codified based on it. In the molecular taxonomy, you can research both DNA and RNA, and the main techniques that have been used in the systematics comprise the build of restriction maps, DNA-DNA hybridization, DNA-RNA hybridization, sequencing of DNA sequencing of sub-units 16S and 23S of rRNA, RAPD, RFLP, PFGE etc. Techniques such as base sequencing, though they are extremely sensible and greatly precise, are relatively onerous and impracticable to the great majority of the bacterial taxonomy laboratories. Several specialized techniques have been applied to taxonomic studies of microorganisms. In the last years, these have included preliminary electrophoretic analysis of soluble proteins and isoenzymes, and subsequently determination of deoxyribonucleic acid base composition and assessment of base sequence homology by means of DNA-RNA hybrid experiments beside others. These various techniques, as expected, have generally indicated a lack of taxonomic information in microbial systematics. There are numberless techniques and methodologies that make bacteria identification and classification study possible, part of them described here, allowing establish different degrees of subspecific and interspecific similarity through phenetic-genetic polymorphism analysis. However, was pointed out the necessity of using more than one technique for better establish similarity degrees within microorganisms. Obtaining data resulting from application of a sole technique isolatedly may not provide significant information from Bacterial Systematics viewpoint

SIMPLIFIED DIAGNOSIS OF MALARIA INFECTION: GFM/PCR/ELISA A SIMPLIFIED NUCLEIC ACID AMPLIFICATION TECHNIQUE BY PCR/ELISA

We report an adaptation of a technique for the blood sample collection (GFM) as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.

ANALYSIS OF Treponema pallidum RECOMBINANT ANTIGENS FOR DIAGNOSIS OF SYPHILIS BY WESTERN BLOTTING TECHNIQUE

Three GST fusion recombinant antigen of Treponema pallidum, described as GST-rTp47, GST-rTp17 and GST-rTp15 were analyzed by Western blotting techniques. We have tested 53 serum samples: 25 from patients at different clinical stages of syphilis, all of them presenting anti-treponemal antibody, 25 from healthy blood donors and three from patients with sexually transmitted disease (STD) other than syphilis. Almost all samples from patients with syphilis presented a strong reactivity with GST-rTp17 antigen. Some samples were non-reactive or showed a weak reaction with GST-rTp47 and/or GST-rTp15, and apparently there was no correlation with the stage of disease. There was no seropositivity among blood donors. No sample reacted with purified GST. We concluded that due to their specificity these recombinant antigens can be used as GST fusion protein for development of syphilis diagnostic assays.

Trypanosoma cruzi parasitemia observed in immunocompromised patients: the importance of the artificial xenodiagnosis

Trypanosoma cruzi parasitemia observed in immunocompromised patients (transplant or positive HIV) occurred more frequently by the artificial xenodiagnosis method (10/38) compared with hemoculture (2/38), given the same quantity of blood. Other ways of diagnosis, like mice inoculation (5/38), QBC and buffy coat (2/38), were evaluated also. This result showed the importance of the artificial xenodiagnosis. The other techniques increased only one more patient positive.

Evaluation of the formalin-Tween concentration technique for parasitic detection

The formalin-Tween sedimentation method was compared with the formalin-ether sedimentation for parasitic detection. Of a total 297 fecal specimens examined, 72.1% were positive. The formalin-tween technique was effective for ascertaining helminths, particularly Ascaris lumbricoides, Trichuris trichiura and hookworm eggs; however it has less capability for protozoa detection. This method is simple, inexpensive, less time consuming and highly sensitive when detecting the parasitic infection, particularly when focusing on helminth eggs.

Assessment of the rapid test based on an immunochromatography technique for detecting anti-Treponema pallidum antibodies

A rapid test based on an immunochromatography assay - Determine™ Syphilis TP (Abbott Lab.) for detecting specific antibodies to Treponema pallidum was evaluated against serum samples from patients with clinical, epidemiological and serological diagnosis of syphilis, patients with sexually transmitted disease other than syphilis, and individuals with negative serology for syphilis. The Determine™ test presented the sensitivity of 93.6%, specificity of 92.5%, and positive predictive value and negative predictive value of 95.2% and 93.7%, respectively. One serum sample from patient with recent latent syphilis showed a prozone reaction. Determine™ is a rapid assay, highly specific and easy to perform. This technique obviates the need of equipment and its diagnostic features demonstrate that it may be applicable as an alternative assay for syphilis screening under some emergency conditions or for patients living in remote localities.

Histoplasma capsulatum fungemia in patients with acquired immunodeficiency syndrome: detection by lysis-centrifugation blood-culturing technique

Progressive disseminated histoplasmosis (PDH) is an increasingly common cause of infection in patients with acquired immune deficiency syndrome (AIDS). We report 21 cases of PDH associated with AIDS diagnosed by lysis-centrifugation blood culture method. The most prevalent clinical findings were fever, weight loss, respiratory symptoms, and mucocutaneous lesions. Chest roentgenogram showed diffuse pulmonary infiltrates in 13 of 21 patients (62%). Brochoalveolar fluid has yelded positive culture in four patients only in medium with cycloheximide.

Immunoperoxidase technique using an anti-Leishmania (L.) chagasi hyperimmune serum in the diagnosis of culture-confirmed American tegumentary leishmaniasis

The present study reports the production of the rabbit anti-Leishmania (L.) chagasi hyperimmune serum, the standardization of the immunohistochemistry (IHC) technique and the evaluation of its employment in cutaneous leishmaniasis (CL) lesions diagnosed by Leishmania sp. culture isolation. Thirty fragments of active CL lesions were examined as well as 10 fragments of cutaneous mycosis lesions as control group. IHC proved more sensitive in detecting amastigotes than conventional hematoxylin-eosin (HE) stained slides: the former was positive in 24 (80%) biopsies whereas the latter, in 16 (53%) (p = 0.028). The reaction stained different fungus species causing cutaneous mycosis. Besides, positive reaction was noticed in mononuclear and endothelial cells. Nevertheless, this finding was present in the control group biopsies. It is concluded that IHC showed good sensitivity in detecting amastigotes.

Influence of variables on centrifuge-flotation technique for recovery of Toxocara canis eggs from soil

The purpose of this study was to evaluate the influence of variables in a flotation technique for the recovery of Toxocara canis eggs from soil. The trials were done under standardized conditions on one gram of previously sterilized soil samples contaminated with 200 eggs of T. canis. The following variables were evaluated in serial steps: sieving; type of wash; time of stirring; resuspension of sediment; solution flotation. Centrifuge-flotation in sodium nitrate (d = 1.20 g/cm³) was adopted as an initial technique, using Tween 80 (0.2%) and decinormal sodium hydroxide as solutions for washing the samples. Ten tests were done to compare the variables, using counting in triplicate. The sieving of the material reduced significantly the recovery of eggs (p < 0.001) and the number of eggs recovered was higher when the sediment was resuspended (p < 0.05). After standardization, flotation solutions sodium chloride, zinc sulfate, sodium dichromate, magnesium sulfate, and sodium nitrate (d = 1.20g/cm³) were compared. The best results were obtained by using zinc sulfate solution. In conclusion, the chances of recovering T. canis eggs from samples using flotation solutions can be increased by washing of soil twice using distilled water, and resuspension of sediment. On the other hand, the sieving procedure can drastically reduce the number of eggs.

Genetic variability of Triatoma flavida and Triatoma bruneri (Hemiptera: Reduviidae) by RAPD-PCR technique

The Triatominae (Hemiptera:Reduviidae) contains the principal and potential Chagas disease vectors present in Mexico, Central America and South America. Triatoma flavida and T. bruneri are Cuban species. These species are closely related according to morphology and were considered synonyms until 1981, when they were separated on the grounds of external characters of the body and the morphology of male genitalia. The present study seeks to analyze genetic polymorphism of T. flavida and T. bruneri populations using RAPD techniques, and to assess the genetic relationship between these species. Ten random primers were used to evaluate the genetic variability among species using RAPD-PCR. The genetic flow among them was calculated. The dendrogram based on calculated Jaccard distances showed two clearly distinguishable clusters which coincided with the studied species. Within each species, moderate genetic differentiation (Fst 0.05-0.15) and migration rates (N > 1) were found among populations, that reveal gene flow and genetic homogeneity. Between species, the Fst value showed a high genetic differentiation and the migration rate was insufficient to maintain genetic homogeneity, and confirmed the absence of gene flow between them. Our results confirm the genetic variability among T. flavida and T. bruneri species.