A simple method for human peripheral blood monocyte Isolation

We describe a simple method using percoll gradient for isolation of highly enriched human monocytes. High numbers of fully functional cells are obtained from whole blood or buffy coat cells. The use of simple laboratory equipment and a relatively cheap reagent makes the described method a convenient approach to obtaining human monocytes.

A simplified method for sample collection and DNA isolation for polymerase chain reaction detection of Trypanosoma rangeli and Trypanosoma cruzi in triatomine vectors

Due to the overlapping distribution of Trypanosoma rangeli and T. cruzi in Central and South America, sharing several reservoirs and triatomine vectors, we herein describe a simple method to collect triatomine feces and hemolymph in filter paper for further detection and specific characterization of these two trypanosomes. Experimentally infected triatomines feces and hemolymph were collected in filter paper and specific detection of T. rangeli or T. cruzi DNA by polymerase chain reaction was achieved. This simple DNA collection method allows sample collection in the field and further specific trypanosome detection and characterization in the laboratory.

An improved method for concentrating rotavirus from water samples

A modified adsorption-elution method for the concentration of seeded rotavirus from water samples was used to determine various factors which affected the virus recovery. An enzyme-linked immunosorbent assay was used to detect the rotavirus antigen after concentration. Of the various eluents compared, 0.05M glycine, pH 11.5 gave the highest rotavirus antigen recovery using negatively charged membrane filtration whereas 2.9% tryptose phosphate broth containing 6% glycine; pH 9.0 was found to give the greatest elution efficiency when a positively charged membrane was used. Reconcentration of water samples by a speedVac concentrator showed significantly higher rotavirus recovery than polyethylene glycol precipitation through both negatively and positively charged filters (p-value <0.001). In addition, speedVac concentration using negatively charged filtration resulted in greater rotavirus recovery than that using positively charged filtration (p-value = 0.004). Thirty eight environmental water samples were collected from river, domestic sewage, canals receiving raw sewage drains, and tap water collected in containers for domestic use, all from congested areas of Bangkok. In addition, several samples of commercial drinking water were analyzed. All samples were concentrated and examined for rotavirus antigen. Coliforms and fecal coliforms (0->1,800 MPN/100 ml) were observed but rotavirus was not detected in any sample. This study suggests that the speedVac reconcentration method gives the most efficient rotavirus recovery from water samples.

Evaluation by ELISA of Anisakis simplex Larval Antigen Purified by Affinity Chromatography

In order to improve the specificity and sensitivity of the techniques for the human anisakidosis diagnosis, a method of affinity chromatography for the purification of species-specific antigens from Anisakis simplex third-stage larvae (L3) has been developed. New Zealand rabbits were immunized with A. simplex or Ascaris suum antigens or inoculated with Toxocara canis embryonated eggs. The IgG specific antibodies were isolated by means of protein A-Sepharose CL-4B beads columns. IgG anti-A. simplex and -A. suum were coupled to CNBr-activated Sepharose 4B. For the purification of the larval A. simplex antigens, these were loaded into the anti-A. simplex column and bound antigens eluted. For the elimination of the epitopes responsible for the cross-reactions, the A. simplex specific proteins were loaded into the anti-A. suum column. To prove the specificity of the isolated proteins, immunochemical analyses by polyacrylamide gel electrophoresis were carried out. Further, we studied the different responses by ELISA to the different antigenic preparations of A. simplex used, observing their capability of discriminating among the different antisera raised in rabbits (anti-A. simplex, anti-A. suum, anti-T. canis). The discriminatory capability with the anti-T. canis antisera was good using the larval A. simplex crude extract (CE) antigen. When larval A. simplex CE antigen was loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with A. simplex CE antigen, its capability for discriminate between A. simplex and A. suum was improved, increasing in the case of T. canis. The best results were obtained using larval A. simplex CE antigen loaded into a CNBr-activated Sepharose 4B coupled to IgG from rabbits immunized with adult A. suum CE antigen. When we compared the different serum dilution and antigenic concentration, we selected the working serum dilution of 1/400 and 1 µg/ml of antigenic concentration.

A New Method for Fixing Biomphalaria glabrata for Histologic Studies, Using Shell Perforation

A new technique for fixation of Biomphalaria glabrata for histologic studies is described. It consists in performing several external holes in the shell, before placing the entire snail into the fixative. It is a very practical and quick procedure that showed excellent results when compared to the usual techniques.

Comparison of the Proportion Method With Mycobacteria Growth Indicator Tube and E-test for Susceptibility Testing of Mycobacterium tuberculosis

The aim of this study was to investigate the correlation between proportion method with mycobacteria growth indicator tube (MGIT) and E-test for Mycobacterium tuberculosis. Forty clinical isolates were tested. MGIT and E-test with the first line antituberculous drugs correlated with the proportion method. Our results suggested that MGIT and E-test methods can be routinely used instead of the proportion method.

First characterization of Candida albicans by random amplified polymorphic DNA method in Nicaragua and comparison of the diagnosis methods for vaginal candidiasis in Nicaraguan women

A total of 106 women with vaginitis in Nicaragua were studied. The positive rate for the identification of Candida species was 41% (44 positive cultures out of 106 women with vaginitis). The sensitivity of microscopic examination of wet mount with the potassium hydroxide (KOH) was 61% and 70% with Gram's stain when using the culture of vaginal fluid as gold standard for diagnosis of candidiasis. Among the 44 positives cultures, isolated species of yeast from vaginal swabs were C. albicans (59%), C. tropicalis (23%), C. glabrata (14%) and C. krusei (4%). This study reports the first characterization of 26 C. albicans stocks from Nicaragua by the random amplified polymorphic DNA method. The genetic analysis in this small C. albicans population showed the existence of linkage disequilibrium, which is consistent with the hypothesis that C. albicans undergoes a clonal propagation.

Detection of telomerase activity in Plasmodium falciparum using a nonradioactive method

A simple, quick and sensitive method was used to detect telomerase activity in Plasmodium falciparum. The telomeric repeat amplification protocol (TRAP assay) was modified using electrophoresis and staining with SYBR-green I to detect telomerase activity in a range of 10² to 10(7) parasites. This might be a useful way to ascertain telomerase activity in different types of nontumor cells.

Development of genital plates in nymphs of Triatoma pallidipennis, Stål 1872, (Hemiptera: Reduviidae) and its application as sexing method

Searching for morphometric differences between sexes in immature forms, the development of genital plates in the exuviae of Triatoma pallidipennis Stål 1872nymphs was studied. Differences were found in the form and size of the 9th genital urosternite, it is larger and wider in males as compared to females. This difference is reported in several South American Triatoma species. From our results it is possible to sex early stages from microscopic observation of genital plates in whole insects.

Evaluation of four methods for detecting the beta-lactamase activity in Neisseria gonorrhoeae isolated in Cuba

Four methods (chromogenic, acidimetric, inhibition, and iodometric) for demonstration of the beta-lactamase production by 70 isolates of Neisseria gonorrhoeae, were evaluated in Cuba. There was 100% correlation between all beta-lactamase methods and the standardized penicillin dilution susceptibility test for penicillinase-non-producing N. gonorrhoeae. For penicillinase-producing N. gonorrhoeae strains, there was a perfect correlation between the chromogenic method and penicillin susceptibility testing, but one and two strains failed to give a positive result for beta-lactamase with the inhibition/acidimetric and the iodometric methods, respectively. There was a high concordance between the chromogenic method, considered as gold standard and the rest of penicillinase tests evaluated: Kappa Index (KI) = 0.98 for inhibition/acidimetric methods and KI = 0.97 for the iodometric method. The four methods evaluated were accurate, reproducible, easily readable, economical, and ease to use for screening primary isolates of N. gonorrhoeae in Cuba. We recommended the use of the inhibition method, when testing the penicillinase activity in gonococcal isolates in provincial and municipal reference laboratories.

Paleopharmacology and pollen: theory, method, and application

Parasitism was a universal human condition. Because of this, people developed herbal medicines to treat parasites as part of their pharmacopoeias. We propose that it is possible to recover evidence of medicinal plants from archaeological sites and link their use to specific health conditions. This is a multidisciplinary approach that must involve at least paleoethnobotanists, archaeoparasitologists, paleopathologists, and pharmacologists.

Viremic blood donor found by a rapid screening method in a season of high human parvovirus B19 activity in Niterói, Rio de Janeiro, Brazil

Erythrovirus B19 infection is usually benign but may have serious consequences in patients with hemolytic anemia (transient aplastic crisis), immunodeficiency (in whom persistent infection can lead to chronic bone marrow failure with anemia), or who are in the first or second trimester of gestation (spontaneous abortion, hydrops fetalis, and fetal death). Being non-enveloped, B19 resists most inactivation methods and can be transmitted by transfusion. B19 is difficult to cultivate and native virus is usually obtained from viremic blood. As specific antibodies may be absent, and there is no reliable immunological method for antigen detection, hybridization or polymerase chain reaction are needed for detecting viremia. A rapid method, gel hemagglutination (Diamed ID-Parvovirus B19 Antigen Test), can disclose highly viremic donations, whose elimination lessens the viral burden in pooled blood products and may even render them non-infectious. In order to obtain native antigen and to determine the frequency of viremic donors, we applied this test to blood donors in a period of high viral activity in our community. Positive or indeterminate results were re-tested by dot-blot hybridization. We tested 472 donors in 1998 and 831 ones in 1999. One viremic donor was found in 1999. We suggest that in periods of high community viral activity the gel hemagglutination test may be useful in avoiding highly viremic blood being added to plasma pools or directly transfused to patients under risk.

Drug susceptibility testing of Mycobacterium tuberculosis by the broth microdilution method with 7H9 broth

In this study, we have evaluated the broth microdilution method (BMM) for susceptibility testing of Mycobacterium tuberculosis. A total of 43 clinical isolates of M. tuberculosis and H37Rv as a control strain were studied. All isolates were tested by the proportion method and the BMM for isoniazid (INH), rifampicin (RIF), streptomycin (STR), and ethambutol (ETM). The proportion method was carried out according to the National Committee for Clinical Laboratory Standards (NCCLS) on Löwenstein-Jensen (LJ) medium. The BMM was carried out using 7H9 broth with 96 well-plates. All strains were tested at 3.2-0.05 µg/ml, 16-0.25 µg/ml, 32-0.5 µg/ml, and 32-0.5 µg/ml concentrations for INH, RIF, STR, and ETM, respectively. When the BMM was compared with the proportion method, sensitivity was 100, 100, 96.9, and 90.2%, while specificity was 100, 85.7, 90.9, and 100% for INH, RIF, STR, and ETM, respectively. The plates were examined 7, 10, 14, and 21 days after incubation. The majority of the result were obtained at 14th days after incubation, while the proportion method result were ended in 21-28 days. According to our results, it may be suggested that the BMM is suitable for early determining of multidrug-resistance-M. tuberculosis strains in developed or developing countries.

Value of morphotyping for the characterization of Candida albicans clinical isolates

Until recently, morphotyping, a method evaluating fringe and surface characteristics of streak colonies grown on malt agar, has been recommended as a simple and unexpensive typing method for Candida albicans isolates. The discriminatory power and reproducibility of Hunter's modified scheme of Phongpaichit's morphotyping has been evaluated on 28 C. albicans isolates recovered from the oral cavity of asymptomatic human immunodeficiency virus-positive subjects, and compared to two molecular typing methods: randomly amplified polymorphic DNA (RAPD) fingerprinting, and contour clamped homogeneous electric field (CHEF) electrophoretic karyotyping. Morphological features of streak colonies allowed to distinguish 11 different morphotypes while RAPD fingerprinting yielded 25 different patterns and CHEF electrophoresis recognized 9 karyotypes. The discriminatory power calculated with the formula of Hunter and Gaston was 0.780 for morphotyping, 0.984 for RAPD fingerprinting, and 0.630 for karyotyping. Reproducibility was tested using 43 serial isolates from 15 subjects (2 to 6 isolates per subject) and by repeating the test after one year storage of the isolates. While genetic methods generally recognized a single type for all serial isolates from each of the subjects studied, morphotyping detected strain variations in five subjects in the absence of genetic confirmation. Poor reproducibility was demonstrated repeating morphotyping after one year storage of the isolates since differences in at least one character were detected in 92.9% of the strains.

Susceptibility of clinical isolates of multiresistant Pseudomonas aeruginosa to a hospital disinfectant and molecular typing

The aim of this study was to evaluate the susceptibility of 35 resistant Pseudomonas aeruginosa clinical isolates to a quaternary ammonium hospital disinfectant. The methodology was the AOAC Use-Dilution Test, with disinfectant at its use-concentration. In addition, the chromosomal DNA profile of the isolates were determined by macro-restriction pulsed field gel electrophoresis (PFGE) method aiming to verify the relatedness among them and the behavior of isolates from the same group regarding the susceptibility to the disinfectant. Seventy one percent of the isolates were multiresistant to antibiotics and 43% showed a reduced susceptibility to the disinfectant. The PFGE methodology detected 18 major clonal groups. We found isolates with reduced susceptibility to the disinfectant and we think that these are worrying data that should be further investigated including different organisms and chemical agents in order to demonstrate that microorganisms can be destroyed by biocide as necessary. We also found strains of the same clonal groups showing different susceptibility to the disinfectant. This is an interesting observation considering that only few works are available about this subject. PFGE profile seems not to be a reliable marker for resistance to disinfectants.