PROTECTIVE EFFECT OF THE PROBIOTIC Saccharomyces boulardii IN Toxocara canis INFECTION IS NOT DUE TO DIRECT ACTION ON THE LARVAE

SUMMARY In a previous study our group found that the probiotic Saccharomyces boulardii was capable of reducing the intensity of infection in mice with toxocariasis. In order to assess whether the mechanism involved would be a direct action of the probiotic on Toxocara canis larvae, this study was designed. Both probiotics were singly cultivated in plates containing RPMI 1640 medium and T. canis larvae. S. boulardii and B. cereus var. toyoi cultures presented 97.6% and 95.7% of larvae with positive motility, respectively, and absence of color by the dye trypan blue, not representing significant difference to the control group (p > 0.05). We conclude that none of the probiotics showed in vitro effects on T. canis larvae and that the interaction with the intestinal mucosa is necessary for the development of the protective effect of S. boulardii.

COMPARISON OF SIX COMMERCIALLY-AVAILABLE DNA POLYMERASES FOR DIRECT PCR

SUMMARYThe use of a “direct PCR” DNA polymerase enables PCR amplification without any prior DNA purification from blood samples due to the enzyme's resistance to inhibitors present in blood components. Such DNA polymerases are now commercially available. We compared the PCR performance of six direct PCR-type DNA polymerases (KOD FX, Mighty Amp, Hemo KlenTaq, Phusion Blood II, KAPA Blood, and BIOTAQ) in dried blood eluted from a filter paper with TE buffer. GoTaq Flexi was used as a standard DNA polymerase. PCR performance was evaluated by a nested PCR technique for detecting Plasmodium falciparum genomic DNA in the presence of the blood components. Although all six DNA polymerases showed resistance to blood components compared to the standard Taq polymerase, the KOD FX and BIOTAQ DNA polymerases were resistant to inhibitory blood components at concentrations of 40%, and their PCR performance was superior to that of other DNA polymerases. When the reaction mixture contained a mild detergent, only KOD FX DNA polymerase retained the original amount of amplified product. These results indicate that KOD FX DNA polymerase is the most resistant to inhibitory blood components and/or detergents. Thus, KOD FX DNA polymerase could be useful in serological studies to simultaneously detect antibodies and DNA in eluents for antibodies. KOD FX DNA polymerase is thus not limited to use in detecting malaria parasites, but could also be employed to detect other blood-borne pathogens.

Evaluation of a direct immunofluorescent antibody (difma) test using Leishmania genus - specific monoclonal antibody in the routine diagnosis of cutaneous leishmaniasis

A direct immunofluorescent antibody (DIFMA) test using a Leishmania genus- specific monoclonal antibody was evaluated in the routine diagnosis of cutaneous leishmaniasis (CL) in Ecuador. This test was compared with the standard diagnostic techniques of scrapings, culture and histology. Diagnostic samples were taken from a total of 90 active dermal ulcers from patients from areas of Ecuador known to be endemic for cutaneous leishmaniasis. DIFMA was positive in all lesions. It was shown to be significantly superior to standard diagnostic methods either alone or in combination. The sensitivity of DIFMA did not diminish with chronicity of lesions. This test proved to be extremely useful in the routine diagnosis of CL because it is highly sensitive, is easy to use and produces rapid results.

Evaluation of a commercial test based on ligase chain reaction for direct detection of Mycobacterium tuberculosis in respiratory specimens

A ligase chain reaction DNA amplification method for direct detection of Mycobacterium tuberculosis (Abbott LCx MTB) in respiratory specimens was evaluated. Results from LCx MTB Assay were compared with those from acid fast bacilli smear, culture, and final clinical diagnosis for each patient. A total of 297 respiratory specimens (sputum and bronchial lavage) from 193 patients were tested. The sensitivity, specificity, positive predictive value and negative predictive value of LCx vs culture were 92.7%, 93%, 67.8% and 98.7%, respectively. When compared to the clinical final diagnosis, the sensitivity, specificity, PPV and NPV for LCx were 88.9%, 96.8%, 86.5% and 97.4%, respectively. The sensitivity of LCx MTB assay was 75% for smear-negative, culture positive samples. The results indicate that LCx MTB assay is a rapid, simple and valuable technique as a complementary tool for the diagnosis of tuberculosis.

Analysis of the direct and indirect costs of treatment of imported malaria in the Slovak Republic

This study analyzed the approximate cost of treatment of patients hospitalized with a diagnosis of imported malaria in Slovakia. Between 2003 and 2007, 15 patients with imported malaria were hospitalized. The mean direct cost of the treatment was 970.75 euros and the mean indirect cost was 53.15 euros. For the patient with the highest cost of treatment, the use of mefloquine prophylaxis would have represented only 0.5% of the total direct cost of treating the disease. Despite the partial resistance of plasmodia, malaria chemoprophylaxis is unequivocally a cheaper choice than subsequent treatment of malaria.

Production of anti-Cryptosporidium polyclonal antibodies and standardization of direct immunofluorescence for detecting oocysts in water

INTRODUCTION: The production of anti-Cryptosporidium polyclonal antibodies and its use in direct immunofluorescence assays to determine the presence of Cryptosporidium in water are described in the present work. METHODS: Two rabbits were immunized with soluble and particulate antigens from purified Cryptosporidium oocysts. The sera produced were prepared for immunoglobulin G extraction, which were then purified and conjugated with fluorescein isothiocyanate (FITC). Slides containing known amounts of oocysts were prepared to determine the sensitivity of the technique. To test the specificity, slides containing Giardia duodenalis cysts were prepared. RESULTS: The conjugate was successfully used in water samples experimentally contaminated with Cryptosporidium oocysts, and it was possible to detect up to five oocysts/spot, corresponding to contamination of 250 oocysts/mL. CONCLUSIONS: The three immunizations performed in the rabbits were enough to produce antibodies against Cryptosporidium, the standard direct immunofluorescence assay permitted the detection of five oocysts in 20% of the samples, and no cross-reaction with Giardia duodenalis cysts occurred.

Comparison of recombinant A2-ELISA with rKE16 dipstick and direct agglutination tests for diagnosis of visceral leishmaniasis in dogs in Northwestern Iran

INTRODUCTION: Various methods are used for the diagnosis of visceral leishmaniasis (VL), such as microscopic examination, culture and inoculation of laboratory animals; however, serological assays are commonly used for the detection of antibodies in serum samples with a wide range of specificity and sensitivity. METHODS: The purpose of this study was to compare three serological methods, including rA2-ELISA, the recombinant KE16 (rKE16) dipstick test and the direct agglutination test (DAT), for the detection of antibodies against VL antigens. The assays utilized 350 statistically based random serum samples from domestic dogs with clinical symptoms as well as samples from asymptomatic and healthy dogs from rural and urban areas of the Meshkinshahr district, northwestern Iran. RESULTS: Samples were assessed, and the following positive rates were obtained: 11.5% by rKE16, 26.9% by DAT and 49.8% by ELISA. The sensitivity among symptomatic dogs was 32.4% with rKE16, 100% with DAT and 52.9% with ELISA. Conversely, rA2-ELISA was less specific for asymptomatic dogs, at 46.5%, compared with DAT, at 88.9%. CONCLUSIONS : This study recommends rA2-ELISA as a parallel assay combined with DAT to detect VL infection among dogs. Further evaluations should be performed to develop an inexpensive and reliable serologic test for the detection of Leishmania infantum among infected dogs.

Performance of direct immunofluorescence assay for the detection of human metapneumovirus under clinical laboratory settings

ABSTRACT INTRODUCTION: Human metapneumovirus (hMPV) is an emergent human respiratory pathogen. This study aimed to evaluate the performance of direct immunofluorescence (DIF) to detect hMPV in a clinical laboratory setting. METHODS: Nasopharyngeal aspirate samples (448) of children and adults with respiratory illness were used to detect hMPV by using DIF and real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays. RESULTS: In all, 36 (8%) samples were positive by DIF and 94 (21%) were positive by qRT-PCR. Direct immunofluorescence specificity was 99% and sensitivity was 38%. CONCLUSIONS: DIF is not very sensitive under clinical laboratory settings.

Mycetoma in an HIV-infected patient

Although oportunistic fungal infections occur commonly in immunocompromised hosts, mycetoma has never been reported in association with HIV infection. The authors present a case that to their knowledge is the first reported case of mycetoma associated with HIV infection. Diagnosis was confirmed by direct examination of grains and histologic examination. Precise identification of the agent, an actinomycete, was not possible. The unusual site of infection may probably be related to the use of contaminated needless and sirynges for HIV drug injection.

Quantitative analysis of collagen and elastic fibers in the transversalis fascia in direct and indirect inguinal hernia

PURPOSE: Our previous studies demonstrated structural and quantitative age-related changes of the elastic fibers in transversalis fascia, which may play a role in inguinal hernia formation. To verify whether there were differences in the extracellular matrix between direct and indirect inguinal hernia, we studied the amount of collagen and elastic fibers in the transversalis fascia of 36 male patients with indirect inguinal hernia and 21 with direct inguinal hernia. MATERIAL AND METHODS: Transversalis fascia fragments were obtained during surgical intervention and underwent histological quantitative analysis of collagen by colorimetry and analysis of elastic fibers by histomorphometry. RESULTS: We demonstrated significantly lower amounts of collagen and higher amounts of elastic fibers in transversalis fascia from patients with direct inguinal hernia compared to indirect inguinal hernia patients. The transversalis fascia from direct inguinal hernia patients showed structural changes of the mature and elaunin elastic fibers, which are responsible for elasticity, and lower density of oxytalan elastic fibers, which are responsible for resistance. These changes promoted loss of resiliency of the transversalis fascia. CONCLUSION: These results improve our understanding of the participation of the extracellular matrix in the genesis of direct inguinal hernia, suggesting a relationship with genetic defects of the elastic fiber and collagen synthesis.

Sperm tail flexibility test: a simple test for selecting viable spermatozoa for intracytoplasmic sperm injection from semen samples without motile spermatozoa

PURPOSE: The objective was to describe the results of the injection of immotile spermatozoa with flexible tails when only immotile spermatozoa are present in the semen sample. METHODS: A retrospective study was conducted to analyze the procedure results for 10 couples who participated in our intracytoplasmic sperm injection program. The sperm tail was considered flexible when it moved up and down independently of the head movement, and it was considered inflexible when the movement occurred together (tail plus head). The fertilization and pregnancy rate were analyzed. RESULTS: The normal fertilization rate (presence of 2 pronuclei) was 30.3% (40/132), and the abnormal fertilization rate (presence of less than or more than 2 pronuclei) was 6.81% (9/132). A total of 52 embryos were obtained with 9 transfer procedures performed (pregnancy rate: 11.12%). CONCLUSIONS: The sperm tail flexibility test (STFT) is an easy and cost-effective way for selecting viable immotile spermatozoa and can be used as an alternative method for determining the viability of spermatozoa. This test seems to be a simple and risk-free method when compared to the swelling test.

Myocardial perfusion by contrast echocardiography. Establishment of normal pattern of intracoronary injection and safety in humans

OBJECTIVE: To establish the normal pattern and safety of echocardiographic contrast in patients with no significant obstruction of epicardial coronary arteries. METHODS: 67 patients with normal coronary arteries or obstructions < 50% were selected from 277 patients who underwent coronary angiography (CA). Mean age was 56 ± 11years and 36 were males. At the end CA, echocardiographic contrast was selectively injected into each coronary artery. The parasternal short axis of the left ventricle (LV) was divided into six segments: anterior (A), antero-lateral (AL), postero-lateral (PL), posterior (P), infero-septal (IS) and antero-septal (AS). Anterolateral (ALPM) and posteromedial papillary muscles (PMPM) were also considered. The pattern and intensity of the appearance of the myocardial contrast was visually analyzed. RESULTS: The right coronary artery (RCA) was dominant in 60 patients. Contrast appearance was sudden and simultaneous in the 3 muscle layers. All segments could be contrasted after the injection in both coronary arteries. 100% of the AS, A and AL segments, 97% of the PL and 98% of the ALPM were perfused by the left coronary artery (LCA). P and IS segments were perfused by the RCA in 85% and 82%, respectively, and by a dominant LCA in 71% of the cases. The PMPM was perfused by a dominant RCA in 77% and by a dominant LCA in 86%. There were no symptoms. CONCLUSION: Intracoronary injection of the sonicated solution is a safe procedure that allows for an excellent opacification of the myocardium and can potentially be used during routine CA.

Experimental Model of Gene Transfection in Healthy Canine Myocardium: Perspectives of Gene Therapy for Ischemic Heart Disease

OBJECTIVE: To assess the transfection of the gene that encodes green fluorescent protein (GFP) through direct intramyocardial injection. METHODS: The pREGFP plasmid vector was used. The EGFP gene was inserted downstream from the constitutive promoter of the Rous sarcoma virus. Five male dogs were used (mean weight 13.5 kg), in which 0.5 mL of saline solution (n=1) or 0.5 mL of plasmid solution containing 0.5 µg of pREGFP/dog (n=4) were injected into the myocardium of the left ventricular lateral wall. The dogs were euthanized 1 week later, and cardiac biopsies were obtained. RESULTS: Fluorescence microscopy showed differences between the cells transfected and not transfected with pREGFP plasmid. Mild fluorescence was observed in the cardiac fibers that received saline solution; however, the myocardial cells transfected with pREGFP had overt EGFP expression. CONCLUSION: Transfection with the EGFP gene in healthy canine myocardium was effective. The reproduction of this efficacy using vascular endothelial growth factor (VEGF) instead of EGFP aims at developing gene therapy for ischemic heart disease.

Influence of angiotensin II receptor subtypes of the paraventricular nucleus on the physiological responses induced by angiotensin II injection into the medial septal area

OBJECTIVE: We determined the effects of losartan and PD 123319 (antagonists of the AT1 and AT2 angiotensin receptors, respectively), and [Sar¹, Ala8] ANG II (a relatively peptide antagonist of angiotensin receptors) injected into the paraventricular nucleus (PVN) on water and 3% NaCl intake, and the diuretic, natriuretic, and pressor effects induced by administration of angiotensin II (ANG II) into the medial septal area (MSA) of conscious rats. METHODS: Holtzman rats were used . Animals were anesthetized with tribromoethanol (20 mg) per 100 grams of body weight, ip. A stainless steel guide cannula was implanted into the MSA and PVN. All drugs were injected in 0.5-mul volumes for 10-15 seconds. Seven days after brain surgery, water and 3% NaCl intake, urine and sodium excretion, and arterial blood pressure were measured. RESULTS: Losartan (40 nmol) and [Sar¹, Ala8] ANG II (40 nmol) completely eliminated whereas PD 123319 (40 nmol) partially blocked the increase in water and sodium intake and the increase in arterial blood pressure induced by ANG II (10 nmol) injected into the MSA. The PVN administration of PD 123319 and [Sar¹, Ala8] ANG II blocked whereas losartan attenuated the diuresis and natriuresis induced by MSA administration of ANG II. CONCLUSION: MSA involvement with PVN on water and sodium homeostasis and arterial pressure modulation utilizing ANGII receptors is suggested.

Contribuição para o conhecimento da intoxicação pelo veneno dos "escorpiões"

Nous avons travaillé à Bello Horizonte, Etat de Minas, avec le venin de 4 espèces de Scorpions: Tityus bahiensis (C. L. KOCK, 1836). Tityus serrulatus (LUTZ-MELLO, 1922). Tityus dorsomaculatus (LUTZ – MELLO, 1922). Bothriurus (espèce em étude), sur un total de 13.640 individus. Nous avons essayé et observe l’action du venin sur 97 espèces differentes d’êtres vivants – depuis les chlamydozoaires jusqu’à l’«Homo sapiens». Nous avons cherché à déterminer une unité toxique «plus précise, plus régulierè». Les étalons dits «unité vésicule», «unité morsure» sont inconstants et sans rigueur. Tout au plus, peuvent ils server à l’étude de l’action générale du venin, et cela meme, dans certains cas seulement. Nous avons employé la pesée pour determiner l’unité toxique. Ce qui est important pour qui étudie ces sujets ce n’est pás lê nombre de vésicules, mais bien la quantité de venin humide ou desséché qu’elles contiennent. La balance, pour notre travail, est um moyen indicateur de bien plus grande précision que la «vésicule» ou la «morsure». Nous sommes parvenus à prouver qu’il existe une relation constante entre le poid brut des vésicules et la quantité de venin humide ou desséché qu’elles contiennent dans leur intérieur. Donc em pesant les vésicules, nous pesons indirectement le venin. Peu nous importe qu’il y ait 10 ou 100 vésicules. Il nous importe seulement de savoir combien elles pèsent, et de déterminer par ce fait, la quantité proportionnelle de vain pur. La technique générale est la suivante: Nous pesons um certain nombre de vésicules. Nous triturons ensuite, dans um mortier stérilisé et nous emulsionnons, par l’addition consécutive d’eau distillée, stérilisée. Nous filtrons l’émulsion sur le papier filtre employé em chimie, préalablement taré et desséché dans une atmosphere de chlorure de calcium. Après le filtrage on sèche à nouveau les papiers filtre employés d'abord à l'étuve et ensuite dans la même atmosphère de chlorure de calcium. Nous pesons plusieurs fois et on obtient la moyenne de ces pesées. On soustrait de cette dernière pesée le taux des substances non venimeuses, glandulaires, également dissoutes et calculées à 23 du poids brut et celles retenues par les papiers,-on obtient ainsi la moyenne réelle du venin pur contenu dans les vésicules utilisèés. Une simple divisiôn suffit pour fixer la moyenne de chacune. Ces données ont été vérifiées par les expériences faites avec du venin pur, largement obtenu dans notre Laboratoire. Nous avons trouvé de la sorte pour une vésicule de Tityus serrulatus: 0,gr.000,386 de T. bahiensis: 0,gr.001.261.24 de venin pur ce qui donne. 7/15,96 pour la 1ère. 1/8,36 pour la 2ème du poids sec de chaque vésicule. Le poids sec, pour une moyenne obtenue de 1.000 vésicules, fut de 0,gr.008,236 pour Tityus bahiensis. Maximum 0,gr.011. Minimum 0,gr.004.4 pour chacun. Pour Tityus serrulatus, en 1.049 vésicules le poids fut de 0,gr.006,08. Maximum 0,gr.014.03. Minimum 0,gr.003,1 pour chacun. C'est pour cette raison que l'unité-vésicule est incertaine. 2 poules A et B.; l'une, A, pesant 2 K.030 gr. reçoit dans une veinè, une émulsion en sèrum physiologique à 8,50/%, stérilisé, de 19 vésicules totales de Tityus serrulatus, et présence de légers phénomènes toxiques. L'autre, B, pesant 2 K.320 gr. meurt avec tous les phénomènes classiques de l'empoisonnement, par l'injection endoveineuse del'émulsion de 16 vésicules totales de venin de Tityus serrulatus! Les premières 19 vésicules pesaient 0,gr.58; les 16 derniéres-84 milligrammes. Les premières contenaient 0,gr.003. 634 et les secondes 0,gr.005.263 de venin pur! La moyenne obtenue de 6346 scorpions, (entre T. bahiensis et T. serrulatus) nous a fourni pour chacun: 0,gr.000,131,53 de venin pur, par piqûre. Si l'on spécifie: Pour 5.197 T. bahiensis. La moyenne pour une piqûre est 0,gr.000.106.15. Pour 1.149 T. serrulatus, la moyenne pour une piqûre est.......0,gr.000.246.30. La quantité a varié, selon les individus, de 0,gr.000.035.71, à 0,gr.000.436.01 de venin pur, pour une piqûre. D'après ce qui vient d'étre dit, on peut voir combien la quantité de venin éjaculé varie, chaque fois, chez les scorpions. L'unité-piqûre ne peut done pas ètre utiliseé pour des expériences dèlicates. Le mieux est de se servir de venin pur, et c'est ce que nous avons fait pour les expériences minutieuses. Quand on n'en possède pas, on peut établir pour chaque série des expériences à tenter-la dose minima mortelle en poids (grammes et fractions) de vésicules. D'après les bases ici consignées, et avec une trés petite erreur, on peut calculer la quantité de venin pur de cette dóse. Ce calcul est d'ailleurs dispensable. On peut s'en rapporter simplement au poids sec des vésicules totales et dire que la D. m. m. est de tant de milligr. secs. Comme le venin se conserve mal dans les vésicules, il faut, dans ce procédé, doser la D. m. m. toutes les fois que l'on veut procéder á une sériê d'expériences. Le venin desséché rappelle, d'après le temps de conservations au Laboratoire, celui de Crotatus terrificus et celui des Lachesis (quand il est vieux). Il est retenu au passage en partie, par les bougies Berkfeld et Chamberland. La conservation en état de dessication est la meilleure. Ainsi gardé, à l'abri de la lumierè, aux approches de 0,gr., pendant 8 mois, il perd à peine 1,2 à 1,4 de sa valeur primitive. L'echauffement à 100 gr. trouble une dissolution de venin dans l'eau distilleé; sans atteindre toutefois son pouvoir toxique, quand on l'injecte par la voie intra-cérébrale. Nous avons fait l'experience par 11 voies diverses. Sur des animaux sensibles, nous n'avons pas obtenu de phénomènes toxiques, apparemment, par les voies suivantes: 1) buccale; 2) gastrique; 3) rectale; 4) chambre oculaire antérieure; 5) cornéenne; 6) trachéenne; 7) meningée {sur; intra; 8) simple contact, bien que direct, avec le systemè nerveux central. La gravité des phènomènes décroît suivant l'échelle ci-dessous: 1) intra-cérébrale...