Browsing by Autor "Michal Svoboda"

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AMNIOTIC FLUID IS NOT USEFUL FOR DIAGNOSIS OF CONGENITAL TRYPANOSOMA CRUZI INFECTION
(American Society of Tropical Medicine and Hygiene, 2006) Myrna Virreira; Sabrina Sales Martínez; Cristina Alonso‐Vega; Faustino Torrico; Marco Solano; Mary Cruz Torrico; Rudy Parrado; Carine Truyens; Yves Carlier; Michal Svoboda
Although Trypanosoma cruzi can be transmitted transplacentally and induce congenital infection, no data are available about the presence of this parasite in human amniotic fluid. We examined 8, 19, and 4 amniotic fluid samples (collected at delivery or by aspiration of gastric content of neonates) from control uninfected mothers (M−B−), infected mothers delivering uninfected newborns (M+B−), and mothers of confirmed congenital cases (M+B+), respectively. Polymerase chain reaction (PCR), using nuclear and kinetoplastic DNA primers (Tcz1-Tcz2 and 121–122), were negative for all control M−B− samples, but positive for 5 of 19 M+B− and 2 of 4 M+B+ samples. To determine the number of parasites in the positive samples, real-time PCR using S35/S36 kinetoplastic DNA was performed. Only one M+B+ sample presented a high parasitic DNA amount, whereas the other six PCR-positive samples displayed traces of T. cruzi DNA. In conclusion, the release of parasites in amniotic fluid is probably a rare event that cannot be helpful for the routine diagnosis of congenital Chagas disease.
COMPARISON OF POLYMERASE CHAIN REACTION METHODS FOR RELIABLE AND EASY DETECTION OF CONGENITAL TRYPANOSOMA CRUZI INFECTION
(American Society of Tropical Medicine and Hygiene, 2003) Myrna Virreira; Faustino Torrico; Carine Truyens; Cristina Alonso‐Vega; Marco Solano; Yves Carlier; Michal Svoboda
The polymerase chain reaction (PCR) is a potentially interesting diagnostic tool for detecting congenital Trypanosoma cruzi infection at birth. We have compared the sensitivity and capacity of a group of T. cruzi PCR primers in detecting the complete spectrum of known T. cruzi lineages, and to improve and simplify the detection of infection in neonatal blood. We found that the two primers, Tcz1/Tcz2 and Diaz1/Diaz2, which target the 195-basepair satellite repeat, detected all parasitic lineages with the same sensitivity. However, the intensity of the amplicon was somewhat higher with Tcz1/Tcz2. For other tested primers (nuclear DNA primers BP1/BP2, O1/O2, Pon1/Pon2, and Tca1/Tca2 and kinetoplast DNA primers S35'/S36' and 121/122), either the intensity of amplicons varied according to T. cruzi lineages or the PCR assay was less sensitive. The use of the Tcz1/Tcz2 primers, which target a tandem repetitive sequence, requires a careful determination of the appropriate amount of Taq polymerase to avoid the formation of smears and multiple amplicon bands. The Tcz1/Tcz2 primers resulted in an intense 200-basepair amplicon with DNA extracted from blood equivalent to 0.02 parasites per assay when used with a simple DNA extraction method and of a low amount of Taq polymerase from a standard PCR kit. To better assess such PCR protocol, we assayed 311 samples of neonatal blood previously tested by parasitologic methods. The reliability of our PCR test was demonstrated, since all the 18 blood samples from newborns with congenital T. cruzi infection were positive, whereas the remaining samples (30 from control newborns of uninfected mothers and 262 of 263 from babies born to infected mothers) were negative. Since our PCR method is simple, reliable, robust, and inexpensive, it appears suitable for the detection of T. cruzi infection in neonatal blood, even in laboratories that are not equipped for performing the PCR.
LIPOLYTIC ENZYME PRODUCTION BY HALOPHILIC/HALOTOLERANT MICROORGANISMS ISOLATED FROM LAGUNA VERDE, BOLIVIA
(2008) Mariana Niño de Guzmán; Virginia A. Vargas; Henry Antezana; Michal Svoboda
Bacterial strains isolated from water samples from Laguna Verde, Potosi-Bolivia and deposited at Biotechnology Center, Cochabamba-Bolivia, were subjected to lipolytic enzyme production studies. Among these, 10 strains were selected as positive in lipolytic enzyme production in solid medium, using olive oil as sole carbon source. Selected strains were subjected to morphological and biochemical studies. Enzyme production in liquid medium identified strain LV01 as the best enzyme producer with 0.079 U/ml lipolytic activity. Partial purification by ion-exchange chromatography of the lipolytic enzyme, produced by strain LV01, presented specific enzyme activity of 0,138 U/mg of eluted protein with approximately 77 KDa size. Partial characterization of the produced enzyme revealed its display maximum activity values at pH 9, 30°C y 1.7M NaCl , thus exhibiting remarkably haloalkalitolerant properties. The 16S rDNA gene sequence analysis showed 99% similarity between strain LV01 and Bacillus pumilus (embAJ494727).
Trypanosoma cruzi: Typing of genotype (sub)lineages in megacolon samples from bolivian patients
(Elsevier BV, 2006) Myrna Virreira; Geovanka Serrano; Luís A. Maldonado; Michal Svoboda