Biochemical characterization of new strains of<Emphasis Type="Italic"> Trypanosoma cruzi</Emphasis> and<Emphasis Type="Italic"> T. rangeli</Emphasis> isolates from Peru and Mexico

Seven trypanosome stocks isolated have been characterized by lectin agglutination, isoenzyme analysis, and the end products excreted. The stocks were isolated from different geographic areas—one from Mexico (TM5), and six from Peru, four of these isolated from different species of triatoma (TP504, TP702, TP704 and TP706), the other two isolated from the salivary glands of Rhodnius ecuadorensis (TRa605 and TRa606). Additionally, one strain of Trypanosoma cruzi isolated from a human case (strain TC-Maracay) and one strain of T. rangeli (TRa, Cajamarca-Peru strain), characterized and maintained in our laboratory, were used as reference strains. According to statistical study, the stocks were grouped into three clusters: (1) cluster I included the reference strain of T. cruzi (TC-Maracay); (2) cluster II was subdivided into two groups—subcluster IIA for the Mexican isolate (TM5) and subcluster IIB for the Peruvian ones, isolated from the salivary glands of Rhodnius ecuadorensis (TRa 605 and TRa 606) and the reference strain T. rangeli (TRa); these two new isolates were classified as T. rangeli; and (3) cluster III for the rest of the Peruvian isolates, which should be considered at least as a different strain from the T. cruzi strain Maracay. We show that the identification of T. cruzi and T. rangeli in mixed infections is readily achieved by biochemical methods. These findings identified three clusters of Mexican and Peruvian stocks that correlate with geographic origin, although assignment to a T. cruzi linage was not possible.     

Multiclonal Leishmania braziliensis population structure and its clinical implication in a region of endemicity for American tegumentary leishmaniasis

In Corte de Pedra (CP), northeastern Brazil, Leishmania braziliensis causes three distinct forms of American tegumentary leishmaniasis (ATL). To test the hypothesis that strain polymorphism may be involved in this disease spectrum and accurately characterize the parasite population structure in CP, we compared one L. major, two non-CP L. braziliensis, one CP L. amazonensis, and 45 CP L. braziliensis isolates, obtained over a 10-year period from localized cutaneous, mucosal, and disseminated leishmaniasis patients, with randomly amplified polymorphic DNA (RAPD). Electrophoretic profiles were mostly unique across species. All typing protocols revealed polymorphism among the 45 CP L. braziliensis isolates, which displayed eight different RAPD patterns and greater than 80% overall fingerprint identity, attesting to the adequacy of the tools to assess strain variability in CP's geographically limited population of parasites. The dendrogram based on the sum of RAPD profiles of each isolate unveiled nine discrete typing units clustered into five clades. Global positioning showed extensive overlap of these clades in CP, precluding geographic sequestration as the mechanism of the observed structuralization. Finally, all forms of ATL presented a statistically significant difference in their frequencies among the clades, suggesting that L. braziliensis genotypes may be accompanied by specific disease manifestation after infection.     

Deposition and passage of transthyretin through the blood-nerve barrier in recipients of familial amyloid polyneuropathy livers

Familial amyloid polyneuropathy (FAP) is characterized by deposition of mutated transthyretin (TTR) in the peripheral nervous system. Prior to amyloid fibrils, nonfibrillar TTR aggregates are deposited inducing oxidative stress with increased nitration (3-NT). As the major source of TTR is the liver, liver transplantation (LT) is used to halt FAP. Given the shortage of liver donors, domino LT (DLT) using FAP livers is performed. The correlation between TTR deposition in the skin and nerve was tested in biopsies from normal individuals, asymptomatic carriers (FAP 0) and FAP patients; in FAP 0, nonfibrillar TTR was observed both in the skin and nerve in the same individuals; in patients, amyloid was detected in both tissues. The occurrence of amyloidosis in recipients of FAP livers was evaluated 1-7 years after DLT: TTR deposition occurred in the skin 3 years after transplantation either as amyloid or aggregates; in one of the recipients, fibrillar TTR was present in the epineurium 6 years after DLT. Deposits were scarce and 3-NT immunostaining was irrelevant. Nerve biopsies from DLT recipients had no FAP-related neuropathy. Our findings suggest that TTR amyloid formation occurs faster than predicted and that TTR of liver origin can cross the blood-nerve barrier. Recipients of FAP livers should be under surveillance for TTR deposition and tissue damage.     

Sporogony and experimental transmission of Babesia equi by Boophilus microplus

The development of Babesia equi in salivary glands of adult female Boophilus microplus was observed under a light microscope using semithin sections stained with toluidine blue. Engorged nymphs were obtained from splenectomized foals experimentally infected with B. equi. As adults, they were then fed on rabbits for 5 days and the salivary glands of manually collected individuals were removed at intervals of 24 h. Sporozoites were found in type III granular acini cells between the 2nd and 5th days following feeding on the rabbits. Sporoblasts and sporozoites were observed in the same or adjacent acini cells in all the glands examined. The formation of the sporozoites occurred following the multiple division of the sporoblasts through a process of radial budding from the periphery of bodies resulting from multiple fission. Sporozoites were detected in smears of adult males stained with Giemsa, between the 2nd and 5th days following feeding by the ticks. Adults of B. microplus, fed during the nymphal phase on foals with patent parasitemia, transmitted sporozoites of B. equi to a splenectomized foal. The role of B. microplus in the transmission and epidemiology of B. equi is discussed. Received: 16 June 1997 / Accepted: 2 September 1997     

Trends in drug resistance, serotypes, and molecular types of Streptococcus pneumoniae colonizing preschool-age children attending day care centers in Lisbon, Portugal: a summary of 4 years of annual surveillance

Of the nasopharyngeal cultures recovered from 942 day care center (DCC) attendees in Lisbon, Portugal, 591 (62%) yielded Streptococcus pneumoniae during a surveillance performed in February and March of 1999. Forty percent of the isolates were resistant to one or more antimicrobial agents. In particular, 2% were penicillin resistant and 20% had intermediate penicillin resistance. Multidrug resistance to macrolides, lincosamides, and tetracycline was the most frequent antibiotype (17% of all isolates). Serotyping and molecular typing by pulsed-field gel electrophoresis were performed for 202 out of 237 drug-resistant pneumococci (DRPn). The most frequent serotypes were 6B (26%), 14 (22%), 19F (16%), 23F (10%), and nontypeable (12%). The majority (67%) of the DRPn strains were representatives of nine international clones included in the Pneumococcal Molecular Epidemiology Network; eight of them had been detected in previous studies. Fourteen novel clones were identified, corresponding to 26% of the DRPn strains. The remaining 7% of the strains were local clones detected in our previous studies. Comparison with studies conducted since 1996 in Portuguese DCCs identified several trends: (i) the rate of DRPn frequency has fluctuated between 40 and 50%; (ii) the serotypes most frequently recovered have remained the same; (iii) nontypeable strains appear to be increasing in frequency; and (iv) a clone of serotype 33F emerged in 1999. Together, our observations highlight that the nasopharynxes of children in DCCs are a melting pot of successful DRPn clones that are important to study and monitor if we aim to gain a better understanding on the epidemiology of this pathogen.     

New studies on the Macushi Indians of northern Brazil

Demographic data and genetic information concerning 40 genetic systems are reported for three populations of Macushi Indians, and have been compared to those already obtained for three other communities of this tribe. These are young populations (mean age, 19 years), with a low sex ratio (90), low percentages of non-Indian ancestry (1-2%) and of marriages between locally born persons (34). Intertribal unions (14%) are less frequent than among their neighbours, the Wapishana. Fertility is high (average of 8.2 children per woman who completed reproduction), but the variance in family size and the frequency of premature deaths relatively low for populations at this cultural level. This conditions the lowest Index of Opportunity for Selection (0.45) calculated thus far among South American Indians. No variation was observed in 20 genetic systems, limited variation in 3, and larger variability in the remaining 17. In 13 of the 29 comparisons (45%), the Macushi gene frequencies present values in the middle third of the range observed among South American Indians. The previously reported private genetic polymorphism of esterase A was encountered in one of the three villages. A comparison of the genetic distances between villages with and without this polymorphism, and a similar comparison for the villages of the neighbouring Wapishana, yields no clue as to the tribe in which this polymorphism originated.     

DNA damage in cytologically normal urothelial cells of patients with a history of urothelial cell carcinoma

In order to determine if patients with a history of previous urothelial cell carcinoma (UCC) but with current normal urinary cytology have DNA damage in urothelial cells, the single-cell gel electrophoresis (comet) assay was conducted with cells obtained by urinary bladder washings from 44 patients (28 with a history of previous UCC). Increased DNA damage was observed in cytologically "normal" urothelial cells of patients with a history of UCC when compared with referents with no similar history and after correcting the data for smoking status and age (P < 0.018). Increased DNA damage also correlated with the highest tumor grade, irrespective of time or course of the disease after clinical intervention (Kendall tau correlation, 0.37, P = 0.016). Moreover, aneuploidy, as assessed by DNA content ratio (DCR; 75th/25th percentile of total DNA fluorescence of 50 comets/patient) was unaltered by smoking status, but increased with UCC grade: 1.39 +/- 0.12 (median +/- 95% confidence interval; referents); 1.43 +/- 0.11 (Grade I UCC; P = 0.264, against referents); 1.49 +/- 0.16 (Grade II UCC; P = 0.057); 1.57 +/- 0.16 (Grade III UCC; P = 0.003). Micronucleated urothelial cells (MNC) were also scored on Giemsa-stained routine cytological smears and were found not to correlate with DNA damage or DCR. MNC frequencies were higher for patients with a history of UCC and/or smoking than referents with neither history, but there was no statistical difference between groups. Taken together, these results suggest that the normal-appearing urothelium of patients resected for UCC still harbor genetically unstable cells.     

Vicker's hardness and Raman spectroscopy evaluation of a dental composite cured by an argon laser and a halogen lamp

We present the results of the Vicker's hardness test and the use of near-infrared Raman spectroscopy (RS) to measure in vitro the degree of conversion (DC) of a bis(phenol)-A-glycidyl-dimethacrylate-based composite resin, photoactivated by both a halogen lamp (power density=478 mW/cm(2); 8-mm diameter spot) and an argon laser (power density=625 mW/cm(2); 7-mm diameter spot). The degree of conversion was estimated by analyzing the relative intensities between the aromatic C=C stretching Raman mode at 1610 cm(-1) and the methacrylate C=C stretching Raman mode (1640 cm(-1)) on top and bottom surfaces. For the hardness evaluation, the samples were embedded in polyester resin and three indentations with a 50-g load for 10 s were made on the top surface. The higher relative DC values achieved by the photoactivation of a composite resin by the argon laser suggest a better biocompatibility in the bottom surface. The correlation test showed that the higher Vicker's hardness number (VHN) values were associated with higher DC values. The derivative analysis showed a greater curing rate from 5 to 20 s of exposure. The comparison of VHN and DC values with both light sources at each curing time showed that a small change in conversion is related to a large change in hardness. Raman spectroscopy is more sensitive to changes in the first stages of curing reaction than later ones, and the Vicker's hardness assay is more sensitive to changes in the last stages.     

Wiedemann‐Steiner syndrome in two patients from Portugal

Wiedemann‐Steiner syndrome (WSS) is a rare genetic disorder characterized by growth retardation, facial dysmorphism, hypertrichosis cubiti and neurodevelopment delay. It is caused by pathogenic variants in the KMT2A gene. This report describes two unrelated Portuguese patients, age 11 and 17 years, with a phenotype concordant with WSS and clinical and molecular diagnosis of WSS by the identification of two novel frameshift variants in the KMT2A gene. This work also highlights the presence of certain clinical features in patients with growth retardation and development delay and should draw attention to the diagnosis of WSS, when hirsutism, particularly hypertrichosis cubiti is present.