No abstract

Possible drug resistance in Mycobacterium leprae strains from Venezuela and three other South American countries was surveyed by molecular methods. None of the 230 strains from new leprosy cases exhibited drug resistance-associated mutations. However, two of the three strains from relapsed cases contained dapsone resistance mutations, and one strain also harbored a rifampin resistance mutation. Single nucleotide polymorphism analysis of these strains revealed five subtypes: 3I (73.8%), 4P (11.6%), 1D (6.9%), 4N (6%), and 4O (1.7%).The success of leprosy control programs relies heavily upon multidrug therapy (MDT) (rifampin, dapsone, and clofazimine). Therefore, it is important that drug resistance trends be monitored periodically and Mycobacterium leprae strains genotyped in order to understand transmission patterns and genetic diversity. In the present study, strains of M. leprae from Bolivia, Brazil, Uruguay, and Venezuela were genotyped using single nucleotide polymorphism (SNP) analysis (17, 18), and their drug resistance-determining regions (DRDRs) in the rpoB, folP1, and gyrA genes were sequenced. In parallel, a real-time PCR assay that targets the most frequent sites of drug resistance in rpoB and folP1 was established.A total of 233 M. leprae strains were obtained from patient biopsy specimens or from slit skin smears; the majority of the patients were from Venezuela (n ϭ 197), and the remainder were from Bolivia (n ϭ 10), Brazil (n ϭ 24), and Uruguay (n ϭ 2). Reference DNA from drug-susceptible strains TN, Br4923, NHDP63, and Thai53 was used as a control for the sequencing and the real-time PCR-based TaqMan assay. A known rifampinresistant strain, 85054 (French West Indies), with an rpoB mutation (Ser425Leu) (8), was included as a positive control. This strain also has a dapsone resistance mutation (Pro55Leu) in folP1.DNA extraction from skin biopsy specimens/slit skin samples was carried out by the "freeze-boiling" method as described previously (24), followed by PCR amplification and DNA sequencing (18). Primers used for analyzing the drug resistance-associated loci are shown in Table 1. The sequences, obtained from an ABI 3130xl genetic analyzer (Applied Biosystems, Life Technologies Corporation, CA), were aligned against the sequences of the TN reference strain using CodonCode Aligner software (Dedham, MA) to identify polymorphisms. SNP genotyping was done as described previously (17, 18). The frequency of SNP7614 (presence of "T" at this position in subtype 3I strains) (18) was also determined.TaqMan probe assays using real-time PCR were performed in triplicate. Each 20-l reaction mixture contained 100 nM probe, 200 nM primers (Table 2) in 1ϫ TaqMan PCR master mix (Applied Biosystems, CA), and 2 l of DNA. PCR mixtures were subjected to thermal cycling for 2 min at 50°C and 10 min at 95°C followed by 40 cycles of 15 s at 95°C and 1 min at 60°C with a 7900HT Fast real-time PCR system (Applied Biosystems, CA). An increase in fluorescence indicates perfect complementarity between template and probe. ...

To test whether there are differences between living lineages of domestic guinea pigs Cavia porcellus, we studied 118 specimens from six breeds collected along six Andean countries as well as 15 from the wild cavy species (Cavia tschudii). The mean weight and body length of 15 adult wild cavies (295 AE 31 g, 242 AE 8.3 mm) were significantly smaller than 25 creole guinea pigs from Bolivia and Chile (639 AE 157 g, 287 AE 23.7 mm, respectively). Eighteen laboratory/pet guinea pigs (including the English Pirbright breed) were also smaller (900 AE 173 g, 308 AE 21 mm) than 25 improved ones from Peru (Tamborada breed, 1241 AE 75.4 g, 317 AE 12 mm) and Ecuador (Auqui breed, 1138 AE 65.5 g, 307 AE 8 mm). Similar size increases appeared in the first axis of a principal component analysis of six skeletal measurements, recovering 84% of total variation. Phylogenetic and haplotype analyses of complete cytochrome b gene sequences consistently joined all 22 domestic individuals (13 shared unambiguous substitutions, 100% bootstrap in 1000 replicates), probably from a single first ancient domestication in the western Andes. Six laboratory/ pet sequences were also joined within a common branch (six shared substitutions, 96% bootstrap), probably from a documented European second phase. By contrast, those from improved Auqui joined a northern creole subgroup (one shared substitution, 84% bootstrap), and those from Nativa and improved Tamborada clustered together and with a southern creole subgroup (four shared substitutions, 86% bootstrap); this suggests at least two independent modern events during a more complex third phase, producing two improved guinea pigs selected for size and meat. Cavia tschudii sequences showed some unexpected geographic variation.

The aim of this study was to determine the prevalence of Ser315Thr substitution in isoniazid (INH)-resistant strains of Mycobacterium tuberculosis in Uruguay. The katG gene of 62 INH-resistant strains was analysed by an RFLP-PCR assay. PCR products were digested with MspI to detect Ser315Thr and Arg463Leu substitutions. A total of 16 of the 62 (26 %) INH-resistant strains analysed had a Ser315Thr substitution. Only one INH-resistant strain had an Arg463Leu substitution and two strains had a deletion in katG. Of the 16 strains with Ser315Thr, 15 showed different profiles using a double-repetitive-element PCR assay, demonstrating that there was no local dissemination of any particular strain. These findings are in agreement with published data from regions where the prevalence of tuberculosis (TB) is intermediate and may be due in part to the success of the local TB control programme.

A case of cavitary pulmonary disease caused by Mycobacterium heckeshornense in Uruguay is described. This is the first case reported in the Latin America and Caribbean region, showing that this species is a worldwide opportunistic human pathogen.