BackgroundThere is large body of evidence that states that invasion of Plasmodium vivax requires the Duffy antigen, but the universality of this specificity is certainly now under question with recent reports showing that in some parts of the world P. vivax infects and causes disease in Duffy-negative people. These findings reinforce the idea that this parasite is rapidly evolving, being able to use other receptors than Duffy to invade the erythrocytes, which may have an enormous impact in P. vivax current distribution. The presence of P. vivax infection in Duffy-negative individuals was investigated in a cross-sectional study conducted in Anajás, Archipelago of Marajó, State of Pará, which is an area of malaria transmission in the Brazilian Amazonia.MethodsDuffy genotyping and Plasmodium species diagnostic assays were performed successfully in 678 individuals. An allele-specific primer polymerase chain reaction (PCR) technique was used for Duffy blood group genotyping. Identification of Plasmodium species was achieved by conventional blood smear light microscopy and a TaqMan-based real-time PCR method to detect mitochondrial genome of Plasmodium falciparum and P. vivax.ResultsPlasmodium spp. infection was detected in 137 samples (20.2%). Prevalence of each Plasmodium species was 13.9% P. vivax, 5.8% P. falciparum, and 0.6% P. vivax plus P. falciparum. Overall, 4.3% (29/678) were genotyped as Duffy-negative (FY*BES/*BES). Among Duffy-negative individuals 6.9% were P. vivax PCR positive and among Duffy-positive 14.2% were P. vivax PCR positive. Although lower, the risk of Duffy-negatives to experience a P. vivax blood stage infection was not significantly different to that of Duffy-positives. Furthermore, the genotypic and allelic frequencies of the Duffy blood group among P. vivax-infected patients and in the control group did not differ significantly, also suggesting no reduction in infection rates among the carriers of FY*BES allele.ConclusionsThe data obtained in Anajás showed no differential resistance vivax malaria among Duffy-negative and Duffy-positive individuals. This result needs additional confirmation through a deeper evaluation in a larger sample of patients with P. vivax malaria and molecular parasite characterization. Nonetheless, this genetic profile of the parasite may be contributing to the high incidence of malaria in the municipality.

We sequenced the oldest bla KPC-2 -bearing plasmid isolated in Brazil and another plasmid also carried by a Klebsiella pneumoniae strain of sequence type 442 (ST442) , Even though the first description of KPC-producing Klebsiella pneumoniae in Brazil was published in 2009, describing a strain isolated from a clinical sample in 2006 (1), a later publication reported the presence of a KPC-bearing strain isolated in May 2005 (2). The latter strain, FCF1305, was isolated in the state of São Paulo, Brazil, from a blood culture and belongs to sequence type 442 (ST442). We completely sequenced the bla KPC-2 -bearing plasmid isolated from FCF1305 and also another bla KPC-2 -bearing plasmid isolated from another K. pneumoniae strain, FCF3SP, also belonging to ST442 and also isolated from a blood culture from a patient in the state of São Paulo, but 52 months later, in September 2009. We chose to sequence the bla KPC-2 -bearing plasmids of these strains because FCF1305 was the oldest strain-bearing KPC in Brazil and strain FCF3SP was the strain most geographically, clinically, and epidemiologically similar to FCF1305 that we could find, but isolated 52 months later, so that differences accumulated in the wild during this period could be evaluated.Because strain FCF1305 bore three plasmids, whereas strain FCF3SP bore five, isolation of the bla KPC-2 -bearing plasmids of each strain was carried out by transformation into Escherichia coli TOP10 (Invitrogen) as described previously (3). Plasmid DNA isolated from E. coli TOP10(pFCF1305) and E. coli TOP10 (pFCF3SP) by alkaline lysis miniprep (3) was used to construct genomic fragment libraries (one for each plasmid) which were sequenced using the Roche GS-FLX sequencer (Roche Life Sciences, Branford, CT, USA) according to the manufacturer's instructions.Sequencing adapters were clipped from the reads and used for de novo assembly using Mira 4.0 (4). Plasmid de novo contigs were then merged by assembling de novo using Geneious (5) with stringent parameters (maximum gap size ϭ 1; minimum overlap identity ϭ 95%). Circular (with large overlapping ends) contigs of ϳ50 kbp were considered to be the plasmids. All other contigs generated which had no identity to the circular contigs were then subjected to a BLAST search (6) against GenBank's nonredundant (nr) database, and 100% of them had as top hits Enterobacteriaceae chromosomes or phages and so were discarded. The plasmid sequences were automatically annotated using the RAST server (7) and manually curated. Repeat regions were identified using UGENE (8) and insertion sequences were identified by online analysis using IS finder (9). The complete sequences of both plasmids were successfully obtained by de novo assembly yielding a 53,081-bp sequence for pFCF1305 (mean coverage of 261ϫ Ϯ 48ϫ; minimum coverage of 39ϫ; maximum coverage of 445ϫ) and a 54,605-bp sequence for pFCF3SP (mean coverage of 1,013ϫ Ϯ 374ϫ; minimum coverage of 67ϫ; maximum coverage of 2,383ϫ).Both plasmids were found to have IncN plasmid backbones and carry a versi...

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