A Two-Year Surveillance in Five Colombian Tertiary Care Hospitals Reveals High Frequency of Non-CG258 Clones of Carbapenem-Resistant Klebsiella pneumoniae with Distinct Clinical Characteristics.

The global spread of carbapenem-resistant Klebsiella pneumoniae (CR-Kp) has been largely associated with sequence type 258 (ST258) and its related variants (clonal group 258 [CG258]). Here we describe the molecular epidemiology of CR-Kp from five tertiary care hospitals in Medellín, the second largest city in Colombia. All CR-Kp-infected patients admitted from June 2012 to June 2014 were included (n = 193). Patients' clinical information was obtained from medical records. Carbapenemase KPC, VIM, IMP, NDM, and OXA-48 genes were detected by PCR. A CG258-tonB79 cluster-specific real-time PCR (targeting the multilocus sequence type [MLST] tonB79 allele), pulsed-field gel electrophoresis (PFGE), and MLST analysis were performed for typing. Remarkably, 62.2% (n = 120) of isolates were from STs unrelated to CG258 (non-CG258). KPC-3 predominated in CG258 isolates (86.3%), while KPC-2 prevailed in non-CG258 isolates (75.5%) (P < 0.001). Multidrug resistance (MDR) frequency was significantly higher in CG258 strains (91.4% versus 56.1%; P < 0.001). ST512 (a single-locus variant of ST258) is the main ST in CG258 (96.3%), and isolates in this group showed closely related pulsotype and similar resistance gene profiles, suggesting the clonal spread of this strain. In contrast, high heterogeneity of STs (34/54), including eight novel STs, was found in non-CG258 isolates. Among non-CG258 isolates, ST14 (13.3%; n = 16) and ST307 (14.2%; n = 17) were the most frequent, and they showed distinct molecular and clinical characteristics in comparison to CG258 isolates. Our results suggest that the dissemination of carbapenem resistance in Medellín is due to heterogeneous K. pneumoniae clones, likely the result of horizontal transmission of KPC in different unrelated lineages, further highlighting the challenge in CR-Kp infection control and the need for a multifocal intervention.

Similar frequencies of Pseudomonas aeruginosa isolates producing KPC and VIM carbapenemases in diverse genetic clones at tertiary-care hospitals in Medellín, Colombia.

Carbapenem-resistant Pseudomonas aeruginosa has become a serious health threat worldwide due to the limited options available for its treatment. Understanding its epidemiology contributes to the control of antibiotic resistance. The aim of this study was to describe the clinical and molecular characteristics of infections caused by carbapenem-resistant P. aeruginosa isolates in five tertiary-care hospitals in Medellín, Colombia. A cross-sectional study was conducted in five tertiary-care hospitals from June 2012 to March 2014. All hospitalized patients infected by carbapenem-resistant P. aeruginosa were included. Clinical information was obtained from medical records. Molecular analyses included PCR for detection of bla(VIM), bla(IMP), bla(NDM), bla(OXA-48), and bla(KPC) genes plus pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) for molecular typing. A total of 235 patients were enrolled: 91.1% of them were adults (n = 214), 88.1% (n = 207) had prior antibiotic use, and 14.9% (n = 35) had urinary tract infections. The bla(VIM-2) and bla(KPC-2) genes were detected in 13.6% (n = 32) and 11.5% (n = 27), respectively, of all isolates. Two isolates harbored both genes simultaneously. For KPC-producing isolates, PFGE revealed closely related strains within each hospital, and sequence types (STs) ST362 and ST235 and two new STs were found by MLST. With PFGE, VIM-producing isolates appeared highly diverse, and MLST revealed ST111 in four hospitals and five new STs. These results show that KPC-producing P. aeruginosa is currently disseminating rapidly and occurring at a frequency similar to that of VIM-producing P. aeruginosa isolates (approximately 1:1 ratio) in Medellín, Colombia. Diverse genetic backgrounds among resistant strains suggest an excessive antibiotic pressure resulting in the selection of resistant strains.

Colombian Anopheles triannulatus (Diptera: Culicidae) Naturally Infected with Plasmodium spp.

The role of Anopheles triannulatus as a local vector has not yet been defined for malaria-endemic regions of Colombia. Therefore, the aim of this work was to detect An. triannulatus naturally infected with Plasmodium spp., as an approximation to determining its importance as malaria vector in the country. A total of 510 An. triannulatus were collected in six malaria-endemic localities of NW and SE Colombia from January 2009 to March 2011. In the NW, two specimens were naturally infected; one with Plasmodium vivax VK247, collected biting on humans and the other with Plasmodium falciparum, collected resting on cattle. In the SE, two specimens were positive for P. falciparum. Although these results show An. triannulatus naturally infected with Plasmodium, further studies are recommended to demonstrate the epidemiological importance of this species in malaria-endemic regions of Colombia.

Evaluation of a PCR-RFLP-ITS2 assay for discrimination of Anopheles species in northern and western Colombia.

Anopheles mosquitoes are routinely identified using morphological characters of the female that often lead to misidentification due to interspecies similarity and intraspecies variability. The aim of this work was to evaluate the applicability of a previously developed PCR-RFLP-ITS2 assay for accurate discrimination of anophelines in twelve localities spanning three Colombian malaria epidemiological regions: Atlantic Coast, Pacific Coast, and Uraba-Bajo Cauca-Alto Sinu region. The evaluation of the stability of the PCR-RFLP patterns is required since variability of the ITS2 has been documented and may produce discrepancies in the patterns previously reported. The assay was used to evaluate species assignation of 939 mosquitoes identified by morphology. Strong agreement between the morphological and molecular identification was found for species Anopheles albimanus, Anopheles aquasalis, Anopheles darlingi and Anopheles triannulatus s.l. (p≥0.05, kappa=1). However, disagreement was found for species Anopheles nuneztovari s.l., Anopheles neomaculipalpus, Anopheles apicimacula and Anopheles punctimacula (p≤0.05; kappa ranging from 0.33 to 0.80). The ITS2-PCR-RFLP assay proved valuable for discriminating anopheline species of northern and western Colombia, especially those with overlapping morphology in the Oswaldoi Group.

Population structure analyses and demographic history of the malaria vector Anopheles albimanus from the Caribbean and the Pacific regions of Colombia.

BACKGROUND: Anopheles albimanus is an important malaria vector in some areas throughout its distribution in the Caribbean and the Pacific regions of Colombia, covering three biogeographic zones of the neotropical region, Maracaibo, Magdalena and Chocó. METHODS: This study was conducted to estimate intra-population genetic diversity, genetic differentiation and demographic history of An. albimanus populations because knowledge of vector population structure is a useful tool to guide malaria control programmes. Analyses were based on mtDNA COI gene sequences and four microsatellite loci of individuals collected in eight populations from the Caribbean and the Pacific regions of Colombia. RESULTS: Two distinctive groups were consistently detected corresponding to COI haplotypes from each region. A star-shaped statistical parsimony network, significant and unimodal mismatch distribution, and significant negative neutrality tests together suggest a past demographic expansion or a selective sweep in An. albimanus from the Caribbean coast approximately 21,994 years ago during the late Pleistocene. Overall moderate to low genetic differentiation was observed between populations within each region. However, a significant level of differentiation among the populations closer to Buenaventura in the Pacific region was observed. The isolation by distance model best explained genetic differentiation among the Caribbean region localities: Los Achiotes, Santa Rosa de Lima and Moñitos, but it could not explain the genetic differentiation observed between Turbo (Magdalena providence), and the Pacific region localities (Nuquí, Buenaventura, Tumaco). The patterns of differentiation in the populations from the different biogeographic provinces could not be entirely attributed to isolation by distance. CONCLUSION: The data provide evidence for limited past gene flow between the Caribbean and the Pacific regions, as estimated by mtDNA sequences and current gene flow patterns among An. albimanus populations as measured by MS loci which may be mainly influenced by semi-permeable natural barriers in each biogeographical region that lead to the genetic differences and effective population sizes detected. The relatively high genetic differentiation in the port city of Buenaventura may be the result of specific ecological conditions, human migration and activities and/or differences in effective population sizes. This knowledge could serve to evaluate and coordinate vector control strategies in these regions of Colombia.

Discrimination of seven Anopheles species from San Pedro de Uraba, Antioquia, Colombia, by polymerase chain reaction-restriction fragment length polymorphism analysis of its sequences.

Accurate identification of anopheline species is essential for vector incrimination and implementation of appropriate control strategies. Several anopheline species are considered important malaria vectors in Colombia; however, species determination is complicated by cryptic morphology and intra-individual variation. We describe polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) of internal transcribed spacer 2 (ITS2) sequences for differentiation of seven Anopheles species collected in a locality in Antioquia, Colombia, with high levels of malaria transmission. Each of these seven species can be identified by unique AluI PCR-RFLP restriction patterns. Comparisons of morphologic identification with molecular identification of voucher specimens confirmed species designation for 886 wild-caught anophelines. This new method can be used as a diagnostic tool for discrimination of anopheline species of medical importance in this region, some of which have overlapping morphologic characters and for conducting complementary studies where rapid and accurate identification of large numbers of specimens is needed.