JC polyomavirus infection in candidates for kidney transplantation living in the Brazilian Amazon Region

This study evaluated the relative occurrences of BK virus (BKV) and JC virus (JCV) infections in patients with chronic kidney disease (CKD). Urine samples were analysed from CKD patients and from 99 patients without CKD as a control. A total of 100 urine samples were analysed from the experimental (CKD patients) group and 99 from the control group. Following DNA extraction, polymerase chain reaction (PCR) was used to amplify a 173 bp region of the gene encoding the T antigen of the BKV and JCV. JCV and BKV infections were differentiated based on the enzymatic digestion of the amplified products using BamHI endonuclease. The results indicated that none of the patients in either group was infected with the BKV, whereas 11.1% (11/99) of the control group subjects and 4% (4/100) of the kidney patients were infected with the JCV. High levels of urea in the excreted urine, low urinary cellularity, reduced bladder washout and a delay in analysing the samples may have contributed to the low prevalence of infection. The results indicate that there is a need to increase the sensitivity of assays used to detect viruses in patients with CDK, especially given that polyomavirus infections, especially BKV, can lead to a loss of kidney function following transplantation.

Ultra high throughput sequencing in human DNA variation detection: a comparative study on the NDUFA3-PRPF31 region.

BACKGROUND: Ultra high throughput sequencing (UHTS) technologies find an important application in targeted resequencing of candidate genes or of genomic intervals from genetic association studies. Despite the extraordinary power of these new methods, they are still rarely used in routine analysis of human genomic variants, in part because of the absence of specific standard procedures. The aim of this work is to provide human molecular geneticists with a tool to evaluate the best UHTS methodology for efficiently detecting DNA changes, from common SNPs to rare mutations. METHODOLOGY/PRINCIPAL FINDINGS: We tested the three most widespread UHTS platforms (Roche/454 GS FLX Titanium, Illumina/Solexa Genome Analyzer II and Applied Biosystems/SOLiD System 3) on a well-studied region of the human genome containing many polymorphisms and a very rare heterozygous mutation located within an intronic repetitive DNA element. We identify the qualities and the limitations of each platform and describe some peculiarities of UHTS in resequencing projects. CONCLUSIONS/SIGNIFICANCE: When appropriate filtering and mapping procedures are applied UHTS technology can be safely and efficiently used as a tool for targeted human DNA variations detection. Unless particular and platform-dependent characteristics are needed for specific projects, the most relevant parameter to consider in mainstream human genome resequencing procedures is the cost per sequenced base-pair associated to each machine.

Prevalence of antibody to hantaviruses in humans and rodents in the Caribbean region of Colombia determined using Araraquara and Maciel virus antigens

We tested sera from 286 agricultural workers and 322 rodents in the department of Córdoba, northeastern Colombia, for antibodies against two hantaviruses. The sera were analysed by indirect ELISA using the lysate of Vero E6 cells infected with Maciel virus (MACV) or the N protein of Araraquara virus (ARAV) as antigens for the detection of antibodies against hantaviruses. Twenty-four human sera were IgG positive using one or both antigens. We detected anti-MACV IgG antibodies in 10 sera (3.5%) and anti-ARAV antibodies in 21 sera (7.34%). Of the 10 samples that were positive for MACV, seven (70%) were cross-reactive with ARAV; seven of the 21 ARAV-positive samples were cross-reactive with MACV. Using an ARAV IgM ELISA, two of the 24 human sera (8.4%) were positive. We captured 322 rodents, including 210 Cricetidae (181 Zygodontomys brevicauda, 28 Oligoryzomys fulvescens and 1 Oecomys trinitatis), six Heteromys anomalus (Heteromyidae), one Proechimys sp. (Echimyidae) and 105 Muridae (34 Rattus rattus and 71 Mus musculus). All rodent sera were negative for both antigens. The 8.4% detection rate of hantavirus antibodies in humans is much higher than previously found in serosurveys in North America, suggesting that rural agricultural workers in northeastern Colombia are frequently exposed to hantaviruses. Our results also indicate that tests conducted with South American hantavirus antigens could have predictive value and could represent a useful alternative for the diagnosis of hantavirus infection in Colombia.

Occult hepatitis B virus infection among injecting drug users in the Central-West Region of Brazil

The prevalence of occult hepatitis B virus (HBV) infection was investigated in 149 hepatitis B surface antigen (HBsAg) negative injecting drug users (IDUs) in the Central-West Region of Brazil. Of these individuals, 19 were positive for HBV DNA, resulting in an occult HBV infection prevalence of 12.7% (19/149); six of these 19 individuals had anti-HBV core and/or anti-HBV surface antibodies and 13 were negative for HBV markers. All IDUs with occult hepatitis B reported sexual and/or parenteral risk behaviours. All HBV DNA-positive samples were successfully genotyped. Genotype D was the most common (17/19), followed by genotype A (2/19). These findings reveal a high prevalence of occult HBV infection and the predominance of genotype D among IDUs in Brazil's Central-West Region.

Seasonal variation and natural infection of Lutzomyia antunesi (Diptera: Psychodidae: Phlebotominae), an endemic species in the Orinoquia region of Colombia

Lutzomyia antunesi has been commonly reported in outbreaks of cutaneous leishmaniasis (CL) in the Orinoquia region of Colombia. The bionomics of this species were studied in the municipality of Villavicencio (Meta, Colombia). Sandflies were captured over the course of one week per month for one year in intradomiciliary, peridomiciliary and extradomiciliary housing areas. The captures were performed from 06:00 pm-06:00 am using CDC light traps and the females were processed for polymerase chain reaction (PCR) to detect Leishmania spp. A total of 22,097 specimens and 19 species were captured of which Lu. antunesi (89%) and Lutzomyia walkeri (5%) were the most abundant. Other species recognised as anthropophilic (Lutzomyia panamensis, Lutzomyia gomezi, Lutzomyia flaviscutellata and Lutzomyia fairtigi) were present in very low abundance (< 2%). Natural infection with Leishmania spp was detected using PCR in Lu. antunesi, Lu. panamensis and Lu. flavicutellata, showing infection rates of 1%, 4.8% and 7.5%, respectively. The present paper provides information on various ecological aspects of Lu. antunesi. An analysis of seasonality shows that this species increases in abundance in the hottest months (December, January and February), directly correlating with the maximum temperature and inversely correlating with precipitation. The natural infection rate is associated with the peaks of highest abundance.

Leishmania infection and host-blood feeding preferences of phlebotomine sandflies and canine leishmaniasis in an endemic European area, the Algarve Region in Portugal

The Algarve Region (AR) in southern Portugal, which is an international tourist destination, has been considered an endemic region of zoonotic leishmaniasis caused by Leishmania infantum since the 1980s. In the present study, phlebotomine and canine surveys were conducted to identify sandfly blood meal sources and to update the occurrence of Leishmania infection in vectors and dogs. Four sandfly species were captured: Phlebotomus perniciosus, Phlebotomus ariasi, Phlebotomus sergenti and Sergentomyia minuta. In one P. perniciosus female, L. infantum DNA was detected. Blood meal tests showed that this species had no host preferences and was an opportunistic feeder. An overall canine leishmaniasis (CanL) seroprevalence of 16.06% was found; the seroprevalence was 3.88% in dogs housed in kennels and 40.63% in dogs that attended veterinary clinics. The simultaneous occurrence of dogs and P. perniciosus infected with L. infantum in the AR indicates that the region continues to be an endemic area for CanL. Our results reinforce the need for the systematic spatial distribution of phlebotomine populations and their Leishmania infection rates and the need to simultaneously perform pathogen monitoring in both invertebrate and vertebrate hosts to investigate the transmission, distribution and spreading of Leishmania infection.

Pyochelin-mediated signalling in Pseudomonas aeruginosa

SUMMARY: Iron is an essential element for nearly all organisms but it is poorly available in most environments and not sufficient to support microbial growth. Bacteria have evolved a range of strategies to acquire this important metal, the most common of these being siderophore-mediated iron uptake. Siderophores are high-affinity iron chelators which are released to the extracellular environment where they complex iron and deliver it to the bacterial cell, via specific uptake systems. The Gram-negative bacterium Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, which both contribute to the virulence of this opportunistic human pathogen. The genes responsible for pyochelin-mediated iron uptake are grouped in the P. aeruginosa chromosome. The pyochelin biosynthetic genes are organized in two divergent operons, pchDCBA and pchEFGHI, which flank the regulatory gene pchR. The fptA gene, encoding the ferric pyochelin outer membrane receptor, occurs immediately downstream of the pchEFGHI genes. The biosynthesis of the siderophore and its receptor is subjected to dual regulation enabling P. aeruginosa to respond not only to the intracellular iron level but also to the presence of the siderophore in the extracellular environment. Negative regulation is mediated by the widespread Fur protein which employs ferrous iron as a corepressor and binds to a consensus sequence in the promoter region of iron-regulated genes. Positive regulation occurs during iron starvation and requires the AraC-type transcriptional regulator PchR. This regulator, together with pyochelin, induces the expression of pyochelin biosynthesis and uptake genes via a mechanism which was partly unraveled during this thesis. A 32-bp conserved sequence element (PchR-box) was identified in promoter regions of pyochelin-controlled genes. The PchR-box in the pchR-pchDCBA intergenic region was found to be essential for the induction of the pchDCBA operon and for the repression of the divergently transcribed pchR gene. PchR was purified as a fusion with maltose-binding protein (MBP). Mobility shift assays demonstrated specific binding of MBP-PchR to the PchR-box in the presence, but not in the absence of pyochelin. PchR-box mutations which interfered with pyochelin-dependent regulation in vivo, also affected pyochelin-dependent PchR-box recognition in vitro. These results show that pyochelin is the intracellular effector required for PchR-mediated regulation. The fact that extracellular pyochelin triggers this regulation implies that the siderophore can enter the cytoplasm. This conclusion was corroborated by analysing the importance of known and putative pyochelin uptake genes for pyochelin-dependent gene regulation. The pyochelin receptor gene fptA is followed by three genes, fptB, fptC, and fptX, which were shown here to be co-transcribed with fPtA. While fPtX encodes an inner membrane pen-I-lease, the functions of FptB and FptC are currently unknown. FptA and FptX, which are both required for pyochelin-mediated iron uptake, were found to be also needed for pyochelin-dependent gene regulation. FptB and FptC however, were not required and their role, if any, in the uptake of the PchR effector pyochelin remains elusive. RESUME Le fer est un élément essentiel pour la quasi-totalité des organismes, mais dans la plupart des environnements, il est difficilement accessible et insuffisant à la croissance microbienne. Les bactéries ont développé de multiples stratégies pour acquérir ce précieux métal, la plus commune étant l'acquisition au moyen de sidérophores. Les sidérophores sont des petites molécules dotées d'une forte affinité pour le fer qui, une fois relâchées dans l'environnement extracellulaire, vont complexer le fer et le délivrer à la cellule bactérienne par l'intermédiaire de systèmes d'acquisition spécifiques. La bactérie Gram-négative Pseudomonas aeruginosa produit deux sidérophores, la pyoverdine et la pyochéline, qui contribuent également à la virulence de ce pathogène opportuniste. Les gènes impliqués dans l'acquisition du fer à l'aide de la pyochéline sont regroupés sur t. le chromosome de P. aeruginosa. Les gènes de biosynthèse de la pyochéline sont organisés en deux opérons divergents, pchDCBA et pchEFGHI, qui flanquent le gène régulateur pchR. Le gène fptA, codant pour le récepteur de la pyochéline dans la membrane externe, est situé immédiatement en aval des gènes pchEFGHL La biosynthèse du sidérophore et de son récepteur est soumise à une double régulation permettant à P. aeruginosa de réagir non seulement à la quantité de fer intracellulaire, mais également à la présence du sidérophore dans le milieu extracellulaire. La répression se fait par l'intermédiaire de la protéine Fur, qui nécessite le fer ferreux comme co-répresseur et se lie à une séquence consensus dans la région promotrice des gènes régulés par le fer. L'induction se produit lorsque le fer est limitant, et requiert PchR, un régulateur transcriptionnel de la famille AraC. En présence de pyochéline, ce régulateur induit l'expression des gènes de biosynthèse et du récepteur de la pyochéline par l'intermédiaire d'un mécanisme partiellement résolu dans ce travail. Une séquence conservée (PchR-box) a été identifiée dans la région promotrice des gènes régulés par la pyochéline. La PchR-box située dans la région intergénique pchR-pchDCBA s'est révélée être importante pour l'induction de l'opéron pchDCBA et la répression du gène divergent pchR. PchR a été purifiée en tant que protéine de fusion avec une protéine liant le maltose (MBP). Des expériences de gel retard ont démontré la liaison spécifique de la protéine MBP-PchR sur la PchR-box en présence, mais non en absence de pyochéline. Les mutations de la PchR-box qui ont affecté la régulation pyochéline-dépendante in vivo, ont également eu un effet sur la liaison de la protéine in vitro. Ces résultats démontrent que la pyochéline est l'effecteur intracellulaire nécessaire à la régulation par PchR. Le fait que la pyochéline extracellulaire soit capable d'activer cette régulation implique que le sidérophore entre dans le cytoplasme. Cette conclusion a été corroborée par l'évaluation du rôle des gènes connus ou putatifs de l'incorporation du fer via la pyochéline sur la régulation pyochéline-dépendente. Le gène fPtA, codant pour le récepteur de la pyochéline, est suivi de trois gènes, fptB,fptC, et fptX, co-transcrits avec,ffitA. Si sffitX code pour une perméase de la membrane interne, la fonction de FptB et FptC reste obscure. FptA et FptX, nécessaires à l'acquisition du fer par l'intermédiaire de la pyochéline, se sont également révélés être requis pour la régulation pyochéline-dépendante des gènes pchDCBA, pchEFGHI et fptABCX. FptB et FptC n'ont quant à eux vraisemblablement pas de rôle majeur à jouer, si ce n'est aucun, dans l'incorporation de la pyochéline.

Characterisation of "fou2", a constitutive wound-activated mutant and analysis of the proteome and leaf physiology in the region proximal to wounds in Arabidopsis

Résumé : Les jasmonates (JA), une famille d'hor1none végétale, jouent un rôle central dans la réponse à la blessure, et aux attaques d'insectes et de pathogènes. Les JA sont principalement dérivés d'un acide gras, l'acide linolénique. L'addition par une lipoxygénase d'une molécule d'oxygène à l'acide linolénique initie la synthèse de JA. Cependant les mécanismes régulant l'activation de la biosynthèse de JA ne sont pas encore connus. C'est pour cette raison que dans ce travail, nous avons caractérisé chez Arabidopsis thaliana (l'Arabette des Dames) un mutant fou2 dont l'activité lipoxygénase est plus élevée que celle d'une plante sauvage. Les niveaux de JA sont constitutivement plus élevés et l'activation de la synthèse de JA après blessure est fortement plus induite chez fou2 que chez le type sauvage. En outre, fou2 est plus résistant au pathogène Botrytis cinerea et à la chenille Spodoptera littoralis. Afin de comprendre quel mécanisme chez fou2 génére ce phénotype, nous avons cloné le gène responsable du phénotype de fou2. Le mutant fou2 porte une mutation dans le gène d'un canal à deux pores transportant probablement du potassium, du lumen de la vacuole végétale vers le compartiment cytosolique. L'analyse du protéome de fou2 a permis d'identifier une expression plus élevée de sept protéines régulées par les JA ou le stress. La découverte de l'implication d'un canal dans le phénotype de fou2 renforce l'hypothèse que les flux de cations pourraient être impliqués dans les étapes précoces de la synthèse des JA. Nous avons également étudié le protéome et la physiologie d'une feuille blessée, Pour évaluer les changements d'expression protéique en réponse à la blessure et contrôlés par les JA, nous avons quantifié l'expression de 5937 protéines chez une plante d'Arabidopsis sauvage et chez un mutant incapable de synthétiser des JA. Parmi ces 5937 protéines, nous avons identifié 99 protéines régulées par la blessure chez le type sauvage. Nous avons observé pour 65% des protéines dont l'expression protéique changeait après blessure une bonne corrélation entre la quantité de transcrits et de protéines. Plusieurs enzymes de la voie des chorismates impliquées dans la biosynthèse des acides aminés phénoliques étaient induites par les JA après blessure. Une quantification des acides aminés a montré que les niveaux d'acides aminés phénoliques augmentaient significativement après blessure. La blessure induisait aussi des changements dans l'expression de protéines impliquées dans la réponse au stress et particulièrement au stress oxydatif. Nous avons quantifié l'état réduit et oxydé du glutathion, un tripeptide qui, sous sa forme réduite, est l'antioxydant majeur des cellules. Nous avons trouvé une quantité significativement plus élevée de glutathion oxydé chez le type sauvage blessé que chez la plante aus blessée. Ce résultat suggère que la génération d'un stress oxydatif et la proportion relative de glutathions réduits et oxydés sont contrôlés par les JA après blessure. Abstract : Plants possess a family of potent fatty acid-derived wound-response and developmental regulators: the jasmonates. These compounds are derived from the tri?unsaturated fatty acid a-linolenic-acid (18:3). Addition of an oxygen molecule to 18:3 by 13-lipoxygenases (13-LOX) initiates JA biosynthesis. Actually components regulating the activation of JA biosynthesis are poorly defined. Therefore we characterized in Arabidopsis thaliana the fatty acid Qxygenation upregulated 2 (fou2) mutant, which was previously isolated in a screen for mutants with an enhanced 13-LOX activity. As a consequence of this increased 13-LOX activity, JA levels in fou2 are higher than in wild type (WT) and wounding strongly increased JA biosynthesis compared to WT. fou2 was more resistant to the fungus Botrytis cinerea and the generalist caterpillar Spodaptera littomlis, The fou2 mutant carries a missense mutation in the Two Pore Channel 1 gene (TPCJ), which encodes a vacuolar cation channel transporting probably K* into the cytosol. Patchclamp analysis of fou2 vacuolar membranes showed faster time-dependent conductivity and activation of the mutated channel at lower membrane potentials than wild-type. Proteomic analysis of fou2 leaves identified increased levels of seven biotic stress- and JA- inducible proteins. The discovery of the implication of a channel in the fou2 phenotype strenghtens the hypothesis that cation fluxes might be implicated in early steps of JA synthesis. We further concentrated on the proteome and leaf physiology in the region proximal to wounds in Arabidopsis using the WT and the aos JA-biosynthesis deficient mutant in order to find JA- induced proteins changes. We used two successive proteomic methods to assess protein changes in response to wounding Arabidopsis leaves, two dimensional electrophoresis (2DE) and linear trap quadrupole ion-trap mass spectrometry. In total 5937 proteins were quantified. We identified 99 wound-regulated proteins in the WT. Most these proteins were also wound-regulated at the transcript level showing a good correlation between transcript and protein abundance. We identified several wound-regulated enzymes involved in amino acid biosynthesis and confirmed this result by amino acid quantification. Proteins involved in stress reponses were upregulated, particularly in redox species regulation. We found a significantly higher quantity of oxidized glutathione in wounded WT relative to wounded aos leaves. This result suggests that levels of reduced glutathione are controlled by JA after wounding.

Detection of the B"-GWGR variant in the southernmost region of Brazil: unveiling the complexity of the human immunodeficiency virus-1 subtype B epidemic

Typical human immunodeficiency virus-1 subtype B (HIV-1B) sequences present a GPGR signature at the tip of the variable region 3 (V3) loop; however, unusual motifs harbouring a GWGR signature have also been isolated. Although epidemiological studies have detected this variant in approximately 17-50% of the total infections in Brazil, the prevalence of B"-GWGR in the southernmost region of Brazil is not yet clear. This study aimed to investigate the C2-V3 molecular diversity of the HIV-1B epidemic in southernmost Brazil. HIV-1 seropositive patients were ana-lysed at two distinct time points in the state of Rio Grande do Sul (RS98 and RS08) and at one time point in the state of Santa Catarina (SC08). Phylogenetic analysis classified 46 individuals in the RS98 group as HIV-1B and their molecular signatures were as follows: 26% B"-GWGR, 54% B-GPGR and 20% other motifs. In the RS08 group, HIV-1B was present in 32 samples: 22% B"-GWGR, 59% B-GPGR and 19% other motifs. In the SC08 group, 32 HIV-1B samples were found: 28% B"-GWGR, 59% B-GPGR and 13% other motifs. No association could be established between the HIV-1B V3 signatures and exposure categories in the HIV-1B epidemic in RS. However, B-GPGR seemed to be related to heterosexual individuals in the SC08 group. Our results suggest that the established B"-GWGR epidemics in both cities have similar patterns, which is likely due to their geographical proximity and cultural relationship.

New records of Mansonella ozzardi: a parasite that is spreading from the state of Amazonas to previously uninfected areas of the state of Acre in the Purus River region

Mansonella ozzardi infections are common in the riverside communities along the Solimões, Negro and Purus Rivers in the state of Amazonas (AM). However, little is known about the presence of this parasite in communities located in regions bordering AM and the state of Acre. The prevalence rate of M. ozzardi infections was determined in blood samples from volunteers according to the Knott method. A total of 355 volunteers from six riverine communities were enrolled in the study and 65 (18.3%) were found to be infected with M. ozzardi. As expected, most of the infections (25%) occurred in individuals involved in agriculture, cattle rearing and fishing and an age/sex group analysis revealed that the prevalence increased beginning in the 40-50-years-of-age group and reached 33% in both sexes in individuals over 50 years of age. Based on the described symptomatology, articular pain and headache were found to be significantly higher among infected individuals (56 and 65% prevalence, respectively, p < 0.05). Sera from volunteers were subjected to ELISA using a cocktail of recombinant proteins from Onchocerca volvulus to evaluate the specificity of the test in an endemic M. ozzardi region. No cross-reactions between M. ozzardi-infected individuals and recombinant O. volvulusproteins were detected, thus providing information on the secure use of this particular cocktail in areas where these parasites are sympatric.

Comparative clinical study of different multiplex real time PCR strategies for the simultaneous differential diagnosis between extrapulmonary tuberculosis and focal complications of brucellosis

Background: Both brucellosis and tuberculosis are chronic-debilitating systemic granulomatous diseases with a high incidence in many countries in Africa, Central and South America, the Middle East and the Indian subcontinent. Certain focal complications of brucellosis and extrapulmonary tuberculosis are very difficult to differentiate clinically, biologically and radiologically. As the conventional microbiological methods for the diagnosis of the two diseases have many limitations, as well as being time-consuming, multiplex real time PCR (M RT-PCR) could be a promising and practical approach to hasten the differential diagnosis and improve prognosis. Methodology/Principal Findings: We designed a SYBR Green single-tube multiplex real-time PCR protocol targeting bcsp31 and the IS711 sequence detecting all pathogenic species and biovars of Brucella genus, the IS6110 sequence detecting Mycobacterium genus, and the intergenic region senX3-regX3 specifically detecting Mycobacterium tuberculosis complex. The diagnostic yield of the M RT-PCR with the three pairs of resultant amplicons was then analyzed in 91 clinical samples corresponding to 30 patients with focal complications of brucellosis, 24 patients with extrapulmonary tuberculosis, and 36 patients (Control Group) with different infectious, autoimmune or neoplastic diseases. Thirty-five patients had vertebral osteomyelitis, 21 subacute or chronic meningitis or meningoencephalitis, 13 liver or splenic abscess, eight orchiepididymitis, seven subacute or chronic arthritis, and the remaining seven samples were from different locations. Of the three pairs of amplicons (senX3-regX3+ bcsp3, senX3-regX3+ IS711 and IS6110+ IS711) only senX3-regX3+ IS711 was 100% specific for both the Brucella genus and M. tuberculosis complex. For all the clinical samples studied, the overall sensitivity, specificity, and positive and negative predictive values of the M RT-PCR assay were 89.1%, 100%, 85.7% and 100%, respectively, with an accuracy of 93.4%, (95% CI, 88.3—96.5%). Conclusions/Significance: In this study, a M RT-PCR strategy with species-specific primers based on senX3-regX3+IS711 sequences proved to be a sensitive and specific test, useful for the highly efficient detection of M. tuberculosis and Brucella spp in very different clinical samples. It thus represents an advance in the differential diagnosis between some forms of extrapulmonary tuberculosis and focal complications of brucellosis.

Genetic diversity and antimicrobial resistance in Streptococcus agalactiae strains recovered from female carriers in the Bucharest area

For the first time, we used multilocus sequence typing (MLST) to understand how Romanian group B streptococcus (GBS) strains fit into the global GBS population structure. Colonising isolates recovered from adult human females were tested for antibiotic resistance, were molecularly serotyped based on the capsular polysaccharide synthesis (cps) gene cluster and further characterised using a set of molecular markers (surface protein genes, pilus-encoded islands and mobile genetic elements inserted in the scpB-lmb intergenic region). Pulsed-field gel electrophoresis was used to complement the MLST clonal distribution pattern of selected strains. Among the 55 strains assigned to six cps types (Ia, Ib, II-V), 18 sequence types (STs) were identified by MLST. Five STs represented new entries to the MLST database. The prevalent STs were ST-1, ST-17, ST-19 and ST-28. Twenty molecular marker profiles were identified. The most common profiles (rib+GBSi1+PI-1, rib+GBSi1+PI-1, PI-2b and alp2/3+PI-1, PI-2a) were associated with the cps III/ST-17 and cps V/ST-1 strains. A cluster of fluoroquinolone-resistant strains was detected among the cps V/ST-19 members; these strains shared alp1 and IS1548 and carried PI-1, PI-2a or both. Our results support the usefulness of implementing an integrated genotyping system at the reference laboratory level to obtain the reliable data required to make comparisons between countries.

Morphology of the larvae, male genitalia and DNA sequences of Anopheles (Kerteszia) pholidotus (Diptera: Culicidae) from Colombia

Since 1984, Anopheles (Kerteszia) lepidotus has been considered a mosquito species that is involved in the transmission of malaria in Colombia, after having been incriminated as such with epidemiological evidence from a malaria outbreak in Cunday-Villarrica, Tolima. Subsequent morphological analyses of females captured in the same place and at the time of the outbreak showed that the species responsible for the transmission was not An. lepidotus, but rather Anopheles pholidotus. However, the associated morphological stages and DNA sequences of An. pholidotus from the foci of Cunday-Villarrica had not been analysed. Using samples that were caught recently from the outbreak region, the purpose of this study was to provide updated and additional information by analysing the morphology of female mosquitoes, the genitalia of male mosquitoes and fourth instar larvae of An. pholidotus, which was confirmed with DNA sequences of cytochrome oxidase I and rDNA internal transcribed spacer. A total of 1,596 adult females were collected in addition to 37 larval collections in bromeliads. Furthermore, 141 adult females, which were captured from the same area in the years 1981-1982, were analysed morphologically. Ninety-five DNA sequences were analysed for this study. Morphological and molecular analyses showed that the species present in this region corresponds to An. pholidotus. Given the absence of An. lepidotus, even in recent years, we consider that the species of mosquitoes that was previously incriminated as the malaria vector during the outbreak was indeed An. pholidotus, thus ending the controversy.

Malaria in Brazil: what happens outside the Amazonian endemic region

Brazil, a country of continental proportions, presents three profiles of malaria transmission. The first and most important numerically, occurs inside the Amazon. The Amazon accounts for approximately 60% of the nation’s territory and approximately 13% of the Brazilian population. This region hosts 99.5% of the nation’s malaria cases, which are predominantly caused by Plasmodium vivax (i.e., 82% of cases in 2013). The second involves imported malaria, which corresponds to malaria cases acquired outside the region where the individuals live or the diagnosis was made. These cases are imported from endemic regions of Brazil (i.e., the Amazon) or from other countries in South and Central America, Africa and Asia. Imported malaria comprised 89% of the cases found outside the area of active transmission in Brazil in 2013. These cases highlight an important question with respect to both therapeutic and epidemiological issues because patients, especially those with falciparum malaria, arriving in a region where the health professionals may not have experience with the clinical manifestations of malaria and its diagnosis could suffer dramatic consequences associated with a potential delay in treatment. Additionally, because the Anopheles vectors exist in most of the country, even a single case of malaria, if not diagnosed and treated immediately, may result in introduced cases, causing outbreaks and even introducing or reintroducing the disease to a non-endemic, receptive region. Cases introduced outside the Amazon usually occur in areas in which malaria was formerly endemic and are transmitted by competent vectors belonging to the subgenus Nyssorhynchus (i.e., Anopheles darlingi, Anopheles aquasalis and species of the Albitarsis complex). The third type of transmission accounts for only 0.05% of all cases and is caused by autochthonous malaria in the Atlantic Forest, located primarily along the southeastern Atlantic Coast. They are caused by parasites that seem to be (or to be very close to) P. vivax and, in a less extent, by Plasmodium malariae and it is transmitted by the bromeliad mosquito Anopheles (Kerteszia) cruzii. This paper deals mainly with the two profiles of malaria found outside the Amazon: the imported and ensuing introduced cases and the autochthonous cases. We also provide an update regarding the situation in Brazil and the Brazilian endemic Amazon.

ESR1 gene promoter region methylation in free circulating DNA and its correlation with estrogen receptor protein expression in tumor tissue in breast cancer patients.

BACKGROUND Tumor expression of estrogen receptor (ER) is an important marker of prognosis, and is predictive of response to endocrine therapy in breast cancer. Several studies have observed that epigenetic events, such methylation of cytosines and deacetylation of histones, are involved in the complex mechanisms that regulate promoter transcription. However, the exact interplay of these factors in transcription activity is not well understood. In this study, we explored the relationship between ER expression status in tumor tissue samples and the methylation of the 5' CpG promoter region of the estrogen receptor gene (ESR1) isolated from free circulating DNA (fcDNA) in plasma samples from breast cancer patients. METHODS Patients (n = 110) with non-metastatic breast cancer had analyses performed of ER expression (luminal phenotype in tumor tissue, by immunohistochemistry method), and the ESR1-DNA methylation status (fcDNA in plasma, by quantitative methylation specific PCR technique). RESULTS Our results showed a significant association between presence of methylated ESR1 in patients with breast cancer and ER negative status in the tumor tissue (p = 0.0179). There was a trend towards a higher probability of ESR1-methylation in those phenotypes with poor prognosis i.e. 80% of triple negative patients, 60% of HER2 patients, compared to 28% and 5.9% of patients with better prognosis such as luminal A and luminal B, respectively. CONCLUSION Silencing, by methylation, of the promoter region of the ESR1 affects the expression of the estrogen receptor protein in tumors of breast cancer patients; high methylation of ESR1-DNA is associated with estrogen receptor negative status which, in turn, may be implicated in the patient's resistance to hormonal treatment in breast cancer. As such, epigenetic markers in plasma may be of interest as new targets for anticancer therapy, especially with respect to endocrine treatment.