Morphological and molecular characterisation of Heliconema hainanensis sp. nov. (Spirurina: Physalopteridae) from congers in the South China Sea, with a key to the species of Heliconema

Heliconema hainanensis sp. nov. collected from Uroconger lepturus (Richardson) (Anguilliformes: Congridae), Muraenesox cinereus (Forsskål) and Congresox talabonoides (Bleeker) (Anguilliformes: Muraenesocidae) in the South China Sea was described using light and scanning electron microscopy. The new species differs from its congeners by the following morphology: pseudolabia, the number and arrangement of caudal papillae (4 pairs of pedunculate precloacal papillae arranged in 2 groups of 2 and 2 pairs and 6 pairs of pedunculate postcloacal papillae arranged in 4 groups of 1, 2, 1 and 2 pairs), the length of spicules [left spicule 0.51-0.69 mm, right spicule 0.20-0.27 mm, spicule (right:left) ratio 1:2.20-2.69] and the morphology of the female tail tip. In addition, specimens of the new species collected from the three different hosts and specimens of an unidentified species of Heliconema collected from U. lepturus were characterised using molecular methods by sequencing the internal transcribed spacer (ITS) of ribosomal DNA. Analyses and comparison of the ITS sequence of H. hainanensis sp. nov. with Heliconema sp. support the validity of the new species based on morphological observations. An identification key to the species of Heliconema is also provided.

Molecular diagnosis of eosinophilic meningitis due to Angiostrongylus cantonensis (Nematoda: Metastrongyloidea) by polymerase chain reaction-DNA sequencing of cerebrospinal fluids of patients

Cerebrospinal fluid (CSF) samples from clinically diagnosed patients with detectable Angiostrongylus canto-nensis-specific antibodies (n = 10), patients with clinically suspected cases that tested negative for A. cantonensis-an-tibodies (n = 5) and patients with cerebral gnathostomiasis (n = 2) and neurocysticercosis (n = 2) were examined by a single-step polymerase chain reaction (PCR) method using the AC primers for the 66-kDa native protein gene. The PCR method detected A. cantonensis DNA in CSF samples from four of 10 serologically confirmed angiostrongyliasis cases. The PCR results were negative for the remaining CSF samples. The nucleotide sequences of three positive CSF-PCR samples shared 98.8-99.2% similarity with the reference sequence of A. cantonensis. These results indicate the potential application of this PCR assay with clinical CSF samples for additional support in the confirmation of eosinophilic meningitis due to A. cantonensis.

Molecular identification of Rickettsia parkeri infecting Amblyomma triste ticks in an area of Argentina where cases of rickettsiosis were diagnosed

Specimens of the hard tick Amblyomma triste were found infected with Rickettsia parkeri in an area of Argentina (General Lavalle, Buenos Aires Province) where cases of human illness attributed to this microorganism have been reported. Molecular detection of R. parkeri was based on polymerase chain reactions that amplify a ca. 400-bp fragment of the 23S-5S intergenic spacer and a ca. 500-bp fragment of the gene encoding a 190-kDa outer membrane protein. Three (6.97%) of 43 A. triste ticks were determined to be positive for R. parkeri. These results provide strong evidence that A. triste is the vector of R. parkeri in the study area. The findings of this work have epidemiological relevance because human parasitism by A. triste ticks has been frequently recorded in some riparian areas of Argentina and Uruguay and new cases of R. parkeri rickettsiosis might arise in the South American localities where humans are exposed to the bites of this tick species.

Phenotypic and molecular characterization of Bruck syndrome (Osteogenesis imperfecta with contractures of the large joints) caused by a recessive mutation in PLOD2

Le Syndrome de Bruck (Bruck Syndrome; BS) est une maladie autosomique récessive assemblant la combinaison inhabituelle de fragilité osseuse semblable à celle de l'Ostéogenèse Imparfaite (0I) avec des contractures congénitales tendineuses et cutanées des grandes articulations («ptérygia»). Les cas décrits jusqu'à ce jour mettent en évidence une grande hétérogénéité du tableau clinique, liée en partie au manque d'un diagnostic biochimique ou moléculaire. Nous savons que dans le BS les gènes codant pour le collagène 1 ne sont pas mutés, mais savons néanmoins, grâce à l'étude du collagène extrait de biopsies osseuses, qu'il y a un déficit d'hydroxylation des résidus de lysine dans les télopeptides du collagène 1 qui servent à la formation des liens intermoléculaires (crosslinks) et donc à la stabilisation des fibres de collagène. Un locus génétique du BS à été mappé sur 17q12, mais le gène responsable sur ce locus reste inconnu; plus récemment, deux mutations dans le gène de la lysyl hydroxylase 2 (PLOD2, position chromosomique 3q23-q24) ont été identifiées, démontrant l'hétérogénéité génétique du ES. La proportion de ES liée à 17p22 (BS type 1) et celle liée à une mutation dans PLOD2 (BS type 2) est encore incertaine et nous manquons de données sur la corrélation phenotype-génotype. Nous avons étudié le cas d'un garçon avec des contractures et des ptérygia dès la naissance, combinées à une ostéopénie sévère de type OI menant à des fractures multiples. Ses urines contenaient une quantité élevée d'hydroxyproline, indiquant un remaniement important du tissu osseux, mais peu de produits de dégradation des crosslinks du collagène, indiquant donc une réduction de la proportion de crosslinks dans le collagène in vivo. Nous avons pu démontrer chez lui la présence d'une nouvelle mutation homozygote dans le gène PLOD2 menant à une substitution Arg598His; les deux parents du sujet étaient hétérozygotes pour la mutation et celle-ci était absente dans notre population témoin. La mutation est adjacente aux deux mutations rapportées précédemment (Gly601Val et Thr608Ile), ce qui suggère la présence d'un ''hotspot'' mutationnel mais aussi d'une région de grande importance fonctionnelle sur PLOD2 : cette observation est importante pour la création d'inhibiteurs de PLOD2, recherchés en ce moment pour le traitement de la fibrose. La combinaison de ptérygia et de fragilité osseuse, comme illustrée par notre patient est apparemment contradictoire et donc difficilement explicable mais indique que l'hydroxylation des résidus lysyl des télopeptides est importante non seulement pour la stabilité osseuse mais aussi dans la morphogénèse et la formation des articulations dans la période prénatale. Finalement, la mesure des produits de dégradation du collagène dans l'urine et l'analyse de mutation de PLOD2 permet le diagnostic du syndrome de Bruck et permet de le différencier de l'Osteogénèse Imparfaite. -- Bruck syndrome (BS) is a recessively-inherited phenotypic disorder featuring the unusual combination of skeletal changes resembling osteogenesis imperfecta (0I) with congenital contractures of the large joints. Clinical heterogeneity is apparent in cases reported thus far. While the genes coding for collagen 1 chains are unaffected in BS, there is biochemical evidence for a defect in the hydroxylation of lysine residues in collagen 1 telopeptides. One BS locus has been mapped at 17p12, but more recently, two mutations in the lysyl hydroxylase 2 gene (PLOD2, 3q23-q24) have been identified in BS, showing genetic heterogeneity. The proportion of BS cases linked to 17p22 (BS type 1) or caused by mutations in PLOD2 (BS type 2) is still uncertain, and phenotypic correlations are lacking. We report on a boy who had congenital contractures with pterygia at birth and severe 0I-like osteopenia and multiple frac-tures. His urine contained high amounts of hydroxyproline but low amounts of collagen crosslinks degradation products; and he was shown to be homozygous for a novel mutation leading to an Arg598His substitution in PLOD2. The mutation is adjacent to the two mutations previously reported (Gly601Val and Thr608Ile), suggesting a functionally important hotspot in PLOD2. The combination of pterygia with bone fragility, as illustrated by this case, is difficult to explain; it suggests that telopeptide lysyl hydroxylation must be involved in prenatal joint formation and morphogenesis. Collagen degradation products in urine and mutation analysis ofPLOD2 maybe used to diagnose BS and differentiate it from M.

Risk for Pneumocystis carinii transmission among patients with pneumonia: a molecular epidemiology study.

We report a molecular typing and epidemiologic analysis of Pneumocystis carinii pneumonia (PCP) cases diagnosed in our geographic area from 1990 to 2000. Our analysis suggests that transmission from patients with active PCP to susceptible persons caused only a few, if any, PCP cases in our setting.

Molecular identification of Giardia duodenalis in Ecuador by polymerase chain reaction-restriction fragment length polymorphism

The aim of this study was to determine the genetic diversity of Giardia duodenalis present in a human population living in a northern Ecuadorian rain forest. All Giardia positive samples (based on an ELISA assay) were analysed using a semi-nested polymerase chain reaction-restriction fragment length polymorphism assay that targets the glutamate dehydrogenase (gdh) gene; those amplified were subsequently genotyped using NlaIV and RsaI enzymes. The gdh gene was successfully amplified in 74 of 154 ELISA positive samples; 69 of the 74 samples were subsequently genotyped. Of these 69 samples, 42 (61%) were classified as assemblage B (26 as BIII and 16 as BIV), 22 (32%) as assemblage A (3 as AI and 19 as AII) and five (7%) as mixed AII and BIII types. In this study site we observe similar diversity in genotypes to other regions in Latin America, though in contrast to some previous studies, we found similar levels of diarrheal symptoms in those individuals infected with assemblage B compared with those infected with assemblage A.

Genetics and molecular epidemiology of multiple myeloma: the rationale for the IMMEnSE consortium (review).

There is strong evidence suggesting the presence of a genetic component in the aetiology of multiple myeloma (MM). However no genetic risk factors have been unequivocally established so far. To further our understanding of the genetic determinants of MM risk, a promising strategy is to collect a large set of patients in a consortium, as successfully done for other cancers. In this article, we review the main findings in the genetic susceptibility and pharmacogenetics of MM and present the strategy of the IMMEnSE (International Multiple Myeloma rESEarch) consortium in contributing to determine the role of genetic variation in pharmacogenetics and in MM risk.

Molecular characterisation of hepatitis B virus in the resident Chinese population in Panama City

Despite the effectiveness of current hepatitis B virus (HBV) vaccines, it is estimated that 350 million individuals suffer from chronic HBV infection and more than 50% of these affected individuals live on the Asian continent. Panama is a country with a great diversity of foreign groups; the Chinese community is a large example of this phenomenon. There is an urgent need to perform studies that evaluate the prevalence and the genetic diversity of HBV in this community. This study aimed to evaluate the prevalence of HBV and its genotypes and mutant variants in the Chinese population residing in Panama. In total, 320 subjects were enrolled in the study. Forty-two subjects (13.1%) were positive for HBsAg and HBV-DNA from 18 subjects revealed the presence of genotypes B2 and C1. Secondary mutations associated with drug resistance at positions rtV207L and rtN239T of the reverse transcriptase gene were identified. Additionally, the mutation pair A1762T/G1764A was found in three samples and the mutation G1896A was detected in an HBeAg-negative subject. In conclusion, to our knowledge, this is the first study to report high HBV prevalence rates in resident ethnic Chinese in Central America and the presence of genotypes B2 and C1 in this region.

Paper de l’ Haemophilus influenzae en les exacerbacions de la Malaltia Pulmonar Obstructiva Crònica (MPOC): tipificació molecular i factors de virulència

Objetivos: 1.-Identificar los factores clínicos y microbiológicos que ayuden a predecir la aparición de exacerbaciones en la EPOC. 2.-Diagnóstico y cuantificación de las especies bacterianas aisladas en esputo (fase de exacerbación y estable) .3.- Tipificación genotípica secuencial de las cepas de H. influenzae y P. aeruginosa. 4.- Impacto del tratamiento antibiótico en la aparición de resistencias en estos patógenos. 5.- Diseño: Estudio prospectivo (3 años). Ámbito del estudio: Hospital Universitario de tercer nivel. Pacientes con EPOC grave atendidos en la Consulta Monográfica de EPOC del Servicio de Neumología. Métodos microbiológicos: Cuantificación de la carga bacteriana en muestras respiratorias en fase estable y en exacerbación. Estudio de la sensibilidad “in vitro”. Tipificación molecular (PFGE y MLST) de H. influenzae y P. aeruginosa. Estudio de los genes de virulencia de H. influenzae mediante PCR. Resultados: Desde Febrero de 2010 a Julio de 2011 se han incluido 77 pacientes. Los microorganismos más frecuentemente aislados en fase de exacerbación fueron: P. aeruginosa (29.3%), H. influenzae (15.92%), M. catarrhalis (12.74%), S. pneumoniae (10.19%) y S. aureus (5.10%). En los 88 episodios por P. aeruginosa se detectaron 38 genotipos diferentes. En los 41 episodios por H. influenzae se detectaron 39 genotipos diferentes. El 10% de los episodios fueron polimicrobianos. Los episodios de EAEPOC y de fase estable tuvieron una distribución de microorganismos similar. Sin embargo, cuando se cuantificaron las cargas bacterianas fueron mayores en EAEPOC (intervalo 4x107 -2x108) que en fase estable (intervalo 2x105 -4x107). Conclusiones: El genotipo de las cepas de P. aeruginosa y H. influenzae aisladas en EAEPOC difieren de un paciente a otro, sin embargo la mayoría de los episodios de cada paciente están causados por un genotipo único.

Novel Chlamydiales strains isolated from a water treatment plant.

Chlamydiae are obligate intracellular bacteria infecting free-living amoebae, vertebrates and some invertebrates. Novel members are regularly discovered, and there is accumulating evidence supporting a very important diversity of chlamydiae in the environment. In this study, we investigated the presence of chlamydiae in a drinking water treatment plant. Samples were used to inoculate Acanthamoeba monolayers (Acanthamoeba co-culture), and to recover autochthonous amoebae onto non-nutritive agar. Chlamydiae were searched for by a pan-chlamydia 16S rRNA gene PCR from both Acanthamoeba co-cultures and autochthonous amoebae, and phylotypes determined by 16S rRNA gene sequencing. Autochthonous amoebae also were identified by 18S rRNA gene amplification and sequencing. From a total of 79 samples, we recovered eight chlamydial strains by Acanthamoeba co-culture, but only one of 28 amoebae harboured a chlamydia. Sequencing results and phylogenetic analysis showed our strains belonging to four distinct chlamydial lineages. Four strains, including the strain recovered within its natural host, belonged to the Parachlamydiaceae; two closely related strains belonged to the Criblamydiaceae; two distinct strains clustered with Rhabdochlamydia spp.; one strain clustered only with uncultured environmental clones. Our results confirmed the usefulness of amoeba co-culture to recover novel chlamydial strains from complex samples and demonstrated the huge diversity of chlamydiae in the environment, by identifying several new species including one representing the first strain of a new family.

Molecular identification of Sporothrix species involved in the first familial outbreak of sporotrichosis in the state of Espírito Santo, southeastern Brazil

Sporotrichosis is a widespread subcutaneous mycosis caused by the dimorphic fungi now known as the Sporothrix schenckii complex. This complex is comprised of at least six species, including Sporothrix albicans, Sporothrix brasiliensis, Sporothrix globosa, Sporothrix luriei, Sporothrix mexicana and S. schenckii. Cases of sporotrichosis have significantly increased in Brazil over the past decade, especially in the state of Rio de Janeiro (RJ), where an epidemic among cat owners has been observed. The zoonotic transmission from cats to humans suggests a common source of infection and indicates that animals can act as vectors. We performed a molecular characterisation of samples collected during the first outbreak of familial sporotrichosis caused by S. brasiliensis in the state of Espírito Santo, Brazil. These results represent the first description of such an outbreak outside the endemic area of zoonotic sporotrichosis in RJ.

Molecular genetics of rare hereditary diseases via genome-wide and integrated approaches

ABSTRACTThe Online Mendelian Inheritance in Man database (OMIM) reports about 3000 Mendelian diseases of known causal gene and about 2000 that remain to be mapped. These cases are often difficult to solve because of the rareness of the disease, the structure of the family (too big or too small) or the heterogeneity of the phenotype. The goal of this thesis is to explore the current genetic tools, before the advent of ultra high throughput sequencing, and integrate them in the attempt to map the genes behind the four studied cases. In this framework we have studied a small family with a recessive disease, a modifier gene for the penetrance of a dominant mutation, a large extended family with a cardiac phenotype and clinical and/or allelic heterogeneity and we have molecularly analyzed a balanced chromosomal translocation.RESUMELa base de données des maladies à transmission mendélienne, Online Mendelian Inheritance in Man (OMIM), contient environ 3000 affections à caractère mendélien pour lesquelles le gène responsable est connu et environ 2000 qui restent à élucider.Les cas restant à résoudre sont souvent difficiles soit par le caractère intrinsèquement rare de ces maladies soit à cause de difficultés structurelles (famille trop petite ou trop étendue) ou hétérogénéité du phénotype ou génétique. Cette thèse s'inscrit avant l'arrivée des nouveaux outils de séquençage à haut débit. Son but est d'explorer les outils génétiques actuels, et de les intégrer pour trouver les gènes impliqués dans quatre cas représentant chacun une situation génétique différente : nous avons étudié une famille de quatre individus avec une transmission récessive, recherché un gène modificateur de la pénétrance de mutations dominantes, étudié une famille étendue présentant un phénotype cardiaque cliniquement et/ou allèliquement hétérogène et nous avons fait l'analyse moléculaire d'une translocation chromosomique balancée.

Molecular analysis of an odorant-binding protein gene in two sympatric species of Lutzomyia longipalpis s.l.

Lutzomyia longipalpis s.l. is the main vector of American visceral leishmaniasis (AVL) and occurs as a species complex. DNA samples from two Brazilian sympatric species that differ in pheromone and courtship song production were used to analyse molecular polymorphisms in an odorant-binding protein ( obp29 ) gene. OBPs are proteins related to olfaction and are involved in activities fundamental to survival, such as foraging, mating and choice of oviposition site. In this study, the marker obp29 was found to be highly polymorphic in Lu. longipalpis s.l. , with no fixed differences observed between the two species. A pairwise fixation index test indicated a moderate level of genetic differentiation between the samples analysed.

Risk assessment of recurrence in sporadic retinoblastoma using a molecular-based algorithm

Le rétinoblastome (Rb) est une tumeur provenant des cellules rétiniennes progénitrices des photorécepteurs. C'est la tumeur pédiatrique maligne la plus fréquente avec une incidence par naissance évaluée entre 1/15Ό00 et 1/20Ό00. Les enfants atteints de Rb sont diagnostiqué dans leur grande majorité avant l'âge de 4 ans, soit le temps nécessaire à la différentiation et à la maturation des photorécepteurs et donc à la disparition de la cellule d'origine du Rb. La survie du patient, la sauvegarde oculaire et le pronostic visuel restent excellents pour autant que le traitement ne soit pas différé. Dans sa variante non héréditaire (60%) le Rb est toujours unilatéral et sporadique. Le Rb héréditaire de transmission dominante autosomique (40%), se décline sous toutes les formes, familiale (10%) ou sporadique (30%), que l'atteinte soit unilatérale ou bilatérale. La majorité des mutations causales sont uniques et distribuées de façon aléatoire sur la totalité du gène RB1 sans région prédisposante. La détection de ces mutations est couteuse et chronophage, tout en présentant un taux de détection relativement bas; surtout dans les cas de Rb sporadiques unilatéraux. Dans le but d'identifier les patients présentant un risque réel de développer un Rb, et de réduire le nombre d'examens sous narcose requis pour le dépistage de la maladie chez les sujets à risque, nous avons développé une stratégie sensible, rapide, efficace et peu couteuse basée sur une analyse de l'haplotype intragénique. Cet algorithme prend en compte a) la perte d'hétérozygotie intratumorale du gène RB1, b) l'origine paternelle préférentielle des nouvelles mutations germinales et c) un risque a priori dérivé des données empiriques de Vogel. Pendant la période allant de janvier 1994 à décembre 2006, nous avons comparé l'apparition de nouveau Rb parmi la fratrie et la descendance de patient atteints au nombre de nouveaux cas attendus calculé par notre algorithme. 134 familles ont été étudiées. L'analyse moléculaire a été effectuée chez 570 personnes dont 99 patients âgés de moins de 4 ans et donc à risque de développer un Rb. Parmi cette cohorte, nous avons observé l'apparition d'un cas de Rb, alors que les risques cumulés a posteriori calculé par notre algorithme prédisait l'apparition de 1.77 nouveau cas. Dans cette étude, nous avons pu valider notre algorithme prédisant la récurrence de Rb chez les parents de 1er degré de patients atteints. Cet outil devrait grandement faciliter le conseil génétique ainsi que le suivi des patients à risque de développer un Rb, surtout dans les cas ou le séquençage direct du gène RB1 n'est pas disponible ou est resté non informatif. - Purpose: Most RBI mutations are unique and distributed throughout the RBI gene. Their detection can be time-consuming and the yield especially low in cases of conservatively-treated sporadic unilateral retinoblas-toma (Rb) patients. In order to identify patients with true risk of developing Rb, and to reduce the number of unnecessary examinations under anesthesia in all other cases, we developed a universal sensitive, efficient and cost-effective strategy based on intragenic haplotype analysis. Methods: This algorithm allows the calculation of the a posteriori risk of developing Rb and takes into account (a) RBI loss of heterozygosity in tumors, (b) preferential paternal origin of new germline mutations, (c) a priori risk derived from empirical data by Vogel, and (d) disease penetrance of 90% in most cases. We report the occurrence of Rb in first degree relatives of patients with sporadic Rb who visited the Jules Gonin Eye Hospital, Lausanne, Switzerland, from January 1994 to December 2006 compared to expected new cases of Rb using our algorithm. Results: A total of 134 families with sporadic Rb were enrolled; testing was performed in 570 individuals and 99 patients younger than 4 years old were identified. We observed one new case of Rb. Using our algorithm, the cumulated total a posteriori risk of recurrence was 1.77. Conclusions: This is the first time that linkage analysis has been validated to monitor the risk of recurrence in sporadic Rb. This should be a useful tool in genetic counseling, especially when direct RBI screening for mutations leaves a negative result or is unavailable.