Phylogeny and spatial distribution of Japanese encephalitis virus vector species in Cambodia.

In Southeast Asia, despite the use of Japanese encephalitis vaccines and vaccination coverage, Japanese encephalitis (JE) transmission is still a major public health issue. The main vectors of this virus are mosquitoes from the genus Culex, which diversity and density are important in Southeast Asia. The main vector species of Japanese encephalitis virus (JEV) in Cambodia belong to the Vishnui subgroup. However, their morphological identification solely based on the adult stage remains challenging, making their segregation and detection difficult. In order to identify and describe the distribution of the three main JEV vector species in Cambodia, namely Culex vishnui, Cx. pseudovishnui and Cx. tritaeniorhynchus, mosquito samplings were carried out throughout the country in different environments. Phylogenetic analysis of the cytochrome c oxidase subunit I (coI) gene using maximum-likelihood tree with ultrafast bootstrap and phylogeographic analysis were performed. The three main Culex species are phylogenetically separated, and represent two distinct clades, one with Cx. tritaeniorhynchus and the second with Cx. vishnui and Cx. pseudovishnui, the latter appearing as a subgroup of Cx. vishnui. The phylogeographic analysis shows a distribution of the Vishnui subgroup on the entire Cambodian territory with an overlapped distribution areas leading to a sympatric distribution of these species. The three JEV vector species are geographically well-defined with a strong presence of Cx. pseudovishnui in the forest. Combined with the presence of Cx. tritaeniorhynchus and Cx. vishnui in rural, peri-urban, and urban areas, the presence of JEV-competent vectors is widespread in Cambodia.

Spread of NDM-5 and OXA-181 Carbapenemase-Producing Escherichia coli in Chad.

We detected for the first time blaNDM-5 and blaOXA-181 in Escherichia coli isolates from hospitalized patients and healthy volunteers in Chad. These resistance genes were located on IncX3 and IncF plasmids. Despite the large diversity of E. coli clones, the identified resistant intestinal isolates belonged mainly to the same sequence type.

High prevalence and characterization of extended-spectrum ß-lactamase producing Enterobacteriaceae in Chadian hospitals.

BACKGROUND: Extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) represent a major problem in the management of nosocomial infections. However, ESBL-PE are not systematically monitored in African countries. The aim of this study was to determine ESBL-PE prevalence in patients from three hospitals in N'Djamena, the capital city of Chad, and to characterize the genetic origin of the observed resistance. METHODS: From January to March 2017, 313 non-duplicate isolates were recovered from various clinical specimens obtained from 1713 patients in the three main hospitals of N'Djamena. Bacterial species were identified by matrix-assisted laser desorption ionization-time of flight mass spectrometry. Susceptibility to 28 antibiotics was tested using the disk diffusion method on Müller-Hinton agar, and ESBL production was confirmed with the double-disc synergy test. The most prevalent ESBL genes associated with the observed resistance were detected using multiplex PCR followed by double-stranded DNA sequencing. RESULTS: Among the 313 isolates, 197 belonged to the Enterobacteriaceae family. The overall ESBL-PE prevalence was 47.72% (n = 94/197), with a higher rate among inpatients compared with outpatients (54.13% vs. 34.37%). ESBL-PE prevalence was highest in older patients (≥60 years of age). E. coli was the most common ESBL-producer organism (63.8%), followed by K. pneumoniae (21.2%). ESBL-PE were mainly found in urine samples (75%). The CTX-M-1 group was dominant (96.7% of the 94 ESBL-PE isolates, CTX-M-15 enzyme), followed by the CTX-M-9 group (4.1%). 86% of resistant isolates harbored more than one ESBL-encoding gene. ESBL production was also associated with the highest levels of resistance to non-ß-lactam drugs. CONCLUSIONS: The prevalence of ESBL-PE harboring resistant genes encoding ESBLs of the CTX-M-1 group was high (48%) among clinical isolates of three main hospitals in Chad, suggesting an alarming spread of ESBL-PE among patients.

Colistin resistance in ESBL- and Carbapenemase-producing Escherichia coli and Klebsiella pneumoniae clinical isolates in Cambodia.

OBJECTIVES: Despite the critical importance of colistin as a last-resort antibiotic, limited studies have investigated colistin resistance in human infections in Cambodia. This study aimed to investigate the colistin resistance and its molecular determinants among Extended-spectrum beta-lactamase (ESBL)- and carbapenemase-producing (CP) Klebsiella pneumoniae (K. pneumoniae) and Escherichia coli (E. coli) isolated in Cambodia between 2016 and 2020. METHODS: E. coli (n = 223) and K. pneumoniae (n = 39) were tested for colistin minimum inhibitory concentration (MIC) by broth microdilution. Resistant isolates were subjected to polymerase chain reaction (PCR) for detection of mobile colistin resistance genes (mcr) and chromosomal mutations in the two-component system (TCS). RESULTS: Eighteen isolates (10 K. pneumoniae and 8 E. coli) revealed colistin resistance with a rate of 5.9% in E. coli and 34.8% in K. pneumoniae among ESBL isolates, and 1% in E. coli and 12.5% in K. pneumoniae among CP isolates. The resistance was associated with mcr variants (13/18 isolates, mcr-1, mcr-3, and mcr-8.2) and TCS mutations within E. coli and K. pneumoniae, with the first detection of mcr-8.2 in Cambodia, the discovery of new mutations potentially associated to colistin resistance in the TCS of E. coli (PhoP I47V, PhoQ N352K, PmrB G19R, and PmrD G85R) and the co-occurrence of mcr genes and colistin resistance conferring TCS mutations in 11 of 18 isolates. CONCLUSIONS: The findings highlight the presence of colistin resistance in ESBL- and CP- Enterobacteriaceae involved in human infections in Cambodia as well as chromosomal mutations in TCS and the emergence of mcr-8.2 in E. coli and K. pneumoniae. It underscores the need for continuous surveillance, antimicrobial stewardship, and control measures to mitigate the spread of colistin resistance.

Speciation patterns of Aedes mosquitoes in the Scutellaris Group: a mitochondrial perspective.

The Scutellaris Group of Aedes comprises 47 mosquito species, including Aedes albopictus. While Ae. albopictus is widely distributed, the other species are mostly found in the Asia-Pacific region. Evolutionary history researches of Aedes species within the Scutellaris Group have mainly focused on Ae. albopictus, a species that raises significant public health concerns, neglecting the other species. In this study, we aimed to assess genetic diversity and estimate speciation times of several species within the Scutellaris Group. Mosquitoes were therefore collected from various Asia-Pacific countries. Their mitochondrial cytochrome c oxidase subunit 1 (cox1) and subunit 3 (cox3) sequences were analyzed alongside those of other Scutellaris Group species available in the GenBank database. To estimate the divergence time, we analyzed 1849 cox1 gene sequences from 21 species, using three species (Aedes aegypti, Aedes notoscriptus and Aedes vigilax) as outgroups. We found that most of the speciation dates occurred during the Paleogene and the Neogene periods. A separation between the Scutellaris Subgroup and the Albopictus Subgroup occurred approximately 64-61 million years ago (MYA). We also identified a split between species found in Asia/Micronesia and those collected in Melanesia/Polynesia approximately 36-35 MYA. Our findings suggest that the speciation of Aedes species within the Scutellaris Group may be driven by diversity in mammalian hosts, climate and environmental changes, and geological dynamics rather than human migration.

Asymptomatic Leishmania infantum infection in dogs and dog owners in an endemic area in southeast France.

The prevalence of asymptomatic leishmaniasis in dogs and their owners in the main endemic areas of France has not been studied to date. The objective of this study was to quantify asymptomatic Leishmania infantum infection in southeast France in healthy people and their dogs using molecular and serological screening techniques. We examined the presence of parasitic DNA using specific PCR targeting kinetoplast DNA (kDNA) and specific antibodies by serology (ELISA for dogs and Western blot for humans) among immunocompetent residents and their dogs in the Alpes-Maritimes. Results from 343 humans and 607 dogs were included. 46.9% (n = 161/343) of humans and 18.3% (n = 111/607) of dogs were PCR positive; 40.2% of humans (n = 138/343) and 9.9% of dogs (n = 60/607) were serology positive. Altogether, 66.2% of humans (n = 227) and 25.7% of dogs (n = 156) had positive serologies and/or positive PCR test results. Short-haired dogs were more frequently infected (71.8%, n = 112) than long-haired dogs (12.2%, n = 19) (p = 0.043). Dogs seemed to be more susceptible to asymptomatic infection according to their breed types (higher infection rates in scenthounds, gun dogs and herding dogs) (p = 0.04). The highest proportion of dogs and human asymptomatic infections was found in the Vence Region, corresponding to 28.2% (n = 20/71) of dogs and 70.5% (n = 31/44) of humans (4.5/100,000 people). In conclusion, the percentage of infections in asymptomatic humans is higher than in asymptomatic dogs in the studied endemic area. It is questionable whether asymptomatic infection in humans constitutes a risk factor for dogs.

Title: Infection asymptomatique à Leishmania infantum chez les chiens et propriétaires de chiens dans une zone endémique du sud-est de la France. Abstract: La prévalence de la leishmaniose asymptomatique chez les chiens et leurs propriétaires dans les principales zones d'endémie françaises n'a pas été étudiée à ce jour. L'objectif de cette étude était de quantifier l'infection asymptomatique à Leishmania infantum dans le sud-est de la France chez des personnes saines et leurs chiens à l'aide de techniques de dépistage moléculaire et sérologique. Nous avons examiné chez des résidents immunocompétents et leurs chiens dans les Alpes-Maritimes la présence d'ADN parasitaire par PCR spécifique ciblant l'ADN du kinétoplaste (ADNk) et d'anticorps spécifiques par sérologie (ELISA pour le chien et Western Blot pour l'homme). Les résultats de 343 humains et 607 chiens ont été inclus; 46,9 % (n = 161/343) des humains et 18,3 % (n = 111/607) des chiens étaient positifs à la PCR et 40,2 % des humains (n = 138/343) et 9,9 % des chiens (n = 60/607) avaient une sérologie positive. Au total, 66,2 % des humains (n = 227) et 25,7 % des chiens (n = 156) avaient des sérologies positives et/ou des résultats de tests PCR positifs. Les chiens à poils courts étaient plus fréquemment infectés (71,8 %, n = 112) que les chiens à poils longs (12,2 %, n = 19) (p = 0,043). Les chiens semblaient plus sensibles à l'infection asymptomatique selon leurs races (taux supérieurs chez les chiens de chasse et chiens de berger) (p = 0,04). La plus forte proportion d'infections asymptomatiques chez les chiens et les humains a été observée dans la Région de Vence, correspondant à 28,2 % (n = 20/71) des chiens et 70,5 % (n = 31/44) des humains (4,5/100 000). personnes). En conclusion, le pourcentage d'infections chez les humains asymptomatiques est plus élevé que chez les chiens asymptomatiques dans la zone d'endémie étudiée. On peut se demander si une infection asymptomatique chez l'homme constitue un facteur de risque pour les chiens.

Genomic insights into anthropozoonotic tuberculosis in captive sun bears (Helarctos malayanus) and an Asiatic black bear (Ursus thibetanus) in Cambodia.

Contact between humans and wildlife presents a risk for both zoonotic and anthropozoonotic disease transmission. In this study we report the detection of human strains of Mycobacterium tuberculosis in sun bears and an Asiatic black bear in a wildlife rescue centre in Cambodia, confirming for the first time the susceptibility of these bear species to tuberculosis when in close contact with humans. After genotyping revealed two different strains of M. tuberculosis from cases occurring between 2009 and 2019, 100 isolates from 30 sun bear cases, a single Asiatic black bear case, and a human case were subjected to whole genome sequencing. We combined single nucleotide polymorphism analysis and exploration of mixed base calls with epidemiological data to indicate the evolution of each outbreak. Our results confirmed two concurrent yet separate tuberculosis outbreaks and established a likely transmission route in one outbreak where the human case acted as an intermediatory between bear cases. In both outbreaks, we observed high rates of transmission and progression to active disease, suggesting that sun bears are highly susceptible to tuberculosis if exposed under these conditions. Overall, our findings highlight the risk of bi-directional transmission of tuberculosis between humans and captive bears in high human tuberculosis burden regions, with implied considerations for veterinary and public health. We also demonstrate the use of standard genomic approaches to better understand disease outbreaks in captive wildlife settings and to inform control and prevention measures.

"Everything you always wanted to know about sex (but were afraid to ask)" in Leishmania after two decades of laboratory and field analyses.

Leishmaniases remain a major public health problem today (350 million people at risk, 12 million infected, and 2 million new infections per year). Despite the considerable progress in cellular and molecular biology and in evolutionary genetics since 1990, the debate on the population structure and reproductive mode of Leishmania is far from being settled and therefore deserves further investigation. Two major hypotheses coexist: clonality versus sexuality. However, because of the lack of clear evidence (experimental or biological confirmation) of sexuality in Leishmania parasites, until today it has been suggested and even accepted that Leishmania species were mainly clonal with infrequent genetic recombination (see [1] for review). Two recent publications, one on Leishmania major (an in vitro experimental study) and one on Leishmania braziliensis (a population genetics analysis), once again have challenged the hypothesis of clonal reproduction. Indeed, the first study experimentally evidenced genetic recombination and proposed that Leishmania parasites are capable of having a sexual cycle consistent with meiotic processes inside the insect vector. The second investigation, based on population genetics studies, showed strong homozygosities, an observation that is incompatible with a predominantly clonal mode of reproduction at an ecological time scale (approximately 20-500 generations). These studies highlight the need to advance the knowledge of Leishmania biology. In this paper, we first review the reasons stimulating the continued debate and then detail the next essential steps to be taken to clarify the Leishmania reproduction model. Finally, we widen the discussion to other Trypanosomatidae and show that the progress in Leishmania biology can improve our knowledge of the evolutionary genetics of American and African trypanosomes.

Extreme inbreeding in Leishmania braziliensis.

Leishmania species of the subgenus Viannia and especially Leishmania braziliensis are responsible for a large proportion of New World leishmaniasis cases. The reproductive mode of Leishmania species has often been assumed to be predominantly clonal, but remains unsettled. We have investigated the genetic polymorphism at 12 microsatellite loci on 124 human strains of Leishmania braziliensis from 2 countries, Peru and Bolivia. There is substantial genetic diversity, with an average of 12.4 +/- 4.4 alleles per locus. There is linkage disequilibrium at a genome-wide scale, as well as a substantial heterozygote deficit (more than 50% the expected value from Hardy-Weinberg equilibrium), which indicates high levels of inbreeding. These observations are inconsistent with a strictly clonal model of reproduction, which implies excess heterozygosity. Moreover, there is large genetic heterogeneity between populations within countries (Wahlund effect), which evinces a strong population structure at a microgeographic scale. Our findings are compatible with the existence of population foci at a microgeographic scale, where clonality alternates with sexuality of an endogamic nature, with possible occasional recombination events between individuals of different genotypes. These findings provide key clues on the ecology and transmission patterns of Leishmania parasites.

Non-toxigenic Corynebacterium diphtheriae in hallux ulceration.

INTRODUCTION: Toxigenic Corynebacterium diphtheriae causes classical diphtheria. Skin infections by toxigenic or non-toxigenic Corynebacterium diphtheriae are prevalent in the tropics but are rarely reported. CASE PRESENTATION: We report the identification of a non-toxigenic Corynebacterium diphtheriae (biovar Gravis) isolate in a 52-year-old Cambodian male. The patient presented purulent and non-healing ulcerations on the right hallux. The wound has healed after 7 days of antibiotic therapy with a favourable outcome. CONCLUSIONS: This case represents, to our knowledge, the first report of Corynebacterium diphtheriae in Cambodia in the last 10 years, and highlights the lack of diagnosis and notifications of diphtheria. It is important to raise awareness among clinicians and to set up diphtheria surveillance in Cambodia.

Reproductive strategies and population structure in Leishmania: substantial amount of sex in Leishmania Viannia guyanensis.

Leishmania species of the subgenus Viannia and especially Leishmania Viannia guyanensis are responsible for a large proportion of New World leishmaniasis cases. Since a recent publication on Leishmania Viannia braziliensis, the debate on the mode of reproduction of Leishmania parasites has been reopened. A predominant endogamic reproductive mode (mating with relatives), together with strong Wahlund effects (sampling of strains from heterogeneous subpopulations), was indeed evidenced. To determine whether this hypothesis can be generalized to other Leishmania Viannia species, we performed a population genetic study on 153 human strains of L. (V.) guyanensis from French Guiana based on 12 microsatellite loci. The results revealed important homozygosity and very modest linkage disequilibrium, which is in agreement with a high level of sexual recombination and substantial endogamy. These results also revealed a significant isolation by distance with relatively small neighbourhoods and hence substantial viscosity of Leishmania populations in French Guiana. These results are of epidemiological relevance and suggest a major role for natural hosts and/or vectors in parasite strain diffusion across the country as compared to human hosts.

Epidemiology and prevalence of extended-spectrum ß-lactamase- and carbapenemase-producing Enterobacteriaceae in humans, animals and the environment in West and Central Africa.

Extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-E) and carbapenemase-producing Enterobacteriaceae (CPE) are widespread. Here we used the 'One Health' approach to determine knowledge gaps on ESBL-E and CPE in West and Central Africa. We searched all articles on ESBL-E and CPE in these African regions published in PubMed, African Journals Online and Google Scholar from 2000 onwards. Among the 1201 articles retrieved, we selected 165 studies (West Africa, 118; Central Africa, 47) with data from 22 of the 26 West and Central Africa countries. Regarding the settings, 136 articles focused only on humans (carriage and/or infection), 6 articles on humans and animals, 13 on animals, 1 on humans and the environment, 8 on the environment and 1 on humans, animals and environments. ESBL-E prevalence ranged from 11-72% in humans and 7-79% in aquatic environments (wastewater). In animals, ESBL-E prevalence hugely varied: 0% in cattle, 11-36% in chickens, 20% in rats, 21-71% in pigs and 32-75% in dogs. The blaCTX-M-15 gene was the predominant ESBL-encoding gene and was associated with plasmids of incompatibility groups F, H, K, Y, N, I1 and R. CPE were studied only in humans. Class B metallo-ß-lactamases (NDM) and class D oxacillinases (OXA-48 and OXA-181) were the most common carbapenemases. Our results show major knowledge gaps, particularly on ESBL and CPE in animals and the environment, that might limit antimicrobial resistance management in these regions. The results also emphasise the urgent need to improve active surveillance programmes in each country and to support antimicrobial stewardship.

Resistance to Second-Line Anti-TB Drugs in Cambodia: A Phenotypic and Genetic Study.

BACKGROUND: Due to the emergence of Mycobacterium tuberculosis (M.tb) clinical isolates resistant to most potent first-line drugs (FLD), second-line drugs (SLD) are being prescribed more frequently. We explore the genetic characteristics and molecular mechanisms of M.tb isolates phenotypically resistant to SLD, including pre-extensively drug-resistant (pre-XDR) and extensively drug-resistant (XDR) isolates. METHODS: Drug-resistant (DR) M.tb isolates collected from 2012 to 2017 were tested using sequencing and phenotypic drug susceptibility testing. Genotypes were determined to explore their links with SLD resistance patterns. RESULTS: Of the 272 DR M.tb isolates, 6 non-multidrug resistant (non-MDR) isolates were fluoroquinolones (FQ)-resistant, 3 were XDR and 16 were pre-XDR (14 resistant to FQ and 2 to second-line injectable drugs). The most frequent mutations in FQ-resistant and second-line injectable drugs resistant isolates were gyrA D94G (15/23) and rrs a1401g (3/5), respectively. Seventy-five percent of pre-XDR isolates and 100% of XDR isolates harbored mutations conferring resistance to pyrazinamide. All XDR isolates belonged to the Beijing genotype, of which one, named XDR+, was resistant to all drugs tested. One cluster including pre-XDR and XDR isolates was observed. CONCLUSION: This is the first description of SLD resistance in Cambodia. The data suggest that the proportion of XDR and pre-XDR isolates remains low but is on the rise compared to previous reports. The characterization of the XDR+ isolate in a patient who refused treatment underlines the risk of transmission in the population. In addition, genotypic results show, as expected, that the Beijing family is the main involved in pre-XDR and XDR isolates and that the spread of the Beijing pre-XDR strain is capable of evolving into XDR strain. This study strongly indicates the need for rapid interventions in terms of diagnostic and treatment to prevent the spread of the pre-XDR and XDR strains and the emergence of more resistant ones.

A case of high altitude cutaneous leishmaniasis in a non-endemic region in Nepal.

A case of cutaneous leishmaniasis was discovered in a 32-year old man with a persistent erythematous plaque. The patient resides in a high altitude (~2000 m above sea level) area that is not endemic for cutaneous leishmaniasis in the Dunai village of Dolpa, Nepal. The patient's lesion was initially misdiagnosed as lupus vulgaris. After response failure to initial treatment, additional testing by histological microscopy revealed the presence of Leishmania amastigotes in tissue from the lesion, and the diagnosis of cutaneous leishmaniasis was confirmed by nested PCR DNA assay of tissue from the lesion, and by a positive rK39 test in blood. Sequencing of the kinetoplast region confirmed the presence of Leishmania donovani complex. The patient responded well to treatments for cutaneous leishmaniasis and the skin lesions regressed after 6 months. This is the first known case of cutaneous leishmaniasis in a patient in Nepal who resides at high altitude in a non-endemic region. Increasing temperatures in this region of Nepal may be expanding the range of vectors that transmit cutaneous leishmaniasis.

Development of species-specific PCR and PCR-restriction fragment length polymorphism assays for L.infantum/L.donovani discrimination.

Discrimination of Leishmania infantum and L. donovani, the members of the L. (L.) donovani complex, is important for diagnosis and epidemiological studies of visceral leishmaniasis (VL). We have developed two molecular tools including a restriction fragment length polymorphisms of amplified DNA (PCR-RFLP) and a PCR that are capable to discriminate L. donovani from L. infantum. Typing of the complex was performed by a simple PCR of cysteine protease B (cpb) gene followed by digestion with DraIII. The enzyme cuts the 741-bp amplicon of L. donovani into 400 and 341 bp fragments whereas the 702 bp of L. infantum remains intact. The designed PCR species-specific primer pair is specific for L. donovani and is capable of amplifying a 317 bp of 3' end of cpb gene of L. donovani whereas it does not generate an amplicon for L. infantum. The species-specific primers and the restriction enzyme were designed based on a 39 bp insertion/deletion (indel) in the middle of the cpb gene. Both assays could differentiate correctly the two species and are reliable and high-throughput alternatives for molecular diagnosis and epidemiological studies of VL in various foci.

Phlebotomus perfiliewi transcaucasicus is circulating both Leishmania donovani and L. infantum in northwest Iran.

Leishmania infantum is the causative agent of infantile visceral leishmaniasis (IVL) in the Mediterranean Basin and, based on isoenzyme typing of the parasite isolated from dogs; this parasite was considered to predominate in the all foci of IVL in Iran. However, based on PCR detection and sequencing of parasite Cysteine Protease B (CPB), only one out of seven sandfly infections in Phlebotomus perfiliewi transcaucasicus was found to be L. infantum in the current investigation. The six other infections were haplotypes of Leishmania donovani, the causative agent of anthroponotic visceral leishmaniasis (AVL) in West Africa and India. The deduced amino acid of the L. donovani haplotype was found to be novel and the shortest CPB protein reported within the Leishmania spp. Circulation of both L. donovani and L. infantum by P. perfiliewi transcaucasicus, in addition to previous data indicating its ability to circulate L. tropica, suggests that this species, like other vectors of VL, is a permissive vector. Finding L. donovani infecting P. perfiliewi transcaucasicus in the area demands extensive and intensive typing of natural Leishmania infections in epidemiological investigations in Iran and the Mediterranean Basin in general.

Fecal carriage of extended-spectrum ß-lactamase-producing Enterobacteriaceae in hospital and community settings in Chad.

Background: Fecal carriage of extended-spectrum ß-lactamase-producing Enterobacteriaceae (ESBL-PE) remains poorly documented in Africa. The objective of this study was to determine the prevalence of ESBL-PE fecal carriage in Chad. Methods: In total, 200 fresh stool samples were collected from 100 healthy community volunteers and 100 hospitalized patients from January to March 2017. After screening using ESBL-selective agar plates and species identification by MALDI-TOF mass spectrometry, antibiotic susceptibility was tested using the disk diffusion method, and ESBL production confirmed with the double-disc synergy test. The different ESBL genes in potential ESBL-producing isolates were detected by PCR and double stranded DNA sequencing. Escherichia coli phylogenetic groups were determined using a PCR-based method. Results: ESBL-PE fecal carriage prevalence was 44.5% (51% among hospitalized patients vs 38% among healthy volunteers; p < 0.05). ESBL-producing isolates were mostly Escherichia coli (64/89) and Klebsiella pneumoniae (16/89). PCR and sequencing showed that 98.8% (87/89) of ESBL-PE harbored blaCTX-M genes: blaCTX-M-15 in 94.25% (82/87) and blaCTX-M-14 in 5.75% (5/87). Phylogroup determination by quadruplex PCR indicated that ESBL-producing E. coli isolates belonged to group A (n = 17; 27%), C (n = 17; 27%), B2 (n = 9; 14%), B1 (n = 8; 13%), D (n = 8; 13%), E (n = 1; 1.6%), and F (n = 1; 1.6%). The ST131 clone was identified in 100% (9/9) of E. coli B2 strains. Conclusions: The high fecal carriage rate of ESBL-PE associated with CTX-M-15 in hospital and community settings of Chad highlights the risk for resistance transmission between non-pathogenic and pathogenic bacteria.

Leishmania and the leishmaniases: a parasite genetic update and advances in taxonomy, epidemiology and pathogenicity in humans.

Leishmaniases remain a major public health problem today despite the vast amount of research conducted on Leishmania pathogens. The biological model is genetically and ecologically complex. This paper explores the advances in Leishmania genetics and reviews population structure, taxonomy, epidemiology and pathogenicity. Current knowledge of Leishmania genetics is placed in the context of natural populations. Various studies have described a clonal structure for Leishmania but recombination, pseudo-recombination and other genetic processes have also been reported. The impact of these different models on epidemiology and the medical aspects of leishmaniases is considered from an evolutionary point of view. The role of these parasites in the expression of pathogenicity in humans is also explored. It is important to ascertain whether genetic variability of the parasites is related to the different clinical expressions of leishmaniasis. The review aims to put current knowledge of Leishmania and the leishmaniases in perspective and to underline priority questions which 'leishmaniacs' must answer in various domains: epidemiology, population genetics, taxonomy and pathogenicity. It concludes by presenting a number of feasible ways of responding to these questions.

Leishmania infantum amastigotes resistant to nitric oxide cytotoxicity: Impact on in vitro parasite developmental cycle and metabolic enzyme activities.

Nitric oxide (NO) has been demonstrated to be the principal effector molecule mediating intracellular killing of Leishmania. The free radical characteristic of NO prevented direct induction of resistance in Leishmania wild-type parasites. Starting from the previous observation that antimony-resistant amastigotes of Leishmania infantum were not affected by NO-induced apoptotic death, we used a continuous NO pressure protocol and succeeded in inducing NO resistance in amastigote forms of L. infantum. Two clones resistant to 50 microM (LiNOR50) and 100 microM (LiNOR100) of the NO donor DETA/NONOate, derived from parental clone weakly resistant to trivalent antimony (LiSbIIIR4), were selected and analysed. Both clones were also resistant to other NO donors, particularly SNAP. In the absence of potassium antimonyl tartrate, all clones (LiSbIIIR4, LiNOR50 and LiNOR100) lost their antimony resistance almost totally. Interestingly, the parasitic developmental life cycle of NO-resistant mutants was dramatically disturbed. NO-resistant amastigotes differentiated more rapidly into promastigotes than the wild-type ones. Nevertheless, NO-resistant amastigotes produce a maximal number of parasites 1.5-2 times lower than the wild-type whereas, after differentiation, NO-resistant promastigotes produced more cells than the wild-type. We showed that this last phenomenon could be a consequence of the overexpression of parasitic enzymes involved in both glycolysis and respiration processes. NO-resistant amastigotes overexpressed three enzymes: cis-aconitase, glyceraldehyde-3-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. The two first enzymes are NO molecular targets which could be directly involved in NO resistance and the third one could interfere in modifying Leishmania metabolism.