Population-specific variations in KCNH2 predispose patients to delayed ventricular repolarization upon dihydroartemisinin-piperaquine therapy.

Dihydroartemisinin-piperaquine is efficacious for the treatment of uncomplicated malaria and its use is increasing globally. Despite the positive results in fighting malaria, inhibition of the Kv11.1 channel (hERG; encoded by the KCNH2 gene) by piperaquine has raised concerns about cardiac safety. Whether genetic factors could modulate the risk of piperaquine-mediated QT prolongations remained unclear. Here, we first profiled the genetic landscape of KCNH2 variability using data from 141,614 individuals. Overall, we found 1,007 exonic variants distributed over the entire gene body, 555 of which were missense. By optimizing the gene-specific parametrization of 16 partly orthogonal computational algorithms, we developed a KCNH2-specific ensemble classifier that identified a total of 116 putatively deleterious missense variations. To evaluate the clinical relevance of KCNH2 variability, we then sequenced 293 Malian patients with uncomplicated malaria and identified 13 variations within the voltage sensing and pore domains of Kv11.1 that directly interact with channel blockers. Cross-referencing of genetic and electrocardiographic data before and after piperaquine exposure revealed that carriers of two common variants, rs1805121 and rs41314375, experienced significantly higher QT prolongations (ΔQTc of 41.8 ms and 61 ms, respectively, vs 14.4 ms in controls) with more than 50% of carriers having increases in QTc >30 ms. Furthermore, we identified three carriers of rare population-specific variations who experienced clinically relevant delayed ventricular repolarization. Combined, our results map population-scale genetic variability of KCNH2 and identify genetic biomarkers for piperaquine-induced QT prolongation that could help to flag at-risk patients and optimize efficacy and adherence to antimalarial therapy.

Differential Incidence of Malaria in Neighboring Villages in a High-Transmission Setting of Southern Mali.

Throughout a phase IIIb/IV efficacy study of repeated treatment with four artemisinin-based combination therapies, significant heterogeneity was found in the number of clinical episodes experienced by individuals during the 2-year follow-up. Several factors, including host, parasite, and environmental factors, may contribute to the differential malaria incidence. We aimed to identify risk factors of malaria incidence in the context of a longitudinal study of the efficacy of different artemisinin-based combination therapy regimens in Bougoula-Hameau, a high-transmission setting in Mali. Risk factors including age, residence, and treatment regimen were compared among individuals experiencing eight or more clinical episodes of malaria ("high-incidence group") and individuals experiencing up to three clinical episodes ("low-incidence group"). Consistent with the known association between age and malaria risk in high-transmission settings, individuals in the high incidence group were significantly younger than individuals in the low-risk group (mean age, 7.0 years versus 10.6 years, respectively; t-test, P < 0.0001). Compared with individuals receiving artemether-lumefantrine, those receiving artesunate-amodiaquine had greater odds of being in the high-incidence group (odds ratio [OR], 2.24; 95% CI, 1.03 - 4.83, P = 0.041), while individuals receiving dihydroartemisinin-piperaquine had a lower odds of being in high incidence group (OR: 0.30, 95% CI, 0.11-0.85; P = 0.024). Individuals residing in the forested areas of Sokourani and Karamogobougou had significantly greater odds of being in the high-incidence group compared with individuals residing in the semi-urban area of Bougoula-Hameau 1 (Karamogobougou: OR, 3.68; 95% CI, 1.46-9.31; P = 0.0059; Sokourani: OR, 11.46; 95% CI, 4.49-29.2; P < 0.0001). This study highlights the importance of fine-mapping malaria risks even at sub-district levels for targeted and customized interventions.

SELECTION OF PFCRT 76T AND PFMDR1 86Y MUTANT PLASMODIUM FALCIPARUM AFTER TREATMENT OF UNCOMPLICATED MALARIA WITH ARTESUNATE-AMODIAQUINE IN REPUBLIC OF GUINEA.

The use of Amodiaquine monotherapy is associated with the selection of molecular markers of Plasmodium falciparum resistance to chloroquine (pfcrt and pfmdr1). The decrease in sensitivity and the emergence of P. falciparum resistant to artemisinin-based combination therapy have been reported. Therefore, it is important to assess the impact of treatment of uncomplicated malaria with Artesunate-Amodiaquine (AS+AQ) on molecular markers of antimalarial resistance. We used standard World Health Organization (WHO) protocols to determine the in vivo efficacy of the combination (AS+AQ). In total, 170 subjects were included in the study. The molecular analysis focused on 168 dried blood spots. The aims were to determine the frequency of pfcrt 76T and pfmdr1 86Y mutations and the rates of reinfection using polymorphism markers msp1, msp2, and microsatellite markers (CA1, Ta87, TA99). Nested-PCR was used, followed in some cases by a restriction digestion. The level of P. falciparum clinical response was 92.9% (156/168) of Adequate Clinical and Parasitological Response (ACPR) before molecular correction and 97.0% (163/168) after molecular correction (P = 0.089). The frequency of mutation point pfcrt 76T was 76.2% (128/168) before treatment and 100% (7/7) after treatment (P = 0.1423). For the pfmdr1 mutation, the frequency was 28% (47/168) before treatment and 60% (6/10) after treatment (P = 0.1124). The rate of pfcrt 76T + pfmdr1 86Y was 22% (37/168) before and 50% (6/12) after treatment (P = 0.1465). Despite the presence of AS in the combination, AS+AQ selects for pfcrt 76T and pfmdr1 86Y mutant P. falciparum in Guinea.

Persistent Submicroscopic Plasmodium falciparum Parasitemia 72 Hours after Treatment with Artemether-Lumefantrine Predicts 42-Day Treatment Failure in Mali and Burkina Faso.

A recent randomized controlled trial, the WANECAM (West African Network for Clinical Trials of Antimalarial Drugs) trial, conducted at seven centers in West Africa, found that artemether-lumefantrine, artesunate-amodiaquine, pyronaridine-artesunate, and dihydroartemisinin-piperaquine all displayed good efficacy. However, artemether-lumefantrine was associated with a shorter interval between clinical episodes than the other regimens. In a further comparison of these therapies, we identified cases of persisting submicroscopic parasitemia by quantitative PCR (qPCR) at 72 h posttreatment among WANECAM participants from 5 sites in Mali and Burkina Faso, and we compared treatment outcomes for this group to those with complete parasite clearance by 72 h. Among 552 evaluable patients, 17.7% had qPCR-detectable parasitemia at 72 h during their first treatment episode. This proportion varied among sites, reflecting differences in malaria transmission intensity, but did not differ among pooled drug treatment groups. However, patients who received artemether-lumefantrine and were qPCR positive at 72 h were significantly more likely to have microscopically detectable recurrent Plasmodium falciparum parasitemia by day 42 than those receiving other regimens and experienced, on average, a shorter interval before the next clinical episode. Haplotypes of pfcrt and pfmdr1 were also evaluated in persisting parasites. These data identify a possible threat to the parasitological efficacy of artemether-lumefantrine in West Africa, over a decade since it was first introduced on a large scale.

Clinical evidence of the role of Methanobrevibacter smithii in severe acute malnutrition.

Gut microbial dysbiosis has been shown to be an instrumental factor in severe acute malnutrition (SAM) and particularly, the absence of Methanobrevibacter smithii, a key player in energy harvest. Nevertheless, it remains unknown whether this absence reflects an immaturity or a loss of the microbiota. In order to assess that, we performed a case-control study in Mali using a propensity score weighting approach. The presence of M. smithii was tested using quantitative PCR on faeces collected from SAM children at inclusion and at discharge when possible or at day 15 for controls. M. smithii was highly significantly associated with the absence of SAM, detected in 40.9% controls but only in 4.2% cases (p < 0.0001). The predictive positive value for detection of M. smithii gradually increased with age in controls while decreasing in cases. Among children providing two samples with a negative first sample, no SAM children became positive, while this proportion was 2/4 in controls (p = 0.0015). This data suggests that gut dysbiosis in SAM is not an immaturity but rather features a loss of M. smithii. The addition of M. smithii as a probiotic may thus represent an important addition to therapeutic approaches to restore gut symbiosis.

Draft genome and description of Negativicoccus massiliensis strain Marseille-P2082, a new species isolated from the gut microbiota of an obese patient.

Strain Marseille-P2082, an anaerobic, non-motile, asporogenous, Gram-negative, coccoid bacterium was isolated from the faeces of a 33 year-old obese French woman before bariatric surgery. The isolate exhibits 98.65% 16S rRNA gene nucleotide sequence similarity with Negativicoccus succinicivorans strain ADV 07/08/06-B-1388T, its current closest phylogenetic neighbour with standing in nomenclature. However, the dDDH relatedness between the new isolate and N. succinicivorans type strain ADV 07/08/06-B-1388T is 52.5 ± 2.7%. Strain Marseille-P2082 has a genome of 1,360,589 bp with a 51.1% G+C content. Its major fatty acids were identified as C18:1n9, C18:0 and C16:0. Based on its phenotypic, genomic and phylogenetic characteristics, strain Marseille-P2082T [= CSURP2082 (Collection de Souches de l'Unité des Rickettsies) = DSM 100853] is proposed as the type strain of the novel species Negativicoccus massiliensis sp. nov. The 16S rRNA gene sequence and whole-genome shotgun sequence have been deposited in EMBL-EBI under accession numbers LN876651 and LT700188, respectively.

Culture of Methanogenic Archaea from Human Colostrum and Milk.

Archaeal sequences have been detected in human colostrum and milk, but no studies have determined whether living archaea are present in either of these fluids. Methanogenic archaea are neglected since they are not detected by usual molecular and culture methods. By using improved DNA detection protocols and microbial culture techniques associated with antioxidants previously developed in our center, we investigated the presence of methanogenic archaea using culture and specific Methanobrevibacter smithii and Methanobrevibacter oralis real-time PCR in human colostrum and milk. M. smithii was isolated from 3 colostrum and 5 milk (day 10) samples. M. oralis was isolated from 1 milk sample. For 2 strains, the genome was sequenced, and the rhizome was similar to that of strains previously isolated from the human mouth and gut. M. smithii was detected in the colostrum or milk of 5/13 (38%) and 37/127 (29%) mothers by culture and qPCR, respectively. The different distribution of maternal body mass index according to the detection of M. smithii suggested an association with maternal metabolic phenotype. M. oralis was not detected by molecular methods. Our results suggest that breastfeeding may contribute to the vertical transmission of these microorganisms and may be essential to seed the infant's microbiota with these neglected critical commensals from the first hour of life.

Repertoire of human breast and milk microbiota: a systematic review.

Breastfeeding is a major determinant of human health. Breast milk is not sterile and ecological large-scale sequencing methods have revealed an unsuspected microbial diversity that plays an important role. However, microbiological analysis at the species level has been neglected while it is a prerequisite before understanding which microbe is associated with symbiosis or dysbiosis, and health or disease. We review the currently known bacterial repertoire from the human breast and milk microbiota using a semiautomated strategy. Total 242 articles from 38 countries, 11,124 women and 15,489 samples were included. Total 820 species were identified mainly composed of Proteobacteria and Firmicutes. We report variations according to the analytical method (culture or molecular method), the anatomical site (breast, colostrum or milk) and the infectious status (healthy control, mastitis, breast abscess, neonatal infection). In addition, we compared it with the other human repertoires. Finally, we discuss its putative origin and role in health and disease.

Differential infectivity of gametocytes after artemisinin-based combination therapy of uncomplicated falciparum malaria.

BACKGROUND: Most malaria-endemic countries use artemisinin-based combination therapy (ACT) as their first-line treatment. ACTs are known to be highly effective on asexual stages of the malaria parasite. Malaria transmission and the spread of resistant parasites depend on the infectivity of gametocytes. The effect of the current ACT regimens on gametocyte infectivity is unclear. OBJECTIVES: This study aimed to determine the infectivity of gametocytes to Anopheles gambiae following ACT treatment in the field. METHODS: During a randomised controlled trial in Bougoula-Hameau, Mali, conducted from July 2005 to July 2007, volunteers with uncomplicated malaria were randomised to receive artemether-lumefantrine, artesunate-amodiaquine, or artesunate-sulfadoxine/pyrimethamine. Volunteers were followed for 28 days, and gametocyte carriage was assessed. Direct skin feeding assays were performed on gametocyte carriers before and after ACT administration. RESULTS: Following artemether-lumefantrine treatment, gametocyte carriage decreased steadily from Day 0 to Day 21 post-treatment initiation. In contrast, for the artesunate-amodiaquine and artesunate-sulfadoxine/pyrimethamine arms, gametocyte carriage increased on Day 3 and remained constant until Day 7 before decreasing afterward. Mosquito feeding assays showed that artemether-lumefantrine and artesunate-amodiaquine significantly increased gametocyte infectivity to Anopheles gambiae sensu lato (s.l.) (p < 10-4), whereas artesunate-sulfadoxine/pyrimethamine decreased gametocyte infectivity in this setting (p = 0.03). CONCLUSION: Different ACT regimens could lead to gametocyte populations with different capacity to infect the Anopheles vector. Frequent assessment of the effect of antimalarials on gametocytogenesis and gametocyte infectivity may be required for the full assessment of treatment efficacy, the potential for spread of drug resistance and malaria transmission in the field.

Correction to: Description of Mediterraneibacter massiliensis, gen. nov., sp. nov., a new genus isolated from the gut microbiota of an obese patient and reclassification of Ruminococcus faecis, Ruminococcus lactaris, Ruminococcus torques, Ruminococcus gnavus and Clostridium glycyrrhizinilyticum as Mediterraneibacter faecis comb. nov., Mediterraneibacter lactaris comb. nov., Mediterraneibacter torques comb. nov., Mediterraneibacter gnavus comb. nov. and Mediterraneibacter glycyrrhizinilyticus comb. nov.

Subsequent to the publication of the above article, it has been noticed that the designation of the type strain is not correct. The strain referred to throughout the article as strain AT7T should be designated as strain Marseille-P2086T (= CSUR P2086T = DSM 100837T). The corrected for protologue for the species Mediterraneibacter massiliensis, represented by strain Marseille-P2086T as type strain, is given below.

Description of Mediterraneibacter massiliensis, gen. nov., sp. nov., a new genus isolated from the gut microbiota of an obese patient and reclassification of Ruminococcus faecis, Ruminococcus lactaris, Ruminococcus torques, Ruminococcus gnavus and Clostridium glycyrrhizinilyticum as Mediterraneibacter faecis comb. nov., Mediterraneibacter lactaris comb. nov., Mediterraneibacter torques comb. nov., Mediterraneibacter gnavus comb. nov. and Mediterraneibacter glycyrrhizinilyticus comb. nov.

An anaerobic isolate, strain AT7T, was cultivated from a stool sample of a morbidly obese French woman using a microbial culturomics approach. The 16S rRNA gene sequence analysis showed that strain AT7T exhibited 96% nucleotide sequence similarity with Ruminococcus torques strain JCM 6553T (= ATCC 27756T = VPI B2-51T), currently the closest related species with a validly published name. The strain was observed to be a Gram-stain positive, non-motile, asporogenous and coccobacillary-shaped bacterium. It was found to be catalase positive and oxidase negative. Its major fatty acids were identified as C16:0 (54%) and C18:1n9 (30%). The draft genome of strain AT7T is 3,069,882 bp long with 42.4% G+C content. 2925 genes were predicted, including 2867 protein-coding genes and 58 RNAs. Based on phenotypic, biochemical, phylogenetic and genomic evidence, we propose the creation of the new genus Mediterraneibacter and species, Mediterraneibacter massiliensis, that contains strain AT7T (= CSUR P2086T = DSM 100837T), and the reclassification of Ruminococcus faecis, Ruminococcus lactaris, Ruminococcus torques, Ruminococcus gnavus, Clostridium glycyrrhizinilyticum as Mediterraneibacter faecis comb. nov., with type strain Eg2T (= KCTC 5757T = JCM15917T), Mediterraneibacter lactaris comb. nov., with type strain ATCC 29176T (= VPI X6-29T), Mediterraneibacter torques comb. nov., with type strain ATCC 27756T (= VPI B2-51T), Mediterraneibacter gnavus comb. nov., with type strain ATCC 29149T (= VPI C7-9T) and Mediterraneibacter glycyrrhizinilyticus comb. nov., with type strain ZM35T (= JCM 13368T = DSM 17593T), respectively.

Draft Genome and Description of Eisenbergiella massiliensis Strain AT11T: A New Species Isolated from Human Feces After Bariatric Surgery.

A novel strain of a Gram-stain negative, non-motile, non-spore forming rod-shaped, obligate anaerobic bacterium, designated AT11T, was isolated from a stool sample of a morbidly obese woman living in Marseille, France. This bacterium was characterized using biochemical, chemotaxonomic, and phylogenetic methods. The 16S rRNA gene sequence analysis showed that strain AT11T had a 97.8% nucleotide sequence similarity with Eisenbergiella tayi strain B086562T, the closest species with standing in nomenclature. The major cellular fatty acids of the novel isolate were C16:0 followed by saturated or unsaturated C18 fatty acids (C18:1n9, C18:1n5 and C18:0). The draft genome of strain AT11T is 7,114,554 bp long with 48% G+C content. 6176 genes were predicted, including 6114 protein-coding genes and 62 were RNAs (with 2 5S rRNA genes, two 16S rRNA genes, two 23S rRNA genes, and 56 tRNA genes). The digital DNA-DNA hybridization (dDDH) relatedness between the new isolate and E. tayi strain B086562T was 23.1% ± 2.2. Based on the phenotypic, chemotaxonomic, genomic, and phylogenetic characteristics, Eisenbergiella massiliensis sp. nov., is proposed. The type strain is AT11T (= DSM 100838T = CSUR P2478T).

Hugonella massiliensis gen. nov., sp. nov., genome sequence, and description of a new strictly anaerobic bacterium isolated from the human gut.

The human gut is composed of a large diversity of microorganisms, which have been poorly described. Here, using culturomics, a new concept based on the variation in culture conditions and MALDI-TOF MS identification, we proceed to explore the microbial diversity of the complex ecosystem of the human gut. Using this approach, we isolated strain AT8T (=CSUR P2118 =  DSM 101782) from stool specimens collected from a 51-year-old obese French woman. Strain AT8T is a strictly anaerobic, nonmotile, nonspore-forming gram-positive coccus that do not exhibit catalase and oxidase activities. 16S rDNA-based identification of strain AT8T demonstrated 92% gene sequence similarity with Eggerthella lenta DSM 2243, the phylogenetically closed validly named type species. Here, we present a set of features for the strain AT8T and the description of its complete genome sequence and annotation. The 2,091,845 bp long genome has a G+C content of 63.46% and encodes1,849 predicted genes; 1,781 were protein-coding genes, and 68 were RNAs. On the basis of the characteristics reported here, we propose the creation of a new bacterial genus Hugonella gen. nov., belonging to the Eggerthellaceae family and including Hugonella massiliensis gen. nov., sp. nov., strain AT8T as the type strain.

Fournierella massiliensis gen. nov., sp. nov., a new human-associated member of the family Ruminococcaceae.

An anaerobic bacterium, strain AT2T, was isolated from the fresh stool sample of a healthy French man using the culturomics approach. The 16S rRNA gene sequence analysis showed that strain AT2T had 95.2 % nucleotide sequence similarity with Gemmiger formicilisATCC 27749T, the phylogenetically closest species with standing in nomenclature. Cells are Gram-stain-negative, catalase- and oxidase-negative, obligately anaerobic, non-motile, non-spore-forming, rod-shaped, and the bacilli were mesothermophilic. The major fatty acids were C16 : 0 (43.8 %) and C18 : 1n9 (20 %). The DNA G+C content of the strain based on its genome sequence was 56.8 mol%. Based on the phenotypic, biochemical and phylogenetic analysis, we propose the creation of the genus Fournierella gen. nov., which contains strain AT2T (=CSUR P2014T=DSM 100451T) as the type strain of the type species Fournierella massiliensis gen. nov., sp. nov.

Culture of previously uncultured members of the human gut microbiota by culturomics.

Metagenomics revolutionized the understanding of the relations among the human microbiome, health and diseases, but generated a countless number of sequences that have not been assigned to a known microorganism1. The pure culture of prokaryotes, neglected in recent decades, remains essential to elucidating the role of these organisms2. We recently introduced microbial culturomics, a culturing approach that uses multiple culture conditions and matrix-assisted laser desorption/ionization-time of flight and 16S rRNA for identification2. Here, we have selected the best culture conditions to increase the number of studied samples and have applied new protocols (fresh-sample inoculation; detection of microcolonies and specific cultures of Proteobacteria and microaerophilic and halophilic prokaryotes) to address the weaknesses of the previous studies3-5. We identified 1,057 prokaryotic species, thereby adding 531 species to the human gut repertoire: 146 bacteria known in humans but not in the gut, 187 bacteria and 1 archaea not previously isolated in humans, and 197 potentially new species. Genome sequencing was performed on the new species. By comparing the results of the metagenomic and culturomic analyses, we show that the use of culturomics allows the culture of organisms corresponding to sequences previously not assigned. Altogether, culturomics doubles the number of species isolated at least once from the human gut.

Repeated artemisinin-based combination therapies in a malaria hyperendemic area of Mali: efficacy, safety, and public health impact.

Artemisinin-based combination therapies (ACTs) are the first-line treatment of uncomplicated malaria. The public health benefit and safety of repeated administration of a given ACT are poorly studied. We conducted a randomized trial comparing artemether-lumefantrine, artesunate plus amodiaquine (AS+AQ) and artesunate plus sulfadoxine-pyrimethamine (AS+SP) in patients 6 months of age and older with uncomplicated malaria in Mali from July 2005 to July 2007. The patient received the same initial treatment of each subsequent uncomplicated malaria episode except for treatment failures where quinine was used. Overall, 780 patients were included. Patients in the AS+AQ and AS+SP arms had significantly less risk of having malaria episodes; risk ratio (RR) = 0.84 (P = 0.002) and RR = 0.80 (P = 0.001), respectively. The treatment efficacy was similar and above 95% in all arms. Although all drugs were highly efficacious and well tolerated, AS+AQ and AS+SP were associated with less episodes of malaria.