Wolbachia symbionts infect D. melanogaster populations worldwide. Infection rates vary greatly. Genetic diversity of Wolbachia in D. melanogaster can be subdivided into several closely related genotypes coinherited with certain mtDNA lineages. mtDNA haplotypes have the following global distribution pattern: mtDNA clade I is mostly found in North America, II and IV in Africa, III in Europe and Africa, V in Eurasia, VI is global but very rare, and VIII is found in Asia. The wMel Wolbachia genotype is predominant in D. melanogaster populations. However, according to the hypothesis of global Wolbachia replacement, the wMelCS genotype was predominant before the XX century when it was replaced by the wMel genotype. Here we analyse over 1500 fly isolates from the Palearctic region to evaluate the prevalence, genetic diversity and distribution pattrern of the Wolbachia symbiont, occurrence of mtDNA variants, and finally to discuss the Wolbachia genotype global replacement hypothesis.Maternally inherited D. melanogaster were infected with Wolbachia at a rate of 33–100%. We did not observe any significant correlation between infection rate and longitude or latitude. Five previously reported Wolbachia genotypes were found in Palearctic populations with a predominance of the wMel variant. The mtDNA haplotypes of the I_II_III clade and V clade were prevalent in Palearctic populations. To test the recent Wolbachia genotype replacement hypothesis, we examined three genomic regions of CS-like genotypes. Low genetic diversity was observed, only two haplotypes of the CS genotypes with a ‘CCG’ variant predominance were found.All studied Palearctic populations of Wolbachia infection prevalence and genotype diversity in Palearctic D. melanogaster populations confirm previous studies. Wolbachia is ubiquitous in the Palearctic region. The wMel genotype is dominant with local occurrence of rare genotypes. Together with variants of the V mtDNA clade, the variants of the ‘III+’ clade are dominant in both infected and uninfected flies of Palearctic populations. Based on our data on Wolbachia and mtDNA in different years in some Palearctic localities, we can conclude that flies that survive the winter make the predominant symbiont contribution to the subsequent generation. A comprehensive overview of mtDNA and Wolbachia infection of D. melanogaster populations worldwide does not support the recent global Wolbachia genotype replacement hypothesis. However, we cannot exclude wMelCS genotype rate fluctuations in the past.The results of our survey of The online version of this article (10.1186/s12862-019-1372-9) contains supplementary material, which is available to authorized users. Wolbachia genus are widespread across Drosophila species [Drosophila spp. are popular model organisms for studies of different aspects of Wolbachia biology including microevolution and population dynamics [Wolbachia can affect the Drosophila host in different ways including male killing in D. bifasciata and D. innubila [D. paulistorum, D. simulans and D. melanogaster [Bacteria of the  species , 2. Drosdynamics . Wolbachinnubila , 10, cytnogaster , 11, 12,nogaster , 14, nutnogaster , fecundinogaster  and suppnogaster –19.Wolbachia strains in different Drosophila hosts are non-related. There are, five genetically distant Wolbachia strains in D. simulans, which differ in geographical distribution and CI expression [siI, siII and siIII) [Wolbachia strains have been recently harboured by D. simulans [Many pression , 20, 21.d siIII) , which sD. melanogaster, the single Wolbachia strain, wMel, has been described based on the analysis of some housekeeping genes [Wolbachia genomes in different D. melanogaster strains have revealed differences in chromosomal rearrangements, variation in indels and repetitive sequences [Wolbachia clades I-V and VIII, and the CS genotypes correspond to clade VI [In ng genes –26. Studequences –31. Thesequences , 32. Furequences , 33, 34.clade VI , 32, 34.D. simulans [D. melanogaster mitochondrial DNA is low. Only analysing Wolbachia polymorphisms as genetic marker of maternal inheritance and genome sequence analyses of many D. melanogaster isolates have provided valuable data on D. melanogaster mtDNA variation. D. melanogaster mtDNA and the wMel Wolbachia strain are strictly coinherited. mtDNA lineages have been designated as M- or S-clade according to polymorphisms of some loci [Wolbachia of MEL group/I-V and VIII clades are associated with mtDNA of M/I-V and VIII clades, and Wolbachia of the CS group/VI clade are associated with mtDNA of the S/VI clade. Phylogenetic analyses revealed the divergence of the wMel Wolbachia strain and D. melanogaster mtDNA from a common ancestor several thousand years ago [In comparison with simulans , 22, theome loci , 35, andome loci , 33, 34.ears ago , 32–34.D. melanogaster maternal lineages. The MEL-group genotypes and M-clade mtDNA of uninfected flies have been found in D. melanogaster populations all over the world [Wolbachia group and mtDNA includes lineages of I-V and VIII clades (both mtDNA and Wolbachia) that have different geographical distributions. The I and III clades of mtDNA and Wolbachia seem to spread across all continents [Wolbachia. This clade is found all over the world, but its frequency is very low. Previously, an additional mtDNA clade was proposed (clade VII), which is associated with wMelCS2 genotype and distributed in the Palearctic  [D. melanogaster mtDNA and Wolbachia from South America and Australia and many territories in Eurasia are lacking.There is a global geographic pattern of he world , 36, 37.ntinents –34. The ntinents , 33, 38.Siberia) , 37. HowSiberia)  did not Wolbachia variants of different clades affect D. melanogaster environmental adaptation [D. simulans, Wolbachia in D. melanogaster induce no/low CI or strong CI in the case of ‘young males’ [Wolbachia density in D. melanogaster populations are unclear because the main factors affecting symbiont spreading are weak, and others, such as protection from viral infections and mutation suppression could only have a localised effect.There are data on how aptation , 38–40. g males’ , 41, 42.Wolbachia and mtDNA prevalence in fly populations across the vast Palearctic region. In particular, we were interested in the frequency and distribution of Wolbachia genotypes and mtDNA variants in Palearctic fly populations. We confirmed previous data on widespread Wolbachia infection, high infection rates and predominance of the wMel Wolbachia genotype in D. melanogaster populations, and show 1) low genetic diversity of CS Wolbachia genotypes, 2) predominance of two mtDNA clades in Palearctic D. melanogaster populations and 3) overwintering flies in urbanized localities. Based on our results, we conclude that global Wolbachia genotype replacement has not occurred in the recent past.In the present study, we evaluated D. melanogaster samples from 12 Palearctic regions  collected between 1974 and 2015  Wolbachia prevalence and genotype diversity (n = 1251), ii) genetic diversity of CS-genotypes (n = 22 including nine long-term storage mutant stocks); iii) M/S clades of mtDNA  I-VIII mtDNA clades (n = 143) , WlonF: 5′-CAAGTGATGATCCGTAAAGT-3′, WlonR: 5′-GGCATAGAGAAAGTAAAAAGA-3′ (297780–298135 region), WmaeBF: 5′-CTGTGTGATAAGCAAGGAGT-3′, WmaeBR: 5′-TGGGTCAAATGGAGTAGGTA-3′ (469653–470116 region).DNA extraction was performed according to Ilinsky . Wolbachwsp gene  and 99F/RNA gene . Wolbachr et al. . Accordin et al. , Chrosten et al.  and Versn et al. , we founD. melanogaster mtDNA by PCR with specific primers [D. melanogaster mtDNA. In other words, they are markers of M- and S-clades [M-mitotype (n = 111) were randomly chosen from both infected and uninfected flies with the addition of two samples harbouring wMel2. All available S-mitotype samples (n = 32) were analysed.All samples were analysed for 2187C/T variants of  primers . These vS-clades . The 343S-clades  and sequAmplicons were purified using a Zymoclean™ Gel DNA Recovery Kit  according to the manufacturer’s instructions, and sequenced by BigDye® Terminator v3.1 cycle sequencing kit (Applied Biosystems). Sequences were deposited in GenBank under accession numbers MG197842 – MG197984 for mtDNA analysis, and MG241453 – MG241491, MH010806 – MH010832  with the AtteStat 12.0.5 add-in.Wolbachia prevalence, the 1251 D. melanogaster samples were examined. Wolbachia were found in all studied D. melanogaster Palearctic populations in the range of 0.33–1.0, with an average of 0.56  over a two-year sampling period. There were also no differences in populations over four years in Nalchik including data from Bykov et al. [P = 0.35), or over a two-year period in Sakhalin . The population of Izobilnoe (Crimea), which lives on the grape seed dump of the winery industry, had the greatest density of flies. The grape seed piles were swarmed with flies, and we assumed the population numbered at least hundreds of thousands of D. melanogaster individuals within a limited area. Wolbachia prevalence in this population was not different from the average prevalence rate of Palearctic populations .To estimate v et al.   to check any facts of mtDNA/Wolbachia coinheritance disorders, which would indicate horizontal transmission of Wolbachia or mtDNA paternal passing, and ii) to compare M/S ratios in infected vs. uninfected flies, which would indirectly indicate the origin of uninfected flies.Samples of mtDNA from infected and uninfected Wolbachia coinheritance disorders were revealed. All samples infected with Wolbachia of the MEL genotype were M-mitotype, and all samples from the CS group were S-mitotype. In uninfected samples, the M-mitotype was found in all localities, whereas the S-mitotype was only found in North Caucasus and the Far East , which statistically indicates the recent loss of the infection by ancestors of S-mitotype uninfected flies. These results confirm the previous data of Ilinsky [No mtDNA/st Table . No sign Ilinsky .M-mitotype samples of the I_II_III group, III and V clades were found in similar proportion across the studied Palearctic territory (Table P = 0.55). There was no difference in infection prevalence in ‘III+’ vs. the V clade . Two samples with IV_VIII clades (both wMel2-infected), were found only in the Far East population. All S-mitotype samples from Eastern Europe to the Far East were confirmed to have the canonical VI-clade sequence.To characterize the mitotype diversity in detail, the 343 bp mtDNA region was sequenced for 143 samples. Among Wolbachia endosymbiont of D. melanogaster were examined in population and phylogeographic terms across the vast Palearctic territory. Our data are consistent with previous observations on i) widespread Wolbachia infection in D. melanogaster populations [ii) predominance of the wMel genotype [iii) strict coinheritance of Wolbachia and mtDNA variants [D. melanogaster populations of Central  and Eastern Europe , and Western Siberia (Altai). No geographical pattern of Wolbachia infection rate was observed in the Palearctic, which corresponds well with the results of Kriesner et al. [Wolbachia variants were found in certain regions, viz. wMelCS2 from Western Siberia to Eastern Europe [Here, the mtDNA and ulations , 35, 47,genotype , 35, andvariants , 36, 48.r et al. . Howevern Europe , 35, 36,n Europe , 34, andn Europe .Wolbachia variants of the CS group were identified. The wMelCS2 genotype was detected in Eastern Europe and North Caucasus, and two samples with wMelCS infection were found in Western Siberia and Central Asia. Riegler et al. [Wolbachia group and S-clade mitochondrial DNA should be observed, whereas diversity of the MEL group and M-clade mtDNA should be rather low. In fact, we observed the opposite situation. There were several lineages within the MEL group and M-clade mtDNA, and only one lineage for the CS group and S-clade mtDNA [Wolbachia groups. Moreover, the ‘CCG’ haplotype was found among wMelCS and wMelCS2 genotypes, and other haplotypes seem to be local variants, namely ‘TCG’ in North America and ‘CCT’ in South-East Asia. Several isolates of the ‘CTG’ haplotype, which are found in wMelCS-infected stocks, could be the result of using one or more sources of maternal laboratory stock(s). Thus, we observed low genetic variation within the CS lineage of Wolbachia, which, together with above-mentioned comparison of MEL/M and CS/S diversity, and low diversity of the VI mtDNA lineage contradicts the hypothesis of recent replacement of Wolbachia genotypes. It is difficult to imagine different mitotypes supplanting the VI clade variants. The reason for the high proportion of wMelCS genotypes in populations before 1970 could be a case of sample error. Indeed, the number of stocks established with flies collected before the 1980s is very low  within the M-clade were revealed. The ‘III+’ lineage consists of clades I and III, which are widespread all over the world [D. melanogaster populations [D. melanogaster populations from Western Europe [D. melanogaster populations. D. melanogaster with the V clade of mtDNA were previously shown to be more viable in cold conditions than other clades [Wolbachia infection within D. melanogaster with ‘III+’ and V mtDNA clades may indicate a recent loss of the symbiont.Coinheritance of  species , 53–58.  studies –34. Our he world –34, 38, ulations , 34. Somn Europe , 34, 38,n Europe . In the D. melanogaster populations is consistent with previous studies and expands our knowledge. Prevalence of Wolbachia infection does not have a specific distribution pattern in the Palearctic. The wMel genotype inhabits every population, whereas other genotypes are rare and localised. Variants of V and ‘III+’ mitochondrial clades predominate in infected and uninfected flies across the Palearctic territory. According to symbiont and mtDNA diversity, the fly populations of many regions in temperate zones renew after the cold season, and the contribution of fly migration is not detected. Low genetic polymorphism of CS genotypes together with mitotypes and Wolbachia infection of global D. melanogaster populations do not support the hypothesis of a recent global Wolbachia genotype replacement. However, an increase in the wMelCS genotype rate in global D. melanogaster populations due to interactions with specific factors cannot be excluded.Our in-depth survey of maternal-inherited factors of Palearctic Additional file 1:Drosophila melanogaster stocks used for analysis of 343 bp mtDNA region. GenBank numbers of sequencing samples are indicated. (XLS 55 kb)List of Additional file 2:Drosophila melanogaster (according to GenBank NC001709). (XLS 37 kb)Polymorphic sites of 343 bp mtDNA region of Additional file 3:Wolbachia from CS-group genotypes. GenBank accession numbers are indicated in brackets where available. (DOC 74 kb)Haplotypes of Additional file 4:Wolbachia variants. Positions chosen for analysis performed in the current study are highlighted in yellow. Uncertain nucleotides highlighted in grey. (XLS 43 kb)Nucleotide polymorphisms of CS Additional file 5:Wolbachia genotypes found in worldwide populations of Drosophila melanogaster during the 1925–2015 period. (XLS 72 kb)Dataset of Additional file 6:Wolbachia patterns. (XLS 31 kb)Dataset used for analysis of geographical


Bubaque is the most populous island of the Bijagos archipelago, a group of malaria-endemic islands situated off the coast of Guinea-Bissau, West Africa. Malaria vector control on Bubaque relies almost exclusively on the use of long-lasting insecticidal nets (LLINs). However, there is little information on local vector bionomics and insecticide resistance.Anopheles mosquitoes were identified to species level and assessed for kdr allele frequency by TaqMan PCR. Females were analysed for sporozoite positivity by CSP-ELISA. Resistance to permethrin and α-cypermethrin was measured using the CDC-bottle bioassay incorporating the synergist piperonyl-butoxide.A survey of mosquito species composition was performed at the onset of the wet season (June/July) and the beginning of the dry season (November/December). Sampling was performed using indoor adult light-traps and larval dipping. Anopheles species were found on the island, all belonging to the Anopheles gambiae sensu lato (s.l.) complex, including An. gambiae sensu stricto, Anopheles coluzzii, Anopheles melas, and An. gambiae/An. coluzzii hybrids. Endophagic Anopheles species composition and abundance showed strong seasonal variation, with a majority of An. gambiae (50% of adults collected) caught in June/July, while An. melas was dominant in November/December (83.9% of adults collected). Anopheles gambiae had the highest sporozoite rate in both seasons, with infection rates of 13.9% and 20% in June/July and November/December, respectively. Moderate frequencies of the West African kdr allele were found in An. gambiae (36%), An. coluzzii (35%), An. gambiae/An. coluzzii hybrids (42%). Bioassays suggest moderate resistance to α-cypermethrin, but full susceptibility to permethrin.Several An. gambiae s.l. population in both June/July and November/December. Anopheles gambiae was the primary vector at the onset of the wet season, while An. melas is likely to be responsible for most dry season transmission. There was moderate kdr allele frequency and synergist assays suggest likely metabolic resistance, which could reduce the efficacy of LLINs. Future control of malaria on the islands should consider the seasonal shift in mosquito species, and should employ continuous monitoring for insecticide resistance.The island of Bubaque maintained an  Bubaque is the most populated island and the main commercial centre of the Bijagos archipelago, a group of 88 remote islands and islets situated off the coast of Guinea-Bissau, West Africa. A majority of the approximately 30,000 Bijagos islanders maintain a lifestyle supported by subsistence agriculture, with the port on Bubaque acting as the major hub for the import and export of goods, and the movement of people between the islands and the mainland. Bubaque consists primarily of dense forest and areas of cultivated agricultural land surrounded by stretches of beach and mangrove swamp, although the majority of the island’s approximately 11,000 inhabitants are concentrated in a semi-urbanized area built around the port. The climate is tropical, with a heavy wet season between June and November, and a dry season from December to May.Anopheles gambiae sensu stricto (s.s.) (hereafter Anopheles gambiae), Anopheles coluzzii, An. gambiae/An. coluzzii hybrids, and a low number of the salt-tolerant species Anopheles melas , AR (R) [AAGTGTCCTTCTCCATCCTA], GA (R) [CTGGTTTGGTCGGCACGTTT], ME (R) [TGACCAACCCACTCCCTTGA])  to detec product  to furthAmplified products were run using the QIAxcel capillary electrophoresis system (Qiagen), using the screening cartridge, 15–1000 bp alignment marker and 50–800 bp reference marker. The results were exported and double scored manually by visualizing gel image and the discrepancies sorted out by a third independent scorer.Mosquito’s head and thorax were placed in a 1.5 ml micro centrifuge tube, then ground in BB NP40 solution. Circumsporozoite protein (CSP) Elisa was performed on the mosquito triturate and plates read at 405 nm to detect the presence of CSP antigen .Anopheles larvae were collected from an inland temporary breeding site close to the semi-rural Bubaque village in mid-June 2018 . A sub-set of randomly selected samples  from the June/July collections and all samples from November/December were identified to species level by PCR.A total of 1869 adult mosquitoes (853 anopheline and 1016 culicine), were collected by CDC lights traps. 721 female anopheline mosquitoes were collected in June/July (the onset of the wet season) while 126 were collected in November/December (the end of the wet/onset of the dry season). All An. gambiae was the predominant species (50%), followed by An. coluzzii (12.6%), An. gambiae/An. coluzzii hybrids (27.5%), and Anopheles melas (9.8%). In November/December, An. melas was the predominant species (83.9%), with smaller proportions of An. gambiae (4.2%), An. coluzzii (2.5%) and An. gambiae/An. coluzzii hybrids (9.3%) , An. gambiae/An. coluzzii hybrids (24.1%), An. coluzzii (7.3%) and An. melas (8.4%) larvae. The locations of the Anopheles-positive larval breeding sites are given in Fig. An. gambiae, An. coluzzii and An. gambiae/An. coluzzii hybrids were associated with urban and agricultural habitats, An. melas tended to be associated with coastal breeding sites. Culicine mosquitoes were also collected and identified as Aedes aegypti, Culex rubinotus, Culex theileri, and Toxorhynchites species. A complete list of species sampled including Culicines can be found in Additional file 88 potential mosquito breeding sites were identified and assessed through larval dipping, of which 16 18.2%) were positive for % were poAnopheles samples collected over June/July were tested for the presence of the ‘West African’ 1014F kdr allele (kdr-w)  and An. gambiae/coluzzii hybrids [odds ratio = 24 ].The species and CR Table . There wAn. gambiae was the dominant endophilic species, although significant proportions of An. coluzzii and hybrid forms were also found. Anopheles melas adults constituted the smallest proportion of overall catches. The relative dominance of An. gambiae over An. coluzzii in this period indicates a reliance of the major portion of the wet season vector population on the availability of temporary, rain-dependent larval breeding sites (An. coluzzii tends to be associated with more permanent water bodies [An. melas was the dominant species (comprising > 85% of catches). This is likely a result of its capacity to maintain year-round populations by breeding in the island’s abundant littoral habitats and mangrove swamps . Breeding of An. gambiae and An. coluzzii is limited to sites of low salinity, which are generally rare in the dry season. Catch rates per household suggest a greater absolute abundance of endophagic An. gambiae s.l. mosquitoes during the wet season (~ 8-fold higher per household catch rates compared with November/December), consistent with the increasing availability of temporary rain-dependent larval habitats supporting expanding mosquito populations.The island of Bubaque displays strong seasonal variation in malaria vector species composition and abundance. During June/July, the onset of the rainy season, r bodies , 19). InAn. gambiae mosquitoes (89.5%) (molecular forms not specified), with a lower proportion of An. melas (8.9%) [An. gambiae and An. coluzzii were found to dominate [Anopheles arabiensis adult on Bubaque (1 out of 67 Anopheles adults caught) [Anopheles arabiensis has also been found in the city of Bissau [An. arabiensis in the present survey in either adult or larval collections likely reflects very low densities of this species on Bubaque, but may also result from a bias introduced by the indoor trapping method used in the large majority of mosquito collections; An. arabiensis tends to be less endophagic and endophilic than An. gambiae and An. coluzzii [Anopheles species composition inferred from adult trapping, with a majority of An. gambiae and a smaller proportion of An. gambiae/An. coluzzii hybrids and An. melas. Several culicine species were also identified including the arbovirus vector Aedes aegypti.A previous small-scale wet-season sample collected on Bubaque, reported a high proportion of s (8.9%) , 20. Thiitoes 89.%  , 20. Anof Bissau , and in f Bissau . The laccoluzzii . Larval An. gambiae s.l. species has been reported elsewhere in coastal regions of West Africa. A survey in The Gambia found reductions in overall An. gambiae s.l. numbers during the dry season, with catches dominated by An. melas [An. gambiae was highly seasonal and dependent on rainfall, while An. melas was able to maintain a relatively constant dry season population [Strong seasonal variation in abundance of n. melas . Similarpulation .Anopheles gambiae and An. coluzzii exhibit strong reproductive isolation throughout the majority of their sympatric range, with rates of hybridization typically < 1% [An. gambiae, An. coluzzii and hybrid catches during June-July), with rates exceeding those commonly found on mainland Guinea-Bissau [lly < 1% , 27. Howlly < 1%  and Guinlly < 1% –30. A laa-Bissau  and in Ta-Bissau .Anopheles gambiae had the highest P. falciparum sporozoite rate among all An. gambiae s.l. mosquitoes sampled, with 13.9% and 20% positivity in the dry and wet season, respectively, although the dry season sporozoite rates should be viewed with caution due to the very small sample size. The greater population density and sporozoite positivity of An. gambiae in the wet season suggests it is the primary wet season vector on Bubaque. Although An. gambiae also displayed the highest sporozoite rate of the An. gambiae s.l. species in the dry season, a far greater density of An. melas (with a 20-fold higher median per trap catch rate in this period) combined with a positive sporozoite rate of 6.1%, suggests that this species may be responsible for the majority of dry season transmission. The sporozoite rates found in the present survey are similar to those observed in a previous wet season survey on mainland Guinea-Bissau that included sampling on several of the islands of the Bijagos, where average sporozoite rates of 12.6% in An. gambiae and 11.1% in An. melas were recorded [An. gambiae, An. gambiae/An. coluzzii hybrids, and An. melas, at the onset of the dry season correlates with months of peak malaria prevalence. However, there was a lower average dry season sporozoite rate across the wider An. gambiae s.l., complex (9.7% in the wet season compared to 6.8% in the dry season) combined with ~ 8-fold lower indoor dry season catch rates. A tentative calculation of sporozoite-positive An. gambiae s.l. during both seasons, gives a median of 0.78 positive females per trap per night in the wet season, compared with 0.068 in the dry season.recorded . The higkdr-w allele frequencies of 36% in An. gambiae, 35% in An. coluzzii and 42% in An. gambiae/An. coluzzii hybrids. This contrasts with a survey in Bissau performed over a decade ago, where kdr-w allele frequencies of 7% and 0.8% were recorded in An. gambiae and An. coluzzii, respectively [kdr-w frequencies is consistent with a response to strong selection associated with the wide-spread distribution of LLINs in Guinea-Bissau since 2011.Analysis of the wet-season samples from Bubaque revealed ectively . The shakdr allele and pyrethroid resistance detected by in vivo assays are important for providing functional data on resistance, and may uncover additional mechanisms. Resistance can arise through a variety of processes, including metabolic resistance resulting from the amplification or upregulation of genes encoding detoxification enzymes. CDC-bottle bioassays were used in the present study to measure the resistance of field-caught An. gambiae s.l. females to discriminating doses of permethrin and α-cypermethrin. Although this represents a limited subset of insecticides used in LLINs, it represents two of the most commonly used pyrethroids in LLINs on Bubaque . Full susceptibility to permethrin and partial resistance to α-cypermethrin was found. This permethrin susceptibility contrasts with studies in the neighbouring Gambia, where marked resistance has previously been described, particularly in coastal areas [An. gambiae populations in West Africa, with biochemical assays and microarray studies implicating metabolic resistance in Benin [Parallel comparisons between distributions of the al areas . FSuscepin Benin , Nigeriain Benin , and Ghain Benin . Next gein Benin , althougin Benin .Anopheles species composition and abundance on the island of Bubaque varied significantly by season, with a relatively large wet season population comprising principally An. gambiae, An. coluzzii and An. gambiae/An. coluzzii hybrids, and a smaller dry season population dominated by An. melas. The persistence of An. melas in the dry season, albeit at a low relative density, may be an important consideration for vector control planning. Information dissemination on LLIN use, for example, should stress the potential for malaria transmission and the benefits of LLIN use beyond the rainy season. Moderate frequencies of the West African kdr-mutation were found in both An. gambiae and An. coluzzii populations, and a low level of resistance to α-cypermethrin was detected through bioassay. Pre-exposure to PBO restored α-cypermethrin susceptibility, indicating the co-occurrence of both target-site and metabolic resistance mechanisms. The emergence of pyrethroid resistance is a serious concern on an island dependent on LLINs for malaria control. The results presented here suggest that resistance should be closely monitored by bioassay, and that the active insecticide compounds used in LLINs should be selected based on net-exposure assays with locally caught An. gambiae s.l. mosquitoes.Endophilic Additional file 1: Figure S1. Number of female Anopheles mosquitoes caught per CDC light trap per night during the June-July and November–December trapping.Additional file 2: Table S1. Larval sampling site description and species found.


Escherichia coli is reported in high levels in household soil in low-income settings. When E. coli reaches a soil environment, different mechanisms, including survival, clonal expansion, and genetic exchange, have the potential to either maintain or generate E. coli variants with capabilities of causing harm to people. In this study, we used whole-genome sequencing to identify that E. coli isolates collected from rural Bangladeshi household soils, including pathogenic and antibiotic-resistant variants, are diverse and likely originated from multiple diverse sources. In addition, we observed specialization of the accessory genome of this Bangladeshi E. coli compared to E. coli genomes available in current sequence databases. Thus, to address the high level of pathogenic and antibiotic-resistant E. coli transmission in low-income settings, interventions should focus on addressing the heterogeneous origins and high diversity. Escherichia coli is present in multiple hosts and environmental compartments as a normal inhabitant, temporary or persistent colonizer, and as a pathogen. Transmission of E. coli between hosts and with the environment is considered to occur more often in areas with poor sanitation. We performed whole-genome comparative analyses on 60 E. coli isolates from soils and fecal sources  in households in rural Bangladesh. Isolates from household soils were in multiple branches of the reconstructed phylogeny, intermixed with isolates from fecal sources. Pairwise differences between all strain pairs were large , suggesting high diversity and heterogeneous origins of the isolates. The presence of multiple virulence and antibiotic resistance genes is indicative of the risk that E. coli from soil and feces represent for the transmission of variants that pose potential harm to people. Analysis of the accessory genomes of the Bangladeshi E. coli relative to E. coli genomes available in NCBI identified a common pool of accessory genes shared among E. coli isolates in this geographic area. Together, these findings indicate that in rural Bangladesh, a high level of E. coli in soil is likely driven by contributions from multiple and diverse E. coli sources  that share an accessory gene pool relatively unique to previously published E. coli genomes. Thus, interventions to reduce environmental pathogen or antimicrobial resistance transmission should adopt integrated One Health approaches that consider heterogeneous origins and high diversity to improve effectiveness and reduce prevalence and transmission.IMPORTANCEEscherichia coli is reported in high levels in household soil in low-income settings. When E. coli reaches a soil environment, different mechanisms, including survival, clonal expansion, and genetic exchange, have the potential to either maintain or generate E. coli variants with capabilities of causing harm to people. In this study, we used whole-genome sequencing to identify that E. coli isolates collected from rural Bangladeshi household soils, including pathogenic and antibiotic-resistant variants, are diverse and likely originated from multiple diverse sources. In addition, we observed specialization of the accessory genome of this Bangladeshi E. coli compared to E. coli genomes available in current sequence databases. Thus, to address the high level of pathogenic and antibiotic-resistant E. coli transmission in low-income settings, interventions should focus on addressing the heterogeneous origins and high diversity. Escherichia coli is a commensal bacterium but also a versatile pathogen capable of causing intestinal and extraintestinal infections  and Shiga toxin-producing E. coli (STEC) of diarrheal disease and extraintestinal pathogenic E. coli (ExPEC) of urinary tract infections : seven are part of E. colisensu stricto  and the eighth is known as clade I (E. coli isolated from open environments (such as soils) and whether specific genetic determinants contribute to survival, adaptation outside the host, or subsequent transmission  in soils  in these settings contribute to increased transmission of strains between hosts and with the environment . For exaand food . Contribin soils , and an in soils . Moreovein LMICs , 23. UndE. coli isolates from feces  and soil in households in rural Bangladesh, an area characterized by high disease transmission. E. coli isolates were further characterized by genes associated with virulence, antibiotic resistance, and plasmid replicons. The accessory genome of Bangladeshi E. coli was further analyzed in a broader context by comparison with representative E. coli genomes available in NCBI.The present study used comparative genomics, including phylogenetic reconstruction and pairwise differences analysis, to investigate genetic and population-level relationships between E. coli collected from soil and feces of humans and animals (chicken and cattle) in households in rural Bangladesh were analyzed using whole-genome sequencing (WGS). The size of the genome assemblies from the 60 E. coli isolates sequenced ranged from 4.52 to 5.50 Mb with a mean GC content of 50.6%  (isolates . We founisolates . No ST wnd HH24)  and S2.10.1128/mSphere.00704-19.1TABLE S1Table S1, XLSX file, 0.1 MB.Assembly statistics and GenBank accession numbers of the 60 genome sequences included in this study. Download Copyright © 2020 Montealegre et al.2020Montealegre et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the 10.1128/mSphere.00704-19.2TABLE S2E. coli isolates included in this study. Download Table S2, XLSX file, 0.1 MB.Achtman 7 Gene MLST of the 60 Copyright © 2020 Montealegre et al.2020Montealegre et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the Escherichia clade I  or from the same source compared to those from different sources .Comparative genomic analyses of the 60 isolates indicates that the majority of isolates cluster within phylogroups B1 (60%) and A (28.3%); however, rich phylogenetic diversity among the isolates falling in these phylogroups was observed . A few i clade I . The 19  lineage   virulence factor-related genes included in the virulence factor database , cation efflux (ibeB and ibeC), adhesive curli fimbriae (csgBAC and csgDEFG operons), and the siderophore enterobactin used for iron acquisition were detected in all isolates. Genes related to the type 1 fimbria operon and flagella were also very common , EPEC, and noncategorized diarrheagenic E. coli (DEC) (eae gene indicating EPEC was detected in five isolates (3 chicken and 2 human). One isolate (HH13H) was a putative enterotoxigenic E. coli (ETEC), as indicated by the presence of the eltA and eltB genes, common diagnostic markers for heat-labile ETEC, while one cattle isolate (HH08C) was a putative STEC as indicated by stx1a, stx1b, stx2a, and stx2db genes. The gene aatA  , was detfor EAEC ) was det2 test, df = 3, unadjusted α = 0.05). Specifically, the adhesin tia gene appeared in eight cattle, two chicken, and two soil isolates but in no human isolates , and the adhesin-related cah gene appeared in seven chicken and six soil isolates but only one human and no cattle isolates . Similarly, leoA, a gene linked to secretion of the heat-labile enterotoxin ; ECP_2814, encoding a hypothetical protein, only appeared in four human isolates and two cattle isolates .The observed distribution of virulence factor-related genes across the four isolate sources  appeared random based on overall prevalence rates for all except four genes , found in all isolates] with identity and coverage greater than 90% against the ResFinder database (tet(A) (n = 11) and/or tet(B) genes (n = 4)  was observed in isolate HH13H, harboring blaDHA-1. The sul and dfrA genes, associated with class 1 integrons (aadA and aph variants) were observed in eight isolates, often from chicken origin (aph(3′′)-Ib and aph(6)-Id appeared to not be randomly distributed across the four sources, as they were only detected in chicken but not in any of the other sources . The plasmid-mediated quinolone resistance (PMQR) genes QnrS1 and QnrB4 were detected in eight E. coli; however, no clinical resistance to ciprofloxacin, based on CLSI breakpoints, was observed in these isolates, except for one soil isolate that coharbored both genes. QnrS1 and QnrB4 are known to provide a low level of resistance, while mutations in the genes encoding DNA gyrase and topoisomerase IV are associated with observable resistance to ciprofloxacin and/or nalidixic acid  mph(A) and/or ermB was detected  . ResistaCTX-M-15 . ReducedCTX-M-15 , was obsntegrons  and encontegrons . Genes axic acid , as in tis study . Resistadetected .2 = 8.4, P = 0.038).By using an identity and coverage threshold greater than 90% against the PlasmidFinder database, the numbers of plasmid replicons detected ranged from 1 to 7 among 49 isolates (81.7%), while the other 11 isolates had no hits above the predefined threshold . Thirty-E. coli against 199 representative E. coli genomes , we then observed uniform distance distribution of the Bangladeshi isolates among the E. coli genomes  (P < 0.001) among the protein-coding genes enriched in the Bangladeshi isolates  than the protein-coding genes shared between the Bangladeshi isolates and the nearest neighbors    and 4. N[42.8%]) .1, IS2, and IS3; three (3.5%) related to toxin/antitoxin systems for plasmid maintenance, one related to DNA-binding transcriptional regulator, and one related to DNA base-flipping. Notably, 13 of the proteins were not found in any of the 265 nearest neighbors, including DNA base-flipping and formate dehydrogenase H proteins present in all 60 Bangladeshi isolates, and two toxin/antitoxin proteins present in 58 (97%) and 33 (55%) of the Bangladeshi isolates  protein-coding genes that were both statistically significantly enriched and present in at least half of the 60 Bangladeshi isolates . The 84 isolates .10.1128/mSphere.00704-19.6TABLE S6E. coli. Download Table S6, XLSX file, 0.1 MB.List accessory genome proteins statistically significantly enriched in Bangladeshi Copyright © 2020 Montealegre et al.2020Montealegre et al.Creative Commons Attribution 4.0 International license.This content is distributed under the terms of the E. coli from household soils in a rural Bangladeshi community using WGS and performed comparative analyses with E. coli isolated from feces of potential contributors  to shed light on probable sources and transmission patterns. Our findings are indicative of a rich phylogenetic diversity among the E. coli isolates circulating in this rural community with the E. coli isolates recovered from front yard households located in multiple branches of the phylogeny intermixed with isolates from fecal sources  . This value is far below the minimum number of SNPs (189 SNPs) observed between the most closely related isolates of this study. Therefore, we found no direct evidence to suggest recent clonal transmission from humans or animals to soils or vice versa. Similarly, other studies have failed to detect recent transmission events between human and animals (domestic and livestock)  , 38, onevestock) , 23, eveectively . Howeverectively , 41 or dectively . Furtherectively , 43. Forutbreaks . In addiutbreaks , 40 in aE. coli isolates do not represent a unique population relative to the nearest E. coli neighbors available in the database. Interestingly, when we interrogated their accessory genomes against the nearest E. coli genomes, we observed that approximately one of every six protein-coding genes in the genomes of the Bangladeshi isolates was statistically significantly enriched relative to the nearest E. coli neighbors  are underrepresented in current sequence databases describing E. coli and associated biological functions, as similarly suggested with recent studies of metagenome-assembled genomes (MAGs) from the gut microbiome , including three soil isolates , coharbored aatA and astA  were not detected in E. coli isolated from soils but were found in isolates from human, chicken, and cattle feces. Overall, these findings are indicative of the potential that E. coli isolated from soils has to cause disease in people. Furthermore, the presence of one or more antibiotic resistance genes in soil isolates  is indicative of the risk that soil E. coli may represent for the transmission of resistant determinants. Indeed, at least one E. coli isolate from soil carried a gene associated with each of the antibiotic resistance gene classes encountered (E. coli collection (81.7%), with no significant difference in the numbers of replicons observed across the sources. Salinas et al. showed that human and domestic animals shared plasmid replicons; however, diversity in the sequences indicated that the plasmids compared were not identical  and ColpVC]; however long-read sequencing would be necessary to establish if the same plasmid is circulating across reservoirs. In contrast, other replicons were absent from one or more of the studied sources , which suggests that ecological factors and/or the genetic makeup of the E. coli circulating within specific hosts could affect the distribution of certain plasmids replicons. However, the apparent enrichment by sample source may be random for at least some—if not all—of the four virulence genes, two antibiotic resistance genes, and one plasmid replicon as a consequence of the large data set, liberal statistical significance cutoff, and purposive sampling. Nevertheless, the genes are discussed here to inform potential further investigations of source-specific adaptation of E. coli.Bangladeshi ountries ), were ping soil . Notablydiarrhea ), highlithotypes –50 and ihotypes –. Howeverutbreaks ; therefoountered . Plasmiddentical . SimilarE. coli contamination in low-income settings. First, the pathogenicity potential and acquired antibiotic resistance of environmental strains reaffirm the need for interventions that effectively reduce E. coli across different environmental reservoirs. This represents a major challenge, as multiple previous studies showed no significant impact of sanitation   . Infectiroaches) , 57, areE. coli using the API-20E . The isolates selected were recovered from 22 households and up to four different sources and included E. coli isolated from front yard soils (n = 19) and fecal samples from human (n = 14), chicken (n = 14), and cattle (n = 13) (E. coli isolates included (n = 52) were isolated from three or four of the four sources studied, while the remaining isolates (n = 8) correspond to E. coli isolated from eight different household soils (E. coli isolate from cattle feces in household 03). Disk diffusion against 16 different antibiotic disks was previously performed  . For 14 ld soils . The nomerformed . In addierformed .DNA was extracted from an overnight culture using the DNeasy Blood & Tissue kit  according to the instructions of the manufacturer. Purity and concentration of the DNA were evaluated with a NanoDrop 2000 spectrophotometer (Thermo Scientific) and a Qubit 2.0 fluorometer (Life Technologies), respectively. Libraries were prepared with the Nextera XT kit, and paired-end sequenced was performed using the Illumina HiSeq platform (2 × 150 bp) .https://www.bioinformatics.babraham.ac.uk/projects/fastqc/. Reads were de novo assembled using SPAdes genome assembler version 3.11.1 (https://github.com/tseemann/mlst)  , 63. Phyd method  availablippy 4.0 . A core .embl.de . The pre.embl.de , Virulen.embl.de , and Pla.embl.de  (query d.embl.de . ChromossFinder/ .https://gephi.org/). Analyses were performed using the 60 Bangladeshi soil and fecal E. coli isolates against 199 nonredundant E. coli genomes representative of each branch of the E. coli phylogeny and against 265 nonredundant nearest E. coli genomes, which represent the 10 most closely related E. coli strains for each of the Bangladeshi E. coli isolates, which may be shared among some Bangladeshi E. coli isolates.Phylogenetic distance was estimated using Mash , while tP value of <0.05 was considered significant.Statistical analyses were performed using R, version 1.2.1335. Pairwise differences in the means of the ranks of the number of SNPs among isolates from the same household or different households and from the same source or different sources were evaluated using the Wilcoxon rank-sum test. To investigate enrichment of virulence factors, antibiotic resistance genes, and plasmid replicons by source, a chi-squared test was used and a VNWZ00000000 to VNZG00000000 presented in This whole-genome shotgun project has been deposited at DDBJ/ENA/GenBank under the accession numbers


Helicobacter pylori is known for its colonization of the upper digestive system, where it escapes the harsh acidic environment by hiding in the mucus layer. One factor promoting this colonization is the helical cell shape of H. pylori. Among shape determining proteins are cytoskeletal elements like the recently discovered bactofilins. Bactofilins constitute a widespread family of polymer-forming bacterial proteins whose biology is still poorly investigated. Here we describe the first biochemical analysis of the bactofilin HP1542 of H. pylori reference strain 26695. Purified HP1542 forms sheet-like 2D crystalline assemblies, which clearly depend on a natively structured C-terminus. Polymerization properties and protein stability were investigated. Additionally, we also could demarcate HP1542 from amyloid proteins that share similarities with the bactofilin DUF domain. By using zonal centrifugation of total H. pylori cell lysates and immunfluorescence analysis we revealed peripheral membrane association of HP1542 mostly pronounced near mid-cell. Interestingly our results indicate that H. pylori bactofilin does not contribute to cell wall stability. This study might act as a starting point for biophysical studies of the H. pylori bactofilin biology as well as for the investigation of bactofilin cell physiology in this organism. Importantly, this study is the first biochemical analysis of a bactofilin in a human pathogen.The human pathogen  Caulobacter crescentus adopt a β-helical architecture, which has not been observed before for any other cytoskeletal filaments Helicobacections) . Within ections)  21]. Mu. MuHelicections)  and motiections) . In H. plaxation  24] and andHelicscaffold , 25. In ein MreB . InfectiH. pylori bactofilin indeed exhibits biochemical properties of bactofilins and forms a cytoskeleton in this bacterium and, if so, how this putative cytoskeleton is involved in helical cell shape was still unclear. We thus sought to characterize the biochemical properties of the single bactofilin HP1542 in vitro and investigate its localization in vivo. As such our results provide the first insights into assembly properties and localization of HP1542 and may thus act as a starting point for the investigation of bactofilin cell physiology in this pathogenic organism as well as for designing targeting molecules.However, whether the single H. pylori strains were routinely cultivated on Dent blood agar in a microaerobic atmosphere as described earlier , th, thin viM. xanthus and L. biflexa cells, the absence of bactofilins significantly lowered the tolerance to cell wall-targeting antibiotics . In. InM. xa profile , which wic sites , 55. H. PG layer . Six houH. pylori, which is an important pathogenic organism, the bactofilin homolog has not been biochemically characterized to date indicating a definite paucity of information. Our work provides the first insight into the properties of H. pylori reference strain 26695 bactofilin homolog HP1542 in terms of protein polymerization, protein stability and aggregation. Furthermore we supplemented our in vitro data with localization studies using zonal centrifugation and immunofluorescence and growth experiments considering cell wall integrity.By now it is accepted that microorganisms also have well-organized cellular interiors  includin6-1542. Furthermore truncation of the C-terminus resulted in soluble polymeric fibers and bundles of filaments and the complete loss of the 2-D sheets. Electron microscopy analysis of purified H. pylori bactofilin without tag clearly demonstrated the authenticity of the sheet-like arrangement. Likewise Vasa et al. reported that purified fractions of C. crescentus BacA additionally contained sheet-like 2-D crystalline arrays . In. InH. pyn before . Re-analL. biflexa. C. crescentus and P. mirabilis . Prirabilis  51] . In. InC. crng cells . BacteriS1 FigESI-TOF/MS analysis of affinity purified Strep-HP1542 deconvolated electronspray mass spectrum of affinity purified HP1542, given in relative Intensity (%) against molecular weight (m/z).(TIF)Click here for additional data file.S2 FigComparison of HP1542 (NCBI Reference Sequence: NP_208333.1) to BacA (UniProtKB—Q9A753 (Q9A753_CAUVC)) in terms of primary sequence, secondary structure motifs and the DUF 583 domains as indicated. Conserved residues are marked with asterisks.(TIF)Click here for additional data file.S3 FigWestern blot using HP1542 specific antiserum, strains as indicated above the lanes . Equal amounts of protein were loaded onto each lane.(TIF)Click here for additional data file.


Candida species. Advances in molecular diagnostics have the potential to guide early targeted treatment of high-risk patients.Invasive candidiasis occurs frequently in hospitalized patients, and is associated with high mortality rates due to delays in recognition and initiation of appropriate antifungals. Management of invasive candidiasis must take into account multiple host, pathogen, and drug-related factors, including the site of infection, host immune status, severity of sepsis, resistance and tolerance to antifungal agents, biofilm formation, and pharmacokinetic/pharmacodynamic considerations. Recent treatment directives have been shaped by the widespread introduction of echinocandins, highly potent and safe antifungals, into clinical use, as well as important changes in drug susceptibility patterns and the emergence of known and novel drug-resistant  Candida species are frequent colonizers of the human alimentary tract and skin that have emerged as important nosocomial pathogens, in tandem with advances in modern medical therapeutics [Candida species were found to be the fourth most frequent causes of bloodstream infection in hospitalized patients in the US, and were associated with the highest mortality rate among leading bloodstream isolates [Candida species account for about 1 in 5 nosocomial bloodstream infections [Candida into normally sterile body sites, is often difficult to diagnose and may affect a population as large as that of candidemia [apeutics ,2. Notabfections . Deep-sendidemia ,5,6. Thendidemia , with anndidemia .Candida albicans is the most common cause of invasive candidiasis, infections due to non-albicans Candida species account for an increasing proportion of cases. Specifically, C. glabrata has become an important pathogen in North America, Europe (with the exception of Spain), and Australia, whereas C. parapsilosis is the dominant non-albicans species in South America, Japan, and Spain [C. albicans, which is overwhelmingly susceptible to all systemic antifungals, to species that are more frequently resistant or tolerant to fluconazole, such as C. glabrata and C. krusei, has impacted treatment recommendations [Candida strains with acquired resistance to echinocandins, specifically C. glabrata, while still generally infrequent, are increasing in incidence in some hospitals [C. auris, a novel Candida species first identified in 2009, has emerged since 2013 as a cause of nosocomial outbreaks in multiple countries across 5 continents. C. auris presents a serious challenge to healthcare systems due to its frequent resistance to multiple systemic antifungals and disinfectants and its capacity for rapid spread within healthcare facilities [Although nd Spain ,9,10. Thndations ,12,13. Cospitals . C. auricilities ,16.Candida endocarditis [The armamentarium of drugs for the treatment of candidiasis currently comprises three major drug classes: the polyenes, azoles, and echinocandins. In addition, flucytosine, a pyrimidine analogue, has specific roles as an adjunct in the treatment of central nervous system candidiasis and carditis ,18.d-glucan, the major fungal cell-wall carbohydrate. Disruption of 1,3-β-d-glucan synthesis damages cellular integrity and leads eventually to cell rupture and death. All three licensed echinocandins  are approved for the treatment of invasive candidiasis. These drugs may only be given intravenously. Caspofungin and micafungin are metabolized in the liver, whereas anidulafungin is slowly degraded in plasma due to chemical opening of its ring structure.The echinocandins are cyclic lipopeptides that inhibit the transmembrane glucan synthase complex (Fks1), a biosynthetic enzyme that produces 1,3-β-Candida species, including azole resistant or tolerant species. C. parapsilosis and C. guilliermondii are intrinsically less susceptible to echinocandins, reflecting naturally occurring polymorphisms in the FKS1 hotspot regions of these species [The echinocandins are fungicidal in vitro against a broad range of  species .The echinocandins are remarkably safe, even at higher than standard doses. Rapid infusion has been associated with flushing, dyspnea, hypotension, and urticaria, attributed to histamine release. These drugs should therefore be infused over 1 h. Cardiotoxicity is a rare and severe side effect that has been observed when echinocandins are administered through a central venous catheter. This phenomenon may be less frequent with anidulafungin than with caspofungin and micafungin. No significant hepatotoxicity or nephrotoxicity have been observed.Drug-drug interactions are relatively few with echinocandins. Caspofungin clearance is increased by inducers of liver CYP450 enzymes, such as rifampicin, efavirenz and carbamazepine, requiring a caspofungin dose increase from 50 mg/day to 70 mg/day if co-administered with those drugs. Caspofungin exposure is increased if co-administered with cyclosporin. Caspofungin decreases tacrolimus exposure, requiring drug level monitoring and dose adjustment. Micafungin and anidulafungin are not modulators or substrates of CYP450 enzymes, and were not found to have important drug-drug interactions.C. albicans infection [Prospective studies comparing echinocandins with amphotericin B for the treatment of candidiasis have shown similar efficacy of caspofungin and amphotericin B deoxycholate in the intention to treat analysis but superiority of caspofungin in the per-protocol analysis , and nonnfection , indicatnfection . A studynfection . Taken tCandida growth by binding to and inhibiting lanosterol 14α demethylase, a cytochrome P450 enzyme encoded by the ERG11 gene [Azoles inhibit G11 gene . LanosteFluconazole is the principle azole drug used in the treatment of candidiasis. Fluconazole has excellent bioavailability and may be given either intravenously or orally at identical doses. The main adverse effect of fluconazole is hepatotoxicity. In addition, fluconazole is a cytochrome P450 inhibitor and may interact with drugs that undergo metabolism via that route.C. albicans [In randomized controlled clinical trials of nonneutropenic patients with candidemia, outcomes were similar for patients treated with fluconazole versus amphotericin B, but toxicity was more frequent with amphotericin B ,27. Treaalbicans . A studyalbicans . Patientalbicans .Candida species, was as effective as amphotericin B followed by fluconazole for the treatment of candidemia [C. krusei.Voriconazole, a broad spectrum triazole active against filamentous fungi as well as ndidemia . VoriconThe polyene amphotericin B is a lipophilic molecule that binds to ergosterol within the fungal cytoplasmic membrane. Amphotericin B self-assembles into oligomers that insert into the cytoplasmic membrane, forming aqueous pores that cause ion leakage and rapid cell lysis. Amphotericin B is formulated with deoxycholate or in one of three lipid formulations . The specific formulation has no effect on the drug’s spectrum of activity, but does alter its distribution to different organs.Once the mainstay of antifungal treatment, amphotericin B has been demoted to a second-line treatment of invasive candidiasis due to its toxicity. The nephrotoxicity caused by amphotericin B may be divided into early reversible azotemia due to afferent arterial vasoconstriction, and late kidney damage due to nephron loss and tubular basement membrane damage. The latter is irreversible and correlates with the total dose of amphotericin B infused. Overall, acute kidney injury occurs in 30% of adults treated with amphotericin B, and is associated with significantly increased mortality rates, length of hospital stay, and costs [In vitro susceptibility testing is performed using broth microdilution  or European Committee on Antimicrobial Susceptibility Testing (EUCAST) methodology) ,32. LessEchinocandin minimal inhibitory concentrations (MICs) are read on broth microdilution assays as the drug concentration that produces significant (~50%) reduction in turbidity after 24 h . CaspofuCandida growth is detected at higher than MIC drug concentrations. This effect is more pronounced with caspofungin than with other echinocandins, is unrelated to MIC, appears to be most common with C. albicans and C. dubliniensis, and is strikingly absent in C. glabrata [C. tropicalis infection using a strain showing paradoxical growth demonstrated impaired fungal clearance with high caspofungin concentrations [A paradoxical (or eagle) echinocandin effect has been observed in which increased glabrata ,38,39. Ttrations , suggestCandida species to echinocandins has, thus far, always been associated with single amino acid substitutions arising from mutations within conserved (“hot spot”) sequences of the gene FKS1, and in C. glabrata FKS2, which encode for glucan synthase. In multivariate analyses, FKS mutations, rather than echinocandin MIC, were the only significant predictor of clinical echinocandin treatment failure [FKS mutations may be the most accurate way of classifying echinocandin resistant isolates. As there are currently no commercially available assays for FKS genotyping, molecular resistance testing is limited to reference laboratories.Clinically significant acquired resistance of  failure . TherefoCandida species, but is most frequently encountered in C. glabrata, where an association with echinocandin treatment failure has been described [Candida isolates have impaired fitness as compared with isogenic FKS wild-type strains [FKS mutants [Candida species carrying FKS hotspot mutations are almost invariably recovered from patients with extensive recent exposure to echinocandins, and usually after more than 3 weeks of treatment [Acquired echinocandin resistance has been described in all major escribed ,42. Echi strains ,44. This mutants . Perhapsreatment .C. albicans, C. tropicalis, and C. parapsilosis were lowered, so that MICs over 4 mg/L are considered resistant. For C. glabrata, an inherently fluconazole-tolerant species, an MIC greater than 32 mg/L is considered resistant, whereas an MIC of 32 mg/L or less is considered susceptible dose-dependent (S-DD), a term indicating that maximal drug dosage is required for optimal treatment.Susceptibility testing to fluconazole using CLSI or EUCAST broth microdilution methodology produces MIC values that correlate reasonably well with the likelihood of successful treatment outcomes ,47. The Candida species. Drug efflux is the most frequent mechanism, and is mediated by transmembrane transporter molecules of either the ATP-binding cassette (ABC) or major facilitator (MFS) superfamilies [C. albicans ERG11 coding sequence, resulting in reduced drug-to-target affinity, and gain-of-function mutations in the gene encoding transcription factor UPC2 that lead to enhanced ERG11 expression [Candida species following prolonged exposure to the drug, as previously shown for AIDS patients with oropharyngeal and esophageal candidiasis [Candida species, C. glabrata is the most prone to acquire fluconazole resistance, usually associated with upregulation of the ABC efflux pumps CDR1 and CDR2 [PDR1 leads to coordinated upregulation of multiple ABC-type drug transporters [C. krusei is intrinsically resistant to fluconazole because of ERG11 polymorphism, but is usually susceptible to other azoles such as voriconazole and posaconazole. C. auris is intrinsically resistant to fluconazole, and cannot reliably be treated with other azoles [Unlike the canonical resistance mechanism to echinocandins, acquired resistance to fluconazole may arise by various mechanisms that differ among the families ,49. Resipression . Acquireand CDR2 ,52. A gasporters ,55,56,57r azoles ,58,59.C. albicans, C. tropicalis, C. parapsilosis, C. glabrata, and C. krusei isolates with MIC > 1 mg/L are assumed to be resistant to amphotericin B [Amphotericin B MIC values are determined as the drug concentration that produces complete or near-complete reduction in growth at 24 h or 48 h. Correlation between amphotericin B MIC values and clinical response has not been consistently established ,61. Baseericin B ,62.C. lusitaniae and C. auris, display higher MICs for amphotericin B than other species. Acquired resistance is also rare, and involves reduced ergosterol biosynthesis and the production of alternative sterols to which amphotericin B has low affinity. Candida strains resistant to amphotericin B were found to harbor mutations in ERG2, ERG3, ERG5, and ERG11, and were co-resistant to fluconazole [Primary resistance to amphotericin B is uncommon. Two related species, conazole ,64,65,66C. glabrata is uniquely predisposed to become resistant to antifungals during treatment. Recent work has revealed both genetic and epigenetic mechanisms underpinning this propensity. Specific mutations in the DNA mismatch repair gene MSH2 lead to a hyper-mutable phenotype and were found to be highly prevalent in clinical C. glabrata isolates [MSH2 deletion significantly increases breakthrough infection and the emergence of resistance to all systemic antifungal drug classes in in vitro and animal experimental systems [C. glabrata strains, and is associated with failure of fluconazole to clear C. glabrata from tissues of experimentally infected immunocompetent mice [isolates . MSH2 de systems . Heterorent mice . Heterorent mice . Heterorent mice , a methoCandida strains.The goals of treatment of patients with invasive candidiasis are to prevent three types of adverse outcomes: 1. Death, usually within the first 7 days after onset of infection; 2. Late complications from metastatic and persistent deep-seated infection; and 3. Emergence of drug-resistant C. glabrata [C. glabrata versus other species [There is extensive clinical evidence that delayed initiation of appropriate antifungal treatment is associated with increased mortality in patients with invasive candidiasis ,71,72. Tglabrata . Moreove species . ConsequCandida score, assigns points for total parenteral nutrition, surgery, multifocal Candida colonization (1 point each), and severe sepsis (2 points) [Candida score is simple, accessible, and inexpensive. Real-life use of this prediction rule in the intensive care setting reveals a high negative predictive value (80% to 97%) but modest positive predictive value (57% to 70%) [Candida score [Risk factors for invasive candidiasis have been incorporated into a number of clinical prediction rules. The best known of these, the  points) . A total points) . The Can to 70%) ,78. The da score .d-glucan is a cell wall component of Candida species and other pathogenic fungi. A meta-analysis of 16 trials found a pooled sensitivity of 76% and specificity of 85% [Candida DNA in full blood or serum are both sensitive and specific [Candida PCR assays that underwent extensive clinical validation. The T2Candida assay  combines real time PCR with amplicon-induced agglomeration of supermagnetic nanoparticles and T2 magnetic resonance detection. In a multicenter study of patients with candidemia, the sensitivity of T2Candida was double that of conventional blood cultures when both were tested on follow-up blood samples [Candida species.A number of non-culture based biomarkers have been utilized to guide therapy of invasive candidiasis. The performance of all biomarkers is improved when multiple test results are analyzed sequentially over time. -β-y of 85% ; false py of 85% . Finallyspecific . However samples . PatientCandida species; 2. Excellent safety profile; and 3. Clinical superiority when compared to other systemic antifungals. A patient-level meta-analysis of 7 clinical trials with 1915 patients found that treatment with an echinocandin was associated with decreased mortality [For most forms of invasive candidiasis, echinocandins are recommended as front-line agents, for the following reasons: 1. Broad spectrum activity against the majority of clinically important ortality . Lipid fortality . Their uortality .C. glabrata with fluconazole remains an area of clinical uncertainty. Not only does C. glabrata have higher median MICs of fluconazole compared with other common Candida species, but treatment failure and the emergence of fluconazole-resistant strains occur frequently during treatment of initially susceptible isolates [C. glabrata isolates [C. glabrata infection with fluconazole should be reserved for cases where: 1. Bloodstream infection has been cleared with a fungicidal agent, such as an echinocandin or amphotericin B formulation; 2. The patient is clinically stable, infected catheters have been removed and deep-seated foci of undrained infection have been ruled out; and 3. The isolate is fluconazole non-resistant. Fluconazole should always be used at maximal doses  in such cases.Treatment of isolates . In addiisolates . With thC. parapsilosis is intrinsically less susceptible to echinocandins than other Candida species; however, there are conflicting epidemiological and clinical data regarding the significance of this observation. Recent exposure to an echinocandin was found to significantly increase the risk of infection with C. parapsilosis [C. parapsilosis in some medical centers has been linked to increased echinocandin use [C. parapsilosis with anidulafungin versus fluconazole [C. parapsilosis in the standard dose regimen (50 mg/day) versus the high-dose regimen (150 mg/day)  [C. parapsilosis infection. In contrast, observational clinical data did not show an association between echinocandin treatment of C. parapsilosis bloodstream and treatment failure [C. parapsilosis infection, but alternative agents should be considered in patients with persistent infection while receiving an echinocandin.psilosis . Indeed,ndin use ,87. Clinconazole  and withconazole , althougctively) . These t failure . EchinocC. auris has not yet been defined. For most clinical isolates, MICs of echinocandins are in the susceptible range. Echinocandins are currently recommended by the US centers for disease control as empiric treatment of C. auris infections [C. auris is tolerant to echinocandins. Exposure to echinocandins was consistently found to be a risk factor for infection with C. auris, and breakthrough infection on echinocandins has been noted [in vitro and animal data support the use of liposomal amphotericin B, alone or in combination with flucytosine, for patients with known or suspected C. auris infection [Optimal drug treatment of fections . Howeveren noted ,90,91. Pnfection .Candida from the bloodstream. In general, once the patient is clinically stable, repeat blood cultures are negative and the Candida isolate is found to be sensitive to fluconazole, treatment may be safely switched to fluconazole. This strategy was shown to be safe and feasible for multiple Candida species, including C. glabrata [Two weeks of antifungal treatment seem to be sufficient to prevent late complications from clinically important metastatic foci of infection in the vast majority of cases . Blood cglabrata . De-escaC. parapsilosis is particularly frequent as a cause of CVC infection, and neutropenic patients are relatively more likely than non-neutropenic patients to have Candida bloodstream infection that originates in the gut [The majority of patients with candidemia have indwelling central venous catheters (CVC) when the diagnostic blood culture is obtained ,95, but  the gut . For ind the gut ,88,96,97 the gut , it is g the gut ,12,84,96 the gut . For cliCandida CVC infection, but is not useful in cases of C. glabrata bloodstream infection [C. glabrata, a breakpoint of 48 h may be used [As for bacterial bloodstream infection, differential time to positivity, i.e., the time difference between detection of growth in blood drawn from the CVC lumen to peripherally drawn blood, is a readily-available metric that may be used to predict the likelihood of CVC infection . A diffenfection . Periphenfection . For the be used .Candida species from difficult to extract CVCs [Catheter lock therapy has been used to salvage long-term CVCs infected with low-pathogenicity microorganisms . There iact CVCs . Ethanolact CVCs .Management of other foci of deep-seated infection is equally important. Intra-abdominal candidiasis tends to progress to abscesses that are persistent and recalcitrant, and if undrained may predispose for the development of drug-resistance ,104. EchEndocarditis is a rare but devastating complication of candidemia. In an observational single center cohort study, the authors reported findings suggestive of endocarditis in 2.9% of patients who underwent transthoracic echocardiography (TTE) and 11.4% of patients who underwent transesophageal echocardiography (TEE) . ImportaCandida species to the eyes occurs in 9% to 16% of patients with candidemia [Metastatic spread of ndidemia ,107, andndidemia . Clinicandidemia . Howeverndidemia , suggestndidemia . For flundidemia ,110. DetCandida-active compounds are currently in various stages of drug development and clinical trials. These include agents with potent activity against challenging Candida species, such as echinocandin- and azole-resistant C. glabrata and multidrug-resistant C. auris.A number of exciting new Candida species in in vitro assays and animal models [Candida strains, including C. auris [C. glabrata strains with acquired resistance to SCY-078 have already been described, harboring partially novel FKS mutations [C. auris infections (NCT03363841).Ibrexafungerp  is a first-in-class oral glucan synthase inhibitor that has shown fungicidal activity against a wide range of l models . ImportaC. auris ,112,113.utations . ResultsRezafungin  is an echinocandin that is notable for its long half-life (80 h), allowing once weekly dosing. Potential advantages include simpler management of patients requiring long courses of treatment as outpatients. A phase 2 study is underway, comparing rezafungin with caspofungin followed by fluconazole in patients with invasive candidiasis (NCT02734862) .C. glabrata strains [Oteseconazole  is a next-generation azole (tetrazole) with improved specific affinity towards fungal versus mammalian CYP51. As a consequence, VT-1161 is active against fluconazole-resistant  strains , and hasC. auris in vitro, and increased the survival of C. auris infected mice  [APX001 , another first-in-class drug, is a small molecule that inactivates the fungal enzyme Gwt1. This prevents post-translational modification of glycosylphosphatidylinositol (GPI) and disrupts the anchoring of mannoproteins to the fungal cell wall, causing cell wall damage and impaired growth and biofilm formation. APX001 was shown to be active against afungin) . IntraveMAT2203  is a novel encochleated amphotericin B formulation that allows oral administration. Orally administered MAT2203 prevented death and reduced tissue fungal burden in mice with invasive candidiasis ,120. CliCandida isolates at some hospitals. Treatment options for drug-resistant C. glabrata and C. auris are poorly defined, and may well change following results from ongoing clinical trials with novel antifungal agents.Treatment of invasive candidiasis is changing. Sensitive molecular diagnostics have the potential to shift treatment strategies from current, risk-based empirical therapy to early targeted treatments. Echinocandins have transformed the care of patients with invasive candidiasis, but their widespread use is leading to rising rates of resistance in


Castanopsis sclerophylla (Lindl.) Schott is an important species of evergreen broad-leaved tree in subtropical areas and has high ecological and economic value. However, there are few studies on its chloroplast genome. In this study, the complete chloroplast genome sequence of C. sclerophylla was determined using the Illumina HiSeq 2500 platform. The complete chloroplast genome of C. sclerophylla is 160,497 bp long, including a pair of inverted repeat (IR) regions  separated by a large single-copy (LSC) region of 90,255 bp and a small single-copy (SSC) region of 18,892 bp. The overall GC content of the chloroplast genome is 36.82%. A total of 131 genes were found; of these, 111 genes are unique and annotated, including 79 protein-coding genes, 27 transfer RNA genes (tRNAs), and four ribosomal RNA genes (rRNAs). Twenty-one genes were found to be duplicated in the IR regions. Comparative analysis indicated that IR contraction might be the reason for the smaller chloroplast genome of C. sclerophylla compared to three congeneric species. Sequence analysis indicated that the LSC and SSC regions are more divergent than IR regions within Castanopsis; furthermore, greater divergence was found in noncoding regions than in coding regions. The maximum likelihood phylogenetic analysis showed that four species of the genus Castanopsis form a monophyletic clade and that C. sclerophylla is closely related to Castanopsis hainanensis with strong bootstrap values. These results not only provide a basic understanding of Castanopsis chloroplast genomes, but also illuminate Castanopsis species evolution within the Fagaceae family. Furthermore, these findings will be valuable for future studies of genetic diversity and enhance our understanding of the phylogenetic evolution of Castanopsis. Castanopsis (Lindl.) Schott. is a monoecious, broad-leaved tree of the genus Castanopsis belonging to the Fagaceae family. The genus contains approximately 120 known species, of which 58 are native and 30 are endemic to China. However, C. sclerophylla is widely distributed in East and South Asia, and the tree has been introduced to North America[C. sclerophylla is a canopy tree widely distributed in subtropical evergreen forests[C. sclerophylla is severely fragmented and constantly threatened, requiring urgent conservation and restoration[n forests. In Jiann forests, biomassn forests, morpholn forests, and genn forests of this storation. Phylogestoration.Chloroplasts are essential organelles in plant cells that play very important roles in photosynthesis, carbon fixation, and synthesis of pigments, starch, fatty acids, and amino acids, 17. TheC. sclerophylla chloroplast genome using Illumina technology. This is the first comprehensive analysis of the C. sclerophylla chloroplast genome in conjunction with the previously published whole-chloroplast genome sequences of three congeneric species. In addition, we used 22 complete chloroplast genome sequences from GenBank to analyze phylogenetic relationships and infer the phylogenetic position of C. sclerophylla. The results not only provide basic knowledge about the characteristics of C. sclerophylla but also enhance our understanding of Castanopsis species evolution within the family Fagaceae. Our data will contribute to our understanding of the genetic resources and evolution of C. sclerophylla based on the diversity in its chloroplast genome and also facilitate the exploration, utilization and application of conservation genetics of this species.In this study, we sequenced the C. sclerophylla were collected from the Jiangxi Agricultural University Arboretum in Nanchang, China . Total genomic DNA was extracted using a Plant Genomic DNA Kit . Agarose gel electrophoresis and a microplate spectrophotometer  were used to measure DNA quality and concentration, respectively. Shotgun libraries with an average insert size of 350 bp were constructed using pure DNA and sequenced from 150 bp paired-end read lengths with the Illumina HiSeq 2500 platform . To obtain high-quality clean data, raw reads were filtered by removing the connector sequence and low-quality reads using NGS QC Toolkit_v.2.3.3[Fresh young leaves of t_v.2.3.3.C. hainanensis, C. echinocarpa, and C. concinna) were used to extract chloroplast-like reads, which were assembled by NOVOPlasty[C. sclerophylla were drawn with the OGDRAW v1.2 program[The high-throughput raw reads were trimmed by FastQC. Next, the trimmed paired-end reads and references , C. echinocarpa (KJ001129.1), and C. hainanensis (MG383644.1), in Shuffle-LAGAN mode, adopting the annotation of C. concinna as a reference.MUMmer was emplC. sclerophylla and all of the published chloroplast genomes (data present in NCBI GenBank on 31.12.2018). The other 19 chloroplast (cp) genomes species are from the specises Castanea henryi voucher CHEN20160703 (KX954615.1), Castanea mollissima (HQ336406.1), Castanopsis concinna voucher Strijk_1489 (KT793041.1), Castanopsis echinocarpa (KJ001129.1), Castanopsis hainanensis (MG383644.1), Fagus engleriana (KX852398.1), Lithocarpus balansae (KP299291.1), Quercus aliena (KP301144.1), Quercus aquifolioides (KP340971.1), Quercus baronii (KT963087.1), Quercus dolicholepis (KU240010.1), Quercus glauca (KX852399.1), Quercus rubra (JX970937.1), Quercus sichourensis (MF787253.1), Quercus spinosa (KM841421.1), Quercus tarokoensis (MF135621.1), Quercus tungmaiensis (MF593893.1), Quercus variabilis (KU240009.1), and Trigonobalanus doichangensis (KF990556.1). Phylogenies were constructed by maximum likelihood (ML) using the 20 cp genomes of the Fagaceae species in GenBank. Corylus fargesii (KX822767.2) and Eucalyptus umbra (KC180778.1) were used as outgroups. Sequences were initially aligned using MAFFT[In total, 20 chloroplast genomes belonging to Fagaceae were analyzed in this study, including the newly generated chloroplast genome ing MAFFT, followeissima HQ6406.1, Cioides KP0971.1, Q1489 KT7941.1, Casensis MF1621.1, Qussima HQ3406.1, CaC. sclerophylla is 160,497 bp in length; it has been deposited in GenBank under accession number MK387847. The genome has a typical quadripartite structure including a pair of IR (IRa and IRb) regions of 25,675 bp that are separated by an LSC region of 90,255 bp and an SSC region of 18,892 bp ; and trnL-UAA the shortest (485 bp)  . A similcutissima. ycf3 geomplexes . TherefoC. sclerophylla chloroplast genome using protein-coding sequences (Relative synonymous codon usage frequency (RSCU) valueswere computed for the equences , as codoequences . SimilarCastanopsis genus  were selected for comparison with that of C. sclerophylla. C. sclerophylla has the smallest chloroplast genome ; C. echinocarpa has the largest chloroplast genome  with the smallest IR region . Additionally, the lengths of LSC regions varied among these four species, from 90,255 bp in C. sclerophylla to 90,394 bp in C. echinocarpa ; the IRb region includes 1,131, 1,157, 1,107, and 1,092 bp of the ycf1 gene. The SSC/IRa junction also extends into the SSC region by different lengths depending on the genome ; the IRa region includes 1,092, 1,114, 1,107, and 1,092 bp of the ycf1 gene.The expansion and contraction of IR regions at the borders are the major reason for chloroplast genome size variation and play vital roles in evolution–49. A dent genes . Overalls Quercus. The rpsabidopsis, 51. TheC. fargesii and E. umbra were used as outgroups. The ML-based phylogenetic analysis showed that these four species of the genus Castanopsis form a monophyletic clade and that C. sclerophylla is closely related to C. hainanensis with strong bootstrap values. The ML tree indicated that Castanopsis is closely related to Castanea. Surprisingly, Quercus species do not form a clade, and Quercus is not divided into two clusters containing either evergreen or deciduous tree species. The phylogenetic status of these genera is consistent with a previous report[Quercus may be related to the widely distributed range which need to local adaptation to different environments. Notably, F. engleriana is the first to diverge in Fagaceae, which indicates the relatively high genetic divergence between F. engleriana and others, followed by T. doichangensis, which indicates that they are early diverging taxa in Fagaceae[Fagus was confirmed by the research based on nuclear marker[Phylogenetic analysis was performed by ML based on the 22 aligned sequences of chloroplast genomes . C. fargus report, 52, 53. Fagaceae. Moreovear marker.Castanopsis, and only three chloroplast genome sequences of Castanopsis species can be found in GenBank, which has greatly hampered the study of the phylogenetic relationships of this genus. Therefore, more research on the complete chloroplast genomes of Castanopsis species needs to be conducted in the future.Little is known to date about the chloroplast genome of C. sclerophylla is an important evergreen broad-leaved species in the Castanopsis genus of the Fagaceae family. In this study, the complete chloroplast genome sequence of C. sclerophylla was determined using the Illumina HiSeq 2500 platform. The C. sclerophylla chloroplast genome exhibits a typical quadripartite and circular structure similar to that of the chloroplast genome of three congeneric species. Compared to the chloroplast genomes of the three other Castanopsis species, that of C. sclerophylla is the smallest . In the ML phylogenetic tree, the phylogenetic relationships among 22 angiosperms strongly support the known classification of C. sclerophylla, and ML analysis showed that these four Castanopsis species form a monophyletic clade and that C. sclerophylla is closely related to C. hainanensis with strong bootstrap values. In addition, Castanopsis is closely related to Castanea. The genus Castanopsis contains approximately 120 known species, nearly half of which are native to China. Indeed, China has a large amount of Castanopsis germplasm resources, and the availability of chloroplast genomes provides a powerful genetic resource for phylogenetic analysis and biological study. Therefore, further research of the complete chloroplast genome of the genus Castanopsis is necessary in the future. The data will contribute to the development of genetic resources and the identification of evolutionary relationships and also facilitate the exploration, utilization and application of conservation genetics for the genus.S1 Table(DOCX)Click here for additional data file.S2 Table(DOCX)Click here for additional data file.


Trypanosoma cruzi TcII/V/VI infection. Positive test response is associated with severity of Chagas cardiomyopathy. Repeated T. cruzi infection may exacerbate disease and boost immune response to this TSSApep-II/V/VI epitope.We describe a novel, point-of-care, rapid diagnostic test for  Trypanosoma cruzi causes Chagas disease in the Americas. The outcome of infection ranges from lifelong asymptomatic status to severe disease. Relationship between T. cruzi lineage (TcI-TcVI) infection history and prognosis is not understood. We previously described peptide-based lineage-specific enzyme-linked immunosorbent assay (ELISA) with trypomastigote small surface antigen (TSSA).A novel rapid diagnostic test  that incorporates a peptide that corresponds to the TSSA II/V/VI common epitope was developed and validated by comparison with ELISA. Patients from Bolivia and Peru, including individuals with varying cardiac pathology, and matched mothers and neonates, were then tested using Chagas Sero K-SeT.P = .007). In Peru, Chagas Sero K-SeT detected the sporadic TcII/V/VI infections.Chagas Sero K-SeT and ELISA results, with a Bolivian subset of cardiac patients, mothers, and neonates, were in accord. In adult chronic infections (n = 121), comparison of severity class A (no evidence of Chagas cardiomyopathy) with class B (electrocardiogram suggestive of Chagas cardiomyopathy) and class C/D  revealed a statistically significant increase in Chagas Sero K-SeT reactivity with increasing severity (χ2 for trend, 7.39; We developed a low cost RDT that can replace ELISA for identification of TSSA II/V/VI immunoglobulin G. Most importantly, we show that response to this RDT is associated with severity of Chagas cardiomyopathy and thus may have prognostic value. Repeated challenge with T. cruzi infection may both exacerbate disease progression and boost the immune response to the TSSApep-II/V/VI epitope. Trypanosoma cruzi is the agent of Chagas disease, considered the most important human parasitic disease in Latin America. Trypanosoma cruzi is transmitted via contamination with infected feces of blood-feeding triatomine bugs through mucous membranes, abraded skin, or oral ingestion. The parasite can also be transmitted congenitally and by contaminated blood components or organs. The initial acute phase may be mild and unrecognized, but the disease can be fatal, particularly in immunocompromised hosts. The subsequent chronic phase may be asymptomatic for life, but around 30% of those infected progress to Chagas cardiomyopathy, and a smaller percentage may develop gastrointestinal megasyndromes. A recent World Health Organization report  7.2). RDT results were positive in 68.7% (57/83) and 66.7% (32/48) of CS+ mothers from Camiri and Santa Cruz, respectively (P = .008) and slightly less likely to be male . There was no difference in the prevalence of positive RDT by participant age (P = .91) or sex (P = 1.0). However, 52.5% (21/40) of those without evidence of cardiomyopathy had positive RDT results compared with 74.1% (60/81) of those with cardiomyopathy . There was a significant trend for increasing frequency of RDT reactivity with increasing cardiac severity stage  were male and the mean age was 57.1 years (SD 12.3). CS− participants were significantly younger . However, the prevalence of positive RDT was markedly lower in Peru compared with Bolivia .A total of 84 specimens (65 CS+ and 19 CS−) were tested, 61 from southern Peru and 23 from northern Peru. The mean age of participants was 41.7 years (SD 19.8) and 59.5% were female. There was no significant difference in the prevalence of positive RDT in CS+ specimens from southern vs northern Peru  were detected by Chagas Sero K-SeT RDT as TcII/V/VI seropositive, corresponding to a specificity of 96.5%  compared with conventional serology.T. cruzi are remarkably diverse. Understanding the relationship between an individual’s history of T. cruzi lineage infection and clinical outcome has been a research interest since the discovery of disparate geographical distributions of TcI and TcII between patients in Venezuela and Brazil, where prevalence of gastrointestinal syndromes also appeared strikingly divergent [T. cruzi diversity and clinical prognosis of Chagas disease, including human genetic diversity, immune competence, and exposure to coinfections [T. cruzi lineages, partially circumventing limitations of selective isolation and genotyping of T. cruzi strains.The genetic lineages of ivergent , 6. Neveivergent . Multiplfections . DevelopT. cruzi lineage genomes, we demonstrated the serological specificity of TSSApep-II/V/VI and the absence of response in control infections [The antigenicity of the TcII/V/VI isoform of TSSA has been established by serology of patients and animals in conjunction with bioinformatic and epitope mapping , 20, 21.fections . Howeverfections , and unpT cruzi antibodies have been reported (set 3).Previous lineage-specific serology with TSSA has used Western blot, ELISA, or other complex protocols that require skilled technical staff, laboratory facilities, and usually a cold storage chain . We deveT. cruzi from neonates born to Bolivian mothers identified predominantly TcV; however, no association has been shown between T. cruzi lineage and congenital transmission risk [To our knowledge, this is the first report of lineage-specific serology with matched maternal and cord blood. Bisio et al  found seion risk . Balouz ion risk  used recOur analysis of results from older adults in Santa Cruz (set 2) demonstrates a significant association between the prevalence of positive results by this RDT and increasing severity of cardiomyopathy. We found evidence of a similar relationship in a previous study in southern Brazil, in which ECG abnormalities were more frequent among TSSApep-II/V/VI seropositive patients compared with TSSApep-II/V/VI seronegative patients . CytokinT. cruzi superinfection in those living in infested houses may increase inflammatory responses and the consequent risk of immunologically mediated cardiac damage [T. cruzi infection in triatomine-infested dwellings may both enhance the inflammatory response and boost the immune response to the TSSApep-II/V/VI epitope. Prolonged residence in an infested house was associated with increased risk of ECG abnormalities in our earlier analysis of data from Bolivian mothers [The mechanism underlying the association we observed between positive RDT and cardiac disease severity may be related to repeated parasite exposure over time. Investigators have previously postulated that repeated c damage . Thus, r mothers .T. cruzi immune responses among patients in southern Peru [We found a much lower prevalence of TcII/V/VI seropositivity among Peruvian patients than Bolivian patients. These results are in agreement with the widely reported low prevalence of these lineages outside the Southern Cone , 3, 25. ern Peru , 26; eveT. cruzi lineages, as well as for improved RDTs that are equally applicable across all diverse T. cruzi endemic regions. Furthermore, we have shown that the Chagas Sero K-Set RDT may provide an indicator of presence and risk of Chagas cardiomyopathy. We have proposed that this is explicable by concomitant exacerbation of disease and boosting of immune response to the TSSApep-II/V/VI epitope.Here, we have produced a highly informative, low-cost, point-of-care, lineage-specific RDT. Thus, there is justification to search for antigens applicable to each of the