Asymptomatic Isolates Research Articles

Genetic variability of grapevine Pinot gris virus (GPGV) in an organically cultivated vineyard in Hungary

Grapevine Pinot gris virus (GPGV) is a recently identified trichovirus infecting grapevines. Despite wide distribution, there is limited available information on epidemiology, transmission, and associated symptoms of grapevine leaf mottling and deformation. Occurrence and genetic diversity of GPGV variants were surveyed in an organically cultivated Hungarian vineyard that was planted between 1996 and 2014. Sequence analysis demonstrated the widespread presence and high variability of GPGV, and according to phylogenetic analyses, the Hungarian virus isolates were classified into three groups. Most of the identified variants clustered with the representative asymptomatic isolates, but all isolates from one grapevine cultivar grouped with representative isolates of clade B. Furthermore, one isolate clustered with representative isolates of clade C, and the identified clade C variant had previously undescribed polymorphisms.

Turbidimetric bioassays: A solution to antimicrobial activity detection in asymptomatic bacteriuria isolates against uropathogenic Escherichia coli.

Traditional bacteriocin screening methods often face limitations due to diffusion-related challenges in agar matrices, which can prevent the peptides from reaching their target organism. Turbidimetric techniques offer a solution to these issues, eliminating diffusion-related problems and providing an initial quantification of bacteriocin efficacy in producer organisms. This study involved screening the cell-free supernatant (CFS) from eight uncharacterized asymptomatic bacteriuria (ABU) isolates and Escherichia coli 83972 for antimicrobial activity against clinical uropathogenic E. coli (UPEC) strains using turbidimetric growth methods. ABU isolates exhibiting activity against five or more UPEC strains were further characterized (PUTS 37, PUTS 58, PUTS 59, S-07-4, and SK-106-1). The inhibition of the CFS by proteinase K suggested that the antimicrobial activity was proteinaceous in nature, potentially bacteriocins. The activity of E. coli PUTS 58 and SK-106-1 was enhanced in an artificial urine medium, with both inhibiting all eight UPECs. A putative microcin H47 operon was identified in E. coli SK-106-1, along with a previously identified microcin V and colicin E7 in E. coli PUTS 37 and PUTS 58, respectively. These findings indicate that ABU bacteriocin-producers could serve as viable prophylactics and therapeutics in the face of increasing antibiotic resistance among uropathogens.

Prevalence of non-falciparum malaria infections among asymptomatic individuals in four regions of Mainland Tanzania

BackgroundRecent studies point to the need to incorporate the detection of non-falciparum species into malaria surveillance activities in sub-Saharan Africa, where 95% of the world’s malaria cases occur. Although malaria caused by infection with Plasmodium falciparum is typically more severe than malaria caused by the non-falciparum Plasmodium species P. malariae, P. ovale spp. and P. vivax, the latter may be more challenging to diagnose, treat, control and ultimately eliminate. The prevalence of non-falciparum species throughout sub-Saharan Africa is poorly defined. Tanzania has geographical heterogeneity in transmission levels but an overall high malaria burden.Methods To estimate the prevalence of malaria species in Mainland Tanzania, we randomly selected 1428 samples from 6005 asymptomatic isolates collected in previous cross-sectional community surveys across four regions and analyzed these by quantitative PCR to detect and identify the Plasmodium species.ResultsPlasmodium falciparum was the most prevalent species in all samples, with P. malariae and P. ovale spp. detected at a lower prevalence (< 5%) in all four regions; P. vivax was not detected in any sample.Conclusions The results of this study indicate that malaria elimination efforts in Tanzania will need to account for and enhance surveillance of these non-falciparum species.Graphical

The role and influence of bacteria on the growth of Blastocystis sp. subtype 3.

Blastocystis sp. is an enteric protistan parasite that affects individuals worldwide with gastrointestinal symptoms such as abdominal discomfort, diarrhea, and flatulence. However, its pathogenicity is controversial due to its presence among asymptomatic individuals. Blastocystis sp. subtype 3 (ST3) is the most prevalent subtype among humans that have been associated with irritable bowel syndrome (IBS), Crohn's disease, ulcerative colitis, and colorectal cancer. Axenization of the parasite has been shown to impede its growth thus revealing the importance of accompanying bacteria in ensuring Blastocystis sp. survival. This study aims to identify the influence of accompanying bacteria on the growth of Blastocystis sp. ST3. Blastocystis sp. cultures were treated with Meropenem, Vancomycin, and Amoxicillin-Clavulanic acid (Augmentin). Bacteria-containing supernatant of antibiotic-treated and control cultures were isolated and identified through 16s rRNA sequencing. Morphological changes of antibiotic-treated Blastocystis sp. ST3 were also observed. The cultures treated with meropenem and augmentin exhibited opposing effects with reduced growth of isolates from symptomatic patients and a significant increase in asymptomatic isolates. Whereas, vancomycin-treated cultures had no difference in the growth of Blastocystis sp. ST3 isolates from symptomatic and asymptomatic patients. Isolates from symptomatic and asymtomatic patients had 6 and 2 distinct bacterial species identified with Proteus mirabilis as the common bacteria among both types of isolates. Morphologically, Blastocystis sp. ST3 cultures exposed to meropenem and augmentin demonstrated an increase in pre-cystic forms. These findings demonstrate the effects of accompanying bacteria on the growth of Blastocystis sp. ST3 that could translate into clinical manifestations observed among Blastocystis sp.-infected patients.

Phylogenetic and Evolutionary Studies of Grapevine Pinot Gris Virus Isolates from Canada.

This study investigated the phylogenetic relationship of grapevine Pinot gris virus (GPGV) isolates from Canada with GPGV isolates reported worldwide. Full-length genomes of 25 GPGV isolates representing the main four grape-growing regions in Canada (British Columbia, Ontario, Nova Scotia and Quebec) were sequenced and compared to genomes of 43 GPGV isolates representing eight countries and three continents. Phylogenetic analysis based on full genome sequences revealed an unambiguous separation of North American GPGV isolates with isolates from Europe and Asia. Within the North American clade, GPGV isolates from the USA segregated into a distinct subclade, whereas the relationships amongst GPGV isolates from different regions of Canada were not clearly defined. The phylogenetic analysis of the overlapping regions of MP and CP genes involving 169 isolates from 14 countries resulted in two distinctive clades, which were seemingly independent of their country of origin. Clade 1 included the majority of asymptomatic isolates (81% asymptomatic), whereas clade 2 was predominantly formed of symptomatic isolates (78% symptomatic). This research is the first study focused on the genetic variability and origin of GPGV in Canada.

Distinct Phenotypic Variation of Blastocystis sp. ST3 from Urban and Orang Asli Population-An Influential Consideration during Sample Collection in Surveys.

Simple SummaryBlastocystis sp. is a common intestinal protozoan of humans with the phenotypic characteristics strongly associated with its activity, including pathogenicity. This characteristic varies, but the variation has not been clearly understood. The present study evaluates the variation when a single subtype of Blastocystis sp. was isolated from a population with distinct gut microbial composition, namely, the urban and orang asli(indigenous) population. Blastocystis sp. cells isolated from orang asli individuals had a higher growth rate with elevated resistance to harsh conditions. Distinct surface coats with amoebic forms were noticed in parasite cells from urban individuals. Proteases, commonly a virulent factor in other parasites, showed variation depending on the isolation source. Stimulation of cancer cell proliferation by only Blastocystis sp. isolated from urban individuals is suggestive of the variation at the antigenic level. This phenotypic variation suggests that implicating subtype to pathogenicity may be too early, and a deeper understanding of Blastocystis sp. and microenvironment interaction is essential.Blastocystis sp. is a globally distributed protozoan parasite with uncertain pathogenicity. Phenotypic variation in Blastocystis sp. suggests its adaptation; however, the phenotypic features of Blastocystis sp. ST3 from a distinct source of isolation is unknown. Blastocystis sp. isolated from individuals in urban and orang asli (indigenous population in Selangor, Malaysia) settlements were studied for phenotypic characteristics such as growth profile, morphology, ultrastructure, and resistance to harsh conditions. Subsequently, pathogenic potentials, such as in protease activity and the ability to stimulate the proliferation of cancer cells, were assessed. Higher parasite counts with granular and apoptotic forms were found in Blastocystis sp. from orang asli individuals. Cells with fuzzy coats and amoebic structures which seemingly implicate increased interaction with bacteria were seen predominantly in urban symptomatic persons. Also, Blastocystis sp. from orang asli isolates resisted harsh environments, suggesting longer co-adaptation to the hosts. Urban and orang asli symptomatic isolates possessed a predominance of only cysteine protease, whereas all the asymptomatic isolates showed significantly higher cysteine, serine, or aspartic protease activity. However, only solubilized antigen from urban symptomatic isolates showed significant stimulation of cancer cell proliferation. For the first time, our findings demonstrate significant phenotypic variation in a single subtype, ST3 of Blastocystis sp., isolated from urban and orang asli populations that are known to have distinct gut microbial compositions. The outcome emphasizes the importance of identifying people’s locations and lifestyles during sample collection before forming conclusions on the prevailing data and implicating subtypes to pathogenicity. The environment plays a significant role in Blastocystis sp. infection.

Demonstrating the utility of Escherichia coli asymptomatic bacteriuria isolates' virulence profile towards diagnosis and management-A preliminary analysis.

Asymptomatic bacteriuria (ASB) caused by Escherichia coli (E. coli) is a significant condition associated with pregnancy and is considered as prognostic for the development of symptomatic urinary tract infection (UTI). However, treating all ASB increases the use of antibiotics and leads to the development of multidrug resistance (MDR). Therefore, this study aimed to identify the distribution of UPEC associated virulence genes and antibiotic susceptibility among phylogroups of E. coli isolated from ASB in pregnancy. Moreover, the gene expression of selected virulence genes was also compared among two E. coli isolates (with different pathogenic potential) to determine its pathogenicity. One hundred and sixty E. coli isolates from midstream urine samples of pregnant women with ASB were subjected to PCR-based detection for its phylogroups and virulence genes. The antibiotic susceptibility of isolated strains was determined by the disc diffusion method. Expression of the virulence genes were determined through microarray analysis and quantitative Real-Time PCR. The prevalence of ASB in this study was 16.1%. Within ASB isolates, the occurrence of phylogroup B2 was the highest, and isolates from this group harboured most of the virulence genes studied. Overall, the most identified virulence genes among all phylogroups in descending order were fimH, chuA, kpsMTII, usp, fyuA, hlyA, iroN, cnf, papC, sfa, ompT, and sat. In this study, higher resistance to antibiotics was observed for ampicillin (77.5%), amoxicillin-clavulanate (54.4%), trimethoprim-sulfamethoxazole (46.9%) and amikacin (43.8%) compared to the other tested antibiotics and 51.9% of the tested isolates were MDR. Furthermore, hierarchical clustering and gene expression analysis demonstrated extreme polarization of pathogenic potential of E. coli causing ASB in pregnancy necessitating the need for bacterial isolate focused approach towards treatment of ASB.

Occurrence and Genetic Characterization of Grapevine Pinot Gris Virus in Russia.

Grapevine Pinot gris virus (GPGV) is a widespread grapevine pathogen associated with symptoms of leaf mottling and deformation. In order to study the distribution and genetic diversity of GPGV in Russia, we tested 1347 grapevine samples from 3 regions of Russia–the Krasnodar Krai, Stavropol Krai, and Republic of Crimea—using duplex real-time RT-PCR. GPGV was detected in 993 grapevines, both symptomatic and asymptomatic. In 119 isolates, we sequenced complete movement protein (MP) and coat protein (CP) genes of the GPGV genome. The percentage of identity of the obtained nucleotide MP/CP sequences with the closest isolates from the GenBank was 97.75–99.56%. A phylogenetic analysis showed that these Russian GPGV isolates are mainly grouped with previously described representative asymptomatic isolates. New post-translational modifications of the MP and CP at the positions of polymorphisms in the genomes of Russian isolates were predicted. The present work is the first study on the distribution and genetic diversity of GPGV in Russia.

Virulence gene mutations as a differentiator of clinical phenotypes: insights from community-acquired uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) remains an important cause of urinary tract infection during pregnancy. Multiple molecular virulence determinants and antibiotic resistant genes facilitate its pathogenesis and virulence phenotype. Hence it is hypothesized that there will be considerable variation in genes among the isolates from symptomatic as well as asymptomatic bacteriuria (ABU) during pregnancy. The aim of this study was to decipher the genetic variation among the two phenotypes. Six different UPEC isolates collected from urine specimens of consecutive pregnant females (five, symptomatic bacteriuria and one, ABU) were tested for their growth kinetics, and biofilm formation. A total of 87 virulence determinants and 56 antibiotic resistance genes were investigated using whole-genome sequencing, to identify putative drives of virulence phenotype. In this analysis, we identified eight different types of fully functional toxin antitoxin (TA) systems [HipAB, YefM-YoeB, YeeU-YeeV (CbtA), YhaV-PrlF, ChpBS, HigAB, YgiUT and HicAB] in the isolates from symptomatic bacteriuria; whereas partially functional TA system with mutations were observed in the asymptomatic one. Isolates of both the groups showed equivalent growth characteristics and biofilm-formation ability. Genes for an iron transport system (Efe UOB system, Fhu system except FhuA) were observed functional among all symptomatic and asymptomatic isolates, however functional mutations were observed in the latter group. Gene YidE was observed predominantly associated with the biofilm formation along with few other genes (BssR, BssS, YjgK, etc.). This study outlines putative critical relevance of specific variations in the genes for the TA system, biofilm formation, cell adhesion and colonization among UPEC isolates from symptomatic and asymptomatic bacteriuria among pregnant women. Further functional genomic study in the same cohort is warranted to establish the pathogenic role of these genes.

Distribution of virulence determinants in Streptococcus agalactiae recovered from different clinical sources

Streptococcus agalactiae (group B Streptococcus, GBS) is a pathobiont, a member of human microbiota that can change from commensal to pathogen, causing a large spectrum of diseases. This study assessed virulence determinants of 32 GBS isolates recovered from different clinical sources associated with asymptomatic and symptomatic clinical outcomes that present distinct capsular types and antimicrobial resistance profiles. The ability of a unique strain to colonize and cause infection in different subjects was also evaluated. By PFGE analysis, it was observed that a given strain could be associated with both asymptomatic and symptomatic outcomes. Cell wall anchor proteins β and alpha C encoding genes (bac and bca, respectively) were detected in all capsular type Ib isolates. bca was more frequent among asymptomatic outcome-related isolates, as well as high expression of β-hemolysin/cytolysin (β-H/C). Symptomatic outcome-related isolates produced strong biofilm more frequently. All bacterial isolates recovered from urine were strong biofilm producers. In growth experiments, asymptomatic outcome-related isolates grew faster after 2 h until the end of the log phase. Taken together, these findings show virulence genotypic and phenotypic features of GBS from distinct sources, which may be helpful to understand their pathogenic potential and predict different clinical outcomes.

Association of Blastocystis ST6 with higher protease activity among symptomatic subjects

BackgroundBlastocystis sp. is an anaerobic intestinal protozoan parasite of humans and a wide range of animals worldwide. In the current study the correlation between the cysteine protease activity of clinical samples of Blastocystis sp. ST1–3 and 6 with the levels of pro-inflammatory cytokines was evaluated.MethodsStool samples were collected from subjects with or without clinical symptoms. All samples were cultivated in DMEM medium. The bacteria were eliminated or reduced in Blastocystis sp. positive samples subtypes 1–3 and 6 by a variety of antibiotics and consecutive sub-cultures. To prepare parasite lysate, 1 × 105Blastocystis sp. from each isolate were harvested and lysed using freeze-thaw. Protease activity of each isolate was measured and the gene expression of pro-inflammatory biomarkers in HT-29 cell line sensed by isolates was investigated using quantitative Real-time PCR.ResultsProtease activity assay showed inter- and intra-subtype variations among subtypes regarding the presence of symptoms, while the protease activity of symptomatic isolates was higher than asymptomatic isolates. The highest and lowest levels of protease activity were seen in ST6 and ST2, respectively. However, patterns of the expression of pro-inflammatory biomarkers in HT-29 cell line was different regarding the presence of symptoms and time points. There was no significant correlation between protease activity of different subtypes with the expression levels of pro-inflammatory biomarkers.ConclusionsOur study indicated a higher protease activity among isolates from symptomatic compared to asymptomatic subjects, suggesting functional role for proteases in clinical symptoms due to Blastocystis sp. The lack of correlation between the levels of expression of pro-inflammatory biomarkers with subtypes regarding the presence of clinical symptoms proposes the importance of host-related factors in presentation of clinical symptoms.

Aframomum melegueta [Roscoe] K. Schum) Ethanolic Seed Extract against Asymptomatic Bacteriuria Isolates from Pregnant Women Attending Antenatal Clinic in Ondo State, Nigeria

The purpose of this research work is to compare the activity of medicinal plant(Aframomum melegueta) and conventional antibiotic against Asymptomatic Bacteriuria isolates from pregnant women attending ante-natal clinic in a major primary health center in Akoko, south ,Ondo state Nigeria. The target Asymptomatic Bacteriuria isolates which is inherent in pregnant women with no observable features. The rate of growth/ death of Asymptomatic Bacteriuria Isolates were x-rayed. Bacteria were isolated from Urine of pregnant women attending antenatal clinic in Public health care Akungba Akoko, Ondo state, Nigeria and were identified using conventional method. The antibiotic susceptibility test and antimicrobial screening of ethanol seed extract of Aframomum melegueta were determined using disc diffusion and agar well diffusion methods respectively. The eleven(11) bacteria identified were Klebsiella pneumoniae (three,3), Proteus mirabilis, Proteus vulgaris, Bacillus subtilis(Two,2),Pseudomonas aeruginosa, Enterobacter cloacae (Two,2), Escherichia coli (three,3), Bacillus cereus, Serratia mercesiens, Enterobacter aerogens. E. coli and klebsiella pneumoniae were the most common isolates. The second most common isotales was Enterobacter cloacae.. Klebsiella pneumonia and Proteus mirabilis were resistant to Ampicillin, Ceporex, Nalixadic acid and Septrin. E. coli, the most common isolate was sensitive to Gentamycin and most of the antibiotics used. The antimicrobial screening of ethanol seed extract of Aframomum melegueta shows zones of inhibition with diameter ranging from 1-25mm. Secondary metabolite screening indicates the presence of flavonoid, tannins, saponins, alkaloids, cardiac glycosides. Ultraviolet spectrophotometer was also used to determine the Growth dynamic /Death rate of the isolates, the addition of antibiotics to the organism at the 48th hour speed up the death rate of the isolates, the addition of ethanol seed extract at the 48th hour also speed up the death rate of the isolates from the urine samples. The results of this study validate the use of Aframomum melegueta seed in the traditional treatment of Asymptomatic bacteriuria in pregnant women.

Epidemiologic and molecular characterization of β-lactamase-producing multidrug-resistant uropathogenic Escherichia coli isolated from asymptomatic hospitalized patients.

Uropathogenic Escherichia coli (UPECs) are the predominant cause of asymptomatic bacteriuria (ABU) and symptomatic UTI. In this study, multidrug-resistant (MDR) ABU-UPECs from hospitalized patients of Kolkata, India, were characterized with respect to their ESBL phenotype, acquisition of β-lactamase genes, mobile genetic elements (MGEs), phylotype property, ERIC-PCR profile, sequence types (STs), clonal complexes (CCs) and evolutionary and quantitative relationships and compared to the symptomatic ones to understand their epidemiology and evolutionary origin. Statistically significant incidence of ESBL producers, β-lactamase genes, MGEs and novel phylotype property (NPP) among ABU-UPECs similar to the symptomatic ones indicated the probable incidence of chromosomal plasticity on resistance gene acquisition through MGEs due to indiscriminate drug usage. ERIC-PCR typing and MLST analysis showed clonal heterogeneity and predominance of ST940 (CC448) among asymptomatic isolates akin to symptomatic ones along with the evidence of zoonotic transmissions. Minimum spanning tree analysis showed a close association between ABU-UPEC with known and unidentified STs having NPPs with isolates that belonged to phylogroups clade I, D, and B2. This is the first study that reported the occurrence of MGEs and NPPs among ABU-UPECs with the predominance of ESBL production which displayed the deleterious effect of MDR among this pathogen demanding alternative therapeutic interventions. Moreover, this study for the first time attempted to introduce a new approach to ascertain the phylotype property of unassigned UPECs. Withal, increased recognition, proper understanding and characterization of ABU-UPECs with the implementation of appropriate therapeutic measures against them when necessary are the need of the era which otherwise might lead to serious complications in the vulnerable population.

Latent Upregulation of Nlrp3, Nlrc4 and Aim2 Differentiates between Asymptomatic and Symptomatic Trichomonas vaginalis Infection

ABSTRACT Trichomonas vaginalis is a parasitic protozoan that causes trichomoniasis. The involvement of NLRP3 inflammasome in trichomoniasis has been discussed in recent studies. The present study aimed to find out the involvement of Nlrp3, Nlrc4, and Aim2 in the BALB/c mouse model infected with symptomatic and asymptomatic isolates of T. vaginalis by quantitative real-time PCR and immunohistochemistry. Our results showed a significantly increased expression of Nlrp3 in the vaginal tissue of the symptomatic group on the 2nd dpi and 14th dpi in the asymptomatic group, respectively. The cervical tissue of asymptomatic groups expressed higher Nlrp3 on 14th dpi than the symptomatic group. The Nlrc4 was expressed on 14th dpi in the vaginal and cervical tissues of mice infected with asymptomatic group as compared to the symptomatic group. Aim2 expression in vaginal tissue was highest at early time points in both the infected groups as compared to controls. However, in cervical tissues, a significant increase of Aim2 expression was observed on 14th dpi in asymptomatic as compared to the symptomatic group. The significantly higher expression of caspase-1 and caspase-4 was observed in cervical tissues of the asymptomatic group on 14th dpi as compared to the symptomatic group, respectively. All NLRs together resulted in higher IL-1β expression in the vaginal tissues of the symptomatic and asymptomatic groups. We conclude from this study that early expression of Nlrp3, Nlrc4, and Aim2 was seen in the symptomatic group as compared to the late-onset asymptomatic in the vaginal and cervical tissues.

Antibacterial and Antibiofilm Potential of Leptospermum petersonii F.M.Bailey, Eucalyptus citriodora Hook., Pelargonium graveolens L’Hér. and Pelargonium roseum (Andrews) DC. Essential Oils Against Selected Dental Isolates

The study aimed to investigate the potency of Leptospermum petersonii, Eucalyptus citriodora, Pelargonium graveolens, and Pelargonium roseum essential oils (EOs) against Streptococcus sanguinis and Streptococcus salivarius dental isolates. To examine the variation in sensitivity among strains of different origin, isolates from symptomatic and asymptomatic root canal infections, as well as the reference, strains S. sanguinis ATCC 10556 and S. salivarius ATCC 9222 were used in the study. EOs were chemically characterised (GC/MS). Their antibacterial and antibiofilm properties were detected by microdilution and CV assay respectively. Statistical analysis was performed by One way ANOVA. Examined bacterial strains, regardless clinical or ATCC, exhibited the highest sensitivity to L. petersonii and E. citriodora EOs (MIC ranged 0.53±0.00 - 5.88±1.27 mg/ml and 0.08±0.03 - 9.37±1.25 mg/ml respectively), while two other EOs were less efficient. Comparison of MIC values showed that sensitivity of the isolates recovered from symptomatic infections was the least, while the susceptibility of asymptomatic isolates and especially of reference strains were higher. EO inhibition of biofilm formation differed between clinical isolates of S. salivarius and their reference strain. Inhibition was higher in the case of S. salivarius ATCC 9222 (84 %-89 %) in comparison to that of clinical isolates (24 %-75 %). Regardless of the strains tested, E. citriodora exhibited the highest antibiofilm potential. To conclude: L. petersonii induced notable antibacterial potential, while E. citriodora EO proved promising antibacterial and antibiofilm activity. Due to the variable sensitivity of the isolates to EOs, it is suggested to screen activity on a large number of strains, especially from symptomatic infections.

Incidence and Genetic Diversity of Grapevine Pinot gris Virus in California

Grapevine Pinot gris virus (GPGV) was discovered in 2012 by high throughput sequencing (HTS) analysis of Pinot gris vineyards in Italy. GPGV is often symptomless but has also been associated with chlorotic mottling and leaf deformation. In 2015, this new member of the genus <i>Trichovirus</i> was first reported in the United States, in California. To assess the incidence of GPGV throughout grapegrowing regions in California, we tested multiple vineyards using real-time quantitative reverse transcription PCR. HTS and phylogenetics were used to evaluate the genetic diversity of GPGV strains recovered. GPGV was detected in 170 out of 716 (23%) grapevine samples, including white and red grape varieties, from several commercial vineyards and one nursery increase block. Moreover, GPGV was identified in three different counties (Yolo, Solano, and Napa) situated in northern California. In the case of Napa County, a widespread distribution of GPGV was determined. During the survey, symptomatic and asymptomatic plants tested positive for GPGV; however, characterized isolates shared close homology (&gt;97% identity) with asymptomatic reference isolates, which was confirmed by phylogenetics. HTS revealed that GPGV-positive grapevines were also infected with other viruses and viroids. Finally, the relationship between GPGV infection and symptom expression is discussed.

Subtype distribution and genetic diversity of Blastocystis in asymptomatic and symptomatic individuals from Puducherry, India

Background: Blastocystis, an intestinal parasite, consists of 17 subtypes (ST), of which nine are capable of inhabiting humans. Blastocystis pathogenicity is controversial as it harbors both symptomatic and asymptomatic individuals. In India, molecular studies on Blastocystis are meager. Hence, large scale molecular epidemiological studies are required to unveil genetic diversity and pathogenicity of Blastocystis. Methods & Materials: In this cross-sectional analytical study, a total of 752 stool samples (379 male; 373 female) were collected from asymptomatic (group I, n = 384) and symptomatic (group II, n = 368) individuals. Group II was subcategorized based on the specific clinical symptoms: diarrhea (n = 152), inflammatory bowel disorder (IBD, n = 83), dysentery (n = 75), irritable bowel syndrome (n = 31), and colorectal cancer (n = 27). 18SSU rDNA was targeted in PCR for the detection and sub-typing of Blastocystis. 83 representative samples were sequenced and phylogenetic analysis was performed using MEGA 7 software. The subtypes and allele types were determined using http://www.pubmlst.org/Blastocystis database. Fisher's exact test was employed for the comparison between the groups. At 95% CI, p-value <0.05 was considered statistically significant. Results: Asymptomatic individuals had higher occurrence of Blastocystis (n = 135; 33.7%) compared to the symptomatic patients (n = 117; 31%) but statistically insignificant (p = 0.33, OR = 0.8599). Within-group II, patients having diarrhea had the highest detection rate of Blastocystis (n = 57; 37.1%) and the least in patients having IBD (n = 18; 21.6%). A comparison between subgroup IBD and asymptomatic group yielded statistically significant results (p = 0.016, OR = 0.5114). Out of the 252 samples positive for Blastocystis, ST1 (n = 79), ST2 (n = 23), ST3 (n = 145), and ST1 + ST3 mixed infections (n = 5) were identified. ST3 was predominant subtype in both groups. Sequenced samples were deposited in NCBI GenBank (Accession No. MK719604-MK719686). Alleles 31, 34, and 36 of ST3, alleles 9 and 11 of ST2 and alleles 4 and 80 of ST1 were detected. The phylogenetic tree showed no distinct segregation of sequences of symptomatic and asymptomatic isolates. Conclusion: This study mainly contributes to the genetic diversity aspects of Blastocystis in India. For the first time, ST2 was identified from India. However, the existence of all three subtypes in both symptomatic and asymptomatic individuals infers that inter-subtype variation may not be a reliable marker to assess the pathogenicity.

Survey and Diversity of Grapevine Pinot gris virus in Algeria and Comprehensive High-Throughput Small RNA Sequencing Analysis of Two Isolates from Vitis vinifera cv. Sabel Revealing High Viral Diversity.

Grapevine Pinot gris virus (GPGV) is a putative causal agent of grapevine leaf mottling and deformation disease that has been reported worldwide throughout the grapevine-growing regions. Fifty-four grapevines collected from five Algerian grapevine-growing regions were tested for the presence of GPGV in phloem tissues. Eight of the tested grapevines were infected by GPGV. Viromes of two selected Vitis vinifera cv. Sabel grapevines infected by GPGV and showing virus-like symptoms were analyzed by small RNA sequencing. Phylogenetic analyses of the partial coding sequence (cds) of the RNA-dependent RNA polymerase (RdRp) domain showed that all Algerian GPGV isolates were grouped with some already-described asymptomatic isolates. This study provides the first survey of the occurrence of GPGV in Algeria. Moreover, Grapevine fleck virus, Grapevine rupestris stem pitting-associated virus, Grapevine virus B, Grapevine rupestris vein feathering virus, Hop stunt viroid and Grapevine yellow speckle viroid 1 were detected in Algeria for the first time.

Genetic variability of E. histolytica strains based on the polymorphism of the SREHP gene using nested PCR-RFLP in Erbil, North Iraq

E. histolytica is an intestinal parasite that causes asymptomatic infection mostly; however, it may also cause amoebic dysentery and liver abscess. Molecular identification is required in epidemiological studies due to the presence of morphologically identical nonpathogenic species. Therefore, this study was conducted to first evaluate the prevalence rate of E. histolytica among symptomatic individuals of Erbil city, and to investigate the genetic diversity of the parasite in a limited geographic area. Accordingly, a total of 2026 samples were examined microscopically, and confirmed by nested PCR for 18s rRNA gene. The results showed that the prevalence rate of E. histolytica was 1.97% (40 samples) among symptomatic patients. The SREHP gene was used as a marker to show the genetic polymorphism of E. histolytica; however, to compare the genetic diversity of symptomatic with asymptomatic isolates, 57 asymptomatic samples were obtained from our previous study. The amplified products of the SREHP gene were digested by AluI endonuclease, and DNA banding patterns were analysed. Results showed 29 different DNA patterns among the 97 symptomatic and asymptomatic samples, 62 of which shared similar DNA patterns. However, 8 different DNA patterns were observed among asymptomatic samples, whereas 15 distinct patterns were observed among symptomatic isolates. In conclusion, this study found that the prevalence rate of E. histolytica was relatively low; relatively high genetic diversity was observed in a restricted endemic area; with higher rates of variability in symptomatic rather than in asymptomatic isolates, indicating a possible correlation between the genotype of E. histolytica and their clinical outcome.

Genes Encoding Proteolytic Enzymes Fungalysin and Subtilisin in Dermatophytes of Human and Animal Origin: A Comparative Study.

Dermatophytes are among the most successful fungal pathogens in humans, but their virulence mechanisms have not yet been fully characterized. Dermatophytic fungi secrete proteases in vivo, which are responsible for fungal colonization and degradation of the keratinized tissue during infection. In the present study, we used PCR to investigate the presence of genes encoding fungalysins (MEP) and subtilisins (SUB) in three dermatophyte species whose incidence is increasing in Europe: the anthropophilic Trichophyton rubrum (n = 58), zoophilic Microsporum canis (n = 33), and Trichophyton benhamiae (n = 6). MEP2 and SUB4 genes were significantly correlated with T. rubrum; MEP3 and SUB1 were mostly frequently harbored by M. canis; and MEP1, 2, and 4 and SUB3-7 were most frequently harbored by T. benhamiae isolates (p < 0.05). Furthermore, MEP1-5 and SUB1-3 genes were significantly more prevalent among human clinical isolates of M. canis (n = 17) than among asymptomatic cat isolates of M. canis (n = 16; p < 0.05). Unidentified MEP and/or SUB genes in some isolates in the current study may suggest that other gene repertoires may be involved in the degradation of keratin. The presented analysis of the incidence of MEP and SUB virulence genes in three dermatophyte species of diverse origins provides an insight into the host-fungus interaction and dermatophyte pathogenesis.