Group-specific PCR Research Articles

Molecular Typing of Somatic Coliphage Groups and Their Occurrence and Survival in Sewage

A conventional, group-specific PCR method was developed to identify each of the four previously defined major taxa (Myoviridae, Siphoviridae, Podoviridae and Microviridae) of somatic coliphages and used to classify isolates from sewage. Somatic coliphage infectivity detection, occurrence and survival in primary human sewage effluent was observed over time to further understand the presence and behavior of the groups of somatic coliphages at two environmental temperatures (4 and 25 °C). Over time, the taxonomic composition of the somatic coliphage population in sewage changed, with the Microviridae family becoming the most prevalent family in the sewage population after several weeks. Based on their persistence and prevalence in environmental waters, phages belonging to the Microviridae family provide supporting information on sewage contamination and possibly of human enteric viruses in sewage-contaminated water.

The food additive xylitol enhances the butyrate formation by the child gut microbiota developed in a dynamic colonic simulator

The gut microbiota should be included in the scientific processes of risk assessment of food additives. Xylitol is a sweetener that shows low digestibility and intestinal absorption, implying that a high proportion of consumed xylitol could reach the colonic microbiota. The present study has evaluated the dose-dependent effects of xylitol intake on the composition and the metabolic activity of the child gut-microbiota. The study was conducted in a dynamic simulator of the colonic microbiota (BFBL Gut Simulator) inoculated with a child pooled faecal sample and supplemented three times per day, for 7 days, with increasing xylitol concentrations (1 g/L, 3 g/L and 5 g/L). Sequencing of 16S rRNA gene amplicons and group-specific quantitative PCR indicated a xylitol dose-response effect on the abundance of Lachnospiraceae, particularly the genera Blautia, Anaerostipes and Roseburia. The microbial changes observed with xylitol corresponded with a dose-dependant effect on the butyrate concentration that, in parallel, favoured an increase in epithelial integrity of Caco-2 cells. The study represents a detailed observation of the bacterial taxa that are the main contributors to the metabolism of xylitol by the child gut microbiota and the results could be relevant in the risk assessment re-evaluation of xylitol as a sweetener.

First report of a 'Candidatus Phytoplasma australasiaticum'-related phytoplasma strain associated with shoot proliferation disease of variegated croton in Taiwan.

Codiaeum variegatum (family Euphorbiaceae) is a leaf ornamental commonly known as variegated croton, which is often found in gardens or grown as indoor plants. In December of 2022, two cutting-propagated variegated croton plants exhibiting abnormal shoot proliferation and little leaf symptoms (Fig. S1) were found in a nursery owned by a private breeder in Wanluan Township, Pingtung County, Taiwan. The plants were propagated from a single stock plant, which died during transplanting from a commercial nursery in Changzhi Township, Pingtung County. To determine the potential cause of such symptoms, leaf tissues were collected from the center of the two symptomatic plants. Their DNA were extracted with a Synergy 2.0 Plant DNA Extraction Kit (OPS Diagnostics, Lebanon, NJ) and used for further testing. As controls, two symptomless stock plants were collected from the commercial nursery in Changzhi Township and used to produce cutting-propagated plants; leaf DNA was extracted from each plant as described above. The DNA samples were subjected to PCR testing using the phytoplasma-specific primer pair P1/P7 (Schneider et al. 1995), and only DNA from the symptomatic plants produced the expected 1.8-kb amplicon. The two phytoplasma isolates detected in the plants were designated as CvaA and CvaB. After sequencing and analyzing the data using the iPhyClassifier program (Zhao et al. 2009), both CvaA and CvaB were classified to subgroup 16SrII-A (GenBank accession no. L33765) with a similarity coefficient of 1.0. The near-full-length 16S rDNA fragments of the detected isolates (GenBank accession no. OR794242) were also identical (1,463/1,463 bp) to that of NCHU2014 (GenBank accession no. CP040925, bp 537768-539230), a reference 'Ca. Phytoplasma australasiaticum'-related strain (16SrII-A) found in Taiwan (Chang et al. 2015; Rodrigues Jardim et al. 2023). To validate the results, the DNA samples were also tested with 16SrII group-specific semi-nested PCR targeting the elongation factor Tu gene. The outer and inner primer pairs used were TUF-II-F1/TUF-II-R1 and TUF-II-F2/TUF-II-R1, respectively (Al-Subhi et al. 2017). An expected amplicon was detected in the symptomatic samples but not in the symptomless counterparts. The amplified fragments' sequences (GenBank accession no. OR634931) were identical to that of the elongation factor Tu gene of the 'Ca. Phytoplasma australasiaticum'-related strain NCHU2014 (989/989 bp; GenBank accession no. CP040925, bp 139344-140332). The protein translocase gene secY of the detected phytoplasma was also amplified and sequenced using semi-nested primers SecYF1(II), SecYF2(II) and SecYR1(II) (Lee et al. 2010). Again, the sequences of the detected isolates (GenBank accession no. OR862773) were identical to that of NCHU2014 (1,263/1,263; GenBank accession no. CP040925, bp 192846-194108). The quality of the DNA samples was confirmed by PCR targeting the plant host's 28S rDNA using primer pair 28KJ/28C (Cullings 1992) and all symptomatic and symptomless samples produced the target amplicon (0.7 kb). 16SrII phytoplasmas have been detected in different host plants in Taiwan (Chang et al. 2015). To our knowledge, this is the first record of this group of pathogens infecting variegated croton in Taiwan. Branch-inducing phytoplasma has been used to improve the ornamental values of poinsettias, another Euphorbiaceae species (Lee et al. 1997). Further testing is needed to determine whether the phytoplasma detected in this work could be used for similar purposes.

Molecular Characterization of a Bipartite Begomovirus and an Alphasatellite Associated with Leaf Curl and Mosaic Disease of Bitter Gourd in Punjab

Bitter gourd (Momordica charantia) is an economically important vegetable and medicinal plant that gets infected by a number of viruses. A survey was conducted in and around Jalandhar region of Punjab, India to study viral symptoms in the bitter gourd fields. The plants showed reduced fruit size, upward leaf curling and mosaic symptoms, indicating begomovirus infection. DNA was isolated from the symptomatic leaves and tested for begomovirus infection using group-specific PCR primers. Amplified products were cloned into pMD20-T vector and sequenced. Based on the obtained sequence, new sets of primers were designed and used for amplification of complete DNA-A and DNA-B. Complete DNA-A and DNA-B were cloned in pMD20-T and sequenced. Sequence analysis of the present DNA-A and DNA-B showed their highest identity with Tomato leaf curl New Delhi virus (ToLCNDV). The associated Tomato leaf curl New Delhi alphasatellite was amplified using universal primers, cloned and sequenced. In phylogenetic analysis, the present DNA-A and DNA-B grouped with ToLCNDV isolates and other begomoviruses reported from bitter gourd. Phylogenetic analysis of the present alphasatellite showed its closet identity with Tomato leaf curl New Delhi alphasatellite. Recombination analysis of the DNA-A sequence showed that the present bitter gourd isolate might be a recombinant of Squash leaf curl China virus (SLCChV) as a major parent and Chilli leaf curl virus as a minor parent. Analysis of the DNA-B suggested it to be a recombinant of SLCChV as a major parent and Chyaote enation yellow mosaic virus as a minor parent. The present alphasatellite might be a recombinant of Tomato yellow spot alphasatellite as a major parent and Croton yellow mosaic alphasatellite as a minor parent. The percent identities of the nucleotide and amino acid sequences validate and support the results of the phylogenetic and recombination analyses.

Pneumococcal Serotype Identification by Capsular Sequence Typing (CST): A Modified Novel Approach for Serotyping Directly in Clinical Samples.

As almost 60–70% of Invasive Pneumococcal Disease (IPD) is identified by nonculture methods in Greece, serotyping is of high importance for the better monitoring of pneumococcal serotypes due to the availability of conjugate vaccines. The aim of the study was the modification and direct application of the Capsular Sequence Typing (CST) assay in clinical samples in order to serotype Streptococcus pneumoniae culture-negative, Polymerase Chain Reaction (PCR_-positive samples, followed by CST group specific single-tube PCR assays. A two-step PCR modified assay was applied on a total of 306 samples (such as CSF, blood, pleural and middle ear fluids, isolates) obtained from 283 patients with IPD. The overall performance permits a rapid, accurate and cost-effective method for nonculture pneumococcal serotyping. As the management of IPD is closely related to the continuous monitoring of pneumococcal serotypes, the proposed approach proved to be a valuable tool for the typing and epidemiological monitoring of S. pneumoniae, for the evaluation of the overall impact of vaccination programs in the era of pneumococcal conjugate vaccines, in order to initiate the appropriate vaccination strategy.

First record of the endophytic bacteria of Deschampsia antarctica Ė. Desv. from two distant localities of the maritime Antarctic

Endophytic bacteria, recognized for their beneficial effects on plant development and adaptation, can facilitate the survival of Antarctic plants in severe environments. Here we studied endophytes of the vascular plant Deschampsia antarctica Ė. Desv. from two distantly located regions in the maritime Antarctic: King George Island (South Shetland Islands) and Galindez Island (Argentine Islands). Bacterial group-specific PCR indicated presence of Alphaproteobacteria, Betaproteobacteria, Gammaproteobacteria, Firmicutes, Cytophaga-Flavobacteria and Actinobacteria in root and leaf endosphere of D. antarctica sampled at four distinct sites of both locations. The diversity of endophytic bacteria was significantly higher in the leaves compared to the roots in plants from Galindez Island. Similarly, the diversity of endophytes was higher in the leaves rather than roots of plants from the King George Island. Twelve bacterial species were isolated from roots of D. antarctica of Galindez Island (the Karpaty Ridge and the Meteo Point) and identified by sequencing the 16S rRNA gene. Isolates were dominated by the Pseudomonas genus, followed by the genera Bacillus and Micrococcus. The vast majority of the isolates exhibited cellulase and pectinase activities, however, Bacillus spp. expressed neither of them, suggesting lack of genetic flow of these traits in endophytic bacilli in the maritime Antarctic. Pseudomonas sp. IMBG305 promoted an increase in the leaf number in most of the treated plant genotypes when compared with non-inoculated plants, and a rapid vegetation period of D. antarctica cultured in vitro, albeit the length of leaves in the treated plants was significantly lower, and flavonoid content leveled off in all treated plants. D. antarctica is known to develop diverse ecotypes with regard to ecological conditions, such as organic input, moisture or wind exposition. The D. antarctica phenotype could be extended further through the endophyte colonization, since phenotypic changes were observed in the inoculated D. antarcticaplants grown in vitro in our study. Herewith, endophytes can contribute to plant phenotypic plasticity, potentially beneficial for adaptation of D. antarctica.

Selection of target genes for pcr diagnostics of Xanthomonas arboricola virulent for cereals and brassicas

Plant pathogenic xanthomonads virulent to wheat, rye, barley, tomato, sunflower, and brassicas were isolated in Russia in 2001–2008. Physiological tests and multilocus sequence typing analysis confirmed their position within the Xanthomonas arboricola species. The obtained draft genome sequence of representative strain 3004 from barley plants, which is also virulent to sunflower, brassicas, and chestnut, demonstrated an absence of the Type 3 Secretion System T3SS and an evidence for the lateral gene transfer of some other virulence genes from distantly related bacteria. It was concluded that T4SS genes can be used as the target for group-specific PCR analysis of the emerging pathogen. It was proposed to use virD4, virB3, virB4, and virB9 genes to design a detection system. After preliminary experiments with classic PCR for the chosen genes, primers and TaqMan(R) probe were designed to specifically amplify a 121 bp fragment of the VirD4 gene. Amplification products were obtained for all target Xanthomonas arboricola strains and were not detected in other Xanthomonas species, or in other pathogenic or epiphytic bacteria occurring on these host plants. The assay readily detected Xanthomonas arboricola infection in diseased plants and from bacterial colonies isolated on semi-selective media, and was more sensitive and specific than traditional plating methods.

Spotted Fever Group Rickettsia Infection of Cats and Cat Fleas in Northeast Thailand.

Rickettsia species cause rickettsioses, which are zoonotic diseases found worldwide, and are transmitted by arthropods such as lice, fleas, ticks, and mites. In Thailand, flea infestations are common among cats and dogs. This study aimed at determining the exposure to spotted fever group rickettsiae (SFGR) of cats in surrounding areas of Rajabhat Maha Sarakham University, Muang district, Maha Sarakham province and rickettsial infection among cat fleas, Ctenocephalides felis, collected from dogs of the surrounding area of Waeng Noi district, Khon Kaen province. Forty-two cat sera were assessed for IgG antibody titers against SFGR by a group-specific enzyme-linked immunosorbent assay. The prevalence of seroreactive cats was 4.76% (2/42). DNA preparations from 23 individual cat fleas from three dogs were assessed by Rickettsia genus-specific, group-specific, and species-specific quantitative real-time PCR (qPCR) assays. Positive results were confirmed by ompB gene fragment sequencing. Twenty-one of 23 cat fleas were positive for Rickettsia asembonensis, and the other two DNA preparations were negative for rickettsial DNA. This study's finding indicates that companion cats and dogs in Northeast Thailand are exposed to SFGR and that exposure may be due to infection with R. asembonensis, an organism known to infect humans, monkeys, and dogs. Clinicians for humans and animals in Northeast Thailand should be aware of rickettsial infections among their patients.

Genetically Distinct Acidovorax citrulli Strains Display Cucurbit Fruit Preference Under Field Conditions.

Strains of Acidovorax citrulli, the causal agent of bacterial fruit blotch (BFB) of cucurbits, can be assigned to two groups, I and II. The natural association of group I and II strains with different cucurbit species suggests host preference; however, there are no direct data to support this hypothesis under field conditions. Hence, the objective of this study was to assess differences in the prevalence of group I and II A. citrulli strains on cucurbit species in the field. From 2017 to 2019, we used group I and II strains to initiate BFB outbreaks in field plots planted with four cucurbit species. At different times, we collected symptomatic tissues and assayed them for group I and II strains using a group-specific PCR assay. Binary distribution data analysis revealed that the odds of melon, pumpkin, and squash foliage infection by group I strains were 21.7, 11.5, and 22.1 times greater, respectively, than the odds of watermelon foliage infection by the group I strain (P < 0.0001). More strikingly, the odds of melon fruit infection by the group I strain were 97.5 times greater than watermelon fruit infection by the same strain (P < 0.0001). Unexpectedly, some of the group II isolates recovered from the 2017 and 2019 studies were different from the group II strains used as inocula. Overall, data from these experiments confirm that A. citrulli strains exhibit a preference for watermelon and melon, which is more pronounced in fruit tissues.

Species of the genus Xanthomonas infecting cereals and oilseeds in the Russian Federation and its diagnostics

Plant pathogenic bacteria of the genus Xanthomonas display high levels of genetic diversity and cause remarkable damage to about 400 plant species. In 2001–2008, a new group of strains of Xanthomonas arboricola has been found as pathogens on novel host plants such as wheat, rye, barley, tomato, sunflower, and brassicas in Russia. Physiological tests and multilocus sequence typing (MLST) analysis confirmed their position within the Xanthomonas arboricola species. The obtained draft genome sequence of Xanthomonas arboricola strain 3004 from barley plants, also virulent to sunflower, brassicas, and chestnut, has demonstrated an evidence for the lateral gene transfer (LGT) of the virulence genes. It can be suggested that the virE and other genes of T4SS, obtained due to LGT, may contribute to the host range extension. Thus, T4SS genes can be used as the target for group-specific PCR analysis of this emerging pathogen of cereals and oilseeds. We propose to use virB3, virB4, and virB9 genes to design a detection system.

First detection of “Candidatus Phytoplasma asteris” ‐ and “Candidatus Phytoplasma solani”‐related strains in fig trees

AbstractIn July 2017, a survey was conducted in a fig collection plot at Locorotondo (south of Italy) to investigate the possible presence of phytoplasmas in plants showing yellowing, deformed leaves, short internodes, mottling and mosaic. Samples were collected from symptomatic plants and tested by nested PCR assays using universal and specific primers to amplify the 16S rDNA of these prokaryotes. PCR results detected the presence of phytoplasma sequences in twenty plant samples that resulted clustering two phylogenetically distinct phytoplasmas, i.e., “Candidatus Phytoplasma asteris” and “Candidatus Phytoplasma solani” affiliated to 16SrI and 16SrXII ribosomal groups, respectively. The presence of phytoplasmas belonging to both ribosomal groups was confirmed with group specific quantitative PCR and RFLP assays on 16S ribosomal amplicons. Results of this study indicate for the first time the occurrence of phytoplasmas in fig; however, more work should be carried out to verify their association with the symptoms observed on diseased fig plants.

Genomic Features ofVibrio parahaemolyticusfrom Lebanon and Comparison to Globally Diverse Strains by Whole-Genome Sequencing

Gastroenteritis is a disease that can be caused by virulent strains of Vibrio parahaemolyticus in humans upon the consumption of contaminated seafood. In summer 2017, a sudden increase in the number of patients suffering from gastroenteritis due to a V. parahaemolyticus infection was observed at the Middle East Institute of Health University Hospital in Lebanon. The aim of this study was to analyze the isolates recovered from stool specimens, and to compare them using different phenotypic assays, genomic profiling techniques, and whole-genome sequencing, to achieve a better understanding of the current V. parahaemolyticus strains available in Lebanon. Virulence potential was analyzed based on the detection of the hemolysins: thermostable direct hemolysin (tdh), thermostable direct hemolysin-related hemolysin (trh), and thermolabile hemolysin (tlh). Resistance was determined by testing antibiotic susceptibility and performing PCR assays for β-lactamases and quinolone resistance determinants. Genetic relatedness was verified by multilocus sequence typing, pulsed-field gel electrophoresis, and whole genome-based single nucleotide polymorphism analysis. All of the isolates had the tdh+, trh-, group-specific PCR+ genotype, which is a characteristic of the O3:K6 pandemic clone. The isolates were resistant to ampicillin (100%), ceftazidime (86%), ticarcillin (14%), and amikacin (14%), belonged to the sequence type ST3, and had very similar phylogenetic fingerprints. The isolates undertaken in this study exhibited almost identical resistance, virulence, and phylogenetic patterns, confirming an outbreak linked to the spread of the pandemic O3:K6 serotype in the country.

Standardization and application of immunofluorescence and immuno- peroxidase tests for detection of bluetongue virus antigen

India is enzootic for bluetongue (BT), a predominant disease of small ruminants. The most important task in the control of disease is rapid and sensitive detection of virus. The present study was undertaken to standardize immunofluorescence (IFT) and immunoperoxidase tests (IPT) employing BTV serogroup specific VP7 monoclonal antibodies (MAbs), polyclonal homologous, and polyclonal heterologous antisera against specific serotypes of BTV for detection of BTV antigen. Serial tenfold dilutions of BTV-9 were tested for limit of detection (LoD) of IFT, IPT, and molecular assays by using MAbs against VP7, homologous anti-BTV-9 serum, and heterologous anti-BTV-16 serum. LoD of IFT was found to be 101 TCID50/mL using MAbs against VP7, anti BTV-9 serum, and anti BTV-16 serum. LoD of IPT was found to be 101 TCID50/mL, 102 TCID50/mL, and 102 TCID50/mL using MAbs against VP7, anti BTV-9 serum and anti BTV-16 serum, respectively. LoD of RT-PCR was 101 TCID50/mL and that of real time PCR was 100 TCID50/mL. This standardized assay was then applied for BTV detection in BTV suspected field samples collected from BT outbreaks followed by confirmation with virus isolation and NS3 group specific PCR. The current study shows that IFT and IPT are specific tests for group specific BTV identification. For IFT, monoclonal and polyclonal (homologous and heterologous) source of antibodies had similar sensitivity in the ability of BTV detection; whereas the most sensitive mode of detection by IPT was with MAbs.

P020 Identification of a new C*06 null allele by SBT typing in an arabic family

A 12 y/o female from Saudi Arabia with chronic granulomatous disease was evaluated at Duke for HSCT. Patient and sibling typing were performed by SBT method (Protrans, Germany) which separates alleles by group-specific PCR before sequencing. The patient was identical to her sibling at HLA-A, B, DRB1, DRB3/4/5 and DQB1 loci. However, the analysis software could not assign alleles at HLA-C locus for both the patient and sibling due to the presence of double peaks in exon 3 electropherogram. For both the patient and the sibling, exon 2 and exon 4 sequences showed no polymorphism. Sequence with forward primer of exon 3 was good 1–224 nt in exon 3 but became double peaks after 224. Reverse primer sequence of exon 3, however, had double peaks 1–224 nt then good after 224. Analyzing the results by ignoring the double peak sequences showed a perfect match to C*06:02 in the database. This indicated there were C*06:02 and another variant of C*06:02 with a G deletion at 224 of exon 3. This corresponds to nt 568 in cDNA which changes codon 166 from GAG to.AG and is predicted to be a null allele due to shift of open reading frame. In order to investigate whether the new variant was inherited from the family, we also obtained the samples from the parents. It turned out the C*06 new null allele was carried by the father. The sequence was submitted to GenBank (accession number MH031344). The patient’s sibling was identified to be the 10/10 matched donor and HSCT was performed in 2018 successfully.

Enigmatic Diphyllatea eukaryotes: culturing and targeted PacBio RS amplicon sequencing reveals a higher order taxonomic diversity and global distribution

BackgroundThe class Diphyllatea belongs to a group of enigmatic unicellular eukaryotes that play a key role in reconstructing the morphological innovation and diversification of early eukaryotic evolution. Despite its evolutionary significance, very little is known about the phylogeny and species diversity of Diphyllatea. Only three species have described morphology, being taxonomically divided by flagella number, two or four, and cell size. Currently, one 18S rRNA Diphyllatea sequence is available, with environmental sequencing surveys reporting only a single partial sequence from a Diphyllatea-like organism. Accordingly, geographical distribution of Diphyllatea based on molecular data is limited, despite morphological data suggesting the class has a global distribution. We here present a first attempt to understand species distribution, diversity and higher order structure of Diphyllatea.ResultsWe cultured 11 new strains, characterised these morphologically and amplified their rRNA for a combined 18S–28S rRNA phylogeny. We sampled environmental DNA from multiple sites and designed new Diphyllatea-specific PCR primers for long-read PacBio RSII technology. Near full-length 18S rRNA sequences from environmental DNA, in addition to supplementary Diphyllatea sequence data mined from public databases, resolved the phylogeny into three deeply branching and distinct clades (Diphy I – III). Of these, the Diphy III clade is entirely novel, and in congruence with Diphy II, composed of species morphologically consistent with the earlier described Collodictyon triciliatum. The phylogenetic split between the Diphy I and Diphy II + III clades corresponds with a morphological division of Diphyllatea into bi- and quadriflagellate cell forms.ConclusionsThis altered flagella composition must have occurred early in the diversification of Diphyllatea and may represent one of the earliest known morphological transitions among eukaryotes. Further, the substantial increase in molecular data presented here confirms Diphyllatea has a global distribution, seemingly restricted to freshwater habitats. Altogether, the results reveal the advantage of combining a group-specific PCR approach and long-read high-throughput amplicon sequencing in surveying enigmatic eukaryote lineages. Lastly, our study shows the capacity of PacBio RS when targeting a protist class for increasing phylogenetic resolution.

Prevalence of Vibrio parahaemolyticus in seafood and water environment in the Mekong Delta, Vietnam.

A total of 449 samples including 385 seafood and 64 water samples in the Mekong Delta of Vietnam collected in 2015 and 2016 were examined. Of 385 seafood samples, 332 (86.2%) samples were contaminated with Vibrio parahaemolyticus and 25 (6.5%) samples were pathogenic V. parahaemolyticus carrying tdh and/or trh genes. The tdh gene positive V. parahaemolyticus strains were detected in 22 (5.7%) samples and trh gene positive V. parahaemolyticus strains were found in 5 (1.3%) samples. Of 25 pathogenic V. parahaemolyticus strains, two strains harbored both tdh and trh genes and the other 23 strains carried either tdh or trh gene. Of 64 water samples at aquaculture farms, 50 (78.1%) samples were contaminated with V. parahaemolyticus. No tdh gene positive V. parahaemolyticus strains were detected; meanwhile, trh gene positive V. parahaemolyticus strain was detected in 1 (1.6%) sample. Twenty-six pathogenic V. parahaemolyticus strains isolated were classified into 6 types of O antigen, in which the serotype O3:K6 was detected in 4 strains. All pathogenic strains were group-specific PCR negative except for 4 O3:K6 strains. The result of antimicrobial susceptibility test indicated that pathogenic strains showed high resistance rates to streptomycin (84.6%), ampicillin (57.7%) and sulfisoxazole (57.7%). These findings can be used for understanding microbiological risk of seafood in the Mekong Delta, Vietnam.

Prevalence of occult hepatitis B virus infection and Torque teno virus infection and their association with hepatocellular carcinoma in chronic hepatitis C patients

BackgroundThe role of occult hepatitis B virus (HBV) infection and Torque teno virus (TTV) infection in the development of hepatocellular carcinoma (HCC) in chronic hepatitis C patients is still uncertain. AimThe aim of the present study was to investigate the prevalence and significance of OBI and TTV infection, and to examine the genetic diversity of these viruses, in chronic hepatitis C patients with and without HCC. MethodsSera from 151 hepatitis C virus (HCV)-infected patients (49 patients with HCC and 102 without HCC) negative for HBV surface antigen (HBsAg) were tested for the presence of OBI and TTV infection by semi-nested and group-specific multiplex PCR assays, respectively. Nucleotide sequencing of HBV S region was further performed. ResultsOBI and TTV infection were detected in 5 (3.3%) and 68 (45%) patients, respectively. HBV isolates were classified into genotypes A (4/5, 80%) and D (1/5, 20%), and no HBsAg escape mutation was observed. TTV phylogenetic group 3 was the most prevalent among both HCC and non-HCC patients. OBI and TTV infection were significantly more frequent in patients with HCC than patients without HCC (p=0.003, and p=0.009, respectively). Moreover, TTV infection was associated with HCC (OR=2.23, 95%CI=1.04–4.80, p=0.040), independently of liver cirrhosis. ConclusionsA low prevalence of OBI was observed in patients with HCV-related chronic liver disease, and TTV infection was an independent factor associated with the occurrence of HCC. Whether TTV influences the progression of liver disease in chronic hepatitis C patients remains to be elucidated.

Do Multi-year Applications of Bacillus thuringiensis subsp. israelensis for Control of Mosquito Larvae Affect the Abundance of B. cereus Group Populations in Riparian Wetland Soils?

Bacillus thuringiensis subsp. israelensis (Bti) is a soil-borne bacterium affiliated to the Bacillus cereus group (Bcg) and has been used in biocontrol products against nematoceran larvae for several decades. However, knowledge is limited on whether long-term Bti application can affect the structure of indigenous communities of Bcg and the overall abundance of Bti. Using species- and group-specific quantitative PCR assays, we measured the Bcg- and Bti-abundances in riparian wetlands in the River Dalälven floodplains of central Sweden. On five occasions during one vegetative season, soil samples were collected in alder swamps and wet meadows which had been treated with Bti for mosquito larvae control during the preceding 11years, as well as in untreated control sites and well-drained forests in the same area. The average abundance of Bcg in alder swamps was around three times higher than in wet meadows. Across all sites and habitats, the Bti treatments had no effect on the Bcg-abundance, whereas the Bti-abundance was significantly higher in the treated than in the control sites. However, for individual sampling sites, abundances of Bti and Bcg were not correlated with the number of Bti applications, indicating that added Bti possibly influenced the total population of Bti in the short term but had only a limited effect in the longer term. The findings of this study increase the understanding of the ecology of Bti applications for mosquito control, which can facilitate environmental risk assessment in connection with approval of microbiological control agents.

A strategy to clarify ambiguities during genotyping of functional KIR framework genes by sequencing-based typing among ethnic Hans from southern China

To categorize ambiguous allelic combinations encountered in genotyping of functional KIR genes by sequencing-based typing of the entire coding sequence and develop an efficient approach to identify such ambiguities. Fourteen KIR functional framework genes from 306 ethnic Chinese individuals were genotyped for the entire encoding sequence. The number of ambiguities was directly counted. Based on the differences within each ambiguous allelic combination, group-specific PCR primers and sequencing primers for amplifying the target allelic sequence were designed. The PCR products were then subjected to sequencing in order to identify the ambiguities. The 14 functional KIR genes were subjected to sequencing-based typing (SBT) for the entire encoding sequence. Six ambiguous allelic combinations were identified. The most common ambiguity 3DL2*(002, 007/010, 015) has accounted for 12.09% of the 306 tested samples. The remaining 5 ambiguities were (2DL5A*001, 2DL5B*006/2DL5A*012, 2DL5B*008), 3DL3*(001, 010/009, 048), 3DL2*(007, 008/016, 027), 3DL3*(00801, 048/01001, 026) and 3DL3*(00802, 048/01002, 026) have accounted for 5.88% (18/306), 3.59% (11/306), 2.29% (7/306), 1.31% (4/306) and 1.31% (4/306) of all samples, respectively. For two ambiguities (2DL5A*001, 2DL5B*006/2DL5A*012, 2DL5B*008) and 3DL2*(007, 008/016, 027) subjected to group-specific PCR and re-sequencing, only one demonstrable genotype was identified, While for in each of other four ambiguities subjected to group-specific PCR and re-sequencing, two different genotypes were identified. An efficient approach by group-specific PCR and sequencing retest has been established to clarify the ambiguities during SBT testing for functional KIR framework genes, which may have a broad application in KIR sequencing-based typing.

A novel real-time PCR assay for highly specific detection and quantification of vaginal lactobacilli

PCR detection and quantification of vaginal lactobacilli remains problematic because of the high level of genetic heterogeneity and taxonomic complexity within the genus Lactobacillus. The aim of the present study was to identify conserved sequences among the genomes of major species of vaginal lactobacilli that could be used for the development of a PCR-based method for quantitative determination of vaginal microbiota-specific lactobacilli. Comparative analysis of the genomes of several species of vaginal lactobacilli allowed us to identify conserved regions in the rplK gene, which encodes ribosomal protein L11, and to design group-specific PCR primers and a probe for selected species from the L. acidophilus complex, including major vaginal lactobacilli Lactobacillus crispatus, L. gasseri, L. iners and L. jensenii as well as other species that are less common in vaginal microbiota. The applicability of the new assay in routine diagnostic testing was evaluated using a set of clinical samples. The assay was able to detect and quantify vagina-associated lactobacilli within a wide range of initial DNA template concentrations, indicating promising potential for clinical applications.