Subtyping Tool Research Articles

Transmitted drug resistance in patients with acute/recent HIV infection in Brazil

IntroductionThe widespread use of antiretroviral therapy increased the transmission of antiretroviral resistant HIV strains. Antiretroviral therapy initiation during acute/recent HIV infection limits HIV reservoirs and improves immune response in HIV infected individuals. Transmitted drug resistance may jeopardize the early goals of early antiretroviral treatment among acute/recent HIV infected patients. MethodsPatients with acute/recent HIV infection who underwent resistance test before antiretroviral treatment initiation were included in this analysis. HIV-1 sequences were obtained using an in house protease/reverse transcriptase genotyping assay. Transmitted drug resistance was identified according to the Stanford HIV Database for Transmitted Drug Resistance Mutations, based on WHO 2009 surveillance list, and HIV-1 subtyping according to Rega HIV-1 subtyping tool. Comparison between patients with and without transmitted drug resistance was made using Kruskal–Wallis and Chi-square tests. ResultsForty-three patients were included, 13 with acute HIV infection and 30 with recent HIV infection. The overall transmitted drug resistance prevalence was 16.3% (95% confidence interval [CI]: 8.1–30.0%). The highest prevalence of resistance (11.6%, 95% CI: 8.1–24.5) was against non-nucleoside reverse transcriptase inhibitors, and K103N was the most frequently identified mutation. ConclusionsThe high prevalence of nonnucleoside reverse transcriptase inhibitors resistance indicates that efavirenz-based regimen without prior resistance testing is not ideal for acutely/recently HIV-infected individuals in our setting. In this context, the recent proposal of including integrase inhibitors as a first line regimen in Brazil could be an advantage for the treatment of newly HIV infected individuals. However, it also poses a new challenge, since integrase resistance test is not routinely performed for antiretroviral naive individuals. Further studies on transmitted drug resistance among acutely/recently HIV-infected are needed to inform the predictors of transmitted resistance and the antiretroviral therapy outcomes among these population.

Increasing HIV-1 subtype diversity in seven states, United States, 2006–2013

PurposeThe aim of the analysis was to explore HIV-1 subtype diversity in the United States and understand differences in prevalence of non-B subtypes and circulating recombinant forms (CRFs) between demographic/risk groups and over time. MethodsWe included HIV-1 polymerase sequences reported to the National HIV Surveillance System for HIV infections diagnosed during 2006–2013 in seven states. We assigned subtype or CRF using the automated subtyping tool COMET, assessed subtype/CRF prevalence by demographic characteristics and country of birth, and determined changes in subtype/CRF by HIV diagnosis year. ResultsOf 32,968 sequences, 30,757 (93.3%) were subtype B. The most common non-B subtypes and CRFs were C (1.6%), CRF02_AG (1.4%), A (0.6%), CRF01_AE (0.5%), and G (0.3%). Elevated percentages of non-B infections occurred among persons aged <13 years at diagnosis (40.9%), Asians (32.1%), persons born outside the United States (22.6%), and persons with infection attributable to heterosexual contact (12.0%–15.0%). Prevalence of non-B infections increased from 5.9% in 2006 to 8.5% in 2013. ConclusionsSubtype B continues to predominate in the United States. However, the percentage of non-B infections has grown in recent years, and numerous demographic subgroups have much higher prevalence. Subgroups and areas with high prevalence of non-B infections might represent sub-epidemics meriting further investigation.

Molecular Epidemiology Database for Sequence Management and Data Mining.

A central database to aggregate sequence information from a range of epidemiological aspects including HTLV-1 pathogenesis, origin, and evolutionary dynamic would be useful to scientists and physicians worldwide. This Chapter describes two online tools for studies related to HTLV-1, the HTLV-1 Molecular Epidemiology Database and the HTLV-1 Subtyping Tool. The HTLV-1 Molecular Epidemiology Database is a tool for sequence management and data mining which allows researchers to download sequences with clinical and demographic information. The HTLV-1 Subtyping Tool is an online software used for HTLV-1 genotyping, the algorithm consists in the alignment of a query sequence with a carefully selected set of predefined reference strains, followed by phylogenetic analysis.

Performance of Celera RUO integrase resistance assay across multiple HIV-1 subtypes

BackgroundHIV-1 sequence variation is a major obstacle to developing molecular based assays for multiple subtypes. This study sought to independently assess performance characteristics of the ViroSeq™ HIV-1 Integrase RUO Genotyping Kit (Celera, US) for samples of multiple different HIV-1 subtypes. Methods264 samples were tested in the validation, 106 from integrase inhibitor naïve patients’ sent for routine HIV-1 drug resistance testing after failing a 1st- or 2nd-line regimen, and 158 samples from an external virology quality assurance program (VQA). For the latter, 53 unique VQA samples were tested in two to five different laboratories to assess assay reproducibility. For all assays, viral RNA was extracted using the ViroSeq extraction module, reverse transcribed, and amplified in a one-step reaction. Four sequencing primers were used to span codons 1–288 of integrase. The Rega subtyping tool was used for subtype assignment. Integrase polymorphisms and mutations were determined as differences from the HXB2 sequence and by the Stanford database, respectively. Sequences obtained from the different laboratories were aligned and sequence homology determined. ResultsHIV-1 RNA in the 264 samples ranged from 3.15 to 6.74logcopies/ml. Successful amplification was obtained for 97% of samples (n=256). The 8 samples that failed to amplify were subtype D (n=3), subtype C (n=1), CRF01_AE (n=1), subtype A1 (n=2), and an unassigned subtype (n=1). Of the 256 that successfully amplified samples, 203 (79%) were successfully sequenced with bidirectional coverage. Of the 53 unsuccessful samples, 13 (5%) failed sequencing and 40 (16%) did not have full bidirectional sequence, as a result of failure of sequencing primers: Primer A (n=1); Primer B (n=18); Primer C (n=1); Primer D (n=7) or short sequences (n=16). For the 135 VQA samples (30 unique samples) that were assayed by different laboratories, homology of the sequences obtained ranged from 92.1% to 100%. However, Laboratory 2 detected more mixtures (74%) compared to the other four laboratories, whereas Laboratory 1 detected the least number of mixtures (35%), likely due to differences between the labs in the methods of sequence analysis. Mutations associated with integrase resistance were observed in seven of the 106 (7%) clinical samples [one sample: Q148K; E138K; G140A; two samples: T97A and four samples: L74I]. Of the four samples with L74I, 3 were subtype G. ConclusionOf the total 264 samples tested, 243 (92%) of samples were able to be amplified and sequenced to generate an integrase genotype. Sequencing results were similar between the testing laboratories with the exception of mixture detection. Mutations associated with integrase inhibitor resistance were observed in only 7% of integrase inhibitor naive samples, and some of these mutations are likely to be due to subtype-specific polymorphisms rather than selection by an integrase inhibitor.

English

HIV-1 genetic diversity in sub-Saharan Africa is broad and the AIDS epidemic is driven predominantly by recombinants in Central and West Africa. The classification of HIV-1 strains is therefore necessary to understand diagnostic efficiency, individual treatment responses as well as options for designing vaccines and antiretroviral (ARV) treatment guidelines. More so, accurate subtyping of a partial or full genome would represent the population dynamics of HIV and provide evidence for designing surveillance strategies within a geographic region. Evaluating the performance of rapid subtyping tools with options that incorporate phylogeny could be fast, more user-friendly and of high performance. A total of 570 HIV-1 partial sequences from Cameroon, Angola, Democratic Republic of Congo, Gabon and Senegal were obtained from the Los Alamos National Library (LANL) HIV Sequence Database. Phylogeny was performed using MEGA v6 and the results were used to evaluate the performance of eleven different rapid HIV-1 subtyping tools: REGA v2, REGA v3, NCBI, Stanford HIVDB, SUDI, Geno2Pheno, Euresist, STAR, jpHMM, COMET and SCUEAL. The performance of these subtyping tools differed among HIV-1 clades and across different viral genes. NCBI and SUDI showed the highest performance in subtyping. The discordance observed between the rapid subtyping tools and phylogeny implies that phylogenetic analysis is still the more suitable method for HIV-1 classification. However, the need to update the reference datasets of the subtyping tools, and validate algorithms for rapid subtyping and quality control is imperative as this information is relevant for clinical use and policy-making to the AIDS response. &nbsp; Key words: HIV, phylogeny, performance, subtyping tools, algorithm.

Comparative Evaluation of Genomic and Laboratory Approaches for Determination of Shiga Toxin Subtypes in Escherichia coli

The determination of Shiga toxin (ST) subtypes can be an important element in the risk characterization of foodborne ST-producing Escherichia coli (STEC) isolates for making risk management decisions. ST subtyping methods include PCR techniques based on electrophoretic or pyrosequencing analysis of amplicons and in silico techniques based on whole genome sequence analysis using algorithms that can be readily incorporated into bioinformatics analysis pipelines for characterization of isolates by their genetic composition. The choice of technique will depend on the performance characteristics of the method and an individual laboratory's access to specialized equipment or personnel. We developed two whole genome sequence-based ST subtyping tools: (i) an in silico PCR algorithm requiring genome assembly to replicate a reference PCR-based method developed by the Statens Serum Institut (SSI) and (ii) an assembly-independent routine in which raw sequencing results are mapped to a database of known ST subtype sequence variants (V-Typer). These tools were evaluated alongside the SSI reference PCR method and a recently described PCR-based pyrosequencing technique. The V-Typer method results corresponded closely with the reference method in the analysis of 67 STEC cultures obtained from a World Health Organization National Reference Laboratory. In contrast, the in silico PCR method failed to detect ST subtypes in several cases, a result which we attribute to assembly-induced errors typically encountered with repetitive gene sequences. The V-Typer can be readily integrated into bioinformatics protocols used in the identification and characterization of foodborne STEC isolates.

Development of a multilocus sequence typing tool for high-resolution subtyping and genetic structure characterization of Cryptosporidium ubiquitum

Cryptosporidium ubiquitum is an emerging zoonotic pathogen in humans. Recently, a subtyping tool targeting the 60-kDa glycoprotein (gp60) gene was developed for C. ubiquitum, and identified six subtype families (XIIa-XIIf). In this study, we selected five genetic loci known to be polymorphic in C. hominis and C. parvum for the development of a multilocus subtyping tool for C. ubiquitum, including CP47 (cgd6_1590), MSC6-5 (cgd6_4290), cgd6_60, cgd2_3690, and cgd4_370. PCR primers for these targets were designed based on whole genome sequence data from C. ubiquitum. DNA sequence analyses of 24 C. ubiquitum specimens showed the presence of 18, 1, 5, 4, and 5 subtypes at the CP47, MSC6-5, cgd6_60, cgd2_3690, and cgd4_370 loci, respectively. Altogether, 18 multilocus sequence typing (MLST) subtypes were detected among the 19 specimens successfully sequenced at all polymorphic loci. Phylogenetic analyses of the MLST data indicated that the rodent subtype families of XIIe and XIIf were highly divergent from others, and the ruminant XIIa subtype family formed a monophyletic group genetically distant from other rodent subtype families XIIb, XIIc, and XIId. The latter showed no consistent grouping of specimens and formed one large cluster in phylogenetic analysis of concatenated multilocus sequences. This was supported by results of STRUCTURE and FST analyses, which further suggested that XIIa originated from one common ancestor whereas XIIb, XIIc, and XIId contained mixed ancestral types, reflecting a close relatedness of the three subtype families and the likely occurrence of genetic recombination among them. Thus, an MLST tool was developed for high-resolution subtyping of C. ubiquitum and results of preliminary characterizations of specimens from humans and animals supported the conclusion on the existence of ruminant and rodent-adapted C. ubiquitum groups.

Abstract C54: High caveolin-1 expression in a cohort of African American women with triple-negative breast cancer

Abstract In the U.S., the incidence rates of breast cancer (BC) among Caucasian (CA) women are lower than those of African American (AA), however, AA women have a significantly higher mortality rate. AA women often present at a younger age, with later stage and higher grade tumors. In fact, AA women are 3x more likely to be diagnosed with the more aggressive molecular subtype, triple-negative breast cancer (TNBC), than CA women. Socioeconomic and lifestyle factors may be contributory; however, it is imperative that we investigate the underlying molecular biology that may be the cause of health disparities between AA and CA with TNBC. In this study, gene expression profiling, using the Almac BC DSA Research Tool, was performed on archived FFPE samples, obtained from CA and AA women diagnosed with early stage (Node 0) TNBC. Unsupervised hierarchical clustering revealed a pattern of differential gene expression in the AA cohort compared to CA. Using the TNBC type: A Subtyping Tool for TNBC, we found a distinct distribution pattern of TNBC molecular subtypes in the AA cohort, which was very different than the CA cohort; Basal-like (14%), Immunomodulatory (43%) and Mesenchymal (43%). Gene expression analyses, comparing the AA and CA cohort (fold change &amp;gt; 2.0, p-value &amp;lt;.05), resulted in 190 differentially expressed genes (DEG). Pathway enrichment analysis conducted in MetaCore GeneGo, revealed that the DEGs were over-represented in cytoskeletal remodeling, cell adhesion, tight junctions, and immune response in the AA TNBC cohort. Furthermore, several genes in the Wnt/β-catenin pathways were over-expressed in the top 10-enrichment pathways. We validated our results using RT-qPCR and identified Caveolin-1 (CAV1) as being significantly expressed in the AA-TNBC cohort (p-value 1.22 x 10-05). An independent cohort of FFPE samples, from AA and CA women with early stage TNBC, was used to create a tissue microarray (TMA). Immunohistochemistry results showed no difference in localization of Cav1 between the AA and CA cohorts, however, the AA cohort had significantly higher levels of Cav1 staining (p-value 0.04). Additionally, using RT-qPCR, we demonstrated that CAV1 mRNA was significantly higher in the AA TNBC Cohort (p-value 0.48). Furthermore, endogenous Cav1 was shown to be highly expressed in a cell line panel of TNBC, in particular, those of the mesenchymal and basal-like molecular subtype. Finally, using siRNA, we demonstrated that CAV1 silencing resulted in a significant decrease in cell proliferation, for each of the TNBC cell lines while it showed no effect on the luminal ER+ cell lines. Our combined study results suggest that CAV1 over-expression may be a biological contributor to the observed health disparity between AA women and CA diagnosed with early stage TNBC. Citation Format: Julie E. Getz, Davyd B. Teoh, Sara Nasser, Christophe R. Legendre, Waibhav Tembe, Venkata Yellapantula, Mary Ellen Ahearn, Carmen R. Gomez, Merce Jorda, Shuk Mei Wong, Mark D. Pegram, John D. Carpten, Lisa L. Baumbach-Reardon. High caveolin-1 expression in a cohort of African American women with triple-negative breast cancer. [abstract]. In: Proceedings of the Eighth AACR Conference on The Science of Health Disparities in Racial/Ethnic Minorities and the Medically Underserved; Nov 13-16, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Epidemiol Biomarkers Prev 2016;25(3 Suppl):Abstract nr C54.

Genetic Diversity and Transmitted Drug Resistance of HIV-1 Subtypes in Blood Donors Candidates in Northern Mozambique

In order to evaluate the genetic diversity of HIV-1 subtypes and transmitted drug resistance (TDR) mutations in northern Mozambique, we analyzed 120 plasma specimens obtained from drug naive blood donors candidates, who tested positive during routine HIV screening in three blood banks in that region. The genotyping for HIV-1 resistance was performed using Trugene Genotyping SystemTM. HIV-1 genetic subtypes were defined based on entire PR and partial RT gene regions, using REGA HIV-1 Subtyping Tool version 3.0 algorithm and confirmed by phylogenetic inference, using NJ algorithm. The majority of genotyped samples were classified as HIV-1 subtype C (80.0%), followed by subtype A1 (10.5%), subtype D (3.2%) and subtype G (2.1%). The inter-subtype recombinant forms (A1/C, A1/D and C/D) were identified in four participants (4.2%). TDR mutations associated with nucleoside and nonnucleoside reverse transcriptase inhibitors (K219E, G190A, K101E and K103N) were observed in five (5.3%) subjects. Although a large proportion of HIV-1 subtype C was observed, non-C and recombinant forms together correspond to 20% in this study, which is different from what was described in central and southern regions, where subtype C was almost 100%. However, the profile mutations from this study correlate with the ARVs used both for first line schemes and PMTCT in Mozambique. This data reinforces the necessity of continuous surveillance of HIV-1 diversity, TDR and routes of spread, through inner cities of Mozambique to understand better the dynamics of the HIV-1 epidemic and support national public health policies in the country.

The Genetic Diversity and Evolution of HIV-1 Subtype B Epidemic in Puerto Rico.

HIV-1 epidemics in Caribbean countries, including Puerto Rico, have been reported to be almost exclusively associated with the subtype B virus (HIV-1B). However, while HIV infections associated with other clades have been only sporadically reported, no organized data exist to accurately assess the prevalence of non-subtype B HIV-1 infection. We analyzed the nucleotide sequence data of the HIV pol gene associated with HIV isolates from Puerto Rican patients. The sequences (n = 945) were obtained from our “HIV Genotyping” test file, which has been generated over a period of 14 years (2001–2014). REGA subtyping tool found the following subtypes: B (90%), B-like (3%), B/D recombinant (6%), and D/B recombinant (0.6%). Though there were fewer cases, the following subtypes were also found (in the given proportions): A1B (0.3%), BF1 (0.2%), subtype A (01-AE) (0.1%), subtype A (A2) (0.1%), subtype F (12BF) (0.1%), CRF-39 BF-like (0.1%), and others (0.1%). Some of the recombinants were identified as early as 2001. Although the HIV epidemic in Puerto Rico is primarily associated with HIV-1B virus, our analysis uncovered the presence of other subtypes. There was no indication of subtype C, which has been predominantly associated with heterosexual transmission in other parts of the world.

Lack of integrase inhibitors associated resistance mutations among HIV-1C isolates.

BackgroundAlthough biochemical analysis of HIV-1 integrase enzyme suggested the use of integrase inhibitors (INIs) against HIV-1C, different viral subtypes may favor different mutational pathways potentially leading to varying levels of drug resistance. Thus, the aim of this study was to search for the occurrence and natural evolution of integrase polymorphisms and/or resistance mutations in HIV-1C Ethiopian clinical isolates prior to the introduction of INIs.MethodsPlasma samples from chronically infected drug naïve patients (N = 45), of whom the PR and RT sequence was determined previously, were used to generate population based sequences of HIV-1 integrase. HIV-1 subtype was determined using the REGA HIV-1 subtyping tool. Resistance mutations were interpreted according to the Stanford HIV drug resistance database (http://hivdb.stanford.edu) and the updated International Antiviral Society (IAS)-USA mutation lists. Moreover, rates of polymorphisms in the current isolates were compared with South African and global HIV-1C isolates.ResultsAll subjects were infected with HIV-1C concordant to the protease (PR) and reverse transcriptase (RT) regions. Neither major resistance-associated IN mutations (T66I/A/K, E92Q/G, T97A, Y143HCR, S147G, Q148H/R/K, and N155H) nor silent mutations known to change the genetic barrier were observed. Moreover, the DDE-catalytic motif (D64G/D116G/E152 K) and signature HHCC zinc-binding motifs at codon 12, 16, 40 and 43 were found to be highly conserved. However, compared to other South African subtype C isolates, the rate of polymorphism was variable at various positions.ConclusionAlthough the sample size is small, the findings suggest that this drug class could be effective in Ethiopia and other southern African countries where HIV-1C is predominantly circulating. The data will contribute to define the importance of integrase polymorphism and to improve resistance interpretation algorithms in HIV-1C isolates.

HIV-1 subtype characteristics of infected persons living in southwestern Greece.

BackgroundThe rapid replication rate of HIV-1, coupled with a high mutation rate and recombination, is the underlying force driving its genetic diversity. In the infected individual, a population of highly related but nonidentical strains exists. At the population level, multiple subtypes often cocirculate, leading to the generation of intersubtype recombinant forms. As a result, the geographic distribution of subtypes and recombinant forms is complex and uneven. Genetic subtyping of HIV-1 isolates has been shown to be helpful for understanding the genetic evolution, the worldwide spread of the virus, and the evaluation of drug resistance.Materials and methodsWe determined the genetic heterogeneity of HIV-1 group M in southwestern Greece. Protease and partial reverse-transcriptase sequences were generated from 150 HIV-1-infected individuals attending the Division of Infectious Diseases of Patras University Hospital, Greece, from 2006 to 2012, and analyzed using online subtyping tools and phylogenetic methods.ResultsThe majority of the infected individuals were male (77%). HIV-1 subtype A1 was responsible for 51.3% of infections, followed by subtypes B (34%), G (4%), F1 (2%), and the circulating recombinant forms 02_AG (2.7%), 14_BG (1.3%), 35_AD (1.3%), and 01_AE (0.7%). Additionally, we identified three cases with a recombinant B/CRF02_AG strain (2%) and one with a recombinant G/GRF_AG strain. Sexual transmission was responsible for 96.3% of cases. Heterosexual transmission was responsible for 70.2% of subtype-A1 infections, whereas subtype B was transmitted by men who have sex with men in 75.5% of cases. Protease substitutions I13V, E35D, M36I, R57K, H69K, and L89M, which serve as drug-resistance support mutations in subtype B, were present in the majority of subtype-A1 sequences of the population.ConclusionHIV-1 infection in southwestern Greece is sexually transmitted and highly heterogeneous. Subtype A1 has surpassed subtype B, and is the most prevalent strain. In the population studied, subtype A1 exhibited certain polymorphisms in the protease region, which may serve as drug-resistance support mutations in subtype B.

Draft Genome Sequences from Cyclospora cayetanensis Oocysts Purified from a Human Stool Sample.

The parasite Cyclospora cayetanensis causes foodborne diarrheal illness. Here, we report draft genome sequences obtained from C. cayetanensis oocysts purified from a human stool sample. The genome assembly consists of 865 contigs with a total length of 44,563,857 bases. These sequences can facilitate the development of subtyping tools to aid outbreak investigations.

Usefulness of High-Quality Core Genome Single-Nucleotide Variant Analysis for Subtyping the Highly Clonal and the Most Prevalent Salmonella enterica Serovar Heidelberg Clone in the Context of Outbreak Investigations.

Salmonella enterica serovar Heidelberg is the second most frequently occurring serovar in Quebec and the third-most prevalent in Canada. Given that conventional pulsed-field gel electrophoresis (PFGE) subtyping for common Salmonella serovars, such as S. Heidelberg, yields identical subtypes for the majority of isolates recovered, public health laboratories are desperate for new subtyping tools to resolve highly clonal S. Heidelberg strains involved in outbreak events. As PFGE was unable to discriminate isolates from three epidemiologically distinct outbreaks in Quebec, this study was conducted to evaluate whole-genome sequencing (WGS) and phylogenetic analysis as an alternative to conventional subtyping tools. Genomes of 46 isolates from 3 Quebec outbreaks (2012, 2013, and 2014) supported by strong epidemiological evidence were sequenced and analyzed using a high-quality core genome single-nucleotide variant (hqSNV) bioinformatics approach (SNV phylogenomics [SNVphyl] pipeline). Outbreaks were indistinguishable by conventional PFGE subtyping, exhibiting the same PFGE pattern (SHEXAI.0001/SHEBNI.0001). Phylogenetic analysis based on hqSNVs extracted from WGS separated the outbreak isolates into three distinct groups, 100% concordant with the epidemiological data. The minimum and maximum number of hqSNVs between isolates from the same outbreak was 0 and 4, respectively, while >59 hqSNVs were measured between 2 previously indistinguishable outbreaks having the same PFGE and phage type, thus corroborating their distinction as separate unrelated outbreaks. This study demonstrates that despite the previously reported high clonality of this serovar, the WGS-based hqSNV approach is a superior typing method, capable of resolving events that were previously indistinguishable using classic subtyping tools.

Characteristics and spread to the native population of HIV-1 non-B subtypes in two European countries with high migration rate.

BackgroundNon-B subtypes account for at least 50 % of HIV-1 infections diagnosed in Belgium and Luxembourg. They are considered to be acquired through heterosexual contacts and infect primarily individuals of foreign origin. Information on the extent to which non-B subtypes spread to the local population is incomplete.MethodsPol and env gene sequences were collected from 410 non-subtype B infections. Profound subtyping was performed using 5 subtyping tools and sequences of both pol and env. Demographic information, disease markers (viral load, CD4 count) and viral characteristics (co-receptor tropism) were compared between subtypes. Maximum likelihood phylogenetic trees were constructed and examined for clustering.ResultsThe majority of non-B infections were diagnosed in patients originating from Africa (55.8 %), individuals born in Western Europe represented 30.5 %. Heterosexual transmission was the most frequently reported transmission route (79.9 %), MSM transmission accounted for 12.2 % and was significantly more frequently reported for Western Europeans (25.7 % versus 4.3 % for individuals originating from other regions; p < 0.001). Subtypes A and C and the circulating recombinant forms CRF01_AE and CRF02_AG were the most represented and were included in the comparative analysis. Native Western Europeans were underrepresented for subtype A (14.5 %) and overrepresented for CRF01_AE (38.6 %). The frequency of MSM transmission was the highest for CRF01_AE (18.2 %) and the lowest for subtype A (0 %). No differences in age, gender, viral load or CD4 count were observed. Prevalence of CXCR4-use differed between subtypes but largely depended on the tropism prediction algorithm applied. Indications for novel intersubtype recombinants were found in 20 patients (6.3 %). Phylogenetic analysis revealed only few and small clusters of local transmission but could document one cluster of CRF02_AG transmission among Belgian MSM.ConclusionsThe extent to which non-B subtypes spread in the native Belgian-Luxembourg population is higher than expected, with 30.5 % of the non-B infections diagnosed in native Western Europeans. These infections resulted from hetero- as well as homosexual transmission. Introduction of non-B variants in the local high at risk population of MSM may lead to new sub-epidemics and/or increased genetic variability and is an evolution that needs to be closely monitored.

PCR melting profile as a tool for outbreak studies of Salmonella enterica in chickens.

BackgroundSalmonellosis is of great economic concern in all phases of the poultry industry, from production to marketing, leading to severe economic losses. Monitoring the source of the bacterial contamination has fundamental importance in the spreading of salmonellosis.ResultsWe applied a ligation-mediated PCR method, PCR MP (PCR melting profile), to type S. enterica ssp. enterica ser. Enteritidis (56 strains) and 43 control strains classified to other serovars isolated from poultry. We demonstrated the PCR MP potential for salmonellosis spreading monitoring. Our rapid test presents higher discriminatory power (0.939 vs. 0.608) compared to current molecular subtyping tool such as pulsed-field gel electrophoresis (PFGE), which ineffectiveness underlies the high degree of clonality of S. Enteritidis.ConclusionsPCR MP was found to be a highly discriminating, sensitive and specific method that could be a valuable molecular tool, particularly for analyzing epidemiological links of limited number of S. enterica ser. Enteritidis strains.

Subtyping Novel Zoonotic Pathogen Cryptosporidium Chipmunk Genotype I

Cryptosporidium chipmunk genotype I is an emerging zoonotic pathogen in humans. The lack of subtyping tools makes it impossible to determine the role of zoonotic transmission in epidemiology. To identify potential subtyping markers, we sequenced the genome of a human chipmunk genotype I isolate. Altogether, 9,509,783 bp of assembled sequences in 853 contigs were obtained, with an N50 of 117,886 bp and >200-fold coverage. Based on the whole-genome sequence data, two genetic markers encoding the 60-kDa glycoprotein (gp60) and a mucin protein (ortholog of cgd1_470) were selected for the development of a subtyping tool. The tool was used for characterizing chipmunk genotype I in 25 human specimens from four U.S. states and Sweden, one specimen each from an eastern gray squirrel, a chipmunk, and a deer mouse, and 4 water samples from New York. At the gp60 locus, although different subtypes were seen among the animals, water, and humans, the 15 subtypes identified differed mostly in the numbers of trinucleotide repeats (TCA, TCG, or TCT) in the serine repeat region, with only two single nucleotide polymorphisms in the nonrepeat region. Some geographic differences were found in the subtype distribution of chipmunk genotype I from humans. In contrast, only two subtypes were found at the mucin locus, which differed from each other in the numbers of a 30-bp minisatellite repeat. Thus, Cryptosporidium chipmunk genotype I isolates from humans and wildlife are genetically similar, and zoonotic transmission might play a potential role in human infections.

Sequencing and phylogenetic analysis of near full-length HIV-1 subtypes A, B, G and unique recombinant AC and AD viral strains identified in South Africa.

By the end of 2012, more than 6.1 million people were infected with HIV-1 in South Africa. Subtype C was responsible for the majority of these infections and more than 300 near full-length genomes (NFLGs) have been published. Currently very few non-subtype C isolates have been identified and characterized within the country, particularly full genome non-C isolates. Seven patients from the Tygerberg Virology (TV) cohort were previously identified as possible non-C subtypes and were selected for further analyses. RNA was isolated from five individuals (TV047, TV096, TV101, TV218, and TV546) and DNA from TV016 and TV1057. The NFLGs of these samples were amplified in overlapping fragments and sequenced. Online subtyping tools REGA version 3 and jpHMM were used to screen for subtypes and recombinants. Maximum likelihood (ML) phylogenetic analysis (phyML) was used to infer subtypes and SimPlot was used to confirm possible intersubtype recombinants. We identified three subtype B (TV016, TV047, and TV1057) isolates, one subtype A1 (TV096), one subtype G (TV546), one unique AD (TV101), and one unique AC (TV218) recombinant form. This is the first NFLG of subtype G that has been described in South Africa. The subtype B sequences described also increased the NFLG subtype B sequences in Africa from three to six. There is a need for more NFLG sequences, as partial HIV-1 sequences may underrepresent viral recombinant forms. It is also necessary to continue monitoring the evolution and spread of HIV-1 in South Africa, because understanding viral diversity may play an important role in HIV-1 prevention strategies.

HIV-1 subtype diversity based on envelope C2V3 sequences from Kenyan patients on highly active antiretroviral therapy.

There is a continuous need to genetically characterize the HIV strains in circulation in order to assess interventions and inform vaccine discovery. We partially sequenced the envelope C2V3 gene from a total of 59 Kenyan patients on highly active antiretroviral treatment (HAART) and determined HIV subtypes using both the JPHMM subtyping tool and the phylogenetic method. HIV-1 subtype A1 was the predominant strain in circulation, representing 65.5% and 74.5% of all isolates as determined by JPHMM and phylogenetic methods, respectively. Subtypes C and D were the next most prevalent pure strains at 9.1% each by both methods. JPHMM identified 9.1% of the isolates as recombinant. Four isolates had short sequences not covering the entire C2V3 region and were thus not subtyped. From this study, subtype A viruses are still the predominant HIV-1 strains in local circulation in Kenya. Constant surveillance is needed to update molecular trends under continuing HAART scale-up.

Molecular characterization of HIV-1 infection in Northwest Spain (2009–2013): Investigation of the subtype F outbreak

BackgroundHIV-1 subtype B is the predominant one in European regions several, while other subtypes and recombinants are also circulating with high prevalence. A sub-epidemic of subtype F with specific characteristics and low response to treatment has been recently identified in Galicia. In this study we investigated the characteristics of the HIV-1 subtype F sub-epidemic in A Coruña and Santiago de Compostela in Northwest Spain. Methods420 newly HIV-1 diagnosed patients during 2009–2013 were enrolled in this study. HIV-1 subtyping was carried out using automated subtyping tools and phylogenetic analysis. Molecular epidemiology investigation of subtypes B and F was performed by means of phylogenetic analysis using fast maximum likelihood. Phylodynamic analysis was performed using Bayesian method as implemented in BEAST v1.8. ResultsSubtype B found to be the predominant (61.2% and 70.4%) followed by subtype F (25.6% and 12.0%) in both areas (A Coruña and Santiago de Compostela, respectively). The latter found to mainly spread among men having sex with men (MSM). The vast majority of subtype F lineages from both areas clustered monophyletically, while subtype B sequences clustered in several tree branches. The exponential growth of subtype F sub-epidemic dated back in 2008 by means of phylodynamic analysis. Most of new infections during 2009–2013 occurred within the subtype F transmission cluster. ConclusionsSubtype F circulates at high prevalence in A Coruña and Santiago de Compostela in Northwest Spain, suggesting that the HIV-1 epidemic in this region has distinct characteristics to the rest of Spain. Subtype F has being spreading among MSM and is currently the most actively spreading network. The single cluster spread of this local sub-epidemic might provide an explanation for the distinct characteristics and the low response to antiretroviral treatment.