Translational characterization of the temporal dynamics of metabolic dysfunctions in liver, adipose tissue and the gut during diet-induced NASH development in Ldlr-/-.Leiden mice.

Background: NAFLD progression, from steatosis to inflammation and fibrosis, results from an interplay of intra- and extrahepatic mechanisms. Disease drivers likely include signals from white adipose tissue (WAT) and gut. However, the temporal dynamics of disease development remain poorly understood. Methods: High-fat-diet (HFD)-fed Ldlr-/-.Leiden mice were compared to chow-fed controls. At t = 0, 8, 16, 28 and 38w mice were euthanized, and liver, WAT depots and gut were analyzed biochemically, histologically and by lipidomics and transcriptomics together with circulating factors to investigate the sequence of pathogenic events and organ cross-talk during NAFLD development. Results: HFD-induced obesity was associated with an increase in visceral fat, plasma lipids and hyperinsulinemia at t = 8w, along with increased liver steatosis and circulating liver damage biomarkers. In parallel, upstream regulator analysis predicted that lipid catabolism regulators were deactivated and lipid synthesis regulators were activated. Subsequently, hepatocyte hypertrophy, oxidative stress and hepatic inflammation developed. Hepatic collagen accumulated from t = 16 w and became pronounced at t = 28-38 w. Epididymal WAT was maximally hypertrophic from t = 8 w, which coincided with inflammation development. Mesenteric and subcutaneous WAT hypertrophy developed slower and did not appear to reach a maximum, with minimal inflammation. In gut, HFD significantly increased permeability, induced a shift in microbiota composition from t = 8 w and changed circulating gut-derived metabolites. Conclusion: HFD-fed Ldlr-/-.Leiden mice develop obesity, dyslipidemia and insulin resistance, essentially as observed in obese NAFLD patients, underlining their translational value. We demonstrate that marked epididymal-WAT inflammation, and gut permeability and dysbiosis precede the development of NAFLD stressing the importance of a multiple-organ approach in the prevention and treatment of NAFLD.

DNA metabarcoding using nrITS2 provides highly qualitative and quantitative results for airborne pollen monitoring.

Airborne pollen monitoring is of global socio-economic importance as it provides information on presence and prevalence of allergenic pollen in ambient air. Traditionally, this task has been performed by microscopic investigation, but novel techniques are being developed to automate this process. Among these, DNA metabarcoding has the highest potential of increasing the taxonomic resolution, but uncertainty exists about whether the results can be used to quantify pollen abundance. In this study, it is shown that DNA metabarcoding using trnL and nrITS2 provides highly improved taxonomic resolution for pollen from aerobiological samples from the Netherlands. A total of 168 species from 143 genera and 56 plant families were detected, while using a microscope only 23 genera and 22 plant families were identified. NrITS2 produced almost double the number of OTUs and a much higher percentage of identifications to species level (80.1%) than trnL (27.6%). Furthermore, regressing relative read abundances against the relative abundances of microscopically obtained pollen concentrations showed a better correlation for nrITS2 (R2 = 0.821) than for trnL (R2 = 0.620). Using three target taxa commonly encountered in early spring and fall in the Netherlands (Alnus sp., Cupressaceae/Taxaceae and Urticaceae) the nrITS2 results showed that all three taxa were dominated by one or two species (Alnus glutinosa/incana, Taxus baccata and Urtica dioica). Highly allergenic as well as artificial hybrid species were found using nrITS2 that could not be identified using trnL or microscopic investigation (Alnus × spaethii, Cupressus arizonica, Parietaria spp.). Furthermore, perMANOVA analysis indicated spatiotemporal patterns in airborne pollen trends that could be more clearly distinguished for all taxa using nrITS2 rather than trnL. All results indicate that nrITS2 should be the preferred marker of choice for molecular airborne pollen monitoring.

Environmental DNA metabarcoding reveals comparable responses to agricultural stressors on different trophic levels of a freshwater community.

Freshwater habitats are under stress from agricultural land use, most notably the influx of neonicotinoid pesticides and increased nutrient pressure from fertilizer. Traditional studies investigating the effects of stressors on freshwater systems are often limited to a narrow range of taxa, depending heavily on morphological expertise. Additionally, disentanglement of multiple simultaneous stressors can be difficult in field studies, whereas controlled laboratory conditions do not accurately reflect natural conditions and food webs. To overcome these drawbacks, we investigated the impacts of two agricultural stressors (the neonicotinoid insecticide thiacloprid and fertilizer) in full-factorial design in a semi-natural research site, using environmental DNA sampling to study three different taxonomic groups representing three trophic levels: bacteria (decomposers), phytoplankton (primary producers), and chironomids (consumers). The results show considerable impact of both stressors across trophic levels, with an additive effect of fertilizer and thiacloprid on community composition at all levels. These findings suggest that agricultural stressors affect the entire food web, either directly or through cascade reactions. They are also consistent with morphological assessments that were performed in the same study site, even at a lower number of replicates. The study presented shows that the use of multimarker environmental DNA provides a more comprehensive assessment of stressor impacts across multiple trophic levels, at a higher taxonomic resolution than traditional surveys. Additionally, many putative novel bioindicators for both agricultural stressors were discovered. We encourage further investigations into stressors impacts at different trophic levels, which will lead to more effective monitoring and management of freshwater systems.

The mitochondrial genome of Nemalecium lighti (Hydrozoa, Leptothecata).

The hydrozoan species Nemalecium lighti (Hargitt, 1924) is widely distributed in tropical marine waters around the world. Here we report the complete linear mitochondrial genome of N. lighti from Sint Eustatius (Lesser Antilles). The mitochondrial genome with a length of 14,320 bp encodes for 13 protein-coding genes, two tRNA genes, and two rRNA genes. Gene arrangement differs from that found in other species of the same taxonomic order and a phylogenetic analysis shows that based on mitochondrial genes, N. lighti clusters outside of the Leptothecata, rendering the order paraphyletic.

Microbiome and environment explain the absence of correlations between consumers and their diet in Bornean microsnails.

Classical ecological theory posits that species partition resources such that each species occupies a unique resource niche. In general, the availability of more resources allows more species to co-occur. Thus, a strong relationship between communities of consumers and their resources is expected. However, correlations may be influenced by other layers in the food web, or by the environment. Here we show, by studying the relationship between communities of consumers (land snails) and individual diets (from seed plants), that there is in fact no direct, or at most a weak but negative, relationship. However, we found that the diversity of the individual microbiome positively correlates with both consumer community diversity and individual diet diversity in three target species. Moreover, these correlations were affected by various environmental variables, such as anthropogenic activity, habitat island size, and a possibly important nutrient source, guano runoff from nearby caves. Our results suggest that the microbiome and the environment explain the absence of correlations between diet and consumer community diversity. Hence, we advocate that microbiome inventories are routinely added to any community dietary analysis, which our study shows can be done with relatively little extra effort. Our approach presents the tools to quickly obtain an overview of the relationships between consumers and their resources. We anticipate our approach to be useful for ecologists and environmentalists studying different communities in a local food web.

Microbiota in Dung and Milk Differ Between Organic and Conventional Dairy Farms.

Organic farming is increasingly promoted as a means to reduce the environmental impact of artificial fertilizers, pesticides, herbicides, and antibiotics in conventional dairy systems. These factors potentially affect the microbial communities of the production stages (soil, silage, dung, and milk) of the entire farm cycle. However, understanding whether the microbiota representative of different production stages reflects different agricultural practices - such as conventional versus organic farming - is unknown. Furthermore, the translocation of the microbial community across production stages is scarcely studied. We sequenced the microbial communities of soil, silage, dung, and milk samples from organic and conventional dairy farms in the Netherlands. We found that community structure of soil fungi and bacteria significantly differed among soil types, but not between organic versus conventional farming systems. The microbial communities of silage also did not differ among conventional and organic systems. Nevertheless, the dung microbiota of cows and the fungal communities in the milk were significantly structured by agricultural practice. We conclude that, while the production stages of dairy farms seem to be disconnected in terms of microbial transfer, certain practices specific for each agricultural system, such as the content of diet and the use of antibiotics, are potential drivers of shifts in the cow's microbiota, including the milk produced. This may reflect differences in farm animal health and quality of dairy products depending on farming practices.

Increased performance of DNA metabarcoding of macroinvertebrates by taxonomic sorting.

DNA-based identification through the use of metabarcoding has been proposed as the next step in the monitoring of biological communities, such as those assessed under the Water Framework Directive (WFD). Advances have been made in the field of metabarcoding, but challenges remain when using complex samples. Uneven biomass distributions, preferential amplification and reference database deficiencies can all lead to discrepancies between morphological and DNA-based taxa lists. The effects of different taxonomic groups on these issues remain understudied. By metabarcoding WFD monitoring samples, we analyzed six different taxonomic groups of freshwater organisms, both separately and combined. Identifications based on metabarcoding data were compared directly to morphological assessments performed under the WFD. The diversity of taxa for both morphological and DNA-based assessments was similar, although large differences were observed in some samples. The overlap between the two taxon lists was 56.8% on average across all taxa, and was highest for Crustacea, Heteroptera, and Coleoptera, and lowest for Annelida and Mollusca. Taxonomic sorting in six basic groups before DNA extraction and amplification improved taxon recovery by 46.5%. The impact on ecological quality ratio (EQR) scoring was considerable when replacing morphology with DNA-based identifications, but there was a high correlation when only replacing a single taxonomic group with molecular data. Different taxonomic groups provide their own challenges and benefits. Some groups might benefit from a more consistent and robust method of identification. Others present difficulties in molecular processing, due to uneven biomass distributions, large genetic diversity or shortcomings of the reference database. Sorting samples into basic taxonomic groups that require little taxonomic knowledge greatly improves the recovery of taxa with metabarcoding. Current standards for EQR monitoring may not be easily replaced completely with molecular strategies, but the effectiveness of molecular methods opens up the way for a paradigm shift in biomonitoring.

The effects of spatial and temporal replicate sampling on eDNA metabarcoding.

BACKGROUND: The heterogeneous nature of environmental DNA (eDNA) and its effects on species detection and community composition estimates has been highlighted in several studies in the past decades. Mostly in the context of spatial distribution over large areas, in fewer occasions looking at spatial distribution within a single body of water. Temporal variation of eDNA, similarly, has mostly been studied as seasonality, observing changes over large periods of time, and often only for small groups of organisms such as fish and amphibians. METHODS: We analyzed and compared small-scale spatial and temporal variation by sampling eDNA from two small, isolated dune lakes for 20 consecutive weeks. Metabarcoding was performed on the samples using generic COI primers. Molecular operational taxonomic unit (MOTUs) were used to assess dissimilarities between spatial and temporal replicates. RESULTS: Our results show large differences between samples taken within one lake at one point in time, but also expose the large differences between temporal replicates, even those taken only 1 week apart. Furthermore, between-site dissimilarities showed a linear correlation with time frame, indicating that between-site differences will be inflated when samples are taken over a period of time. We also assessed the effects of PCR replicates and processing strategies on general patterns of dissimilarity between samples. While more inclusive PCR replicate strategies lead to higher richness estimations, dissimilarity patterns between samples did not significantly change. CONCLUSIONS: We conclude that the dissimilarity of temporal replicates at a 1 week interval is comparable to that of spatial replicate samples. It increases, however, for larger time intervals, which suggests that population turnover effects can be stronger than community heterogeneity. Spatial replicates alone may not be enough for optimal recovery of taxonomic diversity, and cross-comparisons of different locations are susceptible to inflated dissimilarities when performed over larger time intervals. Many of the observed MOTUs could be classified as either phyto- or zooplankton, two groups that have gained traction in recent years as potential novel bio-indicator species. Our results, however, indicate that these groups might be susceptible to large community shifts in relatively short periods of time, highlighting the need to take temporal variations into consideration when assessing their usability as water quality indicators.

Uncovering bacterial and functional diversity in macroinvertebrate mitochondrial-metagenomic datasets by differential centrifugation.

PCR-free techniques such as meta-mitogenomics (MMG) can recover taxonomic composition of macroinvertebrate communities, but suffer from low efficiency, as >90% of sequencing data is mostly uninformative due to the great abundance of nuclear DNA that cannot be identified with current reference databases. Current MMG studies do not routinely check data for information on macroinvertebrate-associated bacteria and gene functions. However, this could greatly increase the efficiency of MMG studies by revealing yet overlooked diversity within ecosystems and making currently unused data available for ecological studies. By analysing six 'mock' communities, each containing three macroinvertebrate taxa, we tested whether this additional data on bacterial taxa and functional potential of communities can be extracted from MMG datasets. Further, we tested whether differential centrifugation, which is known to greatly increase efficiency of macroinvertebrate MMG studies by enriching for mitochondria, impacts on the inferred bacterial community composition. Our results show that macroinvertebrate MMG datasets contain a high number of mostly endosymbiont bacterial taxa and associated gene functions. Centrifugation reduced both the absolute and relative abundance of highly abundant Gammaproteobacteria, thereby facilitating detection of rare taxa and functions. When analysing both taxa and gene functions, the number of features obtained from the MMG dataset increased 31-fold ('enriched') respectively 234-fold ('not enriched'). We conclude that analysing MMG datasets for bacteria and gene functions greatly increases the amount of information available and facilitates the use of shotgun metagenomic techniques for future studies on biodiversity.

Microbiomes of gall-inducing copepod crustaceans from the corals Stylophora pistillata (Scleractinia) and Gorgonia ventalina (Alcyonacea).

Corals harbor complex and diverse microbial communities that strongly impact host fitness and resistance to diseases, but these microbes themselves can be influenced by stresses, like those caused by the presence of macroscopic symbionts. In addition to directly influencing the host, symbionts may transmit pathogenic microbial communities. We analyzed two coral gall-forming copepod systems by using 16S rRNA gene metagenomic sequencing: (1) the sea fan Gorgonia ventalina with copepods of the genus Sphaerippe from the Caribbean and (2) the scleractinian coral Stylophora pistillata with copepods of the genus Spaniomolgus from the Saudi Arabian part of the Red Sea. We show that bacterial communities in these two systems were substantially different with Actinobacteria, Alphaproteobacteria, and Betaproteobacteria more prevalent in samples from Gorgonia ventalina, and Gammaproteobacteria in Stylophora pistillata. In Stylophora pistillata, normal coral microbiomes were enriched with the common coral symbiont Endozoicomonas and some unclassified bacteria, while copepod and gall-tissue microbiomes were highly enriched with the family ME2 (Oceanospirillales) or Rhodobacteraceae. In Gorgonia ventalina, no bacterial group had significantly different prevalence in the normal coral tissues, copepods, and injured tissues. The total microbiome composition of polyps injured by copepods was different. Contrary to our expectations, the microbial community composition of the injured gall tissues was not directly affected by the microbiome of the gall-forming symbiont copepods.

Lactobacillus iners-dominated vaginal microbiota is associated with increased susceptibility to Chlamydia trachomatis infection in Dutch women: a case-control study.

INTRODUCTION: This prospective study aimed to study the composition and structure of the vaginal microbiota prior to Chlamydia trachomatis infection. METHODS: A nested case-control study was performed in 122 women, half of which acquired C. trachomatis within a year after their first visit. At the first visit, the composition and structure of vaginal microbial communities were analysed using 16S rRNA sequencing in the context of the sociodemographic and sexual risk behaviour information using logistic regression. RESULTS: Five vaginal community state types (CSTs) were identified. Four CSTs were dominated by Lactobacillus spp., of which L. crispatus (37%) and L. iners (33%) were the most common. One CST was characterised by the absence of Lactobacillus spp. (25%) and the presence of an array of strict and facultative anaerobes. Multivariate logistic regression analysis revealed that women with a L. iners-dominated CST had an increased risk of C. trachomatis infection (p=0.04; OR: 2.6, 95% CI 1.0 to 6.6). CONCLUSIONS: The distribution of CSTs dominated by Lactobacillus spp. agreed with previous studies. However, the frequency of dysbiosis among Caucasian women was relatively high (24%). Having vaginal microbiota dominated by L. iners was associated with an increased risk for C. trachomatis infection. Therefore, we hypothesise that specific signatures of vaginal microbiota are indicative of increased host predisposition to acquiring STIs.

Earlier Detection of Hepatitis C Virus Infection Through Routine Hepatitis C Virus Antibody Screening of Human Immunodeficiency Virus-Positive Men Who Have Sex With Men Attending A Sexually Transmitted Infection Outpatient Clinic: A Longitudinal Study.

BACKGROUND: In 2007, routine hepatitis C virus (HCV) antibody testing was introduced for men who have sex with men (MSM) with a human immunodeficiency virus (HIV)-positive or unknown status attending a Dutch sexually transmitted infection (STI) outpatient clinic. We evaluated whether this screening resulted in additional and earlier HCV diagnoses among MSM who also attend HIV clinics. METHODS: At first STI consultation, HIV-positive MSM and MSM opting-out of HIV testing (HIV-status-unknown) were tested for HCV antibodies (anti-HCV). During follow-up consultations, only previously HCV-negative men were tested. Retrospectively, STI clinic and HIV clinic HCV diagnosis dates were compared. RESULTS: One hundred twelve (6.4%) of 1742 (95% confidence interval [CI], 5.3-7.6%) HIV-positive and 3 (0.7%) of 446 (95% CI, 0.2-2.0%) HIV-status-unknown MSM tested anti-HCV-positive at first consultation. During follow-up consultations, 32 HIV-positive (incidence HCV-positive: 2.35/100 person years (PY) (95% CI, 1.66-3.33)) and 0 (1-sided, 97.5% CI, 0.0-3.76) HIV-status-unknown MSM became anti-HCV-positive. Four (11.8%) of 34 HIV-positive MSM notified by their sexual partner of HCV tested anti-HCV-positive.Of 163 HIV-positive MSM with HCV antibodies, 78 reported a history of HCV. HCV diagnosis data at the HIV clinic was requested for the remaining 85 MSM and available for 54 MSM. Of these 54 MSM, 28 (51.9%) had their first HCV diagnosis at the STI clinic, of whom 7 concurrently with HIV. At their next scheduled HIV clinic consultation, 3 HCV cases probably would have been missed. CONCLUSIONS: The introduction of routine anti-HCV testing at the STI outpatient clinic resulted in additional and earlier HCV detection among HIV-positive MSM. Testing should be continued among HIV-positive MSM, at least for those not (yet) under the care of an HIV clinic and those notified of HCV by their sexual partner.

The effect of HIV infection on anal and penile human papillomavirus incidence and clearance: a cohort study among MSM.

OBJECTIVES: A large portion of anogenital cancers is caused by high-risk human papillomavirus (hrHPV) infections, which are especially common in HIV-infected men. We aimed to compare the incidence and clearance of anal and penile hrHPV infection between HIV-infected and HIV-negative MSM. DESIGN: Analyses of longitudinal data from a prospective cohort study. METHODS: MSM aged 18 years or older were recruited in Amsterdam, the Netherlands, and followed-up semi-annually for 24 months. At each visit, participants completed risk-factor questionnaires. Anal and penile self-samples were tested for HPV DNA using the SPF10-PCR DEIA/LiPA25 system. Effects on incidence and clearance rates were quantified via Poisson regression, using generalized estimating equations to correct for multiple hrHPV types. RESULTS: Seven hundred and fifty MSM with a median age of 40 years (interquartile 35-48) were included in the analyses, of whom 302 (40%) were HIV-infected. The incidence rates of hrHPV were significantly higher in HIV-infected compared with HIV-negative MSM [adjusted incidence rate ratio (aIRR) 1.6; 95% confidence interval (CI) 1.3-2.1 for anal and aIRR 1.4; 95%CI 1.0-2.1 for penile infection]. The clearance rate of hrHPV was significantly lower for anal [adjusted clearance rate ratio (aCRR) 0.7; 95%CI 0.6-0.9], but not for penile infection (aCRR 1.3; 95%CI 1.0-1.7). HrHPV incidence or clearance did not differ significantly by nadir CD4 cell count. CONCLUSION: Increased anal and penile hrHPV incidence rates and decreased anal hrHPV clearance rates were found in HIV-infected compared with HIV-negative MSM, after adjusting for sexual behavior. Our findings suggest an independent effect of HIV infection on anal hrHPV infections.

Chlamydia trachomatis load in population-based screening and STI-clinics: implications for screening policy.

OBJECTIVES: If the Chlamydia trachomatis (CT) bacterial load is higher in high-risk populations than in the general population, this negatively affects the efficacy of CT screening incentives. In the largest retrospective study to date, we investigated the CT load in specimens collected from 2 cohorts: (1) attendants of a sexually transmitted infection (STI)-clinic and (2) participants of the Dutch population-based screening (PBS). METHODS: CT load was determined using quantitative PCR in CT-positive male urine and female cervicovaginal swabs. CT loads were converted into tertiles. Using multinominal logistic regression, independent association of cohort, symptoms, risk behaviour and human cell count on load were assessed. RESULTS: CT loads were determined in 889 CT-positives from PBS (n = 529; 71.8% female) and STI-clinics (n = 360; 61.7% female). In men, STI-clinic-cohort, human cell count and urethral discharge were positively associated with CT load. In women, PBS-cohort and cell count were positively associated with CT load. Both cohorts had the same range in CT load. CONCLUSIONS: The general population has a similar range of bacterial CT load as a high-risk population, but a different distribution for cohort and gender, highlighting the relevance of population-based CT-screening. When CT loads are similar, possibly the chances of transmission and sequelae are too.

Rapid and reliable discrimination between Shigella species and Escherichia coli using MALDI-TOF mass spectrometry.

E. coli-Shigella species are a cryptic group of bacteria in which the Shigella species are distributed within the phylogenetic tree of E. coli. The nomenclature is historically based and the discrimination of these genera developed as a result of the epidemiological need to identify the cause of shigellosis, a severe disease caused by Shigella species. For these reasons, this incorrect classification of shigellae persists to date, and the ability to rapidly characterize E. coli and Shigella species remains highly desirable. Until recently, existing matrix-assisted laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF MS) assays used to identify bacteria could not discriminate between E. coli and Shigella species. Here we present a rapid classification method for the E. coli-Shigella phylogroup based on MALDI-TOF MS which is supported by genetic analysis. E. coli and Shigella isolates were collected and genetically characterized by MLVA. A custom reference library for MALDI-TOF MS that represents the genetic diversity of E. coli and Shigella strains was developed. Characterization of E. coli and Shigella species is based on an approach with Biotyper software. Using this reference library it was possible to distinguish between Shigella species and E. coli. Of the 180 isolates tested, 94.4% were correctly classified as E. coli or shigellae. The results of four (2.2%) isolates could not be interpreted and six (3.3%) isolates were classified incorrectly. The custom library extends the existing MALDI-TOF MS method for species determination by enabling rapid and accurate discrimination between Shigella species and E. coli.

Urogenital Chlamydia trachomatis strain types, defined by high-resolution multilocus sequence typing, in relation to ethnicity and urogenital symptoms among a young screening population in Amsterdam, The Netherlands.

INTRODUCTION: Previous studies found conflicting results regarding associations between urogenital Chlamydia trachomatis infections and ethnicity or urogenital symptoms among at-risk populations using either ompA-based genotyping or high-resolution multilocus sequence typing (MLST). This study applied high-resolution MLST on samples of individuals from a selected young urban screening population to assess the relationship of C. trachomatis strain types with ethnicity and self-reported urogenital symptoms. Demographic and sexual risk behaviour characteristics of the identified clusters were also analysed. METHODS: We selected C. trachomatis-positive samples from the Dutch Chlamydia Screening Implementation study among young individuals in Amsterdam, the Netherlands. All samples were typed using high-resolution MLST. Clusters were assigned using minimum spanning tree analysis and were combined with epidemiological data of the participants. RESULTS: We obtained full MLST data for C. trachomatis-positive samples from 439 participants and detected nine ompA genovars. MLST analysis identified 175 sequence types and six large clusters; in one cluster, participants with Surinamese/Antillean ethnicity were over-represented (58.8%) and this cluster predominantly consisted of genovar I. In addition, we found one cluster with an over-representation of participants with Dutch ethnicity (90.0%) and which solely consisted of genovar G. No association was observed between C. trachomatis clusters and urogenital symptoms. CONCLUSIONS: We found an association between urogenital C. trachomatis clusters and ethnicity among young screening participants in Amsterdam, the Netherlands. However, no association was found between C. trachomatis clusters and self-reported urogenital symptoms.

Spontaneous pharyngeal Chlamydia trachomatis RNA clearance. A cross-sectional study followed by a cohort study of untreated STI clinic patients in Amsterdam, The Netherlands.

OBJECTIVES: Pharyngeal Chlamydia trachomatis (chlamydia) might contribute to ongoing chlamydia transmission, yet data on spontaneous clearance duration are rare. We examined the prevalence, spontaneous clearance, chlamydial DNA concentration and genotypes of pharyngeal chlamydia among clinic patients with sexually transmitted infection (STI). METHODS: Female patients at high risk for an STI who reported active oral sex and male patients who have sex with men (MSM) were screened for pharyngeal chlamydia RNA using a nucleic acid amplification test. A repeat swab was obtained to evaluate spontaneous clearance in untreated patients with pharyngeal chlamydia. Quantitative chlamydia DNA load was determined by calculating the chlamydia/human cell ratio. RESULTS: Pharyngeal chlamydia was detected in 148/13 111 (1.1%) MSM and in 160/6915 (2.3%) women. 53% of MSM and 32% of women with pharyngeal chlamydia did not have a concurrent anogenital chlamydia infection. In 16/43 (37%) MSM and in 20/55 (36%) women, the repeat pharyngeal swab was negative (median follow-up 10 days, range 4-58 days). Patients with an initial chlamydial DNA concentration above the median were less likely to clear. Of 23 MSM with pharyngeal chlamydia who had sex with a lymphogranuloma venereum (LGV)-positive partner recently or in the past, two were LGV biovar positive (8.7%). CONCLUSIONS: The pharynx is a reservoir for chlamydia and LGV, and may play a role in ongoing transmission. Although delay in ribosomal RNA decline after resolution of the infection might have led to an underestimation of spontaneous clearance, in high-risk STI clinic patients, testing the pharynx for chlamydia should be considered.

Evaluation of a hepatitis C virus (HCV) antigen assay for routine HCV screening among men who have sex with men infected with HIV.

BACKGROUND: For detection of early HCV infection and reinfection, commercial HCV-RNA tests are available. However, these tests are relatively time-consuming and expensive. A commercially available test that may supplement current screening methods, targets the HCV core protein. METHODS: During five waves of anonymous surveys at the Amsterdam STI clinic between 2009-2012, all HIV-infected MSM (N=439) were tested for HCV-antibodies (AxSYM HCV 3.0, Abbott), and HCV-RNA (TMA Versant, Siemens). To evaluate the potential value of the ARCHITECT HCV antigen (HCV-Ag) assay (Abbott), all HCV-RNA-positive sera (N=31) were tested with this assay, as well as two HIV-infected HCV-RNA-negative controls. In addition, all included samples were tested for alanine aminotransferase (ALT). RESULTS: Among 439 HIV-infected MSM, 31 (7.1%) tested positive for HCV-RNA; the HCV-Ag assay showed concordant positive results for 31/31 (100%). A substantial number of MSM, i.e., 5/31 (16.1%), had detectable HCV-RNA but were HCV-seronegative at the time of screening and were presumed to have been recently infected. Concordant HCV-RNA-negative results were obtained in 57/60 control-samples. Specificity was 95.0% (95% CI: 86.1-99.0). The detection limit was between 3.0 and 3.7 Log10 IU/mL, irrespective of HCV genotype/subtype. ALT concentrations were elevated (i.e.,>40 U/L) in 9/31 (29.0%) HCV-RNA positive MSM, including 1/5 (20.0%) MSM with recent HCV-infection. CONCLUSIONS: The HCV-Ag assay proved a valuable screening tool for detection of active HCV infection among HIV-infected MSM with and without anti-HCV. Adding ALT to current screening methods would improve case finding marginally. We therefore recommend implementation of routine HCV-Ag screening for populations at risk for HCV-(re)infection.

HPV seroconversion following anal and penile HPV infection in HIV-negative and HIV-infected MSM.

BACKGROUND: We assessed human papillomavirus (HPV) seroconversion following anal and penile HPV infection in HIV-negative and HIV-infected men who have sex with men (MSM). METHODS: MSM aged ≥18 years were recruited in Amsterdam, the Netherlands (2010-2011), and followed up semiannually. Antibodies against 7 high-risk HPV types in baseline and 12-month serum samples were tested using a multiplex immunoassay. Baseline, 6-, and 12-month anal and penile samples were tested for HPV DNA using the SPF10-PCR DEIA/LiPA25 system. Statistical analyses were performed using logistic regression with generalized estimating equations. RESULTS: Of 644 MSM included in the analysis, 245 (38%) were HIV-infected. Median age was 38 years for HIV-negative and 47 years for HIV-infected MSM (P < 0.001). Seroconversion against ≥1 of the 7 HPV types was observed in 74 of 396 (19%) HIV-negative and 52 of 223 (23%) HIV-infected MSM at risk (P = 0.2). Incident [adjusted OR (aOR) 2.0; 95% confidence interval (CI), 1.1-3.4] and persistent (aOR 3.7; 95% CI, 1.5-9.5) anal HPV infections were independently associated with type-specific seroconversion in HIV-negative MSM. In HIV-infected MSM, there was a nonsignificant positive association between penile HPV infection at any time point and seroconversion (aOR 1.7; 95% CI, 0.9-3.2). CONCLUSIONS: Incident or persistent anal HPV infection was an independent determinant of seroconversion in HIV-negative MSM. IMPACT: Our data support that seroresponse may vary per anatomic site and that persistent HPV infections are more likely to elicit a detectable humoral immune response.

Perceived HIV status is a key determinant of unprotected anal intercourse within partnerships of men who have sex with men in Amsterdam.

The practice of unprotected anal intercourse (UAI) involves at least two partners. We examined the associations between insertive or receptive UAI and perceived HIV seroconcordance and partnership type in self-perceived HIV-negative and self-perceived HIV-positive men who have sex with men (MSM). MSM (age ≥ 18 years) were recruited for a cross-sectional survey at the sexually transmitted infections clinic in Amsterdam, the Netherlands, in 2008-2009. Participants completed a questionnaire concerning partnerships in the preceding 6 months. Associations were quantified via multinomial logistic regression models using generalized estimating equations. The outcomes were 'no, or safe anal intercourse', 'insertive UAI', and 'receptive UAI'. We included 5,456 partnerships from 1,890 self-perceived HIV-negative men and 1,861 partnerships from 558 self-perceived HIV-positive men. Within the partnerships, perceived HIV status of the partner was an important determinant of UAI (p < 0.001). Among HIV-negative men, perceived HIV discordance was negatively associated with receptive UAI compared with no or safe UAI (OR 0.57; 95 % CI 0.36-0.92); when the partners were more familiar with each other, the risk of receptive UAI was increased relative to no or safe anal intercourse. Among HIV-positive men, perceived HIV discordance was negatively associated with insertive UAI (OR 0.05; 95 % CI 0.03-0.08). Within partnerships, perceived HIV status of the partner was one of the strongest determinants of UAI among self-perceived HIV-negative and HIV-positive MSM, and discordant serostatus was negatively associated with UAI. The findings suggest that serosorting is one of the main strategies when engaging in UAI.