TMA Assay Research Articles

ALYREF-JunD-SLC7A5 axis promotes pancreatic ductal adenocarcinoma progression through epitranscriptome-metabolism reprogramming and immune evasion

Pancreatic ductal adenocarcinoma (PDAC) is a kind of tumor lacking nutrients due to its poor vascularity and desmoplasia. Recent studies have shown that cancer cells might achieve growth advantage through epitranscriptome reprogramming. However, the role of m5C in PDAC was not fully understood. We found that Aly/REF export factor (ALYREF), a reader of m5C modification, was overexpressed in PDAC, and associated with bad prognosis. In addition, the ALYREF expression was negatively related to CD8+ T cells infiltration in clinical samples. ALYREF knockdown decreased tumor growth in vivo partly dependent of immunity. ALYREF silencing decreased SLC7A5 expression and subsequently inactivated mTORC1 pathway, resulting in decreased tumor proliferation. Mechanically, ALYREF specifically recognized m5C sites in JunD mRNA, maintained the stabilization of JunD mRNA and subsequently upregulated transcription of SLC7A5. Since SLC7A5 was a key transporter of large neutral amino acids (LNAAs), overexpression of SLC7A5 on tumor cells depleted amino acid in microenvironment and restricted CD8+ T cells function. Moreover, ALYREF-JunD-SLC7A5 axis was overexpressed and negatively related with survival through TMA assays. In conclusion, this research revealed the relationship between m5C modification, amino acid transportation and immune microenvironment. ALYREF might be a novel target for PDAC metabolic vulnerability and immune surveillance.

The diagnostic accuracy of the ID NOW COVID-19 point of care test in acute hospital admissions

BackgroundPrompt identification of patients with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection on admission to hospital is crucial to ensuring initiation of appropriate treatment, optimising infection control and maintaining patient flow. The Abbott ID NOW™ COVID-19 assay (ID NOW) is a point-of-care, isothermal nucleic acid amplification test, capable of producing a result within minutes, potentially placing it as an invaluable tool in helping to control the coronavirus-disease 2019 (COVID-19) pandemic. ObjectivesTo evaluate the diagnostic accuracy of ID NOW in acute hospital admissions. Study designA prospective approach to data collection was undertaken in consecutive patients with ID NOW and Hologic Aptima™ SARS-CoV-2 transcription-mediated amplification assay (Aptima TMA) results, across three hospitals in the south-west of England between 1st March and 30th September 2021. A nasal swab was taken for ID NOW and a combined nose and throat swab for Aptima TMA. Measures of diagnostic accuracy were calculated for ID NOW against Aptima TMA. This study was conducted during a period of alpha and delta strain predominance. Results19,698 ID NOW assays were performed, of which 12,821 had an Aptima TMA assay performed within 24 hours. ID NOW had sensitivity of 85.2 % (95 % CI, 82.2–87.9) and specificity of 99.6 % (95 % CI, 99.4–99.7) compared with the reference assay. The overall PPV was 91.0 % (95 % CI, 88.5–93.0) and the overall NPV was 99.3 % (95 % CI, 99.1–99.4). ConclusionsID NOW offers a valid diagnostic tool to detect SARS-CoV-2, performing comparably to a reference laboratory-based assay which takes longer to provide results.

Multicenter clinical evaluation of a novel transcription-mediated amplification assay for SARS-CoV-2 molecular testing

IntroductionThe onset and spread of COVID-19 pandemic has forced clinical laboratories to rapidly expand testing capacity for SARS-CoV-2. This study evaluates the clinical performance of the TMA Procleix SARS-CoV-2 assay in comparison to the RT-PCR assay Allplex™ SARS-CoV-2 for the qualitative detection of SARS-CoV-2 RNA. MethodsBetween November 2020 and February 2021, 610 upper-respiratory specimens received for routine SARS-CoV-2 molecular testing were prospectively collected and selected at the Hospital Universitari Vall d’Hebron and the Hospital Universitari Bellvitge in Barcelona, Spain. All samples were processed in parallel with the TMA and the RT-PCR assays, and results were compared. Discrepancies were retested by an additional RT-PCR method and the clinical history of these patients was reviewed. ResultsOverall, the level of concordance between both assays was 92.0% (κ, 0.772). Most discordant results (36/38, 94.7%) corresponded to samples testing positive with the TMA assay and negative with the RT-PCR method. Of these discrepant cases, most (28/36, 77.8%) were finally classified as confirmed or probable SARS-CoV-2 cases according to the discrepant analysis. ConclusionIn conclusion, the TMA Procleix SARS-CoV-2 assay performed well for the qualitative detection of SARS-CoV-2 RNA in a multisite clinical setting. This novel TMA assay demonstrated a greater sensitivity in comparison to RT-PCR methods for the molecular detection of SARS-CoV-2. This higher sensitivity but also the qualitative feature of this detection of SARS-CoV-2 should be considered when making testing algorithm decisions.

Multicenter clinical evaluation of a novel transcription-mediated amplification assay for SARS-CoV-2 molecular testing

IntroductionThe onset and spread of COVID-19 pandemic has forced clinical laboratories to rapidly expand testing capacity for SARS-CoV-2. This study evaluates the clinical performance of the TMA Procleix SARS-CoV-2 assay in comparison to the RT-PCR assay Allplex™ SARS-CoV-2 for the qualitative detection of SARS-CoV-2 RNA. MethodsBetween November 2020 and February 2021, 610 upper-respiratory specimens received for routine SARS-CoV-2 molecular testing were prospectively collected and selected at the Hospital Universitari Vall d’Hebron and the Hospital Universitari Bellvitge in Barcelona, Spain. All samples were processed in parallel with the TMA and the RT-PCR assays, and results were compared. Discrepancies were retested by an additional RT-PCR method and the clinical history of these patients was reviewed. ResultsOverall, the level of concordance between both assays was 92.0% (κ, 0.772). Most discordant results (36/38, 94.7%) corresponded to samples testing positive with the TMA assay and negative with the RT-PCR method. Of these discrepant cases, most (28/36, 77.8%) were finally classified as confirmed or probable SARS-CoV-2 cases according to the discrepant analysis. ConclusionIn conclusion, the TMA Procleix SARS-CoV-2 assay performed well for the qualitative detection of SARS-CoV-2 RNA in a multisite clinical setting. This novel TMA assay demonstrated a greater sensitivity in comparison to RT-PCR methods for the molecular detection of SARS-CoV-2. This higher sensitivity but also the qualitative feature of this detection of SARS-CoV-2 should be considered when making testing algorithm decisions.

Performance evaluation of the Aptima HIV-1 Quant Dx assay for detection of HIV in infants in Kenya

BackgroundEarly infant diagnosis (EID) of HIV-1 exposed infants enables timely initiation of antiretroviral therapy (ART), thereby allowing early diagnosis and treatment to slow disease progression and reduce mortality. Turn-around time to results, partially caused by low to medium throughput technology, remains a hindrance to early treatment. A major solution to this challenge is to incorporate high throughput and accurate technologies in the testing process. The Hologic Aptima Quant Dx Assay (Aptima) is a CE marked Real-Time TMA assay running on the high throughput Panther system. ObjectivesThe objective of this study was to evaluate the performance of Aptima for EID using dried blood spots. Study designThis was a cross-sectional prospective study of 2,048 infants seeking HIV services from health facilities in Western Kenya, Africa. Capillary Dried Blood Spot samples DBS were collected from infants with the consent of their mothers. The qualitative performance of Aptima was compared with the Roche COBAS Ampliprep/ COBAS Taqman HIV-1 Qualitative Test v2.0 (CAP/CTM), using these DBS. Demographic information of the participants was also collected. ResultsA total of 1,975 successful comparisons between the two platforms were included in the analysis. The overall agreement between the assays was 99.65 %. The sensitivity and specificity of Aptima was 95.24 % (95 % CI 88.40–98.19 %) and 99.84 % (95 % CI 99.49–99.92 %) respectively. ConclusionsAptima assay has performance characteristics that are comparable to those of the Roche CAP/CTM for qualitative testing on DBS taken from infants. The two assays can therefore be used interchangeably for Early Infant Diagnosis of HIV.

P020 Comparison of the aptima mycoplasma genitalium tma assay and the fasttrack diagnostics (FTD) urethritis basic assay for detection of M. genitalium in gum specimens

IntroductionTesting forM. genitaliumin the UK is limited and detection has relied on realtime PCR assays. The Hologic AptimaMycoplasma genitaliumTMA assay for use on the Panther® system is now available. This study compared a commercial realtime PCR and the Aptima assay using stored clinical specimens.MethodsClinical specimens (76 urines, 33 vaginal swabs, 2 rectal swabs, 1 pooled sample and 2 unknowns) from men with urethritis and women with pelvic inflammatory disease were tested forM. genitaliumDNA using the FastTrack Diagnostics (FTD) Urethritis Basic assay. Residual specimen was then transferred to an Aptima urine tube and tested for the presence ofM. genitaliumribosomal RNA using the Aptima TMA assay.ResultsOf the 113 specimens tested, 24 (21%) were positive and 87 (77%) negative on both assays. There were two discrepant results (1.7%) in urine specimens that were positive on the Aptima TMA assay and negative on the FTD Urethritis assay. One was confirmed as positive by the Reference Laboratory using their in-house MgPa PCR, indicating a false negative result on the FTD Urethritis assay. The other discrepant result was low level positive on the Aptima TMA assay and negative at the Reference Laboratory.Discussion98% of samples gave concordant results, indicating that both assays are appropriate for use in clinical service. However, the additional positive detected by the Aptima assay, explained by detection of target in multiple copies in each bacterial cell, suggests that this assay is more sensitive.

The New Aptima HCV Quant Dx Real-time TMA Assay Accurately Quantifies Hepatitis C Virus Genotype 1-6 RNA

BackgroundSensitive and accurate hepatitis C virus (HCV) RNA detection and quantification is essential for the management of chronic hepatitis C therapy. Currently available platforms and assays are usually batched and require at least 5hours of work to complete the analyses. Objectives and study designThe aim of this study was to evaluate the ability of the newly developed Aptima HCV Quant Dx assay that eliminates the need for batch processing and automates all aspects of nucleic acid testing in a single step, to accurately detect and quantify HCV RNA in a large series of patients infected with different HCV genotypes. ResultsThe limit of detection was estimated to be 2.3 IU/mL. The specificity of the assay was 98.6% (95% confidence interval: 96.1%-99.5%). Intra-assay and inter-assay coefficients of variation ranged from 0.09% to 5.61%, and 1.05% to 3.65%, respectively. The study of serum specimens from patients infected with HCV genotypes 1 to 6 showed a satisfactory relationship between HCV RNA levels measured by the Aptima HCV Quant Dx assay, and both real-time PCR comparators (Abbott RealTime HCV and Cobas AmpliPrep/Cobas TaqMan HCV Test, version 2.0, assays). Conclusionsthe new Aptima HCV Quant Dx assay is rapid, sensitive, reasonably specific and reproducible and accurately quantifies HCV RNA in serum samples from patients with chronic HCV infection, including patients on antiviral treatment. The Aptima HCV Quant Dx assay can thus be confidently used to detect and quantify HCV RNA in both clinical trials with new anti-HCV drugs and clinical practice in Europe and the US.

Mycoplasma genitalium Prevalence and Variability Based on Gender, Race and Sexual Preference in Patients Attending the Oakland County Health Department Disease Clinic

Background Mycoplasma genitalium is an important emerging pathogen implicated in male urethritis as well as cervicitis and pelvic inflammatory disease in women. The epidemiology of M. genitalium resembles that of C. trachomatis and N. gonorrhea as all are found more frequently in younger, sexually active individuals and African Americans as compared with other races. There is currently no FDA cleared diagnostic test for M. genitalium, although a research-use-only TMA assay exists. The aim of our study was to determine the prevalence of M. genitalium in patients presenting to the Oakland County Health Department (OCHD) and determine any differences in prevalence based on sex, age and sexual preference.Methods We obtained a total of 5,021 urine specimens during single patient visits to the OCHD between August 1, 2016 and January 31, 2017. Of these, 2,323 were from female subjects and 2,698 were from male subjects. Urine samples were tested for M. genitalium (using a research-use-only TMA assay) as well as C. trachomatis, N. gonorrhea, and T. vaginalis (using FDA approved PCR assays). Each patient also had VDRL titers drawn for syphilis testing.Results M. genitalium was the most frequent organism recovered in women with a prevalence of 11.11%, followed by C. trachomatis (7.40%), T. vaginalis (4.43%), N. gonorrhea (2.02%) and syphilis (0.65%). In men, M. genitalium was equally prevalent (12.31%) as C. trachomatis (10.67%), followed by N. gonorrhea (4.86%), T. vaginalis (3.48%) and syphilis (1.93%). All organisms, with the exception of syphilis, were more prevalent in African-American men as compared with Caucasian men. No difference in prevalence was seen in women irrespective of race and there was no difference based on sexual orientation.Conclusion Our analysis showed that Mycoplasma genitalium is highly prevalent in our study population. This is of particular public health concern as this organism can result in a significant disease burden if left untreated. Azithromycin is the first-line agent against M. genitalium but can result in treatment failure in 13-32% of cases according to recent studies. For this reason, we advocate for the need to test for M. genitalium routinely in symptomatic patients at risk for sexually transmitted diseases.Disclosures All authors: No reported disclosures.

Comparison of Procleix Ultrio Elite and Procleix Ultrio NAT Assays for Screening of Transfusion Transmitted Infections among Blood Donors in India.

Background. Introduction of nucleic acid testing (NAT) has helped in decreasing window period donations, resulting in increased safety of blood supplies. NAT combines the advantages of direct and highly sequence-specific detection of viral genomes. We analysed the performance of newer Procleix Ultrio Elite (PUE) and Procleix Ultrio assay (PUA) for the screening of the viral markers in our donor population. Material and Methods. 10,015 donor samples were screened by routine immunoassays and both versions of NAT. NAT yields detected were subjected to viral load estimation and to other serological markers. Results. A total of 21 NAT yields were detected; three were positive by both NAT systems, whereas 18 samples were reactive by PUE only. NAT yields include 18 HBV and 3 HCV yields, of which 17 HBV yields were occult infections and 1 was window period (WP) infection. All 3 HCV yields were WP infections. No HIV-1/HIV-2 yield was found. Conclusion. Efficient target capture chemistry in the new TMA assay version significantly improved sensitivity. NAT is superior to serological immunoassays for screening of the viral markers; and the efficient target capture system in the newer TMA assay, namely, the PUE system, has significantly improved sensitivity over the earlier versions.

P05.13 Prevalence and anatomical distribution ofmycoplasma genitaliummacrolide resistance markers from subjects enrolled in a multi-centre us clinical study

<h3>Introduction</h3> This study evaluated the prevalence and anatomical distribution of <i>Mycoplasma genitalium</i> (Mgen) 23s rRNA mutations (23s-MTs) conferring macrolide resistance among male and female subjects enrolled in a prospective multi-site US clinical study. <h3>Methods</h3> Specimens obtained from symptomatic and asymptomatic men and women enrolled from 7 diverse US clinical sites, including obstetrics and gynaecology, family planning, public health, and sexually transmitted disease clinics, were tested using a research TMA assay for Mgen (Hologic, Inc.) on the DTS System or TIGRIS DTS System. Samples positive for Mgen by TMA were evaluated by nested PCR or RT-PCR and Sanger sequencing of Mgen 23S rRNA to identify the presence of macrolide resistance mutations at position 2058 (A2058G, A2058C, A2058T) or position 2059 (A2059G). <h3>Results</h3> Of 50 male subjects with Mgen 23s rRNA sequence results, 21 (42%) contained 23s-MTs. Slightly more 23s-MTs were found in urethral swabs vs male urine samples (44.8% vs 36.7%, respectively). For female subjects, 65 of 128 (50.8%) harboured 23s-MTs, with higher prevalence found in vaginal swab samples (50.2%) compared to urine (46%), Thinprep liquid Pap (41.7%), and endocervical swabs (37.8%). Sequencing of samples collected from anatomically distinct female urogenital sites (vagina, cervix, urine) revealed 18 of 35 (51.4%) subjects had a unique complement of Mgen 23s-MT and/or wild-type sequences at each anatomic site. For male subjects with both urine and urethral swab samples, 3 of 9 (33.3%) subjects had unique Mgen 23s-MT/WT sequences in each sample type. <h3>Conclusion</h3> Mgen strains harbouring 23s rRNA-mediated macrolide resistance phenotypes were highly prevalent in this US cohort of male and female subjects. Detection of different macrolide-resistant Mgen strains in samples collected from different anatomical locations suggests that previous estimates for resistance rates that relied on a single anatomical site sample collection may have underestimated the extent of Mgen macrolide resistance in the population. <h3>Disclosure of interest statement</h3> D Getman, M O’Donnell, and A Jiang are scientists employed by Hologic. S Cohen is a student at Occidental College and a summer intern at Hologic.

Hepatitis C (HCV) Treatment Progress and SVR of Combination Therapy in Practice

Introduction: Sofosbuvir, an interferon-free, direct-acting antiviral, has been shown to considerably improve SVR rates in HCV-infected patients in clinical trials. This study reports the results of sofosbuvir combined with simeprevir, a NS3/4A protease inhibitor, in a single practice for HCV genotype 1 patients. Methods: The study was conducted in a single practice, for patients with HCV genotype 1 infection. Cirrhotic, non-cirrhotic, treatment experienced, and treatment naïve patients received double therapy with sofosbuvir and simeprevir for 12 weeks. HCV RNA quantitative measurements were taken with a sensitive TMA assay (limit of detection of 7 IU/L) at treatment weeks 4 and 12, and at post-treatment weeks 4 and 12. After week 12, a reappearance of detectable HCV RNA defined viral relapse. Undetectable HCV RNA defined suppressed virologic response (SVR). At 4 weeks post-treatment, this was termed SVR-4, and at 12 weeks, SVR-12. The primary objective was to find how quickly HCV RNA was suppressed, and the proportion of patients who maintained undetectable HCV RNA at 4 and 12 weeks post-therapy. Results: Of the 63 participants, 25 were cirrhotic and 34 were previously treated with pegylated interferon. The overall rate of undetectable HCV RNA after 4 weeks of therapy was 71.4% (45/63). The cirrhotic group reached 64.0% (16/25), while non-cirrhotic achieved 81.6% (31/38). Treatment experienced subjects achieved 73.5% (25/34), while treatment naïve reached 75.9% (22/29). After 12 weeks, 100% (33/33) of patients were undetectable. Cirrhotic and non-cirrhotic subjects reached 100% (14/14 and 19/19, respectively). Treatment experienced and naïve achieved 100% (17/17 and 16/16, respectively). By June 15, 2014, 23 subjects were off therapy for 4 weeks, and 100% (23/23) achieved SVR-4. After 12 weeks off therapy, 100% (2/2) achieved SVR-12. No patients failed to respond to therapy, wished to discontinue, terminated therapy, nor relapsed, but 6/63 failed to follow-up posttherapy. Conclusion: Although the success of Gilead’s clinical trials seemed intangible, the results above suggest that the published outcomes are reproducible in practice. The combination therapy seems more effective in non-cirrhotic subjects than in cirrhotic ones at first. However, all subjects who completed therapy cleared the virus. No subjects who reached 4 weeks post-therapy by the time of collection relapsed, nor did any who reached 12 weeks post-therapy. Deviations may be attributed to low n values or to subjects’ failure to follow-up. Greater n values and more information will be amassed as more patients complete therapy and reach 4 and 12 weeks post-treatment.

Hepatitis C (HCV) Treatment and SVR: The Gap Between Clinical Trials and the Real-World

Introduction: Telaprevir, a NS3A/4 protease inhibitor, in combination with peginterferon-ribavirin [PEG-IFN/RBV] has proven to be associated with significantly improved rates of SVR in patients with HCV genotype 1 infection who have not received any previous treatment (N Engl J Med 2011;364:2405-16). The purpose of this study was to compare our results in a single practice with the results of previously published controlled studies. Methods: A single center retrospective cohort study was conducted on patients with HCV genotype 1 infection who had previously not received treatment for the infection. Fifty-one patients received triple therapy with Telaprevir combined with PEG-IFN/RBV for 12 weeks, followed by PEG-IFN/RBV alone for 12 and 36 weeks in non-cirrhotic and cirrhotic patients, respectively. End of treatment (EOT) was defined as a total of 24 weeks of therapy for non-cirrhotic patients and 48 weeks of therapy for cirrhotic patients. HCV RNA levels were measured at treatment weeks 4, 8, 12, 36, 48 and at post-treatment weeks 4 and 12 using a sensitive TMA assay (lower limit of detection of 5 IU/L). Viral relapse was defined as re-appearance of detectable serum HCV RNA between EOT and post treatment. SVR was defined as undetectable HCV RNA at 12 weeks post treatment. The primary end point was the proportion of patients who had undetectable HCV RNA at 12 weeks post therapy. Results: Of the 51 patients treated, 10 patients had cirrhosis. The overall rate of SVR was 51% (26). Three out of the 10 cirrhotic patients achieved SVR (30%) and 23 out of 41 non-cirrhotic patients achieved SVR (56%). Of the 35 patients who completed therapy, 24 achieved SVR (69%), 4 relapsed (11%) and 7 did not follow up post therapy (20%). Therapy was terminated in 16 patients. Discontinuation was based on adverse events in 18% of patients overall, including severe anemia (4), rash (2), blurred vision (1), intolerance (1), and hallucinations (1). Two patients had virologic breakthrough (13%), 1 did not follow up (6%), 1 did not respond to therapy (6%), and 3 patients did not want to continue with the therapy (19%). Conclusion: Despite the significant improvements in pharmacologic treatment efficacy for HCV reported in published clinical trials, our study suggests that in “real-world” patient care, these medical outcomes may be difficult to achieve. This can primarily be attributed to the fact that a significant number of patients failed to follow up. Also, the overall rate of discontinuation of the therapy owing to adverse events was higher in our study. Nevertheless, it is encouraging that a significant rate of SVR was observed in those who completed the therapy.

Efficacy of Weeks 4 and 12 Post-therapy HCV RNA Levels in Determining Sustained Virological Response [SVR] after Completing 24 or 48 Weeks of Telaprevir-based Triple Therapy for Chronic HCV Infection

Purpose: The FDA has recently approved an end-point SVR12 for use in HCV trials with direct acting antiviral agents [DAA]. However, 4-6% of patients had viral relapse between 12 and 24 weeks post-treatment with Sofosbuvir despite having achieved SVR12 [EASL 2013]. Our study evaluated whether assessment of serum HCV RNA at 4 and 12 weeks post-treatment was as reliable in predicting SVR as serum HCV RNA at 24 weeks post-treatment in HCV genotype 1 patients who received Telaprevir, PEG-IFN and ribavirin therapy. Methods: A single-center retrospective cohort study was conducted on HCV patients who had achieved end-of treatment [EOT] virological response on therapy with Telaprevir, PEG-IFN and ribavirin therapy between June, 2011 and January, 2013. HCV RNA levels were measured at EOT and post-treatment weeks four, 12 and 24 using a sensitive TMA assay (lower limit of detection of 5 IU/L). Viral relapse was defined as re-appearance of detectable serum HCV RNA between the EOT and post-treatment. SVR was defined as undetectable HCV RNA at 24 weeks post-treatment. Results: One hundred patients were treated from June, 2011 to January, 2013. Thirty-three patients completed 24 weeks of therapy [Group 1], and 25 completed 48 weeks of therapy [Group 2] with undetectable HCV RNA EOT response. In Group 1, at four weeks post-therapy, 23 patients remained negative and one relapsed, with nine remaining non-compliant. In the same group after 12 weeks post-therapy, 21 patients remained negative and two relapsed. And after 24 weeks post-therapy, the remaining 21 patients had undetectable serum HCV RNA. In Group 2 at four weeks post-therapy, 12 patients remained negative and two relapsed with a total of 11 patients being non-compliant. After 12 weeks post therapy, 10 patients remained negative and two relapsed. And after 24 weeks post-therapy, just as those in Group 1, all patients remained HCV RNA undetectable. Conclusion: The study supports that the assessment of serum HCV RNA at 12 weeks post-treatment has a 100% positive predictive value [PPV] in predicting SVR24 in genotype 1 patients receiving DAA therapy. Furthermore, SVR24 is unnecessary, or rather, patients should no longer be required to come back for a third post-therapy visit, which, if implemented, will reduce the cost of disease burden. Unfortunately, however, SVR4 may miss post-treatment early relapses, as it only has a PPV of 89%. Although future prospective or larger retrospective studies would be helpful in further validating these conclusions, according to this data that parallels the FDA's findings, SVR12 is needed while SVR24 is not.

P2.100 Clinical Efficacy of Sitafloxacin 100Mg Twice Daily For 7 Days For Patients with Non-Gonococcal Urethritis

To date, the standard treatment for the patients with chlamydial non-gonococcal urethritis (NGU) remains effective; however, conventional quinolone antibiotics have less activity against Mycoplasma genitalium-positive NGU. The purpose of this...

P3.244 Prevalence of Sexually Transmitted Infections in Multiple Sample Types Collected from HIV-1 Positive Men

BackgroundDetection of sexually transmitted infections (STIs) in HIV-1 positive (+) men is essential to ensure appropriate treatment and to reduce HIV and STI transmission. We evaluated the baseline prevalences of...

Prediction of minimal residual viremia in HCV type 1 infected patients receiving interferon-based therapy

Introduction. Complete suppression of viral replication is crucial in chronic HCV treatment in order to prevent relapse and resistance development. We wanted to find out which factors influence the period from being already HCV RNA negative by bDNA assay (< 615 lU/mL) to become undetectable by the more sensitive TMA test (< 5.3 lU/mL).Material and methods. Evaluated were 433 HCV type 1-infected patients. All of them received 1.5 ug/kg Peg-IFNa-2b plus ribavirin for 18-48 weeks. bDNA was performed weekly during the first 8 weeks and thereafter at weeks 12, 24, and 48. Patients who became bDNA undetectable were additionally analysed by TMA.Results. Of the 309 patients with on-treatment response (< 615 lU/mL), 289 also reached undetectable HCV RNA levels by TMA. Multivariate analysis revealed that viremia ≤ 400,000 lU/mL (p = 0.001), fast initial virologic decline (p = 0.004) and absence of fibrosis (p = 0.035) were independent predictors of an accelerated on-treatment response by TMA assay in already bDNA negative patients. bDNA negative patients becoming HCV RNA undetectable by TMA within the following 3 weeks had a frequency of relapse of 21%, whereas those showing TMA negativity after 3 weeks relapsed in 38% (p = 0.001). In RVR patients (bDNA < 615 lU/mL at week 4) the corresponding relapse rates were 15.3% vs. 37.5%, respectively (p = 0.003).Conclusion. Early viral kinetics, baseline viremia and fibrosis stage are important tools to predict persistent minimal viremia during interferon-based therapy. The data have implications for designing a more refined treatment strategy in HCV infection, even in the setting of protease inhibitor-based triple treatment.

Contribution of procoagulant phospholipids, thrombomodulin activity and thrombin generation assays as prognostic factors in intensive care patients with septic and non-septic organ failure

Multiple organ dysfunction syndrome (MODS) observed in patients with sepsis and in nonseptic patients organ failure (OF) is associated with a high mortality rate. We investigated whether new coagulation assays [quantification of procoagulant phospholipids (PPL) activity, functional assays measuring the activity of thrombomodulin (TMa) or tissue factor (TFa) and thrombin generation using calibrated automated thrombography (CAT)] could constitute new tools to better understand the physiopathology of MODS and have any prognostic value. We measured TMa, TFa, PPL and CAT in 32 healthy controls, 24 patients with sepsis and 26 patients with non-septic OF. We compared these parameters with usual coagulation assays [prothrombin time, activated partial thromboplastin time, protein C (PC), protein S, D-Dimers (D-Di), soluble thrombomodulin (sTM)] and markers of inflammation (IL-6, CRP). Samples were collected within 24 h of the diagnosis. TMa, TFa, PPL, the lag time and time to thrombin peak levels were increased in both groups of patients. For both groups D-Di, IL-6, CRP and endogenous thrombin potential (ETP) were higher in non-survivors than in survivors, while PC and PPL were lower in non-survivors than in survivors. TMa increase was more marked in non-survivors patients with OF, while the ratio TMa/sTM was low in non-survivors with sepsis. Received operating characteristic (ROC) curve analysis indicated that thrombin peak and ETP were the more powerful discriminating factors in patients with sepsis or non-septic OF, respectively. PPL, TMa and CAT assays could represent promising tools to identify patients with increased risk of mortality in MODS and could procure insights into pathogenesis of MODS.

High‐dose silibinin rescue treatment for HCV‐infected patients showing suboptimal virologic response to standard combination therapy

Incomplete suppression of hepatitis C virus (HCV) replication with persistence of minimal viremia (partial virologic response) leading to treatment failure can be observed in a significant proportion of HCV type 1-infected patients during antiviral therapy. Recently, high-dose intravenous silibinin has demonstrated strong antiviral activity against HCV. We were therefore interested in whether patients with partial virologic response can be rescued by the on-treatment addition of a short-term course of high-dose intravenous silibinin infusions. Twenty patients who failed to achieve a complete virologic response to different interferon-based regimens qualified for the rescue strategy and received 1400 mg/day silibinin infusions on two consecutive days. Complete viral suppression (below the limit of detection <6 IU/mL, TMA assay) could be induced in 13 of 20 patients within the first week after the short-term silibinin infusion, and all but one of them also remained HCV RNA negative during the subsequent follow-up period on continued peginterferon plus ribavirin treatment. In the remaining seven patients, no complete suppression could be achieved although four showed a significant HCV RNA reduction in response to silibinin. Silibinin infusions were generally well tolerated, and activation of abdominal peristalsis with nausea, diarrhoea and vomiting were the most prominent side effects. Of the twelve patients who exhibited a durable response to peginterferon and ribavirin treatment, three achieved an SVR, two achieved a week 12 SVR and four suffered a viral relapse. Three patients could not complete the assigned antiviral treatment with peginterferon alpha and ribavirin for nonvirological reasons. Short-term administration of high-dose intravenous silibinin might be an interesting approach to rescue patients with ongoing minimal residual viremia while on interferon-based therapy. These preliminary findings may stimulate further studies to evaluate more refined therapeutic strategies.

P3-S1.23 Mycoplasma genitalium in South African men with and without symptoms of urethritis: diagnosis and bacterial load

ObjectivesTo diagnose causes of urethritis in symptomatic men and evaluate the role of Mycoplasma genitalium by calculating bacterial loads in urine specimens from symptomatic and asymptomatic men.MethodsUrine specimens from 94...

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Our article1 reported that viral RNA could not be detected in the peripheral blood mononuclear cells (PBMCs) of aviremic seropositive blood donors, a conclusion supported by another recent article which also concluded that there was no evidence for persistent hepatitis C virus (HCV) infection in PBMCs after clearance of viremia in plasma.2 Decreasing or disappearing anti-HCV antibody titers in aviremic subjects also argue against continued HCV protein expression.3 Whether other reservoirs of HCV RNA, such as hepatocytes, exist in aviremic subjects remains unclear with some4-6 although not all7, 8 studies finding no HCV RNA in the liver of treated sustained viral responders. We clearly acknowledged1 that in chronically viremic subjects, PBMCs could be a site of HCV replication and that in vitro mitogen plus cytokine treatment might increase PBMC-associated HCV levels.8-10 With respect to the relative sensitivities of the HCV RNA assays used, we performed two different tests for HCV RNA in PBMCs (cell-associated transcription-mediated amplification [CA-TMA] and reverse transcription nested polymerase chain reaction [RT-nPCR]). We demonstrated a sensitivity of 2-50 HCV RNAs in 5 million PBMCs for the CA-TMA assay and 15-150 HCV RNAs in 5 million cells using RT-nPCR (adjusted from a sensitivity of 1-10 HCV RNAs for RT-nPCR, because only a fraction of total PBMC RNA and derived complementary DNA is sampled).1 Pham et al. report a sensitivity for their RT-nPCR-NAH (nucleic acid hybridization) assay of <10 viral genome equivalent (vge)/mL for serum (and in their letter, <5 vge/million PBMCs). We quantified the sensitivities of our assays using serial dilutions of plasma samples of known viral loads (prequantified using a commercial viral load assay) in the presence of total cellular RNA, as well as of total RNA from the PBMC of viremic subjects and showed the actual replicate assay results.1 Pham et al. used dilutions of a plasmid without interfering cellular nucleic acids for “semiquantitative estimation” in a “preliminary experiment” whose results where not shown.10 The fraction of total complementary DNA derived from PBMCs or 250 μL serum they used as input for their nPCR was not specified.10 With regard to the total number of cell equivalents analyzed in respective assays, we indeed analyzed only 0.16 million PBMCs by RT-nPCR but in parallel, and with consistent results, also used CA-TMA on 2.5 million PBMCs,1 slightly more than the 1 million to 2 million PBMCs Pham and Michalak reported in their letter. We agree with Pham and Michalak that difference in assay sensitivity is a likely explanation for the reported discrepancy but feel that the more relevant assay is that used for detecting HCV RNA in plasma. In our case, we used an extensively validated plasma HCV RNA assay used for blood-donation screening (Transcription mediated amplification, Procleix human immunodeficieny virus-1/HCV assay, Gen-probe, with a 95% detection sensitivity of 30 HCV RNA/mL).11-13 This TMA assay was used in duplicate assays (consuming a total of 1 mL plasma) to identify a total of 69 plasma aviremic blood donors, none of whom possessed detectable PBMC-associated HCV RNA.1 Pham et al. analyzed and found HCV RNA in the PBMCs of two aviremic subjects (so classified using their RT-nPCR-NAH).10 We suspect that further testing of the plasma from these two subjects using TMA assays might have detected a low-level viremia. Also, mostly consistent with our results, Pham et al. reported in 2008 detecting HCV RNA in only one of 10 PBMCs from aviremic subjects (although stimulation with interleukin-2 plus mitogen and testing of purified immune cell subsets lead to the detection of HCV RNA in all 10 PBMCs).14 Our selection criterion for the absence of plasma viremia (based on a highly sensitive, extensively validated assay used in duplicate tests to increase sensitivity) may therefore have been more stringent than that used by Pham et al. and account for the discrepant results seen in PBMCs. Ultimately, we agree with Pham and Michalak that the question of a residual HCV reservoir in the PBMCs of truly aviremic subjects will best be resolved using the demonstrably most sensitive detection assays for HCV RNA in both plasma and PBMC. The use of the same HCV RNA quantitation standards15 by different groups will help compare assay sensitivities. Eric Delwart Ph.D.*, * Blood Systems Research Institute, Department of Laboratory Medicine, University of California, San Francisco, San Francisco CA.