Genotypic Tropism Testing in HIV-1 Proviral DNA Can Provide Useful Information at Low-Level Viremia

The possibility of performing genotypic tropism testing (GTT) with proviral DNA (pvDNA) even during suppressed viremia would facilitate the use of CCR5 inhibitors as part of switching, simplification, or intensification strategies. Thus, we aimed to evaluate the tropism concordance between plasma RNA and pvDNA samples and to assess which factors could affect possible discrepancies between the two compartments. GTT was performed using both plasma RNA and pvDNA from 55 sample pairs from drug-experienced patients. Potential differences between the two compartments were evaluated by analyzing coreceptor usage and genetic variability. Paired samples were also stratified in three levels of viremia (<50, 51 to 500, and >500 copies/ml). Overall, Geno2Pheno comparisons of false-positive rates in the two compartments showed good correlation (r = 0.72). A high level of concordance in tropism predictions for the two compartments was found (46/55 sample pairs [83.6%]). Among the 9 sample pairs with discordant tropisms, a larger proportion of pvDNA samples harboring CXCR4/dual-mixed-tropic viruses was found, in comparison with plasma RNA samples (88.9% versus 11.1%; P = 0.0034). Discordant samples were characterized by greater genetic variability than were concordant samples. With stratification of the paired samples according to viremia levels, the prevalence of discordant samples decreased with increasing viremia (<50 copies/ml, 21.4%; 51 to 500 copies/ml, 15.4%; >500 copies/ml, 6.7%; P = 0.2). Our findings confirm that prediction of viral tropism using pvDNA is feasible even in low-level viremia and provides useful information for therapy optimization for patients with low or suppressed viremia.     

Performance of Genotypic Algorithms for Predicting HIV-1 Tropism Measured against the Enhanced-Sensitivity Trofile Coreceptor Tropism Assay

The objectives of this study were to assess the performance of genotypic algorithms for predicting CXCR4-using virus, with enhanced sensitivity Trofile HIV coreceptor tropism assay (ES Trofile) as the reference, and to compare the concordance/accuracy of genotypic tests with ES Trofile and with the original Trofile assay. Paired phenotypic and genotypic determinations of HIV-1 coreceptor usage were compared in plasma samples from HIV-1-infected patients. Sequencing of the third hypervariable (V3) loop of the viral gene and phenotypic assays were performed for each sample. Genotypic rules used to predict tropism were Geno2pheno (false-positive rate at 1 to 20%), position-specific scoring matrix X4R5 (PSSM_X4R5) and PSSM_sinsi (where “sinsi” stands for syncytium inducing and non-syncytium inducing), and the 11/25, 11/24/25, and net charge rules. Two hundred forty-four phenotypic and genotypic samples were tested. Coreceptor usage was obtained from ES Trofile for 145 (59%) samples and from Trofile for 99 (41%) samples. The highest concordance (82.6%) was obtained with PSSM_X4R5 when ES Trofile was used as the reference. Geno2pheno at a 20% false-positive rate showed the highest sensitivity (76.7%) for CXCR4-using virus detection with ES Trofile. Samples from naïve subjects and those with CD4 cell counts between 200 and 500 cells/mm³ showed the best predictive performance. Overall, the accuracy of the bioinformatics tools to detect CXCR4-using virus was similar for ES Trofile and Trofile; however, the negative predictive values for genotypic tools with ES Trofile were slightly higher than they were with Trofile. The accuracy of genotypic algorithms for detecting CXCR4-using viruses is high when using ES Trofile as the reference. Results are similar to those obtained with Trofile. The concordance with ES Trofile is better with higher CD4 cell counts and nonexposure to antiretroviral therapy.The determination of HIV-1 tropism is now of clinical interest because the chemokine coreceptors CCR5 and CXCR4 are targets for drugs that block HIV-1 entry. Maraviroc, the first CCR5 antagonist approved for clinical use, specifically inhibits the replication of R5-tropic HIV-1 variants; therefore, viral tropism testing is mandatory before using this drug. Several assays have been developed to determine HIV-1 coreceptor usage (1, 10, 18). Phenotypic assays using either HIV primary isolates or recombinant viruses are considered the gold standard for HIV-1 tropism assessment. Among them, the assay from Trofile (Monogram Biosciences, South San Francisco, CA) is the only clinically proven, commercially available diagnostic test to determine HIV-1 coreceptor usage and therefore the most widely used phenotypic test worldwide. In spite of their accuracy, phenotypic methodologies have the inconvenience of their complexity, expensiveness, and the requirement of special facilities and expertise, which makes them unfeasible to be used as a routine part of clinical diagnosis. An alternative method for tropism determination consists of the genotypic prediction of HIV-1 coreceptor usage through bioinformatics tools based on third hypervariable (V3) loop viral sequences. These genotypic methods have demonstrated good correlation with phenotypic tests, including the Trofile assay, in different studies (5, 8, 14, 16, 19), and preliminary data from prospective clinical studies suggest that they may predict clinical response to maraviroc (12, 21, 24). However, a number of factors are thought to reduce the ability of genotyping to predict HIV-1 tropism, including the presence of minority CXCR4-using variants (9, 16).Because of the low sensitivity of the original Trofile assay to detect minority CXCR4 variants when present, an enhanced version (ES Trofile) has been launched by Monogram Biosciences that has significantly improved the ability to identify low levels of CXCR4-using variants, allowing a 30-fold increase in analytical sensitivity for detecting CXCR4-using variants in env clone mixtures (23). This new test constitutes the current gold standard for tropism determination and has replaced the original version of Trofile, which is currently not available. To date, the performance of genotypic algorithms for the prediction of HIV-1 tropism using ES Trofile as a reference has not yet been explored. The objectives of this study were to evaluate the accuracy of genotypic algorithms for detecting CXCR4-using virus when measured against ES Trofile and to compare the concordance/accuracy of genotypic tests with ES Trofile and with the original Trofile assay.(This work was accepted as a late breaker in the 12th European AIDS Conference. 11 to 14 November 2009, Cologne, Germany [abstract LBPE1.2/10].)     

Evaluation of the Liaison Automated Testing System for Diagnosis of Congenital Toxoplasmosis

Congenital toxoplasmosis is a worldwide health problem, and different screening strategies exist. Testing of toxoplasma-specific antibodies in infants identifies congenital toxoplasmosis during the first year of life. However, experience with commercial available immunoassays is limited. The aim of this study was to evaluate both the performance and analytical characteristics of the Liaison diagnostic system in infants. In a retrospective study, serum Toxoplasma gondii antibodies were measured in samples from 333 infants, including 212 noninfected infants and 121 infants with congenital toxoplasmosis. A total of 1,157 umbilical cord blood and peripheral serum samples were analyzed. Liaison toxoplasma-specific IgG and IgM antibodies and the IgG avidity index were compared to the infection status of the infant, determined by the Sabin-Feldman dye test and immunosorbent agglutination assay—IgM. All noninfected infants were seronegative by Liaison IgG within the first year of life. The Liaison system showed a sensitivity of 81.8%, a specificity of 100.0%, a positive predictive value of 100.0%, a negative predictive value of 90.6%, and overall agreement of 84.4% by comparison with the dye test. Overall agreement of both IgM test systems was 96.0%. In this study cohort, avidity did not show a potential diagnostic benefit for the detection of congenital infection. In conclusion, the Liaison system is a valuable tool to monitor the serologic course of infants at risk. A final serologic confirmatory test is recommended to improve the rate of detection of congenital toxoplasmosis at 1 year of life. Protocols of routine follow-up testing in infants and accurate diagnostic tools after acute gestational infections are needed to improve medical care.     

Results of External Quality Assessment for Proviral DNA Testing of HIV Tropism in the Maraviroc Switch Collaborative Study

The Maraviroc Switch collaborative study (MARCH) is a study in aviremic patients on stable antiretroviral therapy and utilizes population-based sequencing of proviral DNA to determine HIV tropism and susceptibility to maraviroc. An external quality assessment (EQA) program was implemented to ensure competency in assessing the tropism of clinical samples conducted by MARCH laboratories (n = 14). The MARCH EQA has three prestudy phases assessing V3 loop sequencing and tropism determination using the bioinformatic algorithm geno2pheno, which generates a false-positive rate (FPR). DNA sequences with low FPRs are more likely to be from CXCR4-using (X4) viruses. Phase 1 of the EQA involved chromatogram interpretation. Phases 2, 2/3, and 3 involved patient and clonal samples. Clinical samples used in these phases were from treatment-experienced HIV-infected volunteers; 18/20 had viral loads of <50 copies/ml, and 10/15 were CXCR4-tropic on prior phenotyping. All samples were tested in triplicate, and any replicate with a geno2pheno FPR of <10% was designated X4. Performance was deemed adequate if ≤2 R5 and ≤1 X4 specimens were miscalled. For several clinical samples in the EQA, triplicate testing revealed marked DNA variability (FPR range, 0 to 96.7%). Therefore, a consensus-based approach was employed for each sample, i.e., a median FPR across laboratories was used to define sample tropism. Further sequencing analysis showed mixed viral populations in the clinical samples, explaining the differences in tropism predictions. All laboratories passed the EQA after achieving predefined competence thresholds in either of the phase 2 rounds. The use of clinical samples from patients resembling those who were likely to be screened in the MARCH, coupled with triplicate testing, revealed inherent DNA variability that might have been missed if single or duplicate testing and/or clonal samples alone were used. These data highlight the importance of intensive EQA of tropism laboratories before embarking on clinical studies. (This study has been registered at ClinicalTrials.gov under registration no. {"type":"clinical-trial","attrs":{"text":"NCT01384682","term_id":"NCT01384682"}}NCT01384682 [http://www.clinicaltrials.gov/ct2/show/study/{"type":"clinical-trial","attrs":{"text":"NCT01384682","term_id":"NCT01384682"}}NCT01384682?term={"type":"clinical-trial","attrs":{"text":"NCT01384682","term_id":"NCT01384682"}}NCT01384682&rank=1].)     

Genotypic Determination of HIV Tropism in a Cohort of Patients Perinatally Infected With HIV-1 and Exposed to Antiretroviral Therapy

The aim of this study was to determine the coreceptor tropism by performing genotypic HIV-1 tropism testing in a cohort of patients perinatally infected with HIV-1 and exposed to antiretroviral therapy. Genotypic coreceptor tropism was determined in patients with HIV-1 RNA<100 copies/mL using PBMC samples by gp120 V3 sequencing followed by geno2pheno interpretation (set at a false positive rate [FPR] of 20%) and in patients with >100 copies/mL using plasma samples (set at a FPR of 20%), according to European guidelines. Out of 55 patients, 50 had an HIV-1 subtype B strain, and mean (SD) age was 18.2 (4.6) years. The median duration of antiretroviral therapy was 13 years (range, 3–23). Thirty-three (60%) patients harbored the R5 virus. At the time of the testing, the median CD4+ T lymphocyte cell count and percentage were 705 cells/mm³ (474–905) and 32.5% in group R5 and 626 cells/mm³ (450–755) and 31.7% in group X4/D-M, respectively. The nadir of CD4+ T-cell count in groups R5 and X4/D-M were 322 cells/mm³ (230–427) and 340 cells/mm³ (242–356), respectively. These differences were not statistically significant. Fifteen patients had HIV-1 RNA >50 copies/mL. The median HIV-1 RNA and HIV-1 DNA were comparable in both groups without a statistical difference. The study provides an overview of the prevalence of coreceptor tropism in a cohort of patients who were vertically infected with HIV-1. The high prevalence of X4/D-M-tropic strains may simply reflect the long-term exposure to HIV.     

Evaluation of Vancomycin Susceptibility Testing for Methicillin-Resistant Staphylococcus aureus: Comparison of Etest and Three Automated Testing Methods

We evaluated the ability of four commercial MIC testing systems (MicroScan, Vitek 2, Phoenix, and Etest) to detect vancomycin MIC values of ≤1 to ≥2 in 200 methicillin-resistant Staphylococcus aureus (MRSA) strains compared to the Clinical and Laboratory Standards Institute broth microdilution (BMD) reference methods. Compared to the BMD method, absolute agreement (0 ± dilution) was highest for the Phoenix system (66.2%) and the MicroScan turbidity method (61.8%), followed by the Vitek 2 system (54.3%). The Etest produced MIC values 1 to 2 dilutions higher than those produced by the BMD method (36.7% agreement). Of interest, the MicroScan system (prompt method) was more likely to overcall an MIC value of 1 mg/liter (74.1%), whereas the Phoenix (76%) and Vitek 2 (20%) systems had a tendency to undercall an MIC of 2 mg/liter. The ability to correctly identify vancomycin MIC values of 1 and 2 has clinical implications and requires further evaluation.     

Clinical Evaluation of the Automated COBAS AMPLICOR MTB Assay for Testing Respiratory and Nonrespiratory Specimens

We evaluated the COBAS AMPLICOR PCR system (Roche Diagnostics) for the routine detection of Mycobacterium tuberculosis complex (MTBC) in clinical specimens. Diagnostic culture, considered as the reference method, was performed with BACTEC, Löwenstein-Jensen, Stonebrink, and Kirchner media. Occasionally MB-Redox, ESP, or MGIT medium was also used. A total of 643 respiratory and 506 nonrespiratory specimens collected from 807 patients were investigated. Of the 95 culture-positive specimens, 80 were COBAS AMPLICOR MTB positive, and of the 1,054 culture-negative specimens, 1,044 were COBAS AMPLICOR MTB negative. After resolving discrepancies by review of the medical history, the overall sensitivity, specificity, and positive and negative predictive values for the COBAS AMPLICOR MTB assay, respectively, were 83.5, 98.8, 86.7, and 98.6% compared to those of diagnostic culture. In smear-positive specimens, the sensitivity of the COBAS AMPLICOR MTB assay was 96%, versus 48% for smear-negative specimens. No significant differences in the test performance between respiratory and nonrespiratory specimens were observed. The overall inhibition rate was less than 2%, excluding stool specimens. The clear advantages of the COBAS AMPLICOR PCR system are standardized procedures and reagents for specimen processing as well as an internal control for reliable monitoring of PCR inhibitors. By simplifying the work flow through a completely automated amplification and amplicon detection procedure, the COBAS AMPLICOR PCR system proved itself as a very useful component for routine diagnostic procedures.     

Comparative Study on Genotypic and Phenotypic Second-Line Drug Resistance Testing of Mycobacterium tuberculosis Complex Isolates

The mycobacterium growth indicator tube (MGIT960) automated liquid medium testing method is becoming the international gold standard for second-line drug susceptibility testing of multidrug- and extensively drug-resistant Mycobacterium tuberculosis complex isolates. We performed a comparative study of the current gold standard in the Netherlands, the Middlebrook 7H10 agar dilution method, the MGIT960 system, and the GenoType MTBDRsl genotypic method for rapid screening of aminoglycoside and fluoroquinolone resistance. We selected 28 clinical multidrug- and extensively drug-resistant M. tuberculosis complex strains and M. tuberculosis H37Rv. We included amikacin, capreomycin, moxifloxacin, prothionamide, clofazimine, linezolid, and rifabutin in the phenotypic test panels. For prothionamide and moxifloxacin, the various proposed breakpoint concentrations were tested by using the MGIT960 method. The MGIT960 method yielded results 10 days faster than the agar dilution method. For amikacin, capreomycin, linezolid, and rifabutin, results obtained by all methods were fully concordant. Applying a breakpoint of 0.5 μg/ml for moxifloxacin led to results concordant with those of both the agar dilution method and the genotypic method. For prothionamide, concordance was noted only at the lowest and highest MICs. The phenotypic methods yielded largely identical results, except for those for prothionamide. Our study supports the following breakpoints for the MGIT960 method: 1 μg/ml for amikacin, linezolid, and clofazimine, 0.5 μg/ml for moxifloxacin and rifabutin, and 2.5 μg/ml for capreomycin. No breakpoint was previously proposed for clofazimine. For prothionamide, a division into susceptible, intermediate, and resistant seems warranted, although the boundaries require additional study. The genotypic assay proved a reliable and rapid method for predicting aminoglycoside and fluoroquinolone resistance.The emergence of multidrug-resistant tuberculosis (MDR-TB) in the 1990s, and more recently, extensively drug resistant tuberculosis (XDR-TB), has revealed the need for new drugs and alternative, second-line treatment regimens. Many of these second-line drugs are either old drugs that had not been frequently used because of side effects or unproven efficacy or newer drugs intended primarily for treatment of other infections (3). Their use necessitated an evaluation of drug susceptibility testing (DST) and a determination of the critical concentrations of these alternative drugs.A variety of techniques are now available for second-line DST, of which the mycobacterium growth indicator tube automated liquid culture system (MGIT960) is probably the most used and best validated at this moment (13). The latest addition to second-line DST are genotypic methods, which detect mutations in the gyrA gene of Mycobacterium tuberculosis that are associated with fluoroquinolone resistance and mutations in the rrs operon that are associated with resistance to capreomycin and the aminoglycosides (2-5).Despite the arrival of these novel tools, many uncertainties remain. Not all methods have been evaluated in comparative studies. Moreover, the critical concentrations for resistance to several second- and third-line drugs, including moxifloxacin and prothionamide, remain the subject of debate (7, 11, 13).In the Netherlands, the Middlebrook 7H10 agar dilution method has been used for second-line DST for 2 decades (18). In order to comply with international standardization requirements, a switch to the MGIT960 method has been initiated.In this study, we have compared the results of the MGIT960 method and the GenoType MTBDRsl assay, a commercially available genotypic second-line DST method, to our reference method, the Middlebrook 7H10 agar dilution method. For the MGIT960 method, we tested the various critical concentrations published for prothionamide and moxifloxacin but also included clofazimine in our drug panel. For the latter drug, which has become increasingly important in the treatment of MDR and XDR-TB, no in vitro DST data for the MGIT960 method were available.     

骨髓间充质干细胞对脑胶质瘤的趋瘤效应

背景：骨髓间充质干细胞在体内、外均具有较强的向胶质瘤趋向性迁移的能力,可作为基因靶向胶质瘤治疗的理想载体。 目的：在复习相关文献的基础上,探讨骨髓间充质干细胞向脑胶质瘤定向迁移的机制。 方法：由第一作者对CNKI和PUBMED数据库进行检索,英文关键词为“mesenchymal stem cells,bone marrow,glioma”,中文检索词为“间充质干细胞,骨髓,胶质瘤”。共检索到51篇相关文献,根据入选标准最终剩余27篇文章进行归纳总结。 结果与结论：骨髓间充质干细胞对胶质瘤具有较强的趋化性迁移的能力,这与胶质瘤细胞分泌的某些细胞因子和骨髓间充质干细胞表面相应受体之间的相互作用有关。增强骨髓间充质干细胞表面相关受体的表达有助于增强其趋瘤效应,进而增强抗胶质瘤作用。     

Automating HIV drug resistance genotyping with RECall, a freely accessible sequence analysis tool

Genotypic HIV drug resistance testing is routinely used to guide clinical decisions. While genotyping methods can be standardized, a slow, labor-intensive, and subjective manual sequence interpretation step is required. We therefore performed external validation of our custom software RECall, a fully automated sequence analysis pipeline. HIV-1 drug resistance genotyping was performed on 981 clinical samples at the Stanford Diagnostic Virology Laboratory. Sequencing trace files were first interpreted manually by a laboratory technician and subsequently reanalyzed by RECall, without intervention. The relative performances of the two methods were assessed by determination of the concordance of nucleotide base calls, identification of key resistance-associated substitutions, and HIV drug resistance susceptibility scoring by the Stanford Sierra algorithm. RECall is freely available at http://pssm.cfenet.ubc.ca. In total, 875 of 981 sequences were analyzed by both human and RECall interpretation. RECall analysis required minimal hands-on time and resulted in a 25-fold improvement in processing speed (～150 technician-hours versus ～6 computation-hours). Excellent concordance was obtained between human and automated RECall interpretation (99.7% agreement for >1,000,000 bases compared). Nearly all discordances (99.4%) were due to nucleotide mixtures being called by one method but not the other. Similarly, 98.6% of key antiretroviral resistance-associated mutations observed were identified by both methods, resulting in 98.5% concordance of resistance susceptibility interpretations. This automated sequence analysis tool provides both standardization of analysis and a significant improvement in data workflow. The time-consuming, error-prone, and dreadfully boring manual sequence analysis step is replaced with a fully automated system without compromising the accuracy of reported HIV drug resistance data.     

The Effective Method for Investigation Meridian Tropism Theory in Rats

This present work describes an effective new method for study traditional Chinese medicine (TCM) on meridian tropism (MT) theory, which plays an essential role in clinical selection of TCM according to syndromes and strengthens the therapeutic effects. The new thread included material basis foundation and its tissue distribution study. Xiheliu, the most popular TCM on heart tropism, was investigated by simple and accurate high performance liquid chromatography (HPLC) method. The analysis of plasma after oral administration the total flavonoid of Xiheliu (TFX) exhibited that tamarixetin and kaempferide had the highest concentration and approximately the highest level within 25 min. The mixture of them could last accelerating the urine excretion more than 7 h after a single dose and could not cause the disorder of ion in rats, which was observed in diuretic activity experiment. In view of the reported biological activities was consistent with the effects of Xiheliu, tamarixetin and kaempferide were likely to be the material basis of it. Tissue distribution study showed that the highest level of analytes was in heart, lung, kidney and liver, and most tissues reached maximum level at 30 min post-dose. Since liver was the most important blood-supply tissue, the result of this experiment was in accordance with the MT record of Xiheliu and confirmed that tamarixetin and kaempferide was the material bases of it on MT. This is the first report for the illumination of material basis and the mechanism of Xiheliu on MT by analysis the record of Xiheliu in Compendium of Materia Medica and experimental study.     

Multicenter Evaluation of Fully Automated BACTEC Mycobacteria Growth Indicator Tube 960 System for Susceptibility Testing of Mycobacterium tuberculosis

The reliability of the BACTEC Mycobacteria Growth Indicator Tube (MGIT) 960 system for testing of Mycobacterium tuberculosis susceptibility to the three front-line drugs (isoniazid [INH], rifampin [RIF], and ethambutol [EMB]) plus streptomycin (STR) was compared to that of the BACTEC 460 TB system. The proportion method was used to resolve discrepant results by an independent arbiter. One hundred and ten strains were tested with an overall agreement of 93.5%. Discrepant results were obtained for seven strains (6.4%) with INH (resistant by BACTEC MGIT 960; susceptible by BACTEC 460 TB), for one strain (0.9%) with RIF (resistant by BACTEC MGIT 960; susceptible by BACTEC 460 TB), for seven strains (6.4%) with EMB (six resistant by BACTEC MGIT 960 and susceptible by BACTEC 460 TB; one susceptible by BACTEC MGIT 960 and resistant by BACTEC 460 TB), and for 19 strains (17.3%) with STR (resistant by BACTEC MGIT 960 and susceptible by BACTEC 460 TB). After resolution of discrepant results, the sensitivity of the BACTEC MGIT 960 system was 100% for all four drugs and specificity ranged from 89.8% for STR to 100% for RIF. Turnaround times were 4.6 to 11.7 days (median, 6.5 days) for BACTEC MGIT 960 and 4.0 to 10.0 days (median, 7.0 days) for BACTEC 460 TB. These data demonstrate that the fully automated and nonradiometric BACTEC MGIT 960 system is an accurate method for rapid susceptibility testing of M. tuberculosis.     

Gag–Pol Region Determines the Tropism of SIVagm for Human Cells

Simian immunodeficiency virus isolated from African green monkeys (SIVagm) does not grow in many of human cell lines such as CEM×174, H9, and MT-4, but could replicate in some human cell lines. Sequence of SIVagm responsible for its narrow host range was determined by making and monitoring growth potential of chimeric clones between SIVagm and human immunodeficiency virus type 1 (HIV-1). The results obtained indicated that the gag–pol region determines the observed narrow host range. By monitoring virus DNA synthesis and progeny virion production, the defect(s) of SIVagm in the replication in the restricted cells was demonstrated to be located at early phase. This revised version was published online in August 2006 with corrections to the Cover Date.     

Meropenem Resistance in Imipenem-Susceptible Meropenem-Resistant Klebsiella pneumoniae Isolates Not Detected by Rapid Automated Testing Systems

Klebsiella pneumoniae showing high resistance to all β-lactams except imipenem, designated as ISMRK (imipenem-susceptible meropenem-resistant Klebsiella) is emerging in Japan. The carbapenem resistance of ISMRK cannot be screened by the Vitek and the RAISUS rapid automated susceptibility test systems, which may lead to inappropriate antimicrobial therapy, resulting in compromised patient outcomes.