Guy Vernet

Determination of anti–Toxoplasma gondii immunoglobulin G avidity: adaptation to the vidas system (bioMérieux)

The determination of specific anti-Toxoplasma gondii IgG avidity has been proposed to improve the determination of the date of toxoplasmic seroconversion in pregnant women. In this study, we adapted this serological technique to the Vidas system (bioMérieux) using 6 M urea as the dissociating agent. We studied 356 sera, including 42 sequential sera from sero-conversions in pregnant women. Our results show that the test is easy to use, and that an avidity index higher than 0.300 allows the exclusion of a recent infection acquired less than 4 months before serum sampling.

Molecular diagnostics in virology

n Abstractn n Molecular biology has significantly improved diagnosis in the field of clinical virology. Virus discovery and rapid implementation of diagnostic tests for newly discovered viruses has strongly beneficiated from the development of molecular techniques. Viral load and antiviral resistance or subtyping assays are now part of the biological monitoring of patients chronically infected by human immunodeficiency virus (HIV), hepatitis B virus (HBV), hepatitis C virus (HCV) and CMV. It will be important to add to this panel assays for other viruses of the herpesviridae family. Qualitative assays for the detection of blood-borne viruses have increased safety of blood donation and organ transplantation. Screening of other blood-borne viruses (parvovirus B19, HAV), multiplexing of detection and test automation to improve practicability and reduce costs will be the next steps. A major evolution in the near future will be the generalization of NAT for the diagnosis of viral etiology in patients, mostly with respiratory, CNS or gastro-intestinal diseases. Major technical improvements have been made to avoid obstacles that still limit this generalization, i.e. genetic variability of viruses, multiplex detection, contamination risk. Commercial offers already exist but menus must be extended to limit the validation and documentation work associated with home-brew assays. Real-time amplification has allowed the development of new NAT platforms but automation and integration of all steps of the reaction are still required to reduce hands-on-time, time-to-result and costs, and to increase throughput.n n

Species differentiation and antibiotic susceptibility testing with DNA microarrays

1. SUMMARYThere is a need for accurate and rapid species identification,strain typing and antibiotic susceptibility testing for severalbacterial genera that contain some of the major humanpathogens like Staphylococcus aureus or Mycobacterium tuber-culosis. Assays based on molecular biology technologies havebeen introduced in recent years and their clinical value is nowwell established, especially for the identification of thedifficult-to-grow bacteria, for the epidemiological character-ization of strains involved in nosocomial infections and for thedetectionofmutationsassociatedwithresistancetoantibiotics.Analysis with high density microarrays following multi-plex nucleic acid amplification allows for simultaneoustesting of specimens for suspected pathogens. Feasibilitystudies have been conducted on two assays for the Myco-bacterium and Staphylococcus genera, based on the photoli-thography DNA-Chip technology developed by Affymetrix.The Mycobacterium assay combines 16S ribosomal DNAidentification of 80 species, strain typing of species inside theM. tuberculosis (MTB) complex and detection of mutationslinked to resistance to rifampin. Spoligotyping can furtherdiscriminate between species inside the TB complex and, fora given species, perform strain typing. A total of 104rifampin mutations were detected in the rpoB gene ofM. tuberculosis. The assay is performed within a working dayon a single smear-positive sputum sample.The Staphylococcus assay combines 16S ribosomal DNAidentification of 34 species, typing of S. aureus strains bymulti-locus sequence typing and detection of genes ormutations linked to resistance to methicillin, fluoroquinolo-nes and aminoglycosides. The objective of this assay was toperform the multiplex analysis of these different targetswithin a working day starting from a single colony.Results obtained with these two prototype assays, andwith other similar techniques in the fields of clinical virologyor food industry testing, show that the microarray technol-ogy is very promising and will soon be part of the laboratorytools available to microbiologists.2. INTRODUCTIONDNA microarrays are expected to take an important place inthe microbiology laboratory. To date, reagents using DNAmicroarrays are mostly used in research laboratories, forexample to look at up- and down-regulated genes. Thisfundamental work yields a set of gene targets that can beused later on as diagnostic tools to routinely serve clinicianneeds. Only few if any DNA microarray are commercializedfor routine in vitro diagnosis of infectious diseases. DNAmicroarrays offer the possibility of multiplex parallelhybridization on DNA probes of nucleic acids eitherdirectly extracted from a specimen or, more often, generatedby an amplification step. They can be used for the initialdiagnosis of an infection when several pathogens can be thecause of a disease (panel diagnostic) or for the precisecharacterization of this pathogen, when it is required for amore accurate treatment management. Panel diagnosis can1. Summary, 592. Introduction, 593. Applications, 603.1 Species identification, 603.2 Strain typing, 623.3 Antibiotic resistance, 634. Procedures, 654.1 Tiling strategies using high-density DNA micro-arrays, 654.1.1 4L-tiling, 654.1.2 2L-tiling, 654.2 Spoligotyping of MTB species, 654.2.1 Reference spoligotyping, 654.2.2 Comparison of spoligotypes obtained by dot-blot and by the microarray with a strain ofM. bovis,654.3 Complete Mycobacterium assay, 665. Conclusions, 666. References, 67

High incidence of treatment-induced and vaccine-escape hepatitis B virus mutants among human immunodeficiency virus/hepatitis B–infected patients

Anti–hepatitis B virus (HBV) nucleos(t)ides analogs (NA) exert selective pressures on polymerase (pol) and surface (S) genes, inducing treatment resistance and increasing the risk of vaccine escape mutants. The rate of emergence for these mutations is largely unknown in patients coinfected with human immunodeficiency virus (HIV) and HBV undergoing dual‐active therapy. In a 3‐year, repeat‐sampling, prospective cohort study, HBV viral genome sequences of 171 HIV‐HBV coinfected patients, presenting with HBV viremia for at least one visit, were analyzed every 12 months via DNA chip. Logistic and Cox proportional hazard models were used to determine risk factors specifically for S gene mutations at baseline and during follow‐up, respectively. HBV‐DNA levels >190 IU/mL substantially decreased from 91.8% at inclusion to 40.3% at month 36 (Pu2009<u20090.001), while lamivudine (LAM) or emtricitabine (FTC) use remained steady (71.9%) and tenofovir (TDF) use expanded (month 0, 17.5%; month 36, 66.7%; Pu2009<u20090.001). The largest increase of any mutation class was observed in l‐nucleoside–associated pol gene/antiviral‐associated S gene mutations (cumulative incidence at the end of follow‐up, 17.5%) followed by alkyl phosphonate‐associated pol‐gene (7.4%), immune‐associated S gene (specifically any amino acid change at positions s120/s145, 6.4%), and d‐cyclopentane–associated pol‐gene mutations (2.4%). Incidence of l‐nucleoside–associated pol‐gene/antiviral–associated S gene mutations was significantly associated with concomitant LAM therapy (adjusted hazard ratio [HR], 4.61; 95% confidence interval [CI], 1.36‐15.56), but inversely associated with TDF use (adjusted HR/month, 0.94; 95% CI,0.89‐0.98). Cumulative duration of TDF was significantly associated with a reduction in the occurrence of immune‐associated S gene mutations (HR/month, 0.88; 95% CI, 0.79‐0.98). No major liver‐related complications (e.g., fulminant hepatitis, decompensated liver, and hepatocellular carcinoma) were observed in patients with incident mutations. Conclusion: Vaccine escape mutants selected by NA exposure were frequent and steadily increasing during follow‐up. Although the high antiviral potency of TDF can mitigate incident mutations, other antiviral options are limited in this respect. The public health implications of their transmission need to be addressed. (Hepatology 2013;53:912–922)

Use of molecular assays for the diagnosis of influenza

Respiratory infections are the third highest cause of death worldwide and influenza has the highest mortality rate among lower respiratory tract infections (LRTIs). Diagnosis of LRTIs relies mostly on clinical symptoms and is not fully satisfactory. Influenza laboratory diagnosis improves the efficiency of prophylaxis or treatment of influenza by antiviral molecules and has a strong impact on the cost–effectiveness of curative treatment. Inappropriate treatment of patients may result in spreading of resistant strains. Molecular diagnostics play a central role in the surveillance and response of pandemic influenza due to highly pathogenic strains. Real-time assays can be used for diagnosis or surveillance purposes in humans and animals, and microarrays can be used to identify and monitor the spread of dangerous variants. Molecular assays are also useful to identify and distinguish influenza, other respiratory viruses and bacteria, although their cost–effectiveness must be proven on a large scale. As new antiviral options will be available to clinicians, a better treatment choice will benefit the patient and community. Recent progress in molecular techniques will be reviewed. Examples of real-time assays for the detection of influenza viruses, including the highly pathogenic influenza A strains H5N1 and H7N7, will be discussed. Promising new techniques that allow detailed genotyping of viruses or multiplex detection of several respiratory pathogens from a unique specimen will also be discussed. These techniques will, in the near future, significantly improve the quality of diagnosis and surveillance of respiratory pathogens.

Characterization of 26 isolates of Staphylococcus aureus, predominantly from dairy sheep, using four different techniques of molecular epidemiology.

Little information is available regarding the molecular epidemiology of Staphylococcus aureus–induced mastitis in dairy sheep. In this study, 4 different typing techniques were compared in typing 26 S. aureus isolates, predominantly from cases of subclinical mastitis in dairy ewes. The 4 techniques were pulsed-field gel electrophoresis (PFGE), restriction fragment length polymorphism (RFLP) on 2 genes (coagulase and clumping factor B), randomly amplified polymorphic DNA–polymerase chain reaction (PCR) (RAPD-PCR), and multilocus sequence typing (MLST). On the basis of discriminatory power as the key parameter of typing systems, MLST and PFGE were found to be the most powerful techniques. The MLST and PFGE could contribute to epidemiological surveillance and evaluation of mastitis control programs, by documenting prevalence and dissemination of endemic clones in infected populations. The results of this study show that a single clone of S. aureus is widely distributed in infected ewe mammary glands.

Diagnosis of zoonotic viral encephalitis.

Human infections by zoonotic encephalitis viruses are usually asymptomatic or symptoms are not specific to these viruses. Some of them have high mortality and morbidity rates and most often no specific treatment exist. This emphasizes the need for a precise identification of arboviruses in clinical specimens from humans and animals. Because these diseases are frequent in developing countries and tend to emerge or re-emerge in others, diagnostic tools must detect the broadest possible range of viruses with a high sensitivity and this is a key factor for surveillance, control of transmission and prevention through vaccination. In countries with limited diagnostic infrastructures, low-cost and easy-to-use tests are required. The diagnosis of arboviral encephalitis has been significantly improved in the recent years. Sensitive ELISA assay to detect antibodies against many arboviruses in serum or CSF are commercially available and can be used to detect early infections. Immunochromatographic rapid tests for the detection of specific IgM that could be used on fingertip blood would be valuable tools in developing countries. A limitation of these serologic assays is their lack of specificity as many arboviruses are antigenically related. Virus isolation or molecular assays from different human or animal tissues are also important diagnostic tools. Molecular assays have been extensively described in the recent years. They are very sensitive and have the advantage over cell culture that specimen transportation is less critical. Real-time detection has even improved sensitivity and reduced time-to-result. Although the utility of molecular assays for the detection of arboviruses in mosquito pools has been demonstrated, an extensive validation of their pertinence in clinical settings is still required. The use of DNA-microarrays may further extend the range of viruses that can be detected in a single test and allow isolates typing for epidemiology purposes.