Adriana Vince

Hybrid Capture II HPV Test detects at least 15 human papillomavirus genotypes not included in its current high-risk probe cocktail

BACKGROUND Hybrid Capture II HPV Test (HCII) (Digene Corporation, Gaithersburg, MD) is a signal amplified hybridization microplate-based assay designated to detect 18 human papillomavirus (HPV) genotypes using two probe cocktails, for high-risk HPV genotypes 16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58, 59 and 68 and low-risk HPV genotypes 6, 11, 42, 43 and 44. At present, HCII is the only commercially available HPV assay with sufficient scientific data to support its performance in the clinical setting. OBJECTIVES To determine the specificity and accuracy of HCII high-risk probe cocktail for detection of 13 HPV genotypes included in the high-risk probe cocktail. STUDY DESIGN Cervical samples obtained from 325 women recognized as HPV positive using the HCII high-risk probe cocktail were included in the study. HPV genotypes were determined by restriction fragment analysis of PGMY09/PGMY11 polymerase chain reaction (PCR) products using seven restriction endonucleases. RESULTS A 450 bp fragment of HPV L1 gene was successfully amplified from 312 out of 325 samples. Of the 312 PCR-positive samples, 280 samples were associated with the expected high-risk HPV genotypes and 32 samples with the HPV genotypes not included in the HCII high-risk probe cocktail. Thus, HPV53 was detected in 8 samples, HPV66 in 4 samples, HPV54 in 3 samples, HPV6, HPV26, HPV70 each in 2 samples, and HPV11, HPV40, HPV42, HPV61, HPV73, HPV81, MM4, IS39, CP6108 each in 1 sample. In 2 samples, we were not able to determine the HPV genotype. CONCLUSIONS Our study shows that HCII high-risk probe cocktail detects at least 15 HPV genotypes not included in the current HCII high-risk probe cocktail. The potential impact of HCII high-risk probe cocktail cross-reactivity with phylogenetically related and unrelated HPV genotypes, including genotypes currently considered to be low-risk HPVs, remains to be determined.

LightCycler SeptiFast assay as a tool for the rapid diagnosis of sepsis in patients during antimicrobial therapy.

Rapid aetiological diagnosis and early administration of adequate antimicrobial therapy soon after a patient’s arrival at the hospital are crucial for the successful management of sepsis and septic shock (Dombrovskiy et al., 2007; Raghavan & Marik, 2006). Routine aetiological diagnosis of sepsis relies on standard culture methods that take at least 24 h or more to give the initial information. Therefore, quick identification of pathogens causing sepsis within the clinically relevant time frame should be based on state-of-the-art molecular methods that target bacterial and fungal DNA (Abdul-Redha et al., 2007; Breitkopf et al., 2005; Dombrovskiy et al., 2007). In this letter, we describe the clinical utility of the first standardized, CE-certified, multiplex real-time PCR assay for the molecular diagnosis of sepsis that has been approved for in vitro diagnostic use (LightCycler SeptiFast assay; Roche Diagnostics). The aim of our preliminary study was to determine the additional diagnostic value of the LightCycler SeptiFast assay, which enables detection of DNA from 25 human pathogens (Gram-positive and Gram-negative bacteria as well as fungi) in the blood of patients with suspected sepsis even after empirical antimicrobial therapy has been started. The study enrolled 36 patients (a total of 39 samples) with a clinical diagnosis of sepsis that were treated at the University Hospital for Infectious Diseases, Zagreb, and Zagreb University Clinical Center in Croatia. Seventeen patients were hospitalized in the intensive care unit (ICU), nine patients outside the ICU and ten patients in the Department of Haematology following bone marrow or peripheral blood stem cell transplantation (BSCT). All patients enrolled in the study were already receiving empirical antimicrobial therapy at the time of testing. Blood cultures were taken from all patients as well. Thirteen out of 39 (33 %) samples tested positive with the SeptiFast assay for bacterial or fungal DNA (Table 1). Gramnegative bacterial DNA was detected in 11 of 13 samples (Klebsiella pneumoniae/oxytoca n54; Escherichia coli n53; Pseudomonas aeruginosa n54). Gram-positive bacterial DNA (Enterococcus faecium n51; Streptococcus pneumoniae n51) was detected in two patients with polymicrobial sepsis (in combination with K. pneumoniae/ oxytoca in both patients). Aspergillus fumigatus DNA was detected in two patients with fever and pulmonary infiltrates. Additional SeptiFast-positive results (blood culture-negative) were obtained in nine of 36 patients. Four of 39 samples (10.3 %) were negative by the SeptiFast assay but positive by culture and those results were interpreted as false-negatives. Four of 39 samples were positive by both the SeptiFast assay and culture. Twenty-two samples tested negative by both techniques. We observed one discordant result between the SeptiFast assay and culture (Enterobacter cloacae was detected by culture and K. pneumoniae/oxytoca by SeptiFast assay). Incorrect interpretation of Enterobacter aerogenes/cloacae as K. pneumoniae/oxytoca due to the similarities of internal transcribed spacer regions of the target micro-organisms was also described by the manufacturer of the assay (4.2 % error rate in the evaluation study). The SeptiFast assay does not detect Actinobacillus sp. DNA, but this microorganism was detected in one patient by culture only. In the group of 10 haematological patients, SeptiFast results were positive for six of the 10 patients (60 %), whereas blood cultures were positive in only two out of 10 patients (20 %). The declared analytical sensitivity of the SeptiFast assay ranges between 30 and 100 c.f.u. (depending on the micro-organism). The SeptiFast assay provides a rapid identification of the causative microorganism within 6 h (3 h of technician hands-on time) whereas blood cultures usually require 2 days for Gram-staining results and a total of 3 days for the species to be identified. However, the use of the SeptiFast assay is limited to well-established molecular laboratories, requires excellent technical skills and is very expensive compared to culture. Additionally, the SeptiFast assay cannot provide information regarding the antibiotic susceptibility of micro-organisms. We conclude that the SeptiFast assay is a clinically valuable add-on to conventional culture methods for rapid aetiological diagnosis of sepsis in patients where empirical antimicrobial therapy has already been started and pretreatment blood cultures were negative. This assay showed particular sensitivity in haematological patients following BSCT,

DNA extraction from archival Giemsa-stained bone-marrow slides: comparison of six rapid methods.

The ability of six rapid DNA extraction procedures to provide DNA for the polymerase chain reaction from archival Giemsa‐stained bone marrow slides was tested on 120 samples. Boiling in distilled water, freeze–thaw method, boiling in 10% Chelex‐100 resin solution, proteinase K/Tween 20/NP‐40 method coupled with simplified phenol/chloroform/isoamyl alcohol protocol or salting‐out procedure using saturated NaCl and modification of commercial QIAamp procedure (Qiagen, Chatsworth, Calif.) gave DNA extraction efficiencies of 50%, 70%, 85%, 95%, 100% and 100%, respectively. Our results demonstrate that rough DNA extraction methods have decreased efficiencies compared to complete DNA extraction protocols and that the latter are required to ensure highly reproducible results from archival Giemsa‐stained bone marrow slides.

Rapid detection of enterovirus infection by automated RNA extraction and real-time fluorescence PCR

BACKGROUND Molecular detection has been shown to be superior to tissue culture for the detection of enteroviruses in cerebrospinal fluid (CSF) specimens. OBJECTIVES In this study, a qualitative molecular assay based on automated RNA extraction with the MagNA Pure LC and real-time PCR on the LightCycler (LC) instrument was evaluated and compared with an in-house molecular assay. STUDY DESIGN A total of 109 CSF specimens were investigated for the comparative study. The detection limit of the new molecular assay was determined with 10-fold dilutions of two enterovirus strains and with the Third European Union Concerted Action Enterovirus Proficiency Panel. RESULTS With the enterovirus strains, the detection limit of the LC assay was found to be 0.1 TCID(50) (50% tissue culture infective dose). When samples of the Third European Union Concerted Action Enterovirus Proficiency Panel were tested, both molecular assays gave identical results to the expected results, which were based upon the results of three reference laboratories using a total of four different molecular methods before distribution of the panel. When clinical specimens were tested, there was a correlation between the LC assay and the in-house assay in 105 of 109 cerebrospinal fluids. CONCLUSIONS The new molecular assay allows rapid detection of enterovirus RNA in CSF. It was found to be labor saving and showed sufficient sensitivity.

Prevalence of HBV genotypes in Central and Eastern Europe

The importance of hepatitis B virus (HBV) genotypes for disease progression and response to interferon‐alpha‐based treatment is well established. While almost all patients in the Mediterranean area are infected with HBV genotype D, HBV genotype A is dominant in Northern Europe. However, the distribution of HBV genotypes is unknown for several Central and Eastern European countries. Data are described of 1313 HBsAg‐positive patients recruited at 14 referral centers in eight countries. There were only very few cases of HBV genotype B, C, E, F, and H infection while HBV genotypes A and D were found in 42% and 48% of patients, respectively. Eight percent of patients had positive bands for more than one genotype using the hybridization assay. The frequency of genotype A was higher in Poland (77%) and the Czech Republic (67%) as compared to Hungary (47%), Lithuania (41%), Croatia (8%), and Germany (32%). In contrast, HBV genotype D was most frequent in Croatian, Romanian, and Russian patients with 80%, 67%, and 93% of cases, respectively. In conclusion, HBV genotype A versus D showed significantly different distribution patterns in Central and Eastern Europe which deserves consideration for national guidelines and treatment decisions. J. Med. Virol. 80:1707–1711, 2008.

Evaluation of the WHO clinical decision rule for streptococcal pharyngitis

Aims: To prospectively assess the WHO clinical decision rule (CDR) for group A beta haemolytic streptococcal (GABHS) pharyngitis in three countries. Methods: A prospective, observational cohort study in urban outpatient clinics in Rio de Janeiro, Cairo, and Zagreb. There were 2225 children aged 2–12 years with cough, rhinorrhoea, red or sore throat; 1810 of these with sore throat were included in the analysis. Results: The proportion of children presenting with sore throat and found to have GABHS pharyngitis ranged from 24.6% (Brazil) to 42.0% (Croatia). WHO CDR sensitivity was low for all sites in both age groups. In children age 5 or older, sensitivity ranged from 3.8% in Egypt to 10.8% in Brazil. In children under 5, sensitivity was low (0.0–4.6%) Specificity was high in both age groups in all countries (93.8–97.4%). Conclusions: In these populations, the current WHO CDR has high specificity, but low sensitivity; it did not detect up to 96.0% of children who have laboratory confirmed GABHS pharyngitis. A CDR with higher sensitivity should be developed for use in regions where rheumatic fever and rheumatic heart disease are still major health problems.

Chemokines CXCL10 and CXCL11 in the cerebrospinal fluid of patients with tick‐borne encephalitis

Objectives –  The aim of our study was to determine whether cerebrospinal fluid (CSF) of patients with tick‐borne encephalitis (TBE) contains CXCL10, CXCL11, p40 subunit of interleukin‐12 (IL‐12)/IL‐23, IL‐18 and IL‐15. We compared serum and CSF concentrations of CXCL10 and analysed the possible concentration gradient of this chemokine between the periphery and central nervous system.

Increased expression of CXCR3 and CCR5 on memory CD4+ T-cells migrating into the cerebrospinal fluid of patients with neuroborreliosis: The role of CXCL10 and CXCL11

The aim of this study was to evaluate the contribution of chemokine receptor CXCR3 and the corresponding ligands CXCL10 and CXCL11 to the recruitment of peripheral blood (PB) memory CD4+ T-cells into the cerebrospinal fluid (CSF) of patients with acute neuroborreliosis. Percentages of memory CD45RO+CD4+ T-cells expressing CXCR3 and CCR5 were significantly enriched in the CSF compared to the PB. Concentrations of CXCL10 and CXCL11 in the CSF of neuroborreliosis patients were significantly higher compared with the corresponding serum samples. Our results suggest that CXCL10 and CXCL11 create a chemokine gradient between the CSF and serum and recruite CXCR3-expressing memory CD4+ T-cells into the CSF of neuroborreliosis patients and that CCR5 also plays a role in this process.

Restrictions for reimbursement of interferon-free direct-acting antiviral drugs for HCV infection in Europe

All-oral direct-acting antiviral drugs (DAAs) for hepatitis C virus, which have response rates of 95% or more, represent a major clinical advance. However, the high list price of DAAs has led many governments to restrict their reimbursement. We reviewed the availability of, and national criteria for, interferon-free DAA reimbursement among countries in the European Union and European Economic Area, and Switzerland. Reimbursement documentation was reviewed between Nov 18, 2016, and Aug 1, 2017. Primary outcomes were fibrosis stage, drug or alcohol use, prescriber type, and HIV co-infection restrictions. Among the 35 European countries and jurisdictions included, the most commonly reimbursed DAA was ombitasvir, paritaprevir, and ritonavir, with dasabuvir, and with or without ribavirin (33 [94%] countries and jurisdictions). 16 (46%) countries and jurisdictions required patients to have fibrosis at stage F2 or higher, 29 (83%) had no listed restrictions based on drug or alcohol use, 33 (94%) required a specialist prescriber, and 34 (97%) had no additional restrictions for people co-infected with HIV and hepatitis C virus. These findings have implications for meeting WHO targets, with evidence of some countries not following the 2016 hepatitis C virus treatment guidelines by the European Association for the Study of Liver.

The utility of rapid antigen detection testing for the diagnosis of streptococcal pharyngitis in low-resource settings.

OBJECTIVES To evaluate the utility of rapid antigen detection testing (RADT) for the diagnosis of group A streptococcal (GAS) pharyngitis in pediatric outpatient clinics in four countries with varied socio-economic and geographic profiles. METHODS We prospectively evaluated the utility of a commercial RADT in children aged 2-12 years presenting with symptoms of pharyngitis to urban outpatient clinics in Brazil, Croatia, Egypt, and Latvia between August 2001 and December 2005. We compared the performance of the RADT to culture using diagnostic and agreement statistics, including sensitivity, specificity, and positive and negative predictive values. The Centor scores for GAS diagnosis were used to assess the potential effect of spectrum bias on RADT results. RESULTS Two thousand four hundred and seventy-two children were enrolled at four sites. The prevalence of GAS by throat culture varied by country (range 24.5-39.4%) and by RADT (range 23.9-41.8%). Compared to culture, RADT sensitivity ranged from 72.4% to 91.8% and specificity ranged from 85.7% to 96.4%. The positive predictive value ranged from 67.9% to 88.6% and negative predictive value ranged from 88.1% to 95.7%. CONCLUSIONS In limited-resource regions where microbiological diagnosis is not feasible or practical, RADTs should be considered an option that can be performed in a clinic and provide timely results.