A high resolution melting (HRM) technology-based assay for cost-efficient clinical detection and genotyping of herpes simplex virus (HSV)-1 and HSV-2.

Genital herpes can be caused by two very similar viruses, herpes simplex virus (HSV)-1 or HSV-2. These two HSV types cannot be distinguished clinically, but genotyping is recommended in the first-episodes of genital herpes to guide counselling and management. Quantitative polymerase chain reaction (qPCR) is the preferred diagnostic method for HSV typing. However, commercial qPCR methods use expensive fluorescent labeled probes for detection. Furthermore, most low-cost methods are not able to differentiate between HSV-1 and -2. The aim of this study was to develop a high resolution melting (HRM) technology-based assay for sensitive HSV-1 and HSV-2 detection and genotyping. Using a panel of 46 clinical specimens, the performance of the HRM assay was compared to two commercial HSV tests: the HRM assay detected HSV in all 23 positive samples, with no false positive results (100% concordance with HSV I/II Real-TM assay). Additionally, the HRM assay correctly genotyped both HSV types in a subset of these clinical samples, as determined by the Realstar HSV PCR Kit. The HSV HRM assay provides a cost-effective alternative method to conventional more expensive assays and can be used in routine clinical specimens, in cases where it is particularly necessary to detect and distinguish HSV-1 from -2.

Evaluation of a standardised real-time PCR based DNA-detection method (Realstar®) in whole blood for the diagnosis of primary human cytomegalovirus (CMV) infections in immunocompetent patients.

Cytomegalovirus (CMV) DNA detection in blood could, as a supplementary test to serology, improve the accuracy and speed of diagnosis of an acute CMV infection. In this study we evaluated the performance of a commercially available and standardised CMV PCR assay in whole blood for the diagnosis of a primary infection in immunocompetent adults. Moreover, the kinetics of viral DNA was evaluated in order to provide a time frame in which viral DNA could be detected during an acute primary infection. Whole blood samples were collected from 66 patients with an acute CMV infection, 65 patients with an acute Epstein-Barr virus infection, 27 patients with various other acute infections (parvovirus B19, HIV, Toxoplasma gondii), 20 patients with past CMV infections (>1 year) and 20 apparently healthy persons. For CMV DNA detection and quantification a commercially available real-time PCR was applied (RealStar®, altona Diagnostics). The clinical sensitivity of CMV PCR in whole blood for the diagnosis of a recent primary CMV infection was 93.9 % and the diagnostic specificity 99.2 %. In the majority of the patients CMV DNA was not detectable anymore approximately within 4 weeks after the first blood sample was taken. From these data we concluded that, together with a suggestive serological profile, a positive CMV PCR result in whole blood can be regarded as a diagnostic confirmation of a recent CMV infection on a single blood sample in an immunocompetent patient. However, a negative CMV PCR result does not exclude a recent CMV infection.

Evaluation of different confirmatory algorithms using seven treponemal tests on Architect Syphilis TP-positive/RPR-negative sera.

The Architect Syphilis TP is considered to be a suitable screening test due to its high sensitivity and full automation. According to the International Union against Sexually Transmitted Infections (IUSTI) 2014 guidelines, however, positive screening tests need confirmation with Treponema pallidum particle agglutination (TP.PA). Among Architect-positive results, samples with a negative non-treponemal test present the major diagnostic challenge. In this multicenter study, we investigated if other, preferable less labor-intensive treponemal tests could replace TP.PA. A total of 178 rapid plasma reagin (RPR)-negative sera with an Architect value between 1 and 15 S/CO were prospectively selected in three centers. These sera were analyzed with TP.PA and six alternative treponemal tests: three immunoblots and three tests on random-access analyzers. The diagnostic performance of the treponemal tests differed substantially, with the overall agreement between the six alternative tests ranging from 44.6 to 82.0%. Based on TP.PA as the gold standard, the INNO-LIA IgG blot, the BioPlex 2200 IgG, and the Syphilis TPA showed a high sensitivity, while the EUROLINE-WB IgG blot, recomLine Treponema IgG blot, and the Chorus Syphilis screen showed a high specificity. However, an Architect cut-off of 5.6 S/CO can serve as an alternative for these confirmatory treponemal tests in case of an RPR-negative result. Treponemal tests show poor agreement in this challenging group of Architect-positive/RPR-negative sera. The most optimal algorithm is obtained by assigning sera with an Architect value >5.6 S/CO as true-positives and sera with a value between 1 and 5.6 S/CO as undetermined, requiring further testing with TP.PA.

Assessment of plasma anti-elastin antibodies for use as a diagnostic aid for chronic progressive lymphoedema in Belgian Draught Horses.

Diagnosis of chronic progressive lymphoedema (CPL) in draught horses, including the Belgian Draught Horse, is mainly based on clinical evaluation of typical lower limb lesions. A deficient perilymphatic elastic support, caused by a pathological elastin degradation in skin and subcutis, has been suggested as a contributing factor for CPL. Elastin degradation products induce the generation of anti-elastin Ab (AEAb), detectable in horse serum by ELISA. For a clinically healthy group of draught horses, a significantly lower average AEAb-level than 3 clinically affected groups (mild, moderate and severe symptoms) was demonstrated previously. To improve CPL-diagnosis, we evaluated the AEAb-ELISA as an in vitro diagnostic aid in individual horses. Test reproducibility was assessed, performing assays independently in 2 laboratories on a total of 345 horses. Possible factors associated with AEAb-levels (age, gender, pregnancy, test lab and date of blood collection) were analyzed using a mixed statistical model. Results were reproducible in both laboratories. AEAb-levels in moderately and severely affected horses were significantly higher than in healthy horses. Nevertheless, this was only demonstrated in barren mares, and, there was a very large overlap between the clinical groups. Consequently, even when a high AEAb cut-off was handled to obtain a reasonable specificity of 90%, a very low sensitivity (21%) of AEAb for CPL-diagnosis was obtained. Results on the present sample demonstrate that the described ELISA procedure is of no use as a diagnostic test for CPL in individual horses.

Analytical issues possibly affecting the performance of commercial human cytomegalovirus IgG avidity assays.

In the majority of cytomegalovirus (CMV) immunoglobulin G (IgG) avidity assays, avidity determination can be performed on the entire CMV IgG measurable positive concentration range. However, in some exceptional samples with very low IgG levels, inappropriately low avidity indexes have been described. In this study, we addressed some possible causes and the clinical importance of these inappropriately low avidity indexes. We compared VIDAS (bioMérieux), Liaison (DiaSorin), and Architect (Abbott) CMV IgG avidity assays on 129 samples from patients with past CMV infections, focusing on samples with low IgG levels. Inappropriately low avidity samples were further evaluated using seven different urea-based IgG avidity assays. We confirmed that inappropriately low avidity indexes in samples with very low IgG levels occur, but are rare. We could show that this phenomenon is not confined to a single assay and that assays employing chaotropic agents are affected more frequently and profoundly. In situations where the CMV IgG avidity is performed on CMV immunoglobulin M (IgM)-negative samples, the avidity index should be interpreted cautiously in cases of very low CMV IgG levels, whatever the technique used.

BMI-related metabolic composition of the follicular fluid of women undergoing assisted reproductive treatment and the consequences for oocyte and embryo quality.

STUDY QUESTION: Is the metabolic composition of the follicular fluid of women undergoing assisted reproductive treatment (ART) related to serum composition and BMI and is it associated with oocyte and embryo quality? SUMMARY ANSWER: We showed that metabolic alterations in the serum are reflected in the follicular fluid and that some of these alterations may affect oocyte quality, irrespective of BMI. WHAT IS KNOWN ALREADY: Many studies have focused on the effect of metabolic disorders, such as obesity and type 2 diabetes, on assisted reproduction outcomes. There are, however, only few studies focusing on the importance of the correlation between serum and follicular fluid compositions and the composition of the follicular fluid as the oocyte's micro-environment, affecting its development and subsequent embryo quality. DESIGN, PARTICIPANTS AND SETTING: In this prospective cohort study, patient information, fertility treatment outcome data, follicular fluid and serum were obtained from women undergoing ART. Patients were categorized according to their BMI (kg/m(2)) as normal (n = 60), overweight (n = 26) or obese (n = 20). Serum and follicular fluid samples were analyzed for urea, total protein, albumin, cholesterol, high-density lipoprotein cholesterol, triglycerides, non-esterified fatty acids, apolipoprotein A1, apolipoprotein B, glucose, lactate, C-reactive protein, insulin-like growth factor -1 (IGF-1), IGF-binding protein 3 (only in follicular fluid), free carnitine and total carnitine. Metabolite concentrations in serum and follicular fluid samples were correlated and were associated with BMI and fertility treatment outcome. MAIN RESULTS: Most serum metabolite differences between patients were reflected in the follicular fluid (P < 0.05). Follicular fluid apolipoprotein A1 and follicular fluid total protein concentrations negatively affected oocyte quality parameters (P < 0.05). However, overall BMI-related associations were poor. BIAS, CONFOUNDING AND OTHER REASONS FOR CAUTION: In this study, we included every patient willing to participate. Within this cohort, women with a BMI transcending 35 kg/m(2) were scarce (n = 2), because extremely overweight women are mostly advised to lose weight before starting ART. Furthermore, the number of patients in each BMI group was different, possibly masking associations between the metabolic composition of serum and follicular fluid and oocyte quality parameters. GENERALIZABILITY TO OTHER POPULATIONS: There were significant associations indicating that metabolic changes in the serum are reflected in the follicular fluid, potentially affecting oocyte quality, irrespective of the patient's BMI. For ethical reasons, this study only focused on women already in need of artificial reproductive treatment. From a metabolic point of view, we consider this cohort as a representative sample of all women of reproductive age. STUDY FUNDING: This study was funded by the special research fund, university of Antwerp (BOF UA). None of the authors has any conflict of interest to declare.

[The Hypo Ionic Protein Profile (HIPP). Laboratory analytical evaluation in Complementary and Alternative Medicine]. / Het hypo-ionische eiwitprofiel. Evaluatie van een laboratoriumanalyse uit de complementaire en alternatieve geneeskunde.

The hypo ionic protein profile (HIPP) is a test based on the reticulo-endothelial index of Sandor. We evaluated the analytical performance of this test by comparing the obtained data in the HIPP to the concentration of some frequently measured specific serum proteins. The alfa euglobulin zone mainly comprises of ceruloplasmin, complement factor 3, apolipoprotein B and haptoglobin. The beta and gamma euglobulin zone reflect the concentration of the immunoglobulins. Since these proteins cannot be distinguished from each other, the diagnostic value of the HIPP will be limited. The HIPP is an outdated and aspecific assay for protein measurements.

Protein precipitation as a possible important pitfall in the clinical chemistry analysis of blood samples containing monoclonal immunoglobulins: 2 case reports and a review of the literature.

Two case reports are presented, both illustrating an analytical interference caused by monoclonal immunoglobulins. Falsely low results were obtained in the routine analysis of glucose, CRP and HDL-cholesterol. When analysing samples containing paraproteins, various problems can be encountered in the clinical laboratory: next to the antibody effect, pseudohyponatraemia, hyperviscosity, cryoglobulinaemia and gel formation have to be taken into account. In our two cases the interference was caused by paraprotein precipitation, causing an increased turbidity and an apparent increase of light absorbance at every wavelength due to light scattering, including the wavelengths used in the clinical chemistry assays. We review the literature on this sometimes overlooked interference in photometric/turbidimetric assays. This reaction is based on the insolubility of these proteins in specific physico-chemical circumstances in which many variables are involved, among others: pH and ionic strength, presence of preservatives and surfactants in the assays, pI and other specific properties of the monoclonal immunoglobulins. The complexity of the problem makes predicting or preventing this probably infrequent interference usually impossible. This artifact can cause both false positive and false negative results in multiple parameters (e.g. bilirubin, creatinine, iron, urea, uric acid), the most frequently reported analyte being phosphate. The Sia water test (Sia euglobulin precipitation test) can provide a first clue to a paraprotein aggregation; confirmation can be obtained by observing the time/ absorbance curves of the analysis, performing the test manually or setting up a serial dilution of the sample. The problem can be solved by avoiding the presence of the proteins in the assay, performing the analysis using an alternative method or diluting out the interference. Both laboratorians and clinicians should be aware of interferences in the clinical laboratory since the clinical consequences could be important.