Mendelian and complex genetics of susceptibility and resistance to parasitic infections

Uncovering the complex genetic basis of susceptibility and resistance to parasitic infectious diseases is an enormous challenge. It probably involves many different host genes, interacting with multiple parasite genetic and environmental factors. Several genes of interest have been identified by family and association studies in humans and by using mouse models, but more robust epidemiological studies and functional data are needed to authenticate these findings. With new technologies and statistical tools for whole-genome association analysis, the next few years are likely to see acceleration in the rate of gene discovery, which has the potential to greatly assist drug and vaccine development.     

Rapid diagnosis of human brucellosis by SYBR Green I-based real-time PCR assay and melting curve analysis in serum samples

The aim of this study was to develop a LightCycler-based real-time PCR (LC-PCR) assay and to evaluate its diagnostic use for the detection of Brucella DNA in serum samples. Following amplification of a 223-bp gene sequence encoding an immunogenetic membrane protein (BCSP31) specific for the Brucella genus, melting curve and DNA sequencing analysis was performed to verify the specificity of the PCR products. The intra- and inter-assay variation coefficients were 1.3% and 6.4%, respectively, and the detection limit was 5 fg of Brucella DNA (one genome equivalent). After optimisation of the PCR assay conditions, a standard curve was obtained with a linear range (correlation coefficient=0.99) over seven orders of magnitude from 10(7) to 10 fg of Brucella DNA. The LC-PCR assay was found to be 91.9% sensitive and 95.4% specific when tested with 65 negative control samples and 62 serum samples from 60 consecutive patients with active brucellosis. The assay is reproducible, easily standardised, minimises the risk of infection in laboratory workers, and has a total processing time of <2 h. It could therefore form a promising and practical approach for the rapid diagnosis of human brucellosis.     

Evaluation of an alternative confirmatory strategy for the diagnosis of HIV infection in Dar Es Salaam, Tanzania, based on simple rapid assays

Alternative confirmatory strategies for detection of antibodies to HIV using enzyme-linked immunosorbent assays (ELISAs) have been shown to be useful in laboratories with limited resources. Three simple and rapid HIV antibody detection assays (Capillus, Serocard and Determine) were evaluated using 1412 fresh serum samples in order to formulate an alternative confirmatory strategy for the diagnosis of HIV infection. All sera were also tested by an anti-HIV ELISA and all sera reactive by any of the assays were tested by a second ELISA as well as by Western blot. Three hundred and eighty-three sera were found to be HIV-1 antibody positive, while 1017 sera were HIV antibody negative; 12 sera which were reactive by one or more of the simple assays had indeterminate Western blot results and these were considered HIV seronegative during the analysis. All assays had a sensitivity of 100%. The initial specificity of the assays were 98.7, 98.2 and 97.9% for Capillus, Serocard and Determine, respectively. In an alternative confirmatory strategy the use of Capillus followed by Serocard or Determine gave a specificity of 99.9 and 99.8%, respectively. Serocard followed by Determine gave a specificity of 99.3%. A testing strategy with 100% specificity (95% CI; 99.6–100%) could be achieved by the sequential use of all three simple/rapid assays or by repeat testing by Capillus followed by Serocard.     

Molecular pathological analysis of sarcomas using paraffin‐embedded tissue: current limitations and future possibilities

Sarcomas of soft tissue and bone are rare neoplasms that can be separated into a large number of different diagnostic entities. Over the years, a number of diagnostic markers have been developed that aid pathologists in reaching the appropriate diagnoses. Many of these markers are sarcoma‐specific proteins that can be detected by immunohistochemistry in formalin‐fixed, paraffin‐embedded (FFPE) sections. In addition, a wide range of molecular studies have been developed that can detect gene mutations, gene amplifications or chromosomal translocations in FFPE material. Until recently, most sequencing‐based approaches relied on the availability of fresh frozen tissue. However, with the advent of next‐generation sequencing technologies, FFPE material is increasingly being used as a tool to identify novel immunohistochemistry markers, gene mutations, and chromosomal translocations, and to develop diagnostic tests.     

QF-PCR-based prenatal detection of common aneuploidies in the Czech population: five years of experience

We present the results from the largest clinical application of QF-PCR for antenatal rapid aneuploidy detection (RAD) in routine prenatal diagnosis in the Czech Republic. QF-PCR was performed in addition to karyotyping (dual testing) in two settings: the first was a single multiplex reaction testing only trisomy 21 and amelogenin X/Y alleles in the second trimester screened positive cases (T21 test), and the second setting consisted of two multiplexes (2M test) for common aneuploidies (13, 18, 21, X and Y) in cases with other RAD indications such as ultrasound findings, late booking or maternal anxiety.

Dual testing was performed in 6349/12,778 (49.7%) of prenatal samples using either T21 or 2M test between 2002 and 2007. The clinical acceptability of our dual testing policy, methodological efficiency of RAD and residual risks of other chromosomal aberrations (CHAs) were evaluated.

QF-PCR detected 92% (175/190) of significant CHAs. The 2M test identified 93.5% and the T21 test identified 87.5% of the significant CHAs with complete specificity. The residual risk of significant CHA was 1/231 in the 2M test and 1/565 in the T21 test. If RAD for all common aneuploidies is used as the sole prenatal diagnosis method, the odds of missing a CHA of any type are 1:90 and the odds of missing significant CHA with no ultrasound findings are 1:1513. If prenatal karyotyping were used as an additional procedure to RAD in cases only with ultrasound findings, 186/190 (97.8%) of the significant CHAs would be detected when 15.7% cases were karyotyped, according to our data.

We consider RAD directed towards trisomy 21 alone (our T21 test) as an economically and clinically acceptable part of second trimester screening for Down syndrome. Both RAD tests allow fast alleviation of maternal anxiety with low residual risk when the test results are negative, and allow fast decision making if the results are positive. However, replacement of dual testing with only the RAD procedure in specific indications accepted in some countries (Great Britain) remains in the Czech Republic a theme for debate.     

Diagnosis of tuberculosis in an era of HIV pandemic: a review of current status and future prospects

HIV and tuberculosis co-infection interact in fundamentally important ways. This interaction is evident patho-physiologically, clinically and epidemiologically. There are several differences between HIV-infected and HIV-uninfected patients with tuberculosis (TB) that have practical diagnostic implications. TB is more likely to be disseminated in nature and more difficult to diagnose by conventional diagnostic procedures as immunosuppression progresses. As TB rates continue to increase in HIV-endemic regions, improved diagnostic techniques merit consideration as TB-control strategies. There is a need to develop more user friendly techniques, which can be adapted for use in the high-burden and low-income countries. This review focuses on the diagnostic challenges in HIV-TB co-infection with an update on the current techniques and future prospects in an era of HIV pandemic.     

Clinical trials in allergen immunotherapy: current concepts and future needs

Allergen immunotherapy (AIT) is a safe, effective treatment for allergic rhinoconjunctivitis and allergic asthma. However, AIT's clinical effect is still contested—primarily due to heterogeneity in clinical trial designs, study populations, therapeutic formulations, and efficacy criteria. After discussing current concepts and unmet needs, an international panel of experts made several recommendations: (i) explore and validate definitions for (clinical) responders in AIT trials; (ii) use of well‐documented, standardized provocation tests prior to inclusion of subjects with relevant diseases in AIT trials; (iii) monitoring neo‐sensitizations and occurrence of new allergy in extended AIT trials, and exclusion of polyallergic participants; (iv) validation of allergen exposure chambers with regard to natural exposure; (v) in studies of seasonal allergies, focus on peak exposure but also consider organizing two parallel, geographically distinct but otherwise identical trials; (vi) discuss adaptive trial designs with the regulatory authorities; (vii) use e‐health and m‐health technologies to capture more information on individual exposure to allergens; (viii) initiate research on potential psychological, biochemical, immune, neural, and even genomic markers of the placebo response; (ix) identify trial designs and primary endpoints that will give children with allergies easier, faster access to AIT formulations; and (x) promote and apply standardized methods for reporting systemic and local adverse events. The latest technologies and trial designs may provide novel, ethical ways of reducing bias and heterogeneity in AIT clinical trials. There is scope for physicians, patient organizations, companies, and regulators to improve clinical trials in AIT and, ultimately, to provide patients with better treatments.     

Flow-assisted allergy diagnosis: current applications and future perspectives

Physicians predominantly rely upon quantification of serum-specific immunoglobulin E (IgE) and/or skin test to confirm clinically suspected IgE-mediated allergy. However, for various reasons, identification of the offending allergen(s) and potentially cross-reactive structures is not always straightforward. Flow-assisted allergy diagnosis relies upon quantification of alterations in the expression of particular basophilic activation markers. Actually, upon challenge with a specific allergen, basophils not only secrete quantifiable bioactive mediators but also upregulate the expression of different markers which can be detected efficiently by flow cytometry using specific monoclonal antibodies. Currently, the technique has been applied in the investigation of IgE-mediated allergy caused by classical inhalant allergens, food, Hevea latex, hymenoptera venoms and drugs. It is also appreciated; the technique proves valuable in the diagnosis of non-IgE-mediated (anaphylactoid) reactions such drug hypersensitivity and the detection of autoantibodies in certain forms of chronic urticaria. This review will not address immunologic features, characteristics and general pitfalls of flow-assisted analysis of in vitro-activated basophils as summarized elsewhere. After a recapitulation of the principles and some specific technical issues of flow-assisted analysis of in vitro-activated basophils, we principally focus on the current clinical and research applications of the basophil activation tests. Personal experience of both research groups is provided, where appropriate. Finally, a viewpoint on how the field might evolve in the following years is provided.     

Lessons in diagnostic virology: expected and unexpected sources of error

Viral diagnostics have shown continued innovation, with serological and molecular diagnostic assays pushing the limits of sensitivity. Technology has provided new automated shared diagnostic platforms that reduce hands‐on time, while with globalisation of the diagnostic market, commercial assays are applied across epidemiologically diverse settings on different patient and viral populations. However, with these novel developments, new and often unexpected sources of diagnostic error emerge. In this review we will reflect on case studies that highlight these often underappreciated or unexpected diagnostic errors spanning pre‐analytical, analytical, and post‐analytic processes. We will also suggest approaches that could help identify error and reduce the impact on patient management.     

Clinical trials of hematopoietic cell transplantations: current needs and future strategies.

Biol Blood Marrow Transplant 2000;6(2):79-89.     

Immunotherapy of inflammatory bowel diseases: current concepts and future perspectives

The etiology and the pathogenesis of inflammatory bowel diseases (IBD), e.g. Crohn's disease and ulcerative colitis are still not completely understood. However, there is growing evidence that an alteration of the mucosal immune system towards luminal antigens in a genetically susceptible host plays a key role in the pathogenesis of IBD. In particular, cytokines produced by intestinal epithelial cells, lamina propria macrophages and CD4+ T cells appear to contribute to the initiation and perpetuation of intestinal inflammation in IBD. This review focuses on the role of the mucosal immune system in the pathogenesis of IBD and potential novel immunotherapeutic strategies for chronic intestinal inflammation. Such strategies include recombinant antiinflammatory cytokines, neutralizing antibodies or fusion proteins, antisense oligonucleotides and adenoviral gene transfer.     

Rapid diagnosis of plant virus diseases by transmission electron microscopy

A clear and rapid diagnosis of plant virus diseases is of great importance for agriculture and scientific experiments in plant phytopathology. Even though negative staining and transmission electron microscopy (TEM) are often used for detection and identification of viral particles and provide rapid and reliable results, it is necessary to examine ultrastructural changes induced by viruses for clear identification of the disease. With conventional sample preparation for TEM it can take several days to obtain ultrastructural results and it is therefore not suitable for rapid diagnosis of virus diseases of plants. The use of microwave irradiation can reduce the time for sample preparation for TEM investigations. Two model virus–plant systems [Nicotiana tabacum plants infected with Tobacco mosaic virus (TMV), Cucurbita pepo plants infected with Zucchini yellow mosaic virus (ZYMV)] demonstrate that it is possible to diagnose ultrastructural alterations induced by viruses in less than half a day by using microwave irradiation for preparation of samples. Negative staining of the sap of plants infected with TMV and ZYMV and the examination of ultrastructure and size were also carried out during sample preparation thus permitting diagnosis of the viral agent by TEM in a few hours. These methods will contribute towards a rapid and clear identification of virus diseases of plants and will be useful for diagnostic purposes in agriculture and in plant phytopathology.     

Zika virus diagnosis: challenges and solutions

Background

Since its sudden appearance and link to microcephaly in 2015, the number of PubMed references for Zika virus (ZIKV) has risen from 181 to 5163, at time of writing, with a vast proportion focused on the consequences of ZIKV infection during pregnancy. This level of attention underlies increased demand for sensitive and specific diagnostic tools able to assess risk to an unborn child, as well as to understand the dynamics and consequences of viral persistence.

Rapid diagnosis of coccidioidomycosis by nested PCR assay of sputum

Coccidioidomycosis is a deep infection caused by two dimorphic fungi, Coccidioides immitis and Coccidioides posadasii. Diagnosis of the disease requires culture of suspicious clinical samples on mycological media. However, as these species are virulent pathogens, handling of their cultures is a high-risk activity, and is limited to Biosafety Level 3 laboratories. This study describes the direct detection of C. posadasii DNA in an inappropriate sputum sample by PCR amplification of the highly specific Ag2/PRA antigen gene. The results obtained suggest that direct detection of the Ag2/PRA sequence in sputum is an excellent method for rapid and specific diagnosis of coccidioidomycosis.     

Electron microscopy: a method for the diagnosis of inherited metabolic storage diseases. Electron microscopy in diagnosis

In the diagnosis of inherited metabolic diseases, electron microscopy has become an important method complementary to clinical, histological and biochemical assays. The characteristic ultrastructure of stored material as well as the site of accumulation in the cell are shown in a number of metabolic disorders. The most prominent advantages of electron microscopical techniques as compared to alternative techniques are discussed. One of the advantages is the fact that ultrastructural investigation requires only tissue samples of very small size, and another that its results may be obtained within two days. Moreover, transmission electron microscopy permits new and promising analytical methods such as quantitative estimation of organellar changes (morphometry) and energy dispersive X-ray elemental analysis (EDX).