The increased burden of hospital admissions due to adverse drug reactions (ADRs) carries significant implications for patients and healthcare systems. Understanding the correlations between genetics and drug safety may improve clinical outcomes through the realization of personalized medicine. This article outlines a practical approach to pharmacogenomics with examples in clinical practice.
by Dr Marcin Bula and Prof. Sir Munir Pirmohamed
Introduction
The World Health Organization (WHO) defines an adverse drug reaction (ADR) as a response to a drug which is harmful and unintended and which occurs at doses normally used in man for prophylaxis, diagnosis or treatment of disease or the modification of physiological function.
There are several classifications used to describe ADRs taking into account severity, source of reported data, reaction time and location of reaction. In this article, we focus on the most-widely used classification and divide ADRs into two major groups: dose-related (type A – ‘Augmented’) and apparently non-dose-related (type B – ‘Bizarre’). Type A reactions are predictable, more common and usually less serious. They can be managed by simply reducing the dose or withholding the drug. Type B reactions are uncommon, unpredictable and usually more serious. They may either be immunologic or non-immunologic in nature, and because we do not understand pathogenesis, this makes the reactions more difficult to predict and prevent.
The overall incidence of ADR-related hospital admissions is approximately 6.5% [1, 2] although this figure might be an underestimate due to complexity of cases presenting to hospitals, compounded in real-world settings, by the poor reporting of ADRs by healthcare professionals. A previous systematic review of drug-related hospital admissions showed that antiplatelets, NSAIDs and anticoagulants were responsible for more than 50% of the total ADR-related hospitalizations [3]. It has been estimated that ADRs cost the UK National Health Service (NHS) approximately £1 billion annually, and studies in the USA have suggested that ADRs are the fourth to sixth leading cause of death [4].
Type B adverse drug reactions
Type B ADRs are a major concern for healthcare because of their unpredictable multifactorial nature, and potentially life threatening clinical outcomes. The most common organs affected are the skin, liver and blood cells. Some type B ADRs have been found to have a genomic component; the most striking example is the association between abacavir hypersensitivity and human leukocyte antigen (HLA). Abacavir is a guanosine analogue used in combination therapy with other antiretroviral medications in the treatment of human immunodeficiency virus (HIV). Previous studies have shown that approximately 4–8% [5] of patients develop a hypersensitivity reaction (HSR) within the first 6 weeks of treatment, characterized by fever, rash, gastrointestinal symptoms, general malaise, and other less common manifestations, such as headaches, respiratory and musculoskeletal symptoms [6]. The association between abacavir hypersensitivity and the HLA Class I allele, HLA-B*57:01 was first reported in 2002 by two independent research teams in Australia and North America, followed by a study in the United Kingdom. This has been complemented by functional studies that have shown that abacavir hypersensitive HLA-B*57:01 carriers show increased proliferation of CD8+ T lymphocytes following drug exposure. The exact mechanisms underlying the reaction are still not fully understand but in vitro models have shown how abacavir interacts with HLA-B*57:01, and with T cell receptors forming an immunological synapse that results in an immune response. Interestingly approximately 50% patients who are carriers of HLA-B*57:01 do not develop abacavir hypersensitivity, but the reasons for this are unknown. A study in the NHS (UK) showed that genetic testing before abacavir initiation is cost-effective [7]. Both the Food and Drug Administration (FDA) and European Medicines Agency (EMA) recommend screening for HLA-B*57:01 even though the carriage rate varies according to ethnicity from 5–8% in Europeans to 2.4% in African Americans [8]. Pre-prescription genotyping has been shown to be highly cost-effective and has reduced the incidence of abacavir hypersensitivity from over 5% to less than 1%.
It is estimated that epilepsy affects 1% of the population worldwide. Carbamazepine is an aromatic anticonvulsant that is also used for trigeminal neuralgia and bipolar disease. Cutaneous adverse reactions to carbamazepine are wide-ranging, and can manifest as maculopapular eruptions at the mild end, to the more severe cutaneous adverse reactions (which include drug reactions with eosinophilia and systemic symptoms (DRESS), Stevens–Johnson syndrome (SJS) and toxic epidermal necrolysis (TEN). SJS/TEN are the most serious ADRs with mortality rates of 5% for SJS and 35% for TEN [9]. SJS and TEN represent a continuum of cutaneous reactions, with the degree of skin detachment able to differentiate between the two (SJS involves less than 10% of the body surface area, whereas TEN affects more than 30% of the body surface area). A study in 2004 found a strong association between HLA-B*15:02 and SJS induced by carbamazepine in Han Chinese. This has been replicated by many other studies undertaken in Han Chinese, Thai and Malays, and a prospective study by Chen et al. [10] subsequently showed that genetic testing prior to treatment significantly reduced the incidence of carbamazepine-induced SJS. The association with HLA-B*15:02 is limited to South East Asian populations, and has not been demonstrated in Northern Europeans because the population prevalence of this allele is very low (<0.5%). Currently regulatory bodies including the FDA and EMA recommend genotyping for HLA-B*15:02 in South East Asian populations before starting treatment with carbamazepine, although various commentaries have questioned what is exactly meant by a South East Asian population. This reflects the difficulties in assigning screening based on self-reported ethnicity as it does not take into account admixture that occurs in almost all populations, and can exclude populations that may also be susceptible but would not be considered to be South East Asian. There is some evidence to show that HLA-B*15:02 may also predispose to SJS/TEN with phenytoin although the risk estimates are much less than with carbamazepine.
More recently, the HLA-A*31:01 allele, which is common in most ethnic groups has been associated with a range of carbamazepine hypersensitivity phenotypes including DRESS and SJS/TEN. In a Han Chinese population, an association with HLA-A*31:01 and carbamazepine-induced DRESS was demonstrated but not with SJS/TEN (where HLA-B*15:02 is predominant). In terms of mechanisms, it is not clear why HLA-B*15:02 only predisposes to SJS/TEN with carbamazepine, whereas HLA-A*31:01 predisposes to a wider range of phenotypes; cooperativity between different HLA alleles, for example with the HLA-DRB1*04:04, and with T-cell receptor clonotypes may be important in determining the phenotype (11). Genetic testing of HLA-A*31:01 is not mandatory at the moment; however, a UK study has recently shown that genotyping before initiating carbamazepine in the NHS would be cost effective (12).
Type A adverse drug reactions
Two interesting examples of the modern use of pharmacogenomics to prevent type A ADRs are with eliglustat and warfarin. Gaucher’s disease (GD) is the most common lysosomal storage disorder, which is inherited in an autosomal recessive fashion with an incidence of 1 in 40 000–60 000 in the general population, and 1 in 450 in Ashkenazi Jews [13]. Type 1 GD is the most common variant affecting more than 90% of all patients without neurological involvement, opposite to the manifestations observed with types 2 and 3 GD. Reduced activity of the β-glucocerebrosidase enzyme as a result of the GBA gene mutation leads to lysosomal accumulation of undegraded glucosylceramide causing dysfunction of various organs. For the last 20 years, the standard treatment for GD has been enzyme replacement therapy (ERT) requiring twice weekly intravenous infusions with a recombinant form of human β-glucosidase. Eliglustat represents an example of a new therapeutic strategy in GD – substrate reduction therapy (SRT), which is characterized by inactivation of glucosylceramide synthase involved in glucosylation of ceramide [14]. Eliglustat undergoes extensive metabolism by cytochrome P450 enzymes, in particular by CYP2D6 and to a lesser extent by CYP3A4. Studies have confirmed a strong correlation between the CYP2D6 metabolizer status and drug exposure. Eliglustat has recently been approved by both the FDA and EMA for the treatment of patients with type I GD – interestingly, given the strong effect of the CYP2D6 gene polymorphism on drug exposure patients need to be genotyped for their CYP2D6 metabolizer status, and the dose needs to be reduced by 50% in poor metabolizers. Furthermore, co-administration of drugs inhibiting CYP2D6 needs to be prescribed with extreme caution to prevent dose-dependent ADRs.
Warfarin is a vitamin K antagonist that is a mainstay of anticoagulation treatment in venous thromboembolism (VTE) and stroke prevention in atrial fibrillation (AF). Vitamin K antagonist therapy (despite high clinical effectiveness) has significant disadvantages and limitations including a narrow therapeutic index, drug and food interactions, routine coagulation monitoring and dose adjustments. Polymorphisms in CYP2C9 and VKORC1 genotypes and inter-individual variability can significantly influence warfarin metabolism and pharmacodynamic (PD), hence the increased risk of significant adverse reaction such as hemorrhage (Fig. 1) [15, 16]. The genetic determinants of warfarin metabolism have been heavily investigated since 1990. CYP2C9 and VKORC1 are the two main genes associated with warfarin dose requirements. Additional genetic variants, such as CYP4F2, contribute to warfarin metabolism; however, their role is less significant. The International Warfarin Pharmacogenetics Consortium proved that, based on previous studies, algorithms incorporating genetics factors (CYP2C9 and VKORC1) are more precise in prediction warfarin dosing algorithms. However, two recent large randomized controlled trials, EU-PACT and COAG, showed conflicting evidence of the role of pharmacogenetics compared to clinically guided warfarin dosing [17]. It is estimated that different outcomes in the EU-PACT and COAG trials are due to various factors including ethnic heterogeneity, genotype information on day one dosing and different control arms. The clinical utility of genotype-based warfarin dosing would need further research in particular in populations other than Caucasians.
Conclusions
Pharmacogenomics is an important area of study in understanding and preventing ADRs. It can be used throughout the whole cycle of drug development. During the pre-clinical stages, determination of how a drug is metabolized and eliminated from the body can provide valuable information on how polymorphisms in drug metabolizing enzymes and transporters affect drug pharmacokinetics and will lead to valuable prescribing information in the summary of product characteristics. This could be followed by specific, subsequent studies that may lead to genotype-dependent dosing, as in the case of eliglustat. Such precise dosing is not commonplace now, but is likely to become more important in the future. Dosing is a key determinant of the risk of ADR, and one that is still ignored. Rare and often more serious ADRs such as hypersensitivity are often not detected until phase IV, and this will require post-marketing studies. This is beautifully exemplified by abacavir hypersensitivity and the different studies that showed an association with HLA-B*57:01.
Implementation of pharmacogenomics into clinical practice has been patchy overall. This is because of many reasons, including poorly replicated gene-drug associations. However, even when the associations have been replicated and are biologically convincing, implementation has sometimes not occurred. This may be because pharmacogenetics (and the whole area of personalized medicine) represents a disruptive innovation that changes the whole clinical pathway. Changing behaviour through re-engineering the clinical pathways in a healthcare setting will require changes in the systems currently employed to deliver clinical care, which can be likened to turning around a supertanker – i.e. it will take time, money and cooperation of every part of the whole healthcare system. Of course, further research is also need in many other areas, and it is important that research in pharmacogenomics is combined with other modalities to ensure that we are covering all possible factors that can affect a response to a drug.
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The authors
Marcin Bula* MBBS, MRCP(L); Munir Pirmohamed MB ChB (Hons), PhD, FRCP, FRCP(E), FBPhS, FMedSci
Institute of Translational Medicine, University of
Liverpool, Liverpool L69 3GL2, UK
*Corresponding author
E-mail: m.bula@liverpool.ac.uk
The antinuclear antibody (ANA) test is a standard screening assay for detecting multiple autoantibodies that may be produced by a patient with an ANA associated rheumatic disease (AARD). Patients with these AARD often present with vague symptoms posting challenges to make an early and accurate diagnosis. The presence of ANAs assists physicians in making a definitive diagnosis of AARD. During the past decade laboratories have tried to move ANA testing by IIF to solid-phase assays. However, solid phase technologies such as bead-based or enzyme-linked immune assay (ELISA) have their own limitations [1-4]. Although there are several methodologies available to screen ANA, in 2009 a task force of the American College of Rheumatology (ACR) issued a statement declaring HEp-2 indirect immunofluorescence (IIF) as the preferred method for ANA screening [5,6].
by Deborah S. Stimson CLS1, Claudia A. Ibarra CCS, MB (ASCP)
The ACR declaration was based on the findings of the task force which collected information from physicians to evaluate non-standardization of the various methodologies on the market for evaluating ANA. Using HEp-2 cells as the substrate, IIF allows detection of over 100 autoantibodies to different nuclear and cytoplasmic antigens [7].
There are 5 to 6 nuclear patterns that are commonly reported. These are: homogeneous, speckled, centromere, nucleolar, dense fine speckled, and nuclear dot. The pattern and titre aid the physician when deciding what further tests to order, if any.
Performing IIF is labour-intensive, subjective, and prone to transcription errors and reader bias. Technologists reading IIF must be well trained and experienced in the interpretation of the complex patterns [7-10].
After the ACR’s 2009 statement, the demand for IIF testing has outpaced the typical laboratory’s capability to perform this test manually. Implementing HEp-2 IIF testing to abide by the recommendations issued in the ACR statement presents a challenge to most laboratories. As newer test technologies emerged, the number of laboratories with knowledge and skill to perform ANA IIF declined. The cost of personnel can be prohibitive, considering the number of staff members who must have skills and expertise to run and interpret ANA IIF. There is a need for automation and standardization of ANA IIF. Since 2002 several studies of automated or digital IIF instruments for positive and negative discrimination have been performed. Some systems incorporate pattern recognition algorithms. All conclude that automated IIF analysis will improve inter- and intra-laboratory results [11-19].
To address the increased demand for ANA testing using HEp-2 IIF, and to overcome problems with manual performance of HEp-2 testing, Inova Diagnostics developed the Integrated Lab [11-19]. To automate IIF processing, the Integrated Lab uses QUANTA-Lyser®; to automate IIF interpretation, it uses NOVA View® with a digital IIF microscope (recently cleared through the FDA no. DEN140039); and to simultaneously confirm and report results directly to the LIS, it uses QUANTA Link® software. The new instrument configuration delivers positive patient identification for IIF samples, thereby eliminating the need for manual transcription, it provides a paperless laboratory environment, while reducing variability and hands-on time.
Materials and methods
Following the manual method currently in use by the lab, a single ANA run of 118 samples was performed and then positive samples were titrated. Subsequently, the same 118 samples were processed, read, and titres reported using the automated Integrated Lab. The Integrated Lab configuration implemented at Exagen consists of three primary instruments, QUANTA-Lyser EIA/IIF processor which processes, and reads and interprets NOVA Lite® bar coded slides to allow positive patient identification, NOVA View digital IIF microscope acquires, displays, and suggests interpretation of HEp-2 IIF images, and QUANTA Link a bi-directional software, as shown in Figure A. A single run of 118 samples sent to Exagen for ANA IIF were used for this study. The samples were processed both manually and using the QUANTA Lyser 240. IIF screens and endpoint titres were read manually on an Olympus BX41 halogen microscope and also with digital images captured by NOVA View. Manual results were reported by transcribing them onto a template in the darkroom, then transcribing them a second time into the LIS. Integrated Lab results were automatically reported to the LIS using QUANTA Link. The kit for both manual and automated runs was the NOVA Lite® HEp-2 IgG ANA kit with DAPI, containing barcoded slides. After screening, forty-two of the 118 samples (36%) produced positive results. In the manual method, the forty-two samples were serially diluted to determine the endpoint titre. By comparison, the Integrated Lab configuration utilizes a unique Single Well Titre (SWT) feature on NOVA View to predict an endpoint titre from the screening well result. The SWT function automatically predicts an endpoint titre using a series of standard curves programmed into the software. Each of the 5 recognized patterns is matched to a specific curve. The SWT feature on NOVA View can be used for up to eight of the most common IIF patterns and does not require additional dilution steps. This study was conducted to quantify hands-on time required to perform our ANA IIF testing, comparing tests run manually with tests run on the Integrated Lab. Each step was timed using a stop watch. Subsequently a 5-month retrospective study to quantify reagent cost savings due to using the Integrated Lab was performed.
Results
Both methods examined 118 screens and 42 endpoint titres; the manual method required 288 HEp-2 wells, while the Integrated Lab used 120 wells. Processing samples on QUANTA-Lyser requires two wells per run of slides designated for controls compared to running manually which requires a positive and negative control on each slide. The SWT feature on NOVA View reduced the number of IIF wells by 58% or 168 wells. Screening results: The Integrated Lab reduced the hands-on time from sample processing through confirmation and reporting results by 64%, from 205.2 minutes manual run to 74.5 minutes. Using the NOVA View to predict endpoint titre eliminated the need to make serial dilutions or process additional wells. Processing 42 positive ANAs, this automated feature reduced total hands-on time by 202 minutes compared to the manual method.
Using the Integrated Lab reduced hands-on time by 82% or 5.5 hours per day compared to the manual IIF process. (Table 1) The outcome was a total annual reduction of 1,442 staff hours. Complete details are compared in Figure B. In 5 months 19,321 ANA IIF sera had been run using the Integrated Lab. A breakdown of results is shown in Table 2. Using the manual method the positive screens would be titrated the following day using 5 wells per patient to ensure finding the endpoint and reporting results 24 hours after the screen. (Table 3) With the SWT application the results with pattern and titre were reported out the same day seconds after the ANA screen result was determined. This saved 24 hours per patient in TAT for reporting. It also saved the laboratory 50,665 HEp-2 wells in 5 months.
Discussion
Recent recommendations from the ACR to use ANA HEp-2 IIF as a screening test for ANA as an aid in the diagnoses of AARD have led to an increased number of ANA IIF tests being ordered. The Integrated Lab provided the solution for automating ANA IIF that helped meet these challenges. At Exagen, the time study we conducted demonstrated a reduced hands-on time of 82% from 407.2 to 74.5 minutes and allowed faster turn-around time by delivering same day results for endpoint titre. Endpoint titre results, using NOVA View’s SWT function reduced the number of additional IIF wells and time to process endpoint titre results allowing same day reporting along with cost savings. Using the NOVA View digital images has also provided standardization among IIF readers, who now enjoy the ability to read and consult using the same digital image at any time. This was an added benefit. We found that this sophisticated, automated technology led to workflow efficiencies and a cost effective alternative to the manual IIF procedure in our laboratory.
We redirected labour savings to developing areas and expanded the tests our lab offers, while satisfying the requests of our clients for ANA titre and pattern. This study was focused on the workflow optimization and cost savings not on analytical or clinical performance which have been addressed in previous studies with convincing outcome.
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The authors
Deborah S. Stimson CLS1, Claudia A. Ibarra CCS, MB (ASCP)1, Vice President, Laboratory Operations Exagen Diagnostics
1Exagen Diagnostics, Vista, CA. 92081, USA.
Corresponding author: Claudia Ibarra
1261 Liberty Way, Vista, CA 92081, USA.
Tel. 888-452-1522
E-mail: cibarra@exagen.com
Ebola virus (EBOV) can lead to severe hemorrhagic fever with a high risk of death in humans and other primates. More recently, reverse transcription loop-mediated isothermal amplification (RT-LAMP) has become readily available for the diagnosis of EBOV, and is a suitable tool for clinical screening, diagnosis and primary quarantine purposes.
by H. Li, W. Lin, X. Wang, X. Wei, E. Li, P. Li, J. Chen, S. Qi, Y. Ma, L. Cui, X. Hu, Dr X. Zhao, Prof. J. Yuan
The 2014 Ebola virus (EBOV; one of the world’s most virulent viruses) caused an outbreak of human disease with widespread transmission in multiple West African countries and sporadic cases in Europe and North America [1, 2]. The numbers of people infected and deaths were the most severe in history. However, the massive public health response has been limited, in part, by the inability to rapidly detect the presence of EBOV in potential patients living in remote areas [3].
EBOV, (species Zaire ebolavirus from the family Filoviridae), was first identified in Zaire in 1976 and named after the River Ebola in Zaire [4]. However, EBOV could not be detected rapidly in many potential patients living in remote and developing areas. The EBOV genome is approximately 19 kb, and encodes the seven proteins in the following order from the 3’-UTR: nucleoprotein (NP), viral structural protein (VSP)35, VSP40, glycoprotein (GP), VP30, VP24, and RNA-dependent RNA polymerase (L) [5]. As the NP gene is highly conserved among EBOV species, it is, therefore, recommended by the World Health Organization (WHO) for use as a target gene for the reverse transcription (RT)-PCR assay. The initial symptoms of EBOV infection could be confused with those of other febrile illnesses such as endemic malaria [6].
Current approaches for the laboratory diagnosis of EBOV infection include virus isolation, electron microscopy, immunohistochemistry, antigen-capture ELISA testing, IgM ELISA, RT-PCR, and serologic testing for IgM or IgG virus-specific antibodies. In 2015, Baca et al. presented a rapid detection of EBOV with a reagent-free, point-of-care biosensor. In general, the detection of EBOV antigens by antigen-capture ELISA is suitable as a method of laboratory diagnosis when the viral load in the blood reaches a very much higher case fatality rate. Thus, real-time (q)RT-PCR has taken over as a first choice diagnostic technique for detection of EBOV recommended by WHO [3]. However, Taq DNA polymerase in PCR-based techniques can be inactivated by inhibitors present in crude biological samples. Moreover, these methods are relatively complex and require specialized high-cost instruments.
Loop-mediated isothermal amplification (LAMP) is a one-step nucleic acid detection method developed by Notomi et al., which relies on autocycling strand displacement DNA synthesis [7]. This novel method is highly specific and sensitive, takes advantage of four or six specific primers to recognize six or eight different sequences of the target gene, and is performed under isothermal conditions in less than 1 h using Bst DNA polymerase. Kurosaki et al. developed a simple reverse transcription (RT)-LAMP assay for the detection of EBOV, targeting the trailer region of the viral genome. However, this method has yet to be tested in clinical samples [8].
To develop an RT-LAMP for clinical screening and rapid diagnosis of EBOV, we first selected potential target regions based on the NP sequences of the EBOV variant Mayinga (GenBank Accession no. AF086833), which were further analysed with Primer Explorer V4 software (http:/primerexplorer.jp/lamp) and subsequently the sequences were aligned with other species of EBOV. A total of five sets of primers were initially designed to detect artificially synthesized EBOV RNA using a real-time turbidimeter. To compare the sensitivity and specificity of RT-LAMP, normal RT-PCR was performed with the primers.
The RT-LAMP reactions were carried out in a 25-μl reaction mixture with an RNA amplification kit (Eiken Chemical Co. Ltd), in accordance with the manufacturer’s protocol. The reaction mixture contained the following reagents (final concentration): RT-LAMP mixture and 8 U Bst DNA polymerase. The amount of primer needed for one reaction was 80 pmol of forward and backward inner primers (FIP and BIP), 40 pmol of loop primer (LB), and 10 pmol of outer forward primer (F3) and outer backward primer (B3). Finally, an appropriate amount of genomic template DNA was added to the reaction tube. The reaction was carried out in the reaction tube at 61 °C, 60–80 min, in dry bath incubators.
Two different methods were used to detect RT-LAMP products. For direct visual inspection, 1 μl of calcein (fluorescent detection reagent; Eiken Chemical Co. Ltd) was added to 25 μl of LAMP products. For a positive reaction, the colour changed from orange to green, whereas a negative reaction remained orange. The colour change could be observed by the naked eye under natural light or with the aid of UV light at 365 nm. For monitoring turbidity, real-time amplification by the RT-LAMP assay was monitored by spectrophotometry, recording the optical density at 650 nm every 6 s with the help of a Loopamp Realtime Turbidimeter (LA-230; Eiken Chemical Co. Ltd) [9].
Assay validation
1. Optimal primer choice and reaction temperature conditions for the RT-LAMP assay
As shown in Figure 1A, the EBL-2 primer set amplified the NP gene using the shortest time of about 10min; therefore, this was chosen as the optimal primer set for EBOV detection of RT-LAMP (Table 1). To further optimize the amplification, reaction temperatures were compared ranging from 59 °C to 69 °C at 2 °C intervals. Ultimately, 61 °C was chosen as the optimal reaction temperature (Fig. 1B).
2. Specificity of NP detection by RT-LAMP using the artificial in vitro transcribed RNA
Twenty-five other non-EBOV viruses were also tested. As shown in Figure 2, the EBOV RNA was identified positively by a successful RT-LAMP reaction with EBL-2 primer set using both methods of analysis. All non-EBOV strains tested negative, including the blank control, indicating that the RT-LAMP method was specific for EBOV.
3. Sensitivity of NP detection by RT-LAMP
A 10-fold serial dilution of artificial EBOV RNA was tested by real-time turbidity monitoring (Fig. 3A), visual detection method (Fig. 3B), and qRT-PCR (Fig. 3C). The limit of detection by the visual method was 10-fold lower compared with the qRT-PCR assay.
4. Clinical sample detection
The 417 clinical blood or swab samples were analysed by RT-LAMP and qRT-PCR simultaneously. The RT-LAMP and qRT-PCR detections both showed that 307 patients were confirmed cases of EBOV infections and 106 patients tested negative for EBOV.
Summary
Zaire ebolavirus is a key member of the Filoviridae family and causes highly lethal hemorrhagic fever in human beings with extremely high morbidity and mortality. As a typical negative-sense single-stranded RNA (ssRNA) virus, EBOV possesses a nucleoprotein (NP) to facilitate genomic RNA encapsidation to form a viral ribonucleoprotein complex (RNP) together with genome RNA and polymerase, which plays the most essential role in virus proliferation cycle. EBOV is found in Central Africa, but re-emerged in Western Africa in 2014 to cause an outbreak that threatened to spread worldwide. Up until 10 January 2016, 28 601 total cases (including suspected, probable, and confirmed) and 11 300 deaths were reported in Guinea and Sierra Leone (http://www.cdc.gov/vhf/ebola/outbreaks/2014-west-africa/case-counts.html). Although several chemical agents, antibodies and vaccines are found to inhibit EBOV in animals or humans, there is no therapeutic with high efficacy that can be provided for clinical usage.
To combat the increasing incidence of EBOV infections, we developed and optimized a novel RT-LAMP assay specific for EBOV diagnosis using primers spanning the 663 bp NP sequence of the viral genome. In the RT-LAMP assay, the reverse transcription reaction and DNA amplification proceed in a single step and with incubation of the reaction mixture at a constant 61°C temperature for a given time period using a temperature-controlled water bath (or other devices that can provide a stable heat are also sufficient). Moreover, LAMP reaction primers specifically recognize five independent regions of the target sequence, compared to PCR primers that recognize two independent regions of the target sequence. The sensitivity of the PCR reaction can be greatly reduced by the presence of exogenous DNA and inhibitors. Therefore, the RT-LAMP method is more suitable for rapid detection of NP in clinical samples.
Conclusion
In conclusion, a specific, sensitive, rapid and cost effective RT-LAMP assay for NP detection in EBOV was established, which is as sensitive as other available technologies, highly specific and extremely rapid in the provision of molecular diagnosis of EBOV infections. The assay can provide accurate results in a short time frame. This makes it potentially useful for clinical diagnosis of EBOV in developing countries.
Acknowledgment
This article is based on one previously published by the authors: Li H, Wang X, Liu W, Wei X, Lin W, Li E, Li P, Dong D, Cui L, Hu X, Li B, Ma Y, Zhao X, Liu C, Yuan J. Survey and Visual detection of Zaire ebolavirus in clinical samples targeting the nucleoprotein gene in Sierra Leone. Frontiers in Microbiology 2015; 6: 1332 [10].
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10. Li H, Wang X, Liu W, Wei X, Lin W, Li E, Li P, Dong D, Cui L, Hu X, Li B, Ma Y, Zhao X, Liu C, Yuan J. Survey and Visual detection of Zaire ebolavirus in clinical samples targeting the nucleoprotein gene in Sierra Leone. Frontiers in Microbiology 2015; 6: 1332.
The authors
Huan Li# MMed, Weishi Lin# MMed, Xuesong Wang MMed, Xiao Wei MMed, Erna Li MMed, Puyuan Li MMed, Jun Chen MMed, Silei Qi MMed, Yanyan Ma MMed, Lifei Cui MMed, Xuan Hu MMed, Xiangna Zhao PhD, Jing Yuan PhD*
Institute of Disease Control and Prevention, Academy of Military Medical Sciences, Beijing, 100071, PR China
#These authors contributed equally to this work
*Corresponding author
E-mail: yuanjing6216@163.com
Atherosclerotic cardiovascular diseases (CVD) are the leading cause of death in the West, and dyslipidemia is considered to be one of their key risk factors. The majority of CVD cases could be prevented by effective management of dyslipidemia. The use of new biomarkers like apolipoproteins as part of extended lipid profiles may be among the most significant new tools for such a task.
Dyslipidemias
Dyslipidemias cover a broad spectrum of lipid abnormalities. Clinicians have so far paid maximum attention to elevated levels of total cholesterol (TC) and low-density lipoprotein-cholesterol (LDL-C). Many other types of dyslipidemias, however, also appear to enhance the risk of CVD.
Lipid metabolism can become imbalanced or disturbed in several ways, resulting in changes to plasma lipoprotein function and thereafter to the development of atherosclerosis. Many patients who have high cardiovascular risk still have unfavourable lipid profiles.
Given the fast-growing interest in lipidology, clinicians have sought ways to apply evidence-based medicine daily in dyslipidemia management. There are several lipid guidelines from professional societies in different parts of the world to diagnose and make assessments of dyslipidemia.
The role of apolipoproteins
In recent years, both Europe and the US have witnessed revisions in CVD guidelines and in the approach to lipid profiling. One major new area of attention is the role of apolipoproteins.
Apolipoproteins serve to bind lipids (fat and cholesterol) to form lipoproteins. Lipids are insoluble in water. However, apolipoproteins have amphiphilic (detergent-like) properties, which make them both fat- and water-soluble. As a result, the lipoprotein particle effectively becomes water-soluble, allowing for the transport of lipids through the lymph and circulatory systems. Apolipoproteins also regulate lipoprotein metabolism.
So far, most efforts have been focused on two apolipoproteins, apolipoprotein B (apo B) and apo A-I.
From a technical viewpoint, there are numerous advantages in determining concentrations of apo B and apo A-I. Robust immunochemical methodologies are possible to attain with conventional assays using appropriate reagents. These methodologies have also been shown to provide required levels of analytical performance. Moreover, apo assays do not require fasting conditions and are not sensitive to moderately high levels of triglycerides (TG).
Apo B
Apo B is the main protein in LDL-C and directly associated with cholesterol uptake. Elevated apo B indicates an increased risk of CVD even when total cholesterol and LDL-C levels are otherwise normal.
In the laboratory, apo B concentration provides a good indicator of the number of particles in plasma of VLDL (very low-density lipoprotein), IDL (intermediate-density lipoprotein) and LDL (low-density lipoprotein).
Apo B has aroused especially high interest given its presence in high concentrations of small dense LDL. The latter is seen to be “an important predictor of cardiovascular events and progression of coronary artery disease (CAD)” and it has been endorsed as an emerging cardiovascular risk factor by the US National Cholesterol Education Program Adult Treatment Panel III in 2007.
Some contradictory evidence indicates need for more study
A host of prospective studies have shown apo B to be equal to LDL-C in risk prediction. Post-hoc analyses of numerous statin trials suggest that apo B may be not only a good biomarker but also a better treatment target than LDL-C.
However, verifying this is likely to take some more years. Apo B is yet to be included in risk calculation algorithms. Meanwhile, data about its utility remains contradictory.
For example, a meta-analysis by the Emerging Risk Factor Collaboration in 2009 indicates that apo B does not provide any benefit beyond non-high-density lipoprotein cholesterol (non-HDL-C) or traditional lipid ratios. A year later, apo B showed no benefit compared to traditional lipid markers in diabetics in the so-called FIELD study (Fenofibrate Intervention and Event Lowering in Diabetes). On the other hand, in 2011, another meta-analysis of LDL-C, non-HDL-C and apo B conducted by Canadian researchers found the apolipoprotein to be a superior marker of CV risk. Indeed, the authors, from the Royal Victoria Hospital at McGill University concluded that apo B would prevent more than one-and-a-half times the number of CV events compared to a non-HDL-C strategy alone.
Apo A-I and HDL
Unlike apo B (and LDL cholesterol), apo A-I is the major protein of HDL and provides a good estimate of HDL concentration. One HDL particle could carry several apo A-I molecules. So far, plasma apo A-I correspondences have been established (with levels of <120 mg/dL for men and <140 mg/dL for women) correlating to ‘low’ HDL-C.
Apo A-I is sometimes tested alongside apo B. The ratio between apo B and apo A-I can be used as an alternative to the total cholesterol/HDL cholesterol ratio or non-HDL-C/HDL-C ratio for indicating risk. However, as with the latter, for diagnosis and treatment, the components of the ratio have to be considered separately.
Other apolipoproteins
Research is also under way into the other apolipoproteins. Indeed, several clinical labs already offer analysis of their concentrations. These include apo A-II, apo C-II and C-III and apo E and have also provoked interest in clinical researchers.
Like apo A-I, apo A-II is also a major constituent of HDL-C, and the distribution of the former in HDL is primarily determined by the rate of production of apo A-II. Apo A-II has an important role in reverse cholesterol transport and lipid metabolism. Its increased production promotes atherosclerosis by decreasing the proportion of anti-atherogenic HDL containing Apo A-I.
Apo C-II is a co-factor for lipoprotein lipase, which breaks down lipoproteins and hydrolyses triglycerides and VLDL for absorption into tissue cells. Low concentrations of apo C-II have been linked with hypertriglyceridemia.
Apo C-III modulates uptake of triglyceride-rich lipoproteins by LDL receptor related proteins through inhibition of lipoprotein lipase. Elevated apo C-III levels are associated with both primary and secondary hypertriglyceridemia.
Apo E is found in IDLs and has several functions. These include transporting triglycerides to the liver and distributing cholesterol between cells. Apo B affects the formation of atherosclerotic lesions by inhibiting platelet aggregation and its deficiency provokes high serum cholesterol and triglyceride levels, leading to premature atherosclerosis.
CVD guidelines and apolipoproteins
In spite of the growing interest in other apolipoproteins, the highest level of interest is on apo B and A-1. Both are covered by recent modifications in certian CVD guidelines.
In 2011, the European Atherosclerosis Society (EAS) and the European Society of Cardiology (ESC) updated several CVD guidelines. Changes included doubling the stratification of cardiovascular risk from two to four categories – “very high”, “high”, “moderate” and “low”, along with the tightening of therapeutic targets for each category.
While acceptable LDL-C levels were reduced significantly across risk groups, two new therapeutic targets were recommended for patients in “very high” and “high” risk categories, especially those with combined dyslipidemia. These consisted of non-HDL cholesterol and apolipoprotein (apo) B levels.
In Europe, updated EAS/ESC guidelines recommend baseline lipid evaluation be made either on the basis of TC (total cholesterol), TG, HDL-C, and LDL-C. These are typically calculated by the so-called Friedewald formula. The new guidelines also propose using “apo B and the apo B/apo A1 ratio,” which it acknowledges are “at least as good risk markers compared with traditional lipid parameters.”
Meanwhile, in the US, professional endocrinology bodies have directly enhanced their focus on dyslipidemia since 2012, when the American Association of Clinical Endocrinologists (AACE) released new clinical practice guidelines (CPG) on the ‘Management of Dyslipidemia and Prevention of Atherosclerosis’.
The AACE’s aim was to update its existing CPGs and to complement the Diabetes Mellitus Comprehensive Care Plan CPG. Nevertheless, the AACE emphasizes that the ‘landmark’ National Cholesterol Education Program (NCEP) guidelines of 1993 continued to serve as the ‘backbone’ of its revised recommendations.
Though the new CPGs from the AACE continue to emphasize the importance of LDL-C reduction and support the measurement of inflammatory markers to stratify risk in certain situations, they nevertheless have several noteworthy features. What makes them unique for endocrinologists is their recommendation on the use of apo B or LDL particle number measurements in order to “achieve effective LDL-C lowering, provide screening recommendations for persons of different ages, and identify special issues for pediatric patients.”
Need to harmonize lipid guidelines
In spite of growing enthusiasm about apolipoproteins, some endocrinologists have said there first needs to be more harmony in lipid guidelines. It is no secret that lipid guidelines have critical differences, including recommended lipoprotein levels for risk assessment, CVD risk estimation methodologies and the need for a treatment target or the use of drugs other than statins.
Though LDL-C remains a primary target in most guidelines, the International Atherosclerosis Society (ISA) favours non-HDL-C for dyslipidemia management, as does the AACE in certain conditions. Non-HDL-C is also considered to have higher predictive capability than LDL-C in a wide variety of clinical situations, and be more practical since it can be performed in a non-fasting state.
Yet another source of much debate concerns differences in approach to CVD risk assessment. Time frames for risk vary from 10-years to life time. On their part, the American College of Cardiologists (ACC) and American Heart Association (AHA) recommend measuring 30-year risk in patients aged 20–59.
CVD risk is defined as the risk of both mortality and morbidity in most guidelines. However, the joint (and revised) EAS/ESC guidelines mentioned above calculate fatal CVD risk only. Many guidelines calculate 10-year risk of CVD. However, ISA recommends measuring the lifetime risk.
Physicians ‘bewildered’
A recent issue of ‘European Endocrinology’ poses some candid questions: Today, “physicians are bewildered by a multitude of guidelines written by different professional societies, which have more diversities than commonalities.” The author calls for “organizing a working group in dyslipidemia management and integrating existing guidelines into a general consensus document.” However, he concludes, “owing to the number of controversial areas, this is not likely to occur soon.”
Healthcare portal Medscape puts the ball back in the clinician’s court – in some senses, restating the obvious. Lipid guidelines do not “override the individual responsibility of health professionals to make appropriate decisions in the circumstances of the individual patients, in consultation with that patient, and, where appropriate and necessary, the patient’s guardian or carer”.
Sepsis is one of the major challenges in healthcare today, with some statistics predicting over 1 million cases per year in the United States, with mortality rates of about 10%. In a recently published study, between 36.9% and 55.9% of deaths among hospitalized individuals occurred in septic patients [1]. Two other findings of this study are critical to the laboratory. First, they observed that patients with initially less severe sepsis made up the majority of sepsis deaths. Secondly, most patients were already septic at the time they were admitted to the hospital. Thus laboratory testing during the initial emergency department (ED) encounter could be critical to improve sepsis-related mortality.
A systematic review of the literature [2] looked at nearly 200 proposed biomarkers for sepsis. Quoting the study’s final conclusions: “Our literature review indicates that there are many biomarkers that can be used in sepsis, but none has sufficient specificity or sensitivity to be routinely employed in clinical practice. PCT and CRP have been most widely used, but even these have limited abilities to distinguish sepsis from other inflammatory conditions or to predict outcome.” Here we take a look at the key issues the healthcare industry is facing and why physicians still do not have a reliable marker for sepsis to offer for their patients.
by Fernando Chaves
Key factors which can impact outcome in sepsis
In the last decade since sepsis awareness became more prevalent, many institutions have started implementing sepsis treatment protocols, which have been successful in decreasing mortality [3-4]. These protocols call for the collection of multiple blood cultures plus immediate start of intravenous fluids and antibiotics, and were implemented because studies have clearly demonstrated that the single most important factor in decreasing sepsis mortality was early intervention.
Later, a large prospective study comparing variations of these protocols, including invasive patient monitoring did not show any significant differences in mortality rates [5]. This indicates that there is minimal additional decreases in sepsis mortality that can be attained through improvements in treatment.
In contrast there are still options available to improve sepsis mortality through diagnostic testing. An optimal approach for early detection of sepsis still eludes us. Diagnosis today is still based primarily on the clinical recognition of systemic inflammation — increased heart and respiratory rates, fever, mental confusion, etc. followed by the documentation of a site of infection. When clinicians recognize these signs and symptoms, the window of opportunity to further decrease sepsis mortality by an earlier diagnosis has passed. Therefore, a laboratory test that could allow for earlier recognition of septic patients is a major unmet need for physicians.
What features should a laboratory test have to address this unmet need?
In order to allow for early recognition of septic patients, a laboratory test would need to meet both clinical performance and accessibility criteria. First, it must have sufficient diagnostic performance (measured by area under the curve “AUC” in the receiver operator curve “ROC curve”) to discriminate sepsis not only from healthy individuals, but also from other sick patients with conditions which mimic sepsis, such as systemic inflammatory response syndrome (SIRS). The traditional laboratory tests used during initial evaluation of patients, such as the complete blood count (CBC) fail to achieve this diagnostic performance.
Secondly, it must meet accessibility criteria, meaning it must be a test which can be widely used in all patients coming to the ED, without the need for the clinician to have an initial suspicion for sepsis. As discussed above, waiting for the physician to order the test will likely miss the window of opportunity to further improve mortality. Currently antibiotics and IV fluids are already being initiated before testing, as per the sepsis protocols now becoming increasingly prevalent in hospitals worldwide.
With so many proposed biomarkers for sepsis, which ones have met these criteria?
Unfortunately, although there have been many promises and exciting results in initial studies, results to date have been disappointing.
Early studies often yielded promising results because of their smaller size, and typically they were case-control studies comparing septic patients with healthy individuals [2]. The real challenge is discriminating sepsis from the plethora of mimicking conditions physicians encounter in the ED.
Once the biomarkers were evaluated in real life scenarios, their diagnostic performance, measured by AUC curve, did not match the results of earlier smaller studies. A perfect example was procalcitonin, PCT, the best known proposed biomarker for sepsis which initially showed very good discriminatory ability for sepsis. As PCT became more widely used and systematically studied, it became clear that it was far from the silver bullet it was optimistically thought to be. A systematic literature review and meta-analysis [6] showed an AUC of 0.78, with diagnostic performance upwardly biased in smaller studies, but moving towards a null effect in larger studies. Several years after PCT became available as a reportable test worldwide, its adoption among hospital laboratories is still sporadic, and when it is used, the most common clinical objective is the monitoring of antibiotic therapy for safe discontinuation, rather than initial diagnosis of the sepsis.
Even if the performance of these tests had been excellent, the accessibility challenge would still limit their ability to positively impact patient outcomes. As mentioned above, if the test for sepsis is ordered by a physician based on observation, the opportunity to start antibiotics sooner was missed, and the positive outcome reduced. Thus, real improvements in patient mortality will only be seen when tests are ordered routinely during initial patient care in the ED, such as the complete blood count with differential (CBC-diff).
Can we diagnose sepsis sooner using only data from a CBC-diff?
To date, there have been multiple attempts to improve the early detection of sepsis using CBC data, either via new parameters or via the creation of index values combining results from multiple traditional parameters. But so far no significant improvement in performance has been achieved —in great part due to the fact that cell counts are also elevated in inflammatory conditions mimicking sepsis. Thus, what is lacking is a parameter which is less sensitive to the inflammatory process, and more specific for the sepsis infection.
Cellular morphologic changes may be the critical tipping point in this quest. As key players in the fight against infection, white blood cells, such as monocytes and neutrophils, get activated and change their morphology. In fact, such changes have been used for years by pathologists and technologists when making their diagnostic decisions at the microscope.
Certain hematological analysers collect cellular morphologic data in their quest to recognize and count cells. Multiple studies have been published over the last decade discussing these parameters and their potential value for the early diagnosis of sepsis. However, as was true for multiple other proposed sepsis biomarkers, the small sample size and retrospective design of these studies limited their value to reliably assess their potential diagnostic performance for sepsis. This limitation has been addressed in a recent large prospective trial, probing the clinical value of morphologic parameters in the early diagnosis of sepsis in the general ED population, as well as in the discrimination between sepsis and its key mimic, SIRS.
The results of this trial will be presented at the Society for Critical Care Medicine (SCCM) annual meeting, and will not be in the public domain in time for publication in this article. But readers are invited to pay close attention to this data once it becomes available in the literature, as this abstract has been selected as one of the “Star Research Presentations” at the upcoming Critical Care Congress in February 2016.
References:
1. Liu V, Escobar GJ, et al. Hospital deaths in patients with sepsis from 2 independent cohorts. JAMA. 2014 Jul 2;312(1):90-2.
2. Pierrakos C, Vincent JL. Sepsis biomarkers: a review. Crit Care. 2010;14(1):R15.
3. Rivers E, Nguyen B, et al. Early Goal-Directed Therapy Collaborative Group. Early goal-directed therapy in the treatment of severe sepsis and septic shock. N Engl J Med. 2001 Nov 8;345(19):1368-77.
4. Jones AE, Shapiro NI, et al. Implementing early goal-directed therapy in the emergency setting: the challenges and experiences of translating research innovations into clinical reality in academic and community settings. Acad Emerg Med 2007;14:1072-8.
5. ProCESS Investigators, Yealy DM, et al. A randomized trial of protocol-based care for early septic shock. N Engl J Med. 2014 May 1;370(18):1683-93.
6. Tang BM, Eslick GD, et al. Accuracy of procalcitonin for sepsis diagnosis in critically ill patients: systematic review and meta-analysis. Lancet Infect Dis. 2007 Mar;7(3):210-7
The author
Fernando Chaves, Director of Global Scientific Affairs, Beckman Coulter Diagnostics.
March 2026
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