Since the mapping of the human genome was completed over a decade ago, our knowledge of genetic drivers of disease continues to evolve at an ever-quickening pace. Consequently, genetic testing and pharmacogenomics have become common within the healthcare system and have generated the knowledge that has empowered us to both understand and influence our lifelong health through pre-emptive intervention.  
Progress in medical genomics and its impact on healthcare cannot be understated; from genotyping patients to predict drug response, to stratifying patients according to the risk of a disease, molecular testing is having a very positive impact on many patient treatment pathways.
Undoubtedly, we are now more aware and in control of our health than ever before. It is no surprise then that the molecular diagnostic market has become the fastest growing segment of the IVD industry with assays serving the gamut of disease areas and breaking new boundaries in personalized healthcare. Despite the public appetite and availability of powerful molecular diagnostic assays that can unequivocally diagnose genetic disorders, their use has not gained universal acceptance. Many traditional diagnostic tests continue to under-diagnose, or diagnostic testing is not attempted, leading to missed opportunities for early and appropriate therapy intervention of potentially life-threatening diseases. One prime example where a molecular diagnostic approach can improve health is mutation profiling for Familial Hypercholesterolemia (FH).

Familial Hypercholesterolemia
Familial Hypercholesterolemia (FH) is a genetic disorder of lipoprotein metabolism. It is a common autosomal dominant, or inherited, disease which affects the plasma clearance of LDL-cholesterol (LDL-C), resulting in premature onset of cardiovascular disease (CVD) and a higher mortality risk.

Early diagnosis of FH is very advantageous as by the time heterozygous FH sufferers enter early adulthood they will have accumulated years of continuous build-up of fatty or lipid masses in arterial walls and are at one hundred-fold greater risk of a heart attack than their non-FH peers. If left untreated, men and women with heterozygous FH with total cholesterol levels of 8–15 mmol/L typically develop coronary heart disease (CHD) before age 55 and 60, while homozygotes with total cholesterol levels of 12–30 mmol/L typically develop CHD very early in life and if untreated die before age 20.
Clinical diagnosis of FH relies on five criteria: family history, clinical history of premature CHD, physical examination for xanthomas and corneal arcus, very high LDL cholesterol on repeated measurements, and / or a causative mutation detected by molecular genetics. To formally quantify this, a number of sets of statistically and genetically validated criteria have been devised; namely the Dutch Lipid Clinic Network Criteria and the Simon Broome Criteria. These classify suspected cases into definite, possible and probable diagnoses of FH. In the absence of definitive diagnosis through detection of a causative mutation using molecular genetics, clinical diagnosis could miss a considerable proportion of FH patients, particularly those with a mild phenotype and the pediatric population in whom the phenotype has not appeared yet.

The UK, US and international guidelines now recommend that probable or possible FH patients undergo a DNA test to confirm the diagnosis of FH. Recommendations also advocate that once an activating mutation has been found in one family member (the index case), cascade screening of that mutation in first degree relatives of the index case should proceed.  Cascade screening using a molecular assay can thus identify index family members who may otherwise be asymptomatic.

The good news is that if detected early, FH can be treated successfully with lipid lowering therapy and lifestyle changes.  In comparison to other hyperlipidemias, FH therapy tends to be more aggressive, so definitive diagnosis has additional benefits in determining care packages.  Statin drug therapy significantly reduces the morbidity and mortality from premature coronary disease in FH, particularly if affected individuals are identified and treated in childhood or early adulthood. Accurate and early diagnosis of specific mutations can result in a better overall outcome for patients through the prescribing of tailored treatments to reduce morbidity and mortality from premature cardiovascular disease. Different mutations can dictate different directions of management, such as the poorer response to lipid-lowering therapy with certain LDLR mutations. The identity of the gene involved can potentially aid the clinician to decide on how aggressive the treatment strategy will be.

Mutation diagnosis also provides clarity, and can help with an individual’s understanding and acceptance of their condition. Also a greater compliance with cholesterol lowering medication is observed with those who have been genetically diagnosed with FH.

Mutational profiling of FH
Currently, ~1200 mutations have been documented worldwide in LDLR; these affect all functional domains of the LDL receptor protein and include single-nucleotide mutations, copy number variations, and splicing mutations throughout the LDLR gene. A single mutation, Arg3500Gln, is the only common FH-related mutation in APOB, while c.1120G>T mutation is predominately detected in PCSK9. Heterozygous LDLR, APOB, and PCSK9 mutations are found in >90%, ~5%, and ~1%, respectively, of heterozygous FH subjects with a causative mutation. Prevalence varies geographically.

The abundance of different FH mutations can make genetic testing labour-intensive and costly, with many laboratories defaulting to performing expensive and lengthy Next Generation Sequencing (NGS) tests in an effort to ensure a comprehensive mutational screen. However, as our understanding of the genetic drivers of FH, as well as common population-specific mutations, increases, novel assays and techniques are being developed to meet the needs facing clinical genetics laboratories, including cost, throughput and time to result.

Randox Laboratories have developed The Familial Hypercholesterolaemia (FH) Arrays I and II that are rapid, simple and accurate diagnostic tests which enable simultaneous detection of 40 FH-causing mutations (20 mutations per array) within the LDLR, ApoB and PCSK9 genes. The assay is based on multiplex PCR followed by biochip array hybridization. Using mutation rate data from a study of 500 UK and Ireland families with genetically-confirmed FH, the Randox FH Arrays are capable of detecting approximately 71% of activating mutations in this population. The mutations will also be detected in other geographical regions.

The assay can be completed from extracted DNA to an easy-to-interpret result report in 3 hours, with the requirement for only 20ng of genomic DNA per array. The system can be used to detect small base changes, insertions and deletions within the same multiplex PCR, allowing addition of new FH mutational targets if required. The arrays are designed for use on the Evidence Investigator (Randox Laboratories Limited, Crumlin, UK). This instrument has been developed alongside Randox’s proprietary Biochip Array Technology (BAT), a multiplex testing platform founded on ELISA principles that currently has application within clinical immunoassays, drug development R&D, clinical research, forensic and clinical toxicology, veterinary drug residues and molecular diagnostics.

FH Array I and II workflow
Randox’s multiplex assays, such as FH Array I and II, have been specifically designed to detect the most common mutations, provide a cost-effective and clinically relevant alternative to NGS testing. Targeting the most commonly detected mutations in a given population enables diagnosis within hours rather than months. Where a mutation is identified in an index patient, cascade testing of family members only requires the mutation in question to be targeted; therefore negating the use of broad profiling approaches such as NGS in this setting.

Conclusion
FH is a common yet underdiagnosed condition that poses a significant risk to public health worldwide. In 2008, cardiovascular diseases were the leading cause of non-communicable deaths worldwide, with an estimated mortality rate of 17 million people. Raised cholesterol was attributed to 2.6 million deaths. Understanding a person’s genetic predisposition to cardiovascular disease through genetic testing will allow patients to receive appropriate therapeutic and interventional treatment to reduce morbidity and mortality associated with cardiovascular disease.
Pioneering multiplex diagnostic assays, tailored to incorporate the relevant FH-causing mutations, provide a promising future for both genetic laboratories, where a rapid, cost-effective approach to determine mutational status in cases of suspected FH is enabled, and the patient, whose treatment and care pathway is managed effectively.

The author
Martin Crockard, PhD
Randox Laboratories Ltd.
55 Diamond Road, Crumlin, Co. Antrim
U.K.

Early diagnosis of sepsis is essential for enabling appropriate treatment. PCT and MR-pro ADM have been shown to be independent biomarkers for sepsis and progression to septic shock, and simultaneous analysis seems to be more effective than the single marker approach.

by Dr S. Angeletti, M. De Cesaris, Dr A. Lo Presti, et al.

Introduction
Sepsis is a severe condition that represents the tenth most common cause of death in the USA. In Europe, sepsis occurs in more than 35% of the patients admitted in the intensive care unit. The mortality associated with sepsis is approximately 28% and it rises to 40–60% in cases of septic shock, despite adequate treatment administration. Nearly 9% of patients with sepsis experience severe sepsis and nearly 3% progress to septic shock leading to multi-organ failure. More than 50% of patients affected by septic shock do not survive [1–3]. Consequently, the rapid recognition and treatment of sepsis is mandatory to reduce both the mortality and the hospitalization with related costs [1-3].

Sepsis is commonly defined as the presence of infection in conjunction with the systemic inflammatory response syndrome (SIRS); severe sepsis, as sepsis complicated by organ dysfunction; and septic shock, as sepsis-induced acute circulatory failure characterized by persistent arterial hypotension despite adequate volume resuscitation and not explained by other causes [1, 4]. The diagnosis of sepsis and evaluation of its severity is complicated by the highly variable and non-specific nature of the signs and symptoms of sepsis [5]. However, the early diagnosis and stratification of the severity of sepsis is very important, increasing the possibility of starting timely and specific treatment [4, 6].

The gold standard for detection of bloodstream infections is blood culture. The time required for a positive blood culture result depends on the incubation time required for the culture to turn positive and the subsequent biochemical identification, along with an antibiotic sensitivity test, both of which usually take 48 h [7]. Furthermore, in some cases, blood culture results remain negative owing to empirical broad-spectrum antibiotics that are frequently started in the presence of SIRS and often continued for a prolonged time course despite the absence of clinical and microbiological data supporting a diagnosis of bacterial infection [4, 8]. Several studies have evaluated the diagnostic utility of various biomarkers, including ferritin, haptoglobin, interleukin 6, C-reactive protein (CRP) and procalcitonin (PCT) for suspected sepsis in the ICU patient population [9–11].

It remains difficult to differentiate sepsis from other non-infectious causes of SIRS [12] and there is a continuous search for better biomarkers of sepsis.

PCT is a polypeptide that has demonstrated the highest reliability in the early diagnosis of sepsis, severe sepsis or septic shock compared to other plasma biomarkers or clinical data alone [13]. Moreover, PCT has been advocated also to clarify the bacterial origin of some localized infections [14–15].

The mid-regional pro-adrenomedullin (MR-proADM) has been shown to play a decisive role in both the induction of hyper-dynamic circulation during the early stages of sepsis and the progression to septic shock [16–18], and recently it has been reported that MR-proADM differentiates sepsis from non-infectious SIRS with high specificity. Moreover, simultaneous evaluation of MR-proADM and PCT in septic patients increased the post-test diagnostic probabilities compared to the independent determination of individual markers [19–20]; probably the multimarker approach seems to be the more effective [14, 19].

The aim of the present study was to perform a focused evaluation of the role of the combination of PCT and MR-proADM in patients with severe sepsis and septic shock (SS) to differentiate it from patients with mild sepsis or SIRS for a prompt and specific treatment administration.

Methods
Patient and control characteristics
One hundred and seventeen patients with SS and 100 patients with SIRS, hospitalized at the University Hospital Campus Bio-Medico of Rome between the years 2012 and 2014, were enrolled in the study. The patients’ details are reported in Table 1.

Sepsis was defined by the American College of Chest Physicians/Society of Critical Care Medicine Consensus Conference definition of sepsis [4] based on the presence of a recognized site of infection and evidence of a SIRS occurring when at least two of the following criteria are present: body temperature higher than 38°C or lower than 36°C, heart rate higher than 90 beats per minute, respiratory rate higher than 20 breaths per minute or hyperventilation as indicated by an arterial partial pressure of carbon dioxide (PaCO2) lower than 32 mm Hg and a white blood cell count of higher than 12,000 cells/mm3 or lower than 4,000.

Patients were classified according to clinical signs into SS and SIRS. Acute physiological and chronic health evaluation (APACHE) II and sequential organ failure assessment (SOFA) scores were computed. APACHE II scores in SS and SIRS patients were calculated by Medscape, APACHE II scoring system calculator [21]. The SOFA score was calculated only for SS patients to better define the severity of the sepsis [22–23]. The study was approved by the Ethics Committee of the University Hospital Campus Bio- Medico, Rome, Italy.

Blood culture
Blood samples for blood culture were collected when patients showed the symptoms and signs of SIRS [1, 2, 4]. Blood culture included three sets (time 0, time 30 and time 60 min) of one aerobic and one anaerobic broth bottles (Bactec Plus Aerobic/F, Bactec Plus Anaerobic/F, Beckton Dickinson) per patient drawn during 1-h period of clinically suspected bloodstream infection. Blood culture vials were incubated in the Bactec 9240 automated system (Beckton Dickinson). Blood culture samples that turned positive were immediately processed for Gram staining and cultivated. Bacterial identification was performed by MALDI-TOF, as previously described [24].

PCT and MR-proADM measurement
The plasma concentrations of PCT and MR-proADM were measured by an automated analyser using a time-resolved amplified emission method (Kryptor, Brahms AG), with commercially available assays (Brahms AG) [25].

Statistical analysis
Data was analysed using MedCalc 11.6.1.0 statistical package (MedCalc Software). Plasma levels of PCT and MR-proADM were log-transformed to achieve a normal distribution. The normal distribution of each marker concentration was tested by the Kolmogorov–Smirnov test. PCT and MR-proADM in patients with SIRS and SS were compared using the Mann–Whitney test. Multiple logistic regression analysis (stepwise method) using SS versus PCT and MR-proADM was performed and the odds ratio (OR) computed. For OR calculation variables were retained for P<0.05 and removed for P>0.1.

Receiver operating characteristic (ROC) analysis was performed among independent variables associated with SS to define the cut-off point for plasma PCT and MR-proADM and their diagnostic accuracy to predict SS [26]. Pre-test odds, post-test odds and the consequent post-test probability were computed to investigate whether the combination of PCT and MR-proADM improves post-test probability. Likelihood ratios were used as these tests are not prone to bias due to prevalence rates [27].

Results
Patients with SS and SIRS characteristics
The mean age of the 117 patients with SS (71 men and 46 women) was 69 ± 3 years (Table 1). The principal comorbidities of patients with SS and SIRS and the sources of bacteremia are summarized in Table 1. In patients with SS the average APACHE II score value was 19.8, corresponding to 24% risk of death and the average SOFA score was 6.8 corresponding to a predicted mortality of <33%. In patients with SIRS the APACHE II score was 7, corresponding to 6% risk of death (Table 1). SS was caused by Gram-negative pathogens in 63/117 (54%) of patients and in Gram-negative sepsis, E. coli (28/63; 44.4%) was the most frequent isolate. Gram-positive SS was present in 24/117 (20.5%) of cases and the most frequent pathogen was S. aureus (14/24; 58.3%), whereas C. albicans was the most frequent isolate in yeast-positive cultures (10/117; 8.5%) and blood cultures were polymicrobial in 20/117 (17%) cases. Bacterial isolates from positive blood culture are reported in Table 2.

PCT and MR-proADM in patients with SS and SIRS
Median values, interquartile ranges (25th percentile and 75th percentile) and Mann–Whitney comparison of PCT and MR-proADM analysed in patients with SS and SIRS are reported in Table 3. PCT and MR-proADM values were significantly higher in patients with SS than SIRS (P<0.0001) (Table 3 and Figure 1). ROC curve and AUC analysis of PCT and MR-proADM in patients with SS
In SS patients, the area under curve (AUC) values of PCT and MR-proADM are reported in Table 4. Based upon ROC curve analysis and AUC characteristics, PCT and MR-proADM were considered applicable for sepsis diagnosis at the cut-off values of 0.5 ng/mL and 1 nmol/L, respectively (Table 4 and Figure 2).
Multiple logistic regression analysis
Multiple logistic regression analysis using SS as the dependent variable and PCT and MR-proADM as independent variables is reported in Table 5. Patients with MR-proADM >1 nmol/L have ‌~195 times the probability of being affected by SS than patients with SIRS, and patients with PCT values >0.5 ng/mL have the probability of developing SS 49 times more than SIRS.

Combined PCT and MR-proADM measurement in SS diagnosis: post-test probability calculation
In patients with SS, PCT and MR-proADM used as single markers have a post-test probability of 0.964 and 0.936, respectively. The combination of PCT and MR-proADM resulted in a higher value of post-test probability, 0.996 (Table 4).

Discussion
The early diagnosis and stratification of the severity of sepsis are essential, increasing the possibility of starting timely the specific treatment, especially in patients affected by SS. In this study, the combined measurement of PCT and MR-proADM in patients with SS was evaluated in order to establish the advantage derived from the use of a multimarker rather than a single marker approach.

PCT has been described as a reliable marker in the early diagnosis of sepsis compared to other plasma biomarkers or clinical data alone [13, 14, 19]. MR-proADM has been used as marker of disease severity in different clinical setting and recently its combination with PCT in bacterial infections and sepsis has been evaluated [28–32, 14, 19]. The combination of PCT and MR-proADM could allow the simultaneous evaluation of the presence of a bacterial infection as well as of the severity of this infection, giving to the ward clinicians a first useful indication waiting for blood culture positivity.

Results from this study demonstrated that in patients with SS, PCT and MR-proADM values are significantly higher than patients with SIRS. ROC curve analysis of PCT and MR-proADM demonstrated a high diagnostic accuracy of these two markers in SS diagnosis at the cut-off value of 0.5 ng/mL and 1 nmol/L, respectively. The logistic regression analysis showed higher OR values for both markers indicating a significant increased risk of having SS when these markers are higher than the cut-off values established. Furthermore, the combination of the two markers leads to a very high post-test probability value of about 99.6%.

These data confirmed the important role of the combination of PCT and MR-proADM in the diagnosis and prognosis of patients with sepsis rather than the single marker approach, because it combines the diagnostic ability of PCT with the prognostic value of MR-proADM, as already described in localized bacterial infections and not complicated sepsis [14, 19].

In conclusion, this study further support the advantage derived from the multi-marker approach in sepsis diagnosis and prognosis, especially in critically ill patients.

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The authors
S. Angeletti*¹ MD, M. De Cesaris¹, A. Lo Presti² PhD, M. Fioravanti¹, F. Antonelli¹, R. Ottaviani¹, L. Pedicino¹, A. Conti¹, A. M. Lanotte¹, M. Fogolari¹ MD, M. Ciccozzi² PhD, G. Dicuonzo¹ MD

¹Clinical Pathology and Microbiology Laboratory, University Hospital Campus Bio-Medico of Rome, Italy
²Department of Infectious, Parasitic, and Immune-Mediated Diseases, Epidemiology Unit, Reference Centre on Phylogeny, Molecular Epidemiology, and Microbial Evolution (FEMEM), National Institute of Health, Rome, Italy

*Corresponding author
E-mail: s.angeletti@unicampus.it