Infectious diseases, in particular lower respiratory infections and diarrhoea are among the top ten causes of death worldwide [1]. Many infectious diseases can be considered ‘syndromes’, i.e. a collection of symptoms that do not point to a specific causative agent. A ‘syndromic approach’ in diagnostics means to achieve, by a single test, a diagnosis without taking into account the symptoms themselves. In molecular diagnostics (MDx) of infectious diseases, this means the use of multiplex PCR panel assays to simultaneously detect genetic material from pathogens of different species and even different taxonomic levels, such as bacteria, viruses, parasites and fungi.
by Dr. Antoinette A. T. P. Brink and Dr. Guus F. M. Simons
Clinical syndromes and their causal pathogens
Respiratory infections
Respiratory infections are highly prevalent and the possible causative agents include several typical and atypical bacteria, as well as many viruses. Among the latter, the influenza virus particularly is associated with morbidity and mortality. As influenza A viruses can infect multiple hosts, including not only humans but also birds and swine, these viruses can undergo antigenic shifts that may result in pandemics such as the 2009 ‘Mexican flu’. Nowadays, many molecular diagnostics (MDx) tests are able to distinguish influenza A virus subtypes associated with non-human hosts, such as H5N7 avian flu. The presence of such a subtype in a patient with respiratory illness may be indicative for zoonosis, which increases the risk of a new pandemic. Besides influenza A virus, syndromic MDx panels generally detect influenza B virus, respiratory syncytial virus (RSV) A and B, adenovirus (AdV), human metapneumovirus (hMPV), parainfluenza virus (PIV) types 1–4 and human coronavirus (hCoV) types 229E, OC43, NL63 and HKU1, rhinovirus (RV) and enterovirus (EV). Bocavirus is not always included, although it is considered an important pathogen especially in children [2].
The merit of testing a broad respiratory panel is illustrated by a study conducted by the Dutch National Institute for Public Health and the Environment (RIVM) among community-dwelling elderly receiving annual influenza vaccination. Vaccine effectiveness was studied by determining the relative contribution of influenza and other respiratory pathogens to influenza-like illness (ILI), using an assay that detects 22 respiratory pathogens simultaneously (RespiFinder®) As expected, vaccination reduced the incidence of influenza, but the number of ILI episodes was similar between vaccinated and non-vaccinated individuals; non-Influenza viruses replaced influenza as a cause of ILI in vaccinated individuals [3].
This finding is in line with the recommendation of the American Society for Microbiology working group for respiratory virus testing not to restrict testing for specific respiratory viruses during certain seasons because with global travel, many ‘seasonal’ viruses can cause disease throughout the year [4]. Moreover, testing should not be restricted to certain patient populations, e.g. testing for RSV/hMPV only in children, because these viruses may cause severe disease in adults as well.
For fastidious bacteria causing atypical pneumonia, such as Legionella pneumophila, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Bordetella pertussis and B. parapertussis, MDx has largely replaced conventional culture.
For Streptococcus pneumoniae and other bacteria associated with typical pneumonia, PCR is not ready to replace conventional culture because these bacteria belong to the normal oral flora. Quantitative detection is necessary to distinguish colonization from infection, but sample quality criteria for this are lacking. Moreover, conventional culture remains necessary for antimicrobial susceptibility testing.
Gastroenteritis
Most gastroenteritis (GE) panels detect an extensive list of viruses, bacteria and parasites. It is self-evident that GE viruses such as norovirus, adenovirus types 40 and 41, and rotavirus are included. Although the clinical course of sapovirus and human astrovirus (HAstV) infections is generally milder than that of, for example, norovirus, they should be included in routine testing to identify outbreaks and to ensure proper care of patients at risk for severe complications, such as infants, elderly and immuno-compromised patients.
The parasites Cryptosporidium spp., Entamoeba histolytica and Giardia lamblia are included in all GE panels because their pathogenicity is well established. For Dientamoeba fragilis this is still a matter of debate, but it should be considered the causal factor of GE symptoms – and treated appropriately – after other causes have been excluded [5].
Regarding GE-causing bacteria, most commercially available tests detect the most common pathogenic Escherichia coli types (EHEC, ETEC, STEC, EPEC, and IEIC), Salmonella spp. and Yersinia enterocolitica. For Campylobacter it is important to detect all pathogenic species, i.e. C. jejuni, C. coli, C. upsaliensis and C. lari [6], the latter two of which are not included in some commercially available assays.
In contrast, for Vibrio cholerae it is important to distinguish the only two serotypes that can cause outbreaks and infections should be treated actively, to avoid overtreatment.
Additional pathogens may be included, but test results may be difficult to interpret owing to (i) frequent contamination from the environment or reagents (e.g. Aeromonas spp.) or (ii) conflicting data regarding pathogenicity (e.g. Plesiomonas shigelloides).
Infections of the central nervous system
The differential diagnosis for patients with suspected meningitis/encephalitis (ME) includes infectious as well as non-infectious causes that cannot be distinguished on the basis of symptoms. A recent study showed that in the majority of patients suspected of a central nervous system (CNS) infection, the etiology could not be found even with the most comprehensive commercially available MDx tests [7]. In line with this, the use of a diagnostic stewardship approach is advocated, including cerebrospinal fluid white blood cell count to prevent unnecessary use of expensive tests [8]. Having said that, true CNS infections are medical emergencies that require rapid pathogen identification to allow timely and appropriate clinical intervention.
Causative agents of CNS infections include enteroviruses, parechoviruses and all eight members of the human herpes virus family, the most prevalent of which are herpes simplex virus types 1 and 2. The other herpes viruses, such as cytomegalovirus, varicella-zoster virus, human herpes virus 6, and Epstein-Barr virus, are mostly seen in immunocompromised patients such as transplant recipients [9].
Measles and mumps viruses may also cause CNS infections, especially in regions where vaccine coverage is sub-optimal due to, for example, a weak healthcare system or vaccine refusal. Of note, when this paper was drafted, four countries in the European Union (Albania, Czechia, Greece and the United Kingdom) had recently lost their measles elimination status previously assigned by the World Health Organization.
Meningitis-causing bacteria usually belong to the normal oropharyngeal flora, and may enter the CNS by anatomic defects in the natural barriers, or defects in the immune system. Streptococcus pneumoniae is the most common cause of community-acquired meningitis in non-neonates worldwide, followed by Neisseria meningitidis. Other bacterial pathogens in meningitis are Listeria monocytogenes, Haemophilus influenzae, Staphylococcus aureus and Borrelia burgdorferi.
Vaccination campaigns against various serogroups of these bacteria are ongoing. As serogroup replacement occurs as a consequence of vaccination, it is important that in vitro diagnostic kits detect all serogroups, and that assay designs are checked periodically for this.
Sexually transmitted infections
Various bacteria, parasites and viruses can cause sexually transmitted infections (STI).
STIs can be non-symptomatic, but if left untreated they can have severe sequelae including permanent infertility or ectopic pregnancy.
STI test panels should at least include Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis. For a syndromic approach, herpes simplex virus types 1 and 2 and Treponema pallidum should be included.
The editorial board of the European STI Guidelines recommends not to test routinely for Mycoplasma hominis and Ureaplasma parvum. In addition, in men with symptomatic urethritis, Ureaplasma urealyticum should only be treated after C. trachomatis, N. gonorrhoeae and M. genitalium have been excluded [10].
Several subtypes of STI require non-standard therapy. For example, the ‘L’ serovars of C. trachomatis can cause lymphogranuloma venereum (LGV) and proctitis, which should be treated differently from other C. trachomatis infections [11].
Furthermore, N. gonorrhoeae strains have emerged that are resistant to all antimicrobials used for treatment, owing to point mutations and/or genetic recombination with commensal Neisseria species [12]. In addition, the change from doxycycline to azithromycin as the first-line treatment for C. trachomatis and non-gonococcal urethritis has resulted in selection of macrolide-resistant M. genitalium strains [13]. Fortunately, such pathogen properties can be distinguished by molecular methods and, in fact, several commercially available multiplex MDx assays can do this already.
MDx methods for syndromic approach
Laboratory-developed tests
Laboratory-developed PCR assays for infectious diseases have been in use since the early 1990s [14].
Generally, laboratory-developed tests (LDTs) run on conventional 96-well realtime PCR systems, allowing the testing of large batches of samples. Most LDTs are TaqMan-based, generally limiting multiplexing to four targets per reaction. A syndromic approach is, therefore, only possible by running several reactions (up to eight to cover a full respiratory panel) per sample, which limits throughput.
Although the cost-of-goods for LDTs is low, testing can be laborious and time-consuming and requires highly skilled laboratory personnel.
Commercial: medium to high throughput
Clinical laboratories needing a syndromic approach and medium to high throughput may use commercial assays instead of LDT. To exceed the multiplexing capacity of LDTs, manufacturers have developed proprietary methods. For example, Luminex’s platform uses fluorescently labelled bead array technology with dedicated instruments. Korean Seegene’s assays include melting curve analysis or differential detection temperatures to allow distinction of multiple targets per fluorescent channel.
Commercial: random access/low throughput
Examples of commercial random access systems are the FilmArray by bioMérieux, the QIAstat-Dx by QIAGEN and the ePlex by GenMark. The high price for the dedicated instruments and high price-per-test can be an argument against implementation in routine use if large amounts of samples are processed. Hence, these systems are particularly suitable for point-of-care purposes or when laboratory skills of personnel are limited. However, the ease-of-use may conceal to inexperienced users that these tests are actually very sensitive, and careful reaction set-up and cleanliness of the environment are needed to avoid false positives. It is self-evident that (unexpected) positive results should be interpreted in the context of clinical symptoms. Moreover, the ISO 15189 standard for medical laboratories recommends users to run additional control materials from independent third parties.
Summary
A syndrome-based approach using broad panel MDx assays may assist in timely diagnosis of respiratory infections, GE, CNS infections and STI.
Syndrome-based MDx results in a decrease in the number of chest radiographs, reduced admission rates, fewer barrier nursing days [15], shorter duration of hospitalization, more appropriate prescription of antivirals, better antibiotic stewardship and decreased duration of antimicrobial use [16–18], which is likely to result in less antibiotic resistance in the long term.
In addition, broad panel tests provide useful information about epidemiology, seasonality and possibly clinically relevant co-infections
Figure 1: Example of high multiplexing (the 2SMARTFinder® principle of Dutch firm PathoFinder)
Schematic representation of the PathoFinder 2SMARTFinder® test principle: (a) Step 1 (pre-amplification): specific multiplex target enrichment. (b) Step 2 (2SMART reaction): signal amplification by means of 2SMART primers each consisting of a targetspecific sequence (yellow/blue) and a universal sequence (grey/green). Each reverse 2SMART primer contains a stuffer/barcode sequence (red) for detection. The combined action of the 2SMART primers and the universal primers, of which the reverse is labelled with fluorescein amidites (FAM), results in the generation of (c) a FAM-labelled PCR product. (d) The reaction mixture contains labelled SMART probes complementary to each stuffer. (e) When a probe hybridizes to its corresponding stuffer in the reaction product, the FAM label acts as a Förster Resonance Energy Transfer (FRET) donor and the label in the SMART probe as an acceptor, resulting in the emission of light that can be measured in real-time. Finally, the temperature is increased, resulting in dissociation of the probe-stuffer hybrid and a sharp decline in fluorescence (f). The negative derivative of this graph shows the actual melting peaks (g). The stuffer/probe sequence determines the position of the melting peak and reveals which pathogen was present in the sample. −Δ(F)/dT, negative derivative of the change in fluorescence (F) as a function of temperature (T).
Figure 2: Typical result read-out on LightCycler 480
Example of the read-out of the RespiFinder® 2SMART assay mix 1 in the carboxyrhodamine (ROX™) channel of a LightCycler 480 II real-time PCR instrument. AdV, adenovirus; hMPV, human metapneumovirus; Inf A, influenza A virus; Inf B, influenza B virus; RSVA, respiratory syncytial virus A; RSVA, respiratory syncytial virus B
References
1. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, Abraham J, Adair T, Aggarwal R, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. The Lancet 2012; 380: 2095–2128.
2. Ma X, Conrad T, Alchikh M, Reiche J, Schweiger B, Rath B. Can we distinguish respiratory viral infections based on clinical features? A prospective pediatric cohort compared to systematic literature review. Rev Med Virol 2018; 28: e1997.
3. van Beek J, Veenhoven RH, Bruin JP, van Boxtel RAJ, de Lange MMA, Meijer A, Sanders EAM, Rots NY, Luytjes W. Influenza-like illness incidence is not reduced by influenza vaccination in a cohort of older adults, despite effectively reducing laboratory-confirmed influenza virus infections. J Infect Dis 2017; 216: 415–424.
4. Ginocchio CC, McAdam AJ. Current best practices for respiratory virus testing. J Clin Microbiol 2011; 49: S44–S48.
5. Van Gestel RSFE, Kusters JG, Monkelbaan JF. A clinical guideline on Dientamoeba fragilis infections. Parasitology 2018; 1–9.
6. Klena JD, Parker CT, Knibb K, Ibbitt JC, Devane PML, Horn ST, Miller WG, Konkel ME. Differentiation of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis by a multiplex PCR developed from the nucleotide sequence of the lipid A gene lpxA. J Clin Microbiol 2004; 42: 5549–5557.
7. Sall O, Thulin Hedberg S, Neander M, Tiwari S, Dornon L, Bom R, Lagerqvist N, Sundqvist M, Molling P. Etiology of central nervous system infections in a rural area of Nepal using molecular approaches. Am J Trop Med Hyg 2019; 101(1): 253–259.
8. Messacar K, Robinson CC, Dominguez SR. Letter to the editor: economic analysis lacks external validity to support universal syndromic testing for suspected meningitis/encephalitis. Future Microbiol 2018; 13: 1553–1554.
9. Chadwick DR. Viral meningitis. Br Med Bull 2005; 75–76: 1–14.
10. Horner P, Donders G, Cusini M, Gomberg M, Jensen JS, Unemo M. Should we be testing for urogenital Mycoplasma hominis, Ureaplasma parvum and Ureaplasma urealyticum in men and women? – a position statement from the European STI Guidelines Editorial Board. J Eur Acad Dermatol Venereol 2018; 32: 1845–1851.
11. Ceovic R, Gulin SJ. Lymphogranuloma venereum: diagnostic and treatment challenges. Infect Drug Resist 2015; 8: 39–47.
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13. Jensen JS. Mycoplasma genitalium: yet another challenging STI. Lancet Infect Dis 2017; 17: 795–796.
14. Claas HC, Wagenvoort JH, Niesters HG, Tio TT, Van Rijsoort-Vos JH, Quint WG. Diagnostic value of the polymerase chain reaction for Chlamydia detection as determined in a follow-up study. J Clin Microbiol 1991; 29: 42–45.
15. Goldenberg SD, Bacelar M, Brazier P, Bisnauthsing K, Edgeworth JD. A cost benefit analysis of the Luminex xTAG Gastrointestinal Pathogen Panel for detection of infectious gastroenteritis in hospitalised patients. J Infect 2015; 70: 504–511.
16. Rappo U, Schuetz AN, Jenkins SG, Calfee DP, Walsh TJ, Wells MT, Hollenberg JP, Glesby MJ. Impact of early detection of respiratory viruses by multiplex PCR assay on clinical outcomes in adult patients. J Clin Microbiol 2016; 54: 2096–2103.
17. Andrews D, Chetty Y, Cooper BS, Virk M, Glass SK, Letters A, Kelly PA, Sudhanva M, Jeyaratnam D. Multiplex PCR point of care testing versus routine, laboratory-based testing in the treatment of adults with respiratory tract infections: a quasi-randomised study assessing impact on length of stay and antimicrobial use. BMC Infect Dis 2017; 17: 671–671.
18. Echavarria M, Marcone DN, Querci M, Seoane A, Ypas M, Videla C, O’Farrell C, Vidaurreta S, Ekstrom J, Carballal G. Clinical impact of rapid molecular detection of respiratory pathogens in patients with acute respiratory infection. J Clin Virol 2018; 108: 90–95.
The authors
Antoinette A.T.P. Brink* PhD, Guus F.M.
Simons PhD PathoFinder B.V., 6229 EG Maastricht,
The Netherlands
*Corresponding author
E-mail: antoinette.brink@pathofinder.com
EurobioPlex – Real Time PCR for infectious diseases
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, /in Featured Articles /by 3wmediaEvaluation of protein induced by vitamin K absence II as a novel tumour marker in pancreatic cancer
, /in Featured Articles /by 3wmediaPancreatic cancer is a highly lethal malignancy: as its poor prognosis is also related to a difficulty of early diagnosis, a reliable and easily detectable biomarker is urgently needed. This article aims to describe a new serum tumour marker for pancreatic cancer, protein induced by vitamin K absence II.
by Dr Sara Tartaglione, Prof. Antonio Angeloni and Prof. Emanuela Anastasi
Pancreatic cancer
Pancreatic cancer (PC) is the 11th most common cancer in the world counting 458.918 new cases and causing 432.242 deaths (4.5.% of all deaths caused by cancer) in 2018 [1].
There are two main causes of PC: (i) pancreatic ductal adeno-carcinoma (PDAC), which occurs in the exocrine glands of the pancreas and is by far the most common (85.% of PC), and (ii) pancreatic neuroendocrine tumours, which arise in pancreatic endocrine tissue, are less common (<5.%) and have a better prognosis owing to its quite specific-symptoms. On the contrary, at its early stages, PDAC is usually clinically silent, and even upon progression of the neoplasm the symptoms are few and non-specific, such as weight loss, abdominal pain, jaundice, dyspepsia, light-colored stools and fatigue. PC has a very poor prognosis: the diverse and delayed symptoms of this disease are in part why 80.% of PDAC patients are usually diagnosed at an advanced or metastatic stage of disease, when the 5-year survival is less than 10.% [2]. At these stages, in fact, only 10–20.% of PDAC patients are treatable with surgery. When PC is identified in stage I, patients have a 5-year survival rate of 25.%, having a chance of successful resection and possible cure [3]. Surgery, chemotherapy and radiotherapy are traditionally used to extend survival and/or relieve the patients’ symptoms but for advanced stage PC cases, there is still no definitive treatment pathway. Indeed, in spite of recent progress in the clinical management of PC, its overall survival rate has not raised during the last two decades [4]. Therefore, PC still represents a major challenge for both research studies and clinical management.
Challenges in PC detection
Detection of PC at the early stages remains a great challenge because of a lack of specific detection tests. To date, there are several imaging tools available, such as abdominal ultrasonography, multidetector computed tomography (the standard for diagnosis), magnetic resonance imaging and endoscopic ultrasound-guided fine-needle aspiration for cytological diagnosis (which has a sensitivity reported to be about 80.%). Each of these techniques has its own advantages and disadvantages but are all susceptive to operator variability and the change in use of various diagnostic modalities differs between developed and undeveloped countries. Since the available diagnostic tests are non-specific and may miss patients with early-stage disease, many studies and clinical trials have sought to identify an inexpensive and minimally invasive biomarker with high sensitivity and specificity for PC to improve early diagnosis and subsequent treatment [2]. Many investigations have been conducted to find an appropriate serum biomarker. At present several tumour markers, such as CA 19-9, CA242, carcino-embryonic antigen (CEA), have been proposed for PC management, even if their benefits remain unclear: although sensitivity is increased, specificity is often sub-optimal [5]. The recommendations in existing clinical practice guidelines on early diagnosis of PC are inconsistent: most of them endorse measuring serum CA 19-9 as a complementary test, but also stated that it is not useful for diagnosing early pancreatic cancer or for screening. For these reasons an abundance of research in recent years has focused on identifying biomarkers for PC and there is a constant ongoing effort to identify additional ones.
A novel serum biomarker: protein induced by vitamin K absence II
In a recent preliminary study investigating a novel serum biomarker in PC, we reported for the first time that protein induced by vitamin K absence II (PIVKA-II) is significantly increased in a cohort of Italian PC patients. PIVKA-II, also known as des-gamma-carboxy prothrombin, is released by the liver in situations of vitamin K insufficiency or as consequence of an acquired defect in the post-translational carboxylation of the prothrombin precursor in cancer cells [6]. PIVKA-II is an abnormal prothrombin containing some glutamic acid (Glu) residues. When prothrombin is generated by liver cells under conditions of reduced vitamin K or in the presence of a vitamin K antagonist, the Glu residue at the N-terminal of the prothrombin precursor is not completely converted to carboxyglutamic acid (Gla) by gamma-carboxylase. Until the 1980s, the resulting protein, PIVKA-II, was mainly used as an indicator of blood coagulability and currently it is a useful tool for detecting subclinical vitamin K deficiency, as it increases before the prothrombin time is affected [7]. Since Liebman et al. in 1984 reported detection of a high rate of serum PIVKA-II in hepatocellular carcinoma (HCC) patients, this biomarker has been widely used for this neoplasm and it presently represents an important tool in its diagnosis and prognosis [8 and references therein]. Additionally, a rise in PIVKA-II above normal limits has been recently reported in literature (mainly from Japan) not only in HCC but also in other gastrointestinal malignancies, including PC. The mechanism of PIVKA-II production in non-HCC tumours is currently unknown. Hepatoid differentiation of tumours has been speculated as one of the mechanisms of PIVKA-II production. However, it has been showed that 10 of 23 reported cases of PIVKA-II-producing tumours (43.4.%) did not have hepatoid differentiation. It has been demonstrated that PIVKA-II-producing tumours show a high rate of liver metastasis and poor prognosis [9]. It is widely assumed that the pancreas and the liver retain a latent capability to transdifferentiate into the other tissue because they originate from the primitive foregut of the embryo [10]. Therefore, PIVKA-II expression, which is characteristic of HCC, can reasonably be present in PC, even if the mechanism of PIVKA-II pancreatic production is still unknown. Recently there is rising attention on the relationship between vitamin K and malignancy: several large population studies have established a relationship between vitamin K intake and cancer mortality. A number of studies have shown a cytotoxicity of vitamin K towards cancer cells. Various mechanisms, responsible for cell growth arrest and suppression of proliferation by vitamin K, have been described, however, almost all of them are focused on modulation of redox balance and induction of oxidative stress in cancer cells due to the quinone structure of vitamin K. In vitro and in vivo studies have suggested anti-carcinogenic effects exerted by vitamin K both directly (given its ability to suppress cancer growth, induce apoptosis and differentiation in cancer cells) and indirectly through post-translational activation of proteins such as PIVKA-II [11]. Multiple studies have recently evaluated the role of vitamin K against PC cell oncogenesis and it has been lately confirmed that apoptosis is mainly involved in vitamin K -induced pancreatic PC cell death. It has been demonstrated that vitamin K causes apoptosis of pancreatic cancer cells through either caspase-dependent mechanisms and induction of ERK phosphorylation, or via intracellular calcium, reactive oxygen species, and wild-type p53, respectively [12].
Pilot study
According to all these recent findings, our study was the first to investigate the role of PIVKA-II as a new biomarker for PC [13]. In this aim, a total of 46 Caucasian patients, 26 with PC (Group 1) and 20 with benign pancreatic diseases (Group 2), matched for age and sex, were enrolled from subjects attending the laboratory of Tumour Markers of the Policlinico Umberto I, Sapienza University of Rome. We aimed to evaluate PIVKA-II performance in comparison to established PC biomarkers CA 19-9, CEA and CA242. PIVKA-II and CEA serum levels were measured on LUMIPULSE G1200 (Fujirebio-Europe), an assay system based on chemiluminescent enzyme immunoassay (CLEIA) technology by a two-step sandwich in immunoreaction cartridges [14]. Serum CA 19-9 was measured by a RIA method ELSA (CisBio Bioassays), a solid-phase two-step sandwich immunometric assay, whereas CA242 levels were determined by an enzyme immunoassay (EIA) technique (Fujirebio Diagnostics AB), a solid-phase, non-competitive immunoassay. All assays were performed according to the manufacturers’ instructions and cut-offs of normality were considered as 6|ng/ml, 37|U/ml, 16|U/ml, and 48|mAU/ml, respectively for CEA, CA 19-9, CA242 and PIVKA-II. In PC patients, we observed high levels of PIVKA-II in the highest percentage of cases compared to other biomarkers, while in patients with benign pancreatic diseases PIVKA-II was increased in the smallest percentage of subjects compared to CA 19-9, CA242 and CEA. All markers showed statistically significant differences between Group 1 and Group 2 patients (Fig. 1). Diagnostic performance of the markers in discriminating malignant from benign gynecologic conditions was verified using receiver operator characteristic (ROC) curve analysis. The PIVKA-II ROC curve analysis showed the best specificity and sensitivity in comparison with CEA, CA 19-9 or CA242 (Table 1). This pilot study showed that PIVKA-II is significantly higher in PC than in benign pancreatic disease: as a serum tumour marker it demonstrated a rather good diagnostic performance compared to traditional PC biomarkers like CA 19-9, CEA and CA242, being less prone to elevation in patients with non-neoplastic pancreatic diseases.
Summary
Our results are promising as the discovery of a biomarker that would facilitate earlier identification of PC would greatly affect patient management and prognosis. Moreover, it has been reported that a combination of serum biomarkers increases specificity and sensitivity of the individual test: in this aim the detection of a biomarker to complement others in diagnostic accuracy would be of real importance for facilitating PC detection [15]. In conclusion, our study suggested that including serum PIVKA-II measurement in the diagnostic work-up for PC could be considered a valuable additional tool in clinical practice.
References
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2. Rawla P, Sunkara T, Gaduputi V. Epidemiology of pancreatic cancer: global trends, etiology and risk factors. World J Oncol 2019; 10(1): 10–27.
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6. Dahlberg S, Nilsson CU, Kander T, Schött U. Detection of subclinical vitamin K deficiency in neurosurgery with PIVKA-II. Scand J Clin Lab Invest 2017; 77: 267–274.
7. Dauti F, Hjaltalin Jonsson M, Hillarp A, Bentzer P, Schött U. Perioperative changes in PIVKA-II. Scand J Clin Lab Invest 2015; 75(7): 562–567.
8. Viggiani V, Palombi S, Gennarini G, D’Ettorre G, De Vito C, Angeloni A, Frati L, Anastasi E Protein induced by vitamin K absence or antagonist-II (PIVKA-II) specifically increased in Italian hepatocellular carcinoma patients. Scand J Gastroenterol. 2016; 51: 1257–1262.
9. Kurohama H, Mihara Y, Izumi Y, Kamata M, Nagashima S, Komori A, Matsuoka Y, Ueki N, Nakashima M, Ito M. Protein induced by vitamin K absence or antagonist II (PIVKA-II) producing large cell neuroendocrine carcinoma (LCNEC) of lung with multiple liver metastases: a case report. Pathol Int 2017; 67(2): 105–109.
10. Gordillo M, Evans T, Gouon-Evans V. Orchestrating liver development. Development 2015; 142: 2094–108.
11. Dahlberg S, Ede J, Schött U. Vitamin K and cancer. Scand J Clin Lab Invest 2017; 77: 555–567.
12. Davis-Yadley AH, Malafa MP. Vitamins in pancreatic cancer: a review of underlying mechanisms and future applications. Adv Nutr 2015; 6: 774–802.
13. Tartaglione S, Pecorella I, Zarrillo SR, Granato T, Viggiani V, Manganaro L, Marchese C, Angeloni A, Anastasi E. Protein Induced by Vitamin K Absence II (PIVKA-II) as a potential serological biomarker in pancreatic cancer: a pilot study. Biochem Med (Zagreb) 2019; 29(2): 020707.
14. Falzarano R, Viggiani V, Michienzi S, Longo F, Tudini S, Frati L, Anastasi E. Evaluation of a CLEIA automated assay system for the detection of a panel of tumor markers. Tumour Biol 2013; 34(5): 3093–3100.
15. Zhang Y, Yang J, Li H, Wu Y, Zhang H, Chen W. Tumor markers CA 19.9, CA242 and CEA in the diagnosis of pancreatic cancer: a meta-analysis. Int J Clin Exp Med 2015; 8(7): 11683–11691.
The authors
Sara Tartaglione*1 MD, Antonio Angeloni2 BS, Prof. Emanuela Anastasi1 BS
1 Department of Molecular Medicine, Policlinico Umberto I, University of Rome “Sapienza”, Rome, Italy
2 Department of Experimental Medicine, Policlinico Umberto I, University of Rome “Sapienza”, Rome, Italy
*Corresponding author
E-mail: sara.tartaglione@uniroma1.it
Biomarkers for improving lung cancer diagnosis, prognosis and treatment
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by Managing Editor Alison Sleigh PhD
Background
In the UK, lung cancer is the second most common cancer in both men and women, accounting for 13.% of all new cancer cases in 2016 [1], and is the leading cause of death from cancer [2]. These statistics are also reflected in the United States of America [3]. In the UK, 5-year survival rates are low; on average, around 9.%. This is mainly because the majority of patients are already at late stage or metastatic disease at the point of diagnosis [1]. The main cause of lung cancer by far is smoking tobacco cigarettes. However, there are a number of other risk factors that should not be ignored. Environmental risk factors include exposure to radon, asbestos, pollution/poor air quality as well as infection. Genetics also plays a part because not all smokers develop lung cancer and a family history of the disease increases risk by around twofold [3]. In addition, genome-wide association studies have identified a number of chromosome regions that are associated with increased risk of lung cancer. Some of the first regions found have the strongest associations and include 5p15, 15q25-26 and 6p21. Mutations in the 15q25-26 region are linked to increased nicotine dependence and susceptibility for lung cancer. The 5p15 region contains the gene for telomerase reverse transcriptase, and mutations within this gene have been associated with adenocarcinomas in both smokers and non-smokers. Single nucleotide polymorphisms in the BAG6 gene on 6p21 are strongly associated with squamous cell carcinoma (see Bossé and Amos 2018 for a thorough review [4]). Interestingly, although smoking is the major primary cause of lung cancer, around 10–15.% of lung cancer patients have never smoked. Lung cancer in never smokers seems to occur most often in women and younger patients, involving
specific driver mutations such as in epidermal growth factor reductase (EGFR) gene and the echinoderm microtubule-associated protein-like 4 (EML4)–anaplastic lymphoma kinase (ALK) gene fusion, which gives rise to the ELM4-ALK fusion protein.
Diagnosis of lung cancer
Diagnosis of lung cancer usually occurs after a patient presents at a GP clinic with symptoms that can commonly include:
• a persistent cough
• coughing up blood
• persistent breathlessness
• unexplained tiredness and weight loss
• an ache or pain when breathing or coughing.
After this, diagnosis is confirmed by imaging (chest X-ray and then CT scan, and possibly also a PET-CT scan) and biopsy to confirm staging [5].
The challenges with diagnosis are that the early stages of the disease are symptomless; once symptoms become apparent, diagnosis often confirms late stage/metastatic disease, which has low survival rates. In addition, the methods of diagnosis are fairly invasive.
Screening programmes
Low-dose computed tomography (LDCT) screening of people with a higher risk of lung cancer has been trialled but has given with mixed results. Three smaller scale European trials showed non-significant effects or even an increase in mortality [6]. The largest trial, the National Lung Screening Trial, in the USA, showed much more promise with a 20.% reduction in lung cancer mortality [7]. However, the authors also reported an 18.% overdiagnosis rate: of the 24.2.% of patients classified as positive, 96.4.% were actually false positives. This means that 320 people need to be screened to prevent 1 lung cancer death, representing an unacceptable level of screening rounds, exposure to radiation, increased patient anxiety and costs.
Biomarkers
The use of biomarkers could, therefore, be a useful, non-invasive adjunct for identifying true/false positives from initial LDCT screening. Biomarkers can be non-invasively collected, and can come from the tumour itself, the tumour microenvironment as well as the host’s response to the tumour. Properly developed and validated, biomarkers can be diagnostic, prognostic and useful for monitoring therapy. There is, needless to say, a vast amount of research being done to discover such biomarkers for lung cancer and it is outwith the scope of this article to review it all. We will, however, discuss certain aspects of showing promise.
TRACERx: understanding the genetic development of lung cancer with circulating tumour DNA
TProfessor Charles Swanton at the Francis Crick Institute in London, UK, and his team have been analysing circulating tumour DNA (ctDNA) from individual non-small-cell lung cancer (NSCLC) patients through time, mapping the genetic evolution of the disease in a study known as TRACERx [Tracking NSCLC Evolution Through Therapy (Rx)]. In 2017, the initial results of 100 patients from a target group of 842 were published [8]. They found that although driver mutations in EGFR, MET, BRAF, and TP53 were almost always clonal, the heterogeneous driver alterations that occurred later in evolution (found in more than 75.% of the tumours) were common in PIK3CA and NF1 and in genes involved in chromatin modification and DNA damage response and repair. They also found that chromosomal instability was associated with intratumour heterogeneity and that elevated copy-number heterogeneity was associated with a significant increase in risk of recurrence or death [8]. Results from a more recent paper from the same consortium suggest that the immune microenvironment exerts a strong selection pressure in early-stage, untreated NSCLCs that produces multiple routes to immune evasion, which indicates a poor prognosis [9].
Early detection of Cancer of the Lung Scotland: diagnosing lung cancer at an earlier stage with a tumour-associated autoantibodies
The Early detection of Cancer of the Lung Scotland (ECLS) study has just this month made public the results of their randomized controlled trial of Oncimmune’s EarlyCDT®–Lung test on over 12.000 volunteers in Scotland (NHS areas of Tayside, Greater Glasgow and Clyde, and Lanarkshire) [10]. The EarlyCDT®–Lung test is a commercially available ELISA-based blood test that measures a panel of seven tumour-associated autoantibodies: p53, NY-ESO-1, CAGE, GBU4–5, SOX2, HuD and MAGE A4. The volunteers were asymptomatic adults aged between 50 amd 75 who had a high risk of developing lung cancer over the next 24 months. Participants who tested positive were followed up with chest X-ray and non-contrast CT scan. During the study period 127 participants were diagnosed with lung cancer; 41.% of patients from the intervention group who went on to develop cancer were diagnosed with early-stage cancer compared with only 26.8.% from the control group. The results showed that using a combination of the blood test with CT imaging gave a significant decrease in the late-stage diagnosis of lung cancer. The patients will now be followed over the next 5 years to determine mortality outcomes.
Immune checkpoint inhibitor therapy: biomarkers for better patient selection
In recent years, immune checkpoint inhibitor (ICI) therapy has been revolutionizing cancer treatment. This ‘cancer immunotherapy’ uses monoclonal antibodies that typically target programmed death 1 (PD-1), programmed death-ligand 1 (PD-L1), or cytotoxic T-lymphocyte-associated protein 4 (CTLA-4), allowing the restoration of the cytotoxic immune response. However, while some patients respond very well to ICIs, many do not and even go on to develop hyper-progressive disease or immune-related adverse events. Hence, there is a need for biomarkers to aid the selection of patients who will benefit from this treatment. The recent review by Costantini et al. [11] discusses progress that is being made with a variety of types of biomarkers for this purpose, including soluble PD-L1, other soluble proteins (granzyme B, PD-L2, interleukine 2, interferon-gamma), ctDNA, the tumour mutational burden as well as effects of the gut microbiome.
Future perspectives
The work discussed here suggests that very positive steps can be taken towards reducing the mortality rate from lung cancer – probably not from any one aspect alone, but by using many approaches in combination: better biomarker testing will allow an initial screening and improvements in the analysis of CT scans (such as by artificial intelligence [12]) will both help to reduce rates of false positives and minimize the need for unnecessary invasive biopsies. These kinds of improvements may help to generate more cost-effective screening therefore encourage increased role out of lung cancer screening programmes. The rise in popularity of electronic nicotine delivery systems (vaping), particularly in the under 35s, is often thought of as a ‘safe’ way to smoke. However, there have now been 450 cases of a vaping-linked lung illness in the USA, perhaps heralding a need for a different sort of biomarker.
References
1. Lung cancer statistics. Cancer Research UK
(https://www.cancerresearchuk.org/health-professional/cancer-statistics/statistics-by-cancer-type/lung-cancer)
2. Smittenaar CR, Petersen KA, Stewart K, Moitt N. Cancer incidence and mortality projections in the UK until 2035. Br J Cancer 2016; 115(9): 1147–1155.
3. de Groot PM, Wu CC, Carter BW, Munden RF.
The epidemiology of lung cancer. Transl Lung Cancer Res. 2018; 7(3): 220–233.
4. Bossé Y, Amos C. A decade of GWAS results in lung cancer. Cancer Epidemiol Biomarkers Prev 2018; 27(4): 363–379.
5. Lung cancer: diagnosis. NHS website 2019.
(https://www.nhs.uk/conditions/lung-cancer/diagnosis/).
6. Sozzi G, Boeri M. Potential biomarkers for lung cancer screening. Transl Lung Cancer Res 2014; 3(3): 139–148.
7. National Lung Screening Trial Research Team, Aberle DR, Adams AM, Berg CD, Black WC, Clapp JD, Fagerstrom RM, Gareen IF, Gatsonis C, et al. Reduced lung cancer mortality with low-dose computed tomographic screening. N Engl J Med 2011; 365(5): 395–409.
8. Jamal-Hanjani M, Wilson GA, McGranahan N, Birkbak NJ, Watkins TBK, Veeriah S, Shafi S, Johnson DH, Mitter R, et al. Tracking the evolution of non-small cell lung cancer.
N Engl J Med 2017; 376(22): 2109–2121.
9. Rosenthal R, Cadieux EL, Salgado R, Bakir MA, Moore DA, Hiley CT, Lund T, Tanić M, Reading JL, et al. Neoantigen-directed immune escape in lung cancer evolution. Nature 2019; 567(7749): 479–485.
10. Sullivan F. PL02.03 – Early Detection of Cancer of the Lung Scotland (ECLS): trial results. Presented at the 2019 World Conference on Lung Cancer, Barcelona, Spain (https://library.iaslc.org/conference-program?product_id=15&author=&category=&date=2019-09-09&session_type=Plenary%20Session&session=&presentation=&keyword=sullivan&cme=undefined&).
11. Costantini A, Takam Kamga P, Dumenil C, Chinet T, Emile JF, Giroux Leprieur E. Plasma biomarkers and immune checkpoint inhibitors in non-small cell lung cancer: new tools for better patient selection? Cancers (Basel) 2019; 11(9): pii: E1269.
12. Ardila D, Kiraly AP, Bharadwaj S, Choi B, Reicher JJ, Peng L, Tse D, Etemadi M, Ye W, et al. End-to-end lung cancer screening with three-dimensional deep learning on low-dose chest computed tomography. Nat Med 2019; 25(6): 954–961.
Syndromic approach for molecular diagnostics of infectious diseases
, /in Featured Articles /by 3wmediaInfectious diseases, in particular lower respiratory infections and diarrhoea are among the top ten causes of death worldwide [1]. Many infectious diseases can be considered ‘syndromes’, i.e. a collection of symptoms that do not point to a specific causative agent. A ‘syndromic approach’ in diagnostics means to achieve, by a single test, a diagnosis without taking into account the symptoms themselves. In molecular diagnostics (MDx) of infectious diseases, this means the use of multiplex PCR panel assays to simultaneously detect genetic material from pathogens of different species and even different taxonomic levels, such as bacteria, viruses, parasites and fungi.
by Dr. Antoinette A. T. P. Brink and Dr. Guus F. M. Simons
Clinical syndromes and their causal pathogens
Respiratory infections
Respiratory infections are highly prevalent and the possible causative agents include several typical and atypical bacteria, as well as many viruses. Among the latter, the influenza virus particularly is associated with morbidity and mortality. As influenza A viruses can infect multiple hosts, including not only humans but also birds and swine, these viruses can undergo antigenic shifts that may result in pandemics such as the 2009 ‘Mexican flu’. Nowadays, many molecular diagnostics (MDx) tests are able to distinguish influenza A virus subtypes associated with non-human hosts, such as H5N7 avian flu. The presence of such a subtype in a patient with respiratory illness may be indicative for zoonosis, which increases the risk of a new pandemic. Besides influenza A virus, syndromic MDx panels generally detect influenza B virus, respiratory syncytial virus (RSV) A and B, adenovirus (AdV), human metapneumovirus (hMPV), parainfluenza virus (PIV) types 1–4 and human coronavirus (hCoV) types 229E, OC43, NL63 and HKU1, rhinovirus (RV) and enterovirus (EV). Bocavirus is not always included, although it is considered an important pathogen especially in children [2].
The merit of testing a broad respiratory panel is illustrated by a study conducted by the Dutch National Institute for Public Health and the Environment (RIVM) among community-dwelling elderly receiving annual influenza vaccination. Vaccine effectiveness was studied by determining the relative contribution of influenza and other respiratory pathogens to influenza-like illness (ILI), using an assay that detects 22 respiratory pathogens simultaneously (RespiFinder®) As expected, vaccination reduced the incidence of influenza, but the number of ILI episodes was similar between vaccinated and non-vaccinated individuals; non-Influenza viruses replaced influenza as a cause of ILI in vaccinated individuals [3].
This finding is in line with the recommendation of the American Society for Microbiology working group for respiratory virus testing not to restrict testing for specific respiratory viruses during certain seasons because with global travel, many ‘seasonal’ viruses can cause disease throughout the year [4]. Moreover, testing should not be restricted to certain patient populations, e.g. testing for RSV/hMPV only in children, because these viruses may cause severe disease in adults as well.
For fastidious bacteria causing atypical pneumonia, such as Legionella pneumophila, Chlamydophila pneumoniae, Mycoplasma pneumoniae, Bordetella pertussis and B. parapertussis, MDx has largely replaced conventional culture.
For Streptococcus pneumoniae and other bacteria associated with typical pneumonia, PCR is not ready to replace conventional culture because these bacteria belong to the normal oral flora. Quantitative detection is necessary to distinguish colonization from infection, but sample quality criteria for this are lacking. Moreover, conventional culture remains necessary for antimicrobial susceptibility testing.
Gastroenteritis
Most gastroenteritis (GE) panels detect an extensive list of viruses, bacteria and parasites. It is self-evident that GE viruses such as norovirus, adenovirus types 40 and 41, and rotavirus are included. Although the clinical course of sapovirus and human astrovirus (HAstV) infections is generally milder than that of, for example, norovirus, they should be included in routine testing to identify outbreaks and to ensure proper care of patients at risk for severe complications, such as infants, elderly and immuno-compromised patients.
The parasites Cryptosporidium spp., Entamoeba histolytica and Giardia lamblia are included in all GE panels because their pathogenicity is well established. For Dientamoeba fragilis this is still a matter of debate, but it should be considered the causal factor of GE symptoms – and treated appropriately – after other causes have been excluded [5].
Regarding GE-causing bacteria, most commercially available tests detect the most common pathogenic Escherichia coli types (EHEC, ETEC, STEC, EPEC, and IEIC), Salmonella spp. and Yersinia enterocolitica. For Campylobacter it is important to detect all pathogenic species, i.e. C. jejuni, C. coli, C. upsaliensis and C. lari [6], the latter two of which are not included in some commercially available assays.
In contrast, for Vibrio cholerae it is important to distinguish the only two serotypes that can cause outbreaks and infections should be treated actively, to avoid overtreatment.
Additional pathogens may be included, but test results may be difficult to interpret owing to (i) frequent contamination from the environment or reagents (e.g. Aeromonas spp.) or (ii) conflicting data regarding pathogenicity (e.g. Plesiomonas shigelloides).
Infections of the central nervous system
The differential diagnosis for patients with suspected meningitis/encephalitis (ME) includes infectious as well as non-infectious causes that cannot be distinguished on the basis of symptoms. A recent study showed that in the majority of patients suspected of a central nervous system (CNS) infection, the etiology could not be found even with the most comprehensive commercially available MDx tests [7]. In line with this, the use of a diagnostic stewardship approach is advocated, including cerebrospinal fluid white blood cell count to prevent unnecessary use of expensive tests [8]. Having said that, true CNS infections are medical emergencies that require rapid pathogen identification to allow timely and appropriate clinical intervention.
Causative agents of CNS infections include enteroviruses, parechoviruses and all eight members of the human herpes virus family, the most prevalent of which are herpes simplex virus types 1 and 2. The other herpes viruses, such as cytomegalovirus, varicella-zoster virus, human herpes virus 6, and Epstein-Barr virus, are mostly seen in immunocompromised patients such as transplant recipients [9].
Measles and mumps viruses may also cause CNS infections, especially in regions where vaccine coverage is sub-optimal due to, for example, a weak healthcare system or vaccine refusal. Of note, when this paper was drafted, four countries in the European Union (Albania, Czechia, Greece and the United Kingdom) had recently lost their measles elimination status previously assigned by the World Health Organization.
Meningitis-causing bacteria usually belong to the normal oropharyngeal flora, and may enter the CNS by anatomic defects in the natural barriers, or defects in the immune system. Streptococcus pneumoniae is the most common cause of community-acquired meningitis in non-neonates worldwide, followed by Neisseria meningitidis. Other bacterial pathogens in meningitis are Listeria monocytogenes, Haemophilus influenzae, Staphylococcus aureus and Borrelia burgdorferi.
Vaccination campaigns against various serogroups of these bacteria are ongoing. As serogroup replacement occurs as a consequence of vaccination, it is important that in vitro diagnostic kits detect all serogroups, and that assay designs are checked periodically for this.
Sexually transmitted infections
Various bacteria, parasites and viruses can cause sexually transmitted infections (STI).
STIs can be non-symptomatic, but if left untreated they can have severe sequelae including permanent infertility or ectopic pregnancy.
STI test panels should at least include Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis. For a syndromic approach, herpes simplex virus types 1 and 2 and Treponema pallidum should be included.
The editorial board of the European STI Guidelines recommends not to test routinely for Mycoplasma hominis and Ureaplasma parvum. In addition, in men with symptomatic urethritis, Ureaplasma urealyticum should only be treated after C. trachomatis, N. gonorrhoeae and M. genitalium have been excluded [10].
Several subtypes of STI require non-standard therapy. For example, the ‘L’ serovars of C. trachomatis can cause lymphogranuloma venereum (LGV) and proctitis, which should be treated differently from other C. trachomatis infections [11].
Furthermore, N. gonorrhoeae strains have emerged that are resistant to all antimicrobials used for treatment, owing to point mutations and/or genetic recombination with commensal Neisseria species [12]. In addition, the change from doxycycline to azithromycin as the first-line treatment for C. trachomatis and non-gonococcal urethritis has resulted in selection of macrolide-resistant M. genitalium strains [13]. Fortunately, such pathogen properties can be distinguished by molecular methods and, in fact, several commercially available multiplex MDx assays can do this already.
MDx methods for syndromic approach
Laboratory-developed tests
Laboratory-developed PCR assays for infectious diseases have been in use since the early 1990s [14].
Generally, laboratory-developed tests (LDTs) run on conventional 96-well realtime PCR systems, allowing the testing of large batches of samples. Most LDTs are TaqMan-based, generally limiting multiplexing to four targets per reaction. A syndromic approach is, therefore, only possible by running several reactions (up to eight to cover a full respiratory panel) per sample, which limits throughput.
Although the cost-of-goods for LDTs is low, testing can be laborious and time-consuming and requires highly skilled laboratory personnel.
Commercial: medium to high throughput
Clinical laboratories needing a syndromic approach and medium to high throughput may use commercial assays instead of LDT. To exceed the multiplexing capacity of LDTs, manufacturers have developed proprietary methods. For example, Luminex’s platform uses fluorescently labelled bead array technology with dedicated instruments. Korean Seegene’s assays include melting curve analysis or differential detection temperatures to allow distinction of multiple targets per fluorescent channel.
Commercial: random access/low throughput
Examples of commercial random access systems are the FilmArray by bioMérieux, the QIAstat-Dx by QIAGEN and the ePlex by GenMark. The high price for the dedicated instruments and high price-per-test can be an argument against implementation in routine use if large amounts of samples are processed. Hence, these systems are particularly suitable for point-of-care purposes or when laboratory skills of personnel are limited. However, the ease-of-use may conceal to inexperienced users that these tests are actually very sensitive, and careful reaction set-up and cleanliness of the environment are needed to avoid false positives. It is self-evident that (unexpected) positive results should be interpreted in the context of clinical symptoms. Moreover, the ISO 15189 standard for medical laboratories recommends users to run additional control materials from independent third parties.
Summary
A syndrome-based approach using broad panel MDx assays may assist in timely diagnosis of respiratory infections, GE, CNS infections and STI.
Syndrome-based MDx results in a decrease in the number of chest radiographs, reduced admission rates, fewer barrier nursing days [15], shorter duration of hospitalization, more appropriate prescription of antivirals, better antibiotic stewardship and decreased duration of antimicrobial use [16–18], which is likely to result in less antibiotic resistance in the long term.
In addition, broad panel tests provide useful information about epidemiology, seasonality and possibly clinically relevant co-infections
Figure 1: Example of high multiplexing (the 2SMARTFinder® principle of Dutch firm PathoFinder)
Schematic representation of the PathoFinder 2SMARTFinder® test principle: (a) Step 1 (pre-amplification): specific multiplex target enrichment. (b) Step 2 (2SMART reaction): signal amplification by means of 2SMART primers each consisting of a targetspecific sequence (yellow/blue) and a universal sequence (grey/green). Each reverse 2SMART primer contains a stuffer/barcode sequence (red) for detection. The combined action of the 2SMART primers and the universal primers, of which the reverse is labelled with fluorescein amidites (FAM), results in the generation of (c) a FAM-labelled PCR product. (d) The reaction mixture contains labelled SMART probes complementary to each stuffer. (e) When a probe hybridizes to its corresponding stuffer in the reaction product, the FAM label acts as a Förster Resonance Energy Transfer (FRET) donor and the label in the SMART probe as an acceptor, resulting in the emission of light that can be measured in real-time. Finally, the temperature is increased, resulting in dissociation of the probe-stuffer hybrid and a sharp decline in fluorescence (f). The negative derivative of this graph shows the actual melting peaks (g). The stuffer/probe sequence determines the position of the melting peak and reveals which pathogen was present in the sample. −Δ(F)/dT, negative derivative of the change in fluorescence (F) as a function of temperature (T).
Figure 2: Typical result read-out on LightCycler 480
Example of the read-out of the RespiFinder® 2SMART assay mix 1 in the carboxyrhodamine (ROX™) channel of a LightCycler 480 II real-time PCR instrument. AdV, adenovirus; hMPV, human metapneumovirus; Inf A, influenza A virus; Inf B, influenza B virus; RSVA, respiratory syncytial virus A; RSVA, respiratory syncytial virus B
References
1. Lozano R, Naghavi M, Foreman K, Lim S, Shibuya K, Aboyans V, Abraham J, Adair T, Aggarwal R, et al. Global and regional mortality from 235 causes of death for 20 age groups in 1990 and 2010: a systematic analysis for the Global Burden of Disease Study 2010. The Lancet 2012; 380: 2095–2128.
2. Ma X, Conrad T, Alchikh M, Reiche J, Schweiger B, Rath B. Can we distinguish respiratory viral infections based on clinical features? A prospective pediatric cohort compared to systematic literature review. Rev Med Virol 2018; 28: e1997.
3. van Beek J, Veenhoven RH, Bruin JP, van Boxtel RAJ, de Lange MMA, Meijer A, Sanders EAM, Rots NY, Luytjes W. Influenza-like illness incidence is not reduced by influenza vaccination in a cohort of older adults, despite effectively reducing laboratory-confirmed influenza virus infections. J Infect Dis 2017; 216: 415–424.
4. Ginocchio CC, McAdam AJ. Current best practices for respiratory virus testing. J Clin Microbiol 2011; 49: S44–S48.
5. Van Gestel RSFE, Kusters JG, Monkelbaan JF. A clinical guideline on Dientamoeba fragilis infections. Parasitology 2018; 1–9.
6. Klena JD, Parker CT, Knibb K, Ibbitt JC, Devane PML, Horn ST, Miller WG, Konkel ME. Differentiation of Campylobacter coli, Campylobacter jejuni, Campylobacter lari, and Campylobacter upsaliensis by a multiplex PCR developed from the nucleotide sequence of the lipid A gene lpxA. J Clin Microbiol 2004; 42: 5549–5557.
7. Sall O, Thulin Hedberg S, Neander M, Tiwari S, Dornon L, Bom R, Lagerqvist N, Sundqvist M, Molling P. Etiology of central nervous system infections in a rural area of Nepal using molecular approaches. Am J Trop Med Hyg 2019; 101(1): 253–259.
8. Messacar K, Robinson CC, Dominguez SR. Letter to the editor: economic analysis lacks external validity to support universal syndromic testing for suspected meningitis/encephalitis. Future Microbiol 2018; 13: 1553–1554.
9. Chadwick DR. Viral meningitis. Br Med Bull 2005; 75–76: 1–14.
10. Horner P, Donders G, Cusini M, Gomberg M, Jensen JS, Unemo M. Should we be testing for urogenital Mycoplasma hominis, Ureaplasma parvum and Ureaplasma urealyticum in men and women? – a position statement from the European STI Guidelines Editorial Board. J Eur Acad Dermatol Venereol 2018; 32: 1845–1851.
11. Ceovic R, Gulin SJ. Lymphogranuloma venereum: diagnostic and treatment challenges. Infect Drug Resist 2015; 8: 39–47.
12. Ameyama S, Onodera S, Takahata M, Minami S, Maki N, Endo K, Goto H, Suzuki H, Oishi Y. Mosaic-like structure of penicillin-binding protein 2 gene (penA) in clinical isolates of Neisseria gonorrhoeae with reduced susceptibility to cefixime. Antimicrob Agents Chemother 2002; 46: 3744–3749.
13. Jensen JS. Mycoplasma genitalium: yet another challenging STI. Lancet Infect Dis 2017; 17: 795–796.
14. Claas HC, Wagenvoort JH, Niesters HG, Tio TT, Van Rijsoort-Vos JH, Quint WG. Diagnostic value of the polymerase chain reaction for Chlamydia detection as determined in a follow-up study. J Clin Microbiol 1991; 29: 42–45.
15. Goldenberg SD, Bacelar M, Brazier P, Bisnauthsing K, Edgeworth JD. A cost benefit analysis of the Luminex xTAG Gastrointestinal Pathogen Panel for detection of infectious gastroenteritis in hospitalised patients. J Infect 2015; 70: 504–511.
16. Rappo U, Schuetz AN, Jenkins SG, Calfee DP, Walsh TJ, Wells MT, Hollenberg JP, Glesby MJ. Impact of early detection of respiratory viruses by multiplex PCR assay on clinical outcomes in adult patients. J Clin Microbiol 2016; 54: 2096–2103.
17. Andrews D, Chetty Y, Cooper BS, Virk M, Glass SK, Letters A, Kelly PA, Sudhanva M, Jeyaratnam D. Multiplex PCR point of care testing versus routine, laboratory-based testing in the treatment of adults with respiratory tract infections: a quasi-randomised study assessing impact on length of stay and antimicrobial use. BMC Infect Dis 2017; 17: 671–671.
18. Echavarria M, Marcone DN, Querci M, Seoane A, Ypas M, Videla C, O’Farrell C, Vidaurreta S, Ekstrom J, Carballal G. Clinical impact of rapid molecular detection of respiratory pathogens in patients with acute respiratory infection. J Clin Virol 2018; 108: 90–95.
The authors
Antoinette A.T.P. Brink* PhD, Guus F.M.
Simons PhD PathoFinder B.V., 6229 EG Maastricht,
The Netherlands
*Corresponding author
E-mail: antoinette.brink@pathofinder.com
Evidence MultiSTAT – An Evidence Series immunoanalyser
, /in Featured Articles /by 3wmediaOffering the most advanced screening technology on the market, Randox has transformed the landscape of emergency point-of-care testing with innovative drug of abuse (DoA) and stroke diagnosis and detection.
The Evidence MultiSTAT – Utilizing Randox’s Biochip Technology
Designed to work across a wide variety of matrices, this revolutionary multi-analyte testing platform allows clinicians to achieve a complete immunoassay profile in the initial screening phase. Offering the most advanced screening technology on the market, Randox has transformed the landscape of emergency point of care testing. The Randox drugs of abuse Biochip Arrays form part of an unrivalled toxicology test menu capable of detecting over 500 drugs and drug metabolites along with the new addition of the Randox Stroke Biochip. Moving away from traditional single analyte assays, Biochip Technology boasts cutting-edge multiplex testing capabilities providing rapid and accurate detection and diagnosis from a single sample.
Based on ELISA principles, the biochip is a solid-state device with discrete test sites onto which antibodies specific to different compounds are immobilized and stabilized, offering a highly sensitive screen.
Using chemiluminescence as a measurement principle, the Evidence MultiSTAT consistently delivers accurate results and offers a highly sensitive method of detecting and diagnosing. Biochip Technology has a proven high standard of accurate test results with CVs typically <10% with a 98% agreement with confirmatory methods.
Each biochip can have up to 49 Discrete Test Regions (DTR). This means that up to 44 tests can be carried out simultaneously. The additional DTRs are reserved for internal quality control and visual reference, a unique Biochip Array Technology feature.
Rapid, accurate stroke diagnosis and differentiation
A ground-breaking new test which improves the accuracy of stroke diagnosis has been developed by Randox scientists. The Randox Stroke Biochip is a rapid and highly sensitive blood test that will complement and enhance existing CT scanning technology to facilitate accurate classification of stroke patients and improve patient care pathways.
With a unique ability to differentiate between ischemic and hemorrhagic stroke, the Randox Stroke Biochip takes less than 30 minutes to complete – making sure patients get the right diagnosis as fast as possible.
Dr Peter FitzGerald, Managing Director of Randox Laboratories, whose team developed the test, commented:
“There is great tragedy in the fact that the majority of stroke damage can be minimized if intervention is delivered on time, yet too often the window closes before a diagnosis is made. For doctors, nothing is more frustrating.
“Excellent work has been undertaken to assist the public in recognizing the signs of a stroke so people can get to hospital as quickly as possible. Our stroke test is the vital next step – assisting clinicians in making a rapid diagnosis and
differentiation between hemorrhagic and ischemic stroke, so their patients get the right treatment at the right time.”
Using Randox revolutionary patented biochips, the Randox Stroke Biochip provides a unique solution for simultaneous detection of multiple stroke biomarkers from a single sample, facili-
tating fast and accurate classification of stroke patients in an emergency setting.
Benefits of the Randox Stroke Biochip
• Results in 30 minutes on the Evidence MultiSTAT
• Unique solution for stroke classification
• Differentiation of ischemic and
hemorrhagic stroke
• Guarantees rapid thrombolytic therapy ensuring better patient outcomes
John Lamont, R&D Director for Randox Laboratories, explained:
“While patients undergo a CT scan to confirm either the presence or lack of a hemorrhagic stroke, a blood test on the Randox Biochip can be run on our innovative point-of-care analyser, the Evidence MultiSTAT, to identify the same for an ischemic stroke.”
“For the almost 90% of stroke patients who are ruled out for hemorrhagic stroke(1), the Randox Biochip will then accelerate decision making for clinicians with regards to thrombolytic therapy.”
“Any treatment is most effective if started as soon as possible after the stroke occurs, and so every minute that passes without a diagnosis is likely to leave a permanent mark on a stroke patient’s future health and lifestyle. The vitally important diagnostic information from the Randox Stroke Biochip facilitates accurate stroke classification, directs the appropriate patient care pathway, and enables rapid thrombolytic therapy, ensuring a better patient outcome for ischemic stroke sufferers, for whom time is of the essence.”
Whilst the Randox Stroke Biochip is currently being used as complementary testing in parallel with CT scanning, Mr Lamont is confident of a more prominent role for the test in the future patient pathway:
“The Biochip has the potential to really revolutionize the stroke diagnosis pathway as we currently know it. The accessibility of this type of blood testing could potentially extend its use beyond the A&E department, to ambulances and even the home, in the form of a hand-held testing device.”
Emergency Drugs of Abuse screening
Randox are world leaders in clinical toxicology solutions with the capability of detecting over 500 drugs and drug metabolites using innovative Biochip Technology. The Evidence MultiSTAT Drugs of Abuse panels have been specifically selected to detect levels of the most common drug types, covering over 200 drugs and drug metabolites.
Designed to work across a wide variety of matrices, this revolutionary multi-analyte testing platform allows clinicians to achieve a complete immunoassay profile in the initial screening phase. Offering the most advanced screening technology on the market, Randox has transformed the landscape of drugs of abuse testing. The Randox DoA toxicology panel simultaneously detects 21 classical, prescription and synthetic drugs from a single patient sample suitable for use with urine and blood samples with as little as 200μl sample volume required.
References
1. Intercollegiate Stroke Working Party. National clinical guideline for stroke, 5th edition. London: Royal
College of Physicians 2016
The author
Martin Conway, Marketing Executive
Martin.Conway@randox.com
Randox Laboratories
55 Diamond Road
Crumlin,
County Antrim,
BT29 4QY
United Kingdom
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