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Featured Articles
SCIENTIFIC LITERATURE REVIEW: Colorectal cancer
Highly sensitive stool DNA testing of Fusobacterium nucleatum as a marker for detection of colorectal tumours in a Japanese population
Suehiro Y, Sakai K, Nishioka M, Hashimoto S, Takami T, Higaki S, Shindo Y, Hazama S, Oka M, Nagano H, Sakaida I, Yamasaki T. Ann Clin Biochem. 2016; pii: 0004563216643970. [Epub ahead of print]
BACKGROUND: Accumulating evidence shows an over-abundance of Fusobacterium nucleatum in colorectal tumour tissues. Although stool DNA testing of Fusobacterium nucleatum might be a potential marker for the detection of colorectal tumours, the difficulty in detecting Fusobacterium nucleatum in stool by conventional methods prevented further explorations. Therefore, we developed a droplet digital polymerase chain reaction (PCR) assay for detecting Fusobacterium nucleatum in stool and investigated its clinical utility in the management of colorectal tumours in a Japanese population.
METHODS: Feces were collected from 60 healthy subjects (control group) and from 11 patients with colorectal non-advanced adenomas (non-advanced adenoma group), 19 patients with colorectal advanced adenoma/carcinoma in situ (advanced adenoma/carcinoma in situ (CIS) group) and 158 patients with colorectal cancer of stages I to IV (colorectal cancer group). Absolute copy numbers of Fusobacterium nucleatum were measured by droplet digital PCR.
RESULTS: The median copy number of Fusobacterium nucleatum was 17.5 in the control group, 311 in the non-advanced adenoma group, 122 in the advanced adenoma/CIS group, and 317 in the colorectal cancer group. In comparison with that in the control group, the Fusobacterium nucleatum level was significantly higher in the non-advanced adenoma group, the advanced adenoma/CIS group and the colorectal cancer group.
CONCLUSIONS: This study illustrates the potential of stool DNA testing of Fusobacterium nucleatum by droplet digital PCR to detect individuals with colorectal tumours in a Japanese population.
A genome-wide assessment of variations of primary colorectal cancer maintained in metastases
Cai Z, Han S, Li Z, He L, Zhou J, Huang W, Xu Y. Gene 2016; 595(1): 18–24.
Colorectal cancer (CRC) is a highly heterogeneous disease that is the third leading cause of cancer-related deaths worldwide. This study presents a genome-wide assessment of variations in primary colorectal cancer maintained in metastases, even in distant metastases. The purpose of this study was to determine whether intratumor heterogeneity is related to disease progression and metastasis in CRC. The results showed that 882 single nucleotide polymorphism (SNP) associated genes and 473 copy number variant (CNV) associated genes specific to metastasis were found. In addition, 57 SNPs mapped to miRNAs showed significant differences between primary tumours and metastases. Functional annotation of metastasis-specific genes suggested that adhesion and immune regulation may be essential in the development of tumours. Moreover, the locus rs12881063 in the fourteenth chromosome was found to have a high rate of the G/C type in metastases. The rate of the G/C type in nearby lymph node metastases was 66.7%, while the rate of the G/C type in distance lymph node metastases was 83.3%. These results indicate that rs12881063 may be the basis for clinical diagnosis of CRC metastasis.
High tumour mast cell density is associated with longer survival of colon cancer patients
Mehdawi L, Osman J, Topi G, Sjölander A. Acta Oncol. 2016; 55(12): 1434–1442.
BACKGROUND: Inflammatory cells and inflammatory mediators play an important role in colorectal cancer (CRC). Previous studies have shown that CRC patients with increased expression of cysteinyl leukotriene receptor 1 (CysLTR1) have a poorer prognosis, and Cysltr1-/- mice display fewer intestinal polyps. However, the role of mast cells (MCs) in colon cancer progression remains unclear. The aim of the present study was to explore the relevance of MCs in CRC.
MATERIAL AND METHODS: A tissue microarray from 72 CRC patients was stained with MC anti-tryptase and -chymase antibodies. Mouse colon tissue was stained with MC anti-tryptase antibody. Immunohistochemistry was used to identify MCs in patients and mice.
RESULTS: Patient colon cancer tissue had in comparison with normal colon tissue a reduced number of MCs, predominantly of chymase-positive cells. Further analysis revealed that patients with a relative high MCD in their cancer tissues showed significantly longer overall survival compared to those with a low MCD [hazard ratio (HR) 0.539; 95% confidence interval (CI), 0.302–0.961]. Similar results were observed in subgroups of patients with either no distant metastasis (p = 0.004), or <75 years (p = 0.015) at time of diagnosis. Multivariate Cox analysis showed that MCD independently correlated with reduced risk of death in colon cancer patients (HR 0.380; 95% CI 0.202-0.713). Additionally, a negative correlation was found between cytoplasmic CysLTR1 expression and number of MCs. In agreement, in the CAC mouse model, Cysltr1-/- mice showed significantly higher MCs in their polyp/tumor areas compared with wild-type mice.
CONCLUSION: A high MCD in cancer tissue correlated with longer patient survival independently from other risk factors for CRC. The concept that MCs have an anti-tumor effect in CRC is further supported by the findings of a negative correlation with CysLTR1 expression in patients and a high MCD in colon polyps/tumors from CysLTR1-/- mice.
Are hemorrhoids associated with false-positive fecal immunochemical test results?
Kim NH, Park JH, Park DI, Sohn CI, Choi K, Jung YS. Yonsei Med J. 2016; 58(1):150–157.
PURPOSE: False-positive (FP) results of fecal immunochemical tests (FITs) conducted in colorectal cancer (CRC) screening could lead to performing unnecessary colonoscopies. Hemorrhoids are a possible cause of FP FIT results; however, studies on this topic are extremely rare. We investigated whether hemorrhoids are associated with FP FIT results.
MATERIALS AND METHODS: A retrospective study was conducted at a university hospital in Korea from June 2013 to May 2015. Of the 34,547 individuals who underwent FITs, 3946 aged ≥50 years who underwent colonoscopies were analysed. Logistic regression analysis was performed to determine factors associated with FP FIT results.
RESULTS: Among 3946 participants, 704 (17.8%) showed positive FIT results and 1303 (33.0%) had hemorrhoids. Of the 704 participants with positive FIT results, 165 had advanced colorectal neoplasia (ACRN) and 539 had no ACRN (FP results). Of the 1303 participants with hemorrhoids, 291 showed FP results, of whom 81 showed FP results because of hemorrhoids only. Participants with hemorrhoids had a higher rate of FP results than those without hemorrhoids (291/1176, 24.7% vs. 248/2361, 10.5%; p<0.001). Additionally, the participants with hemorrhoids as the only abnormality had a higher rate of FP results than those experiencing no such abnormalities (81/531, 15.3% vs. 38/1173, 3.2%; p<0.001). In multivariate analysis, the presence of hemorrhoids was identified as an independent predictor of FP results (adjusted odds ratio, 2.76; 95% confidence interval, 2.24-3.40; p<0.001).
CONCLUSION: Hemorrhoids are significantly associated with FP FIT results. Their presence seemed to be a non-negligible contributor of FP results in FIT-based CRC screening programmes.
Clinical utility of PIVKA-II in the diagnosis of hepatocellular carcinoma
The need for cost-effective and reliable early diagnostic tools for hepatocellular carcinoma (HCC) is more than ever of great interest, as incidence rates are on the rise globally and two thirds of the patients are diagnosed with HCC in advanced stages. Alpha-fetoprotein (AFP), the marker that is currently in widespread use for HCC, lacks sensitivity and specificity. Protein induced by vitamin K absence/antagonist-II (PIVKA-II), a novel tumour marker, has been shown to be of good sensitivity and specificity for the diagnosis of HCC.
by Volha Klimovich, Kieran Voong, Prof. Roy Sherwood and Dr Dominic J. Harrington
Introduction
Primary liver cancer is the seventh most common cancer worldwide and the third most common cause of death from cancer [1]. Seventy-five to eighty-five percent of primary liver cancer cases are associated with HCC. The distinctive features of HCC include relatively large-sized tumours, vascular invasion, intra-hepatic metastasis, low differentiation, common recurrence and poor prognosis [2, 3]. In 70–90% of the cases, development of HCC requires a chronic liver disorder and cirrhosis as a background; these are caused mostly by chronic hepatitis C virus, hepatitis B virus, alcohol abuse, non-alcoholic steatohepatitis and less typically observed in inherited haemochromatosis, autoimmune hepatitis, antitrypsin deficiency, aflatoxin intoxication and also in some cases of oral contraception treatment [4, 5]. In addition, some chronic conditions, such as diabetes mellitus, cholelithiasis, obesity and hormone imbalance, are associated with HCC development [6]. The overall 5-year survival rate is believed to be less than 40%; however, a diagnosis at the early stages, followed by liver resection or transplantation, can improve this rate to 60–70% [7–9].
Taking into account the prevalence and mortality and also poor prognosis of HCC, it is apparent that highly sensitive techniques for diagnosis at the early stages are needed. The main diagnostic tool for HCC screening is radiologic imaging investigations such as ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI). With the development and introduction of contrast-enhanced ultrasound (CEUS) for analysis of intra-nodular vascularisation pattern, the sensitivity and specificity have been reported to be 90.9% and 100% for progressed HCC and 85.7% and 96.1% for early HCC, respectively [10]. The role of MRI and CT in producing reliable three-dimensional images is very important; however, the relationship between the radiographic and pathological tumour sizes is not yet well established. At this point application of tumour markers as supplementary analysis may provide useful information for making a diagnosis and monitoring of confirmed HCC [11, 12].
Protein induced by vitamin K absence/antagonist-II (PIVKA-II), also known as des-gamma carboxyprothrombin (DCP), is an abnormal form of prothrombin formed as a result of impaired or insufficient post-translational γ-carboxylation that occurs in the presence of vitamin K deficiency and leads to the loss of biological activity of the protein. Following synthesis in the liver, prothrombin, alongside the other hepatic vitamin K-dependent proteins undergoes transformation of specific glutamyl (GLU) residues into γ-carboxyl glutamyl (GLA) residues under the influence of vitamin K-dependent γ-glutamyl carboxylase in the presence of reduced vitamin K concentration (Fig. 1) [13]. Interestingly, carboxylation may not occur at all, which results in the formation of different variants of PIVKA-II with various degree of biological activity [14].
The role of PIVKA-II in HCC pathology is still not well established. It has been shown that PIVKA-II induces the malignant potential of HCC through stimulation of cell proliferation owing to a structural resemblance to hepatocyte growth factor [15–17]. Furthermore, PIVKA-II promotes angiogenesis in HCC resulting in local tissue invasion and metastases via stimulation of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) [13, 18].
Primary liver cancer is the seventh most common cancer worldwide and the third most common cause of death from cancer [1]. Seventy-five to eighty-five percent of primary liver cancer cases are associated with HCC. The distinctive features of HCC include relatively large-sized tumours, vascular invasion, intra-hepatic metastasis, low differentiation, common recurrence and poor prognosis [2, 3]. In 70–90% of the cases, development of HCC requires a chronic liver disorder and cirrhosis as a background; these are caused mostly by chronic hepatitis C virus, hepatitis B virus, alcohol abuse, non-alcoholic steatohepatitis and less typically observed in inherited haemochromatosis, autoimmune hepatitis, antitrypsin deficiency, aflatoxin intoxication and also in some cases of oral contraception treatment [4, 5]. In addition, some chronic conditions, such as diabetes mellitus, cholelithiasis, obesity and hormone imbalance, are associated with HCC development [6]. The overall 5-year survival rate is believed to be less than 40%; however, a diagnosis at the early stages, followed by liver resection or transplantation, can improve this rate to 60–70% [7–9].
Taking into account the prevalence and mortality and also poor prognosis of HCC, it is apparent that highly sensitive techniques for diagnosis at the early stages are needed. The main diagnostic tool for HCC screening is radiologic imaging investigations such as ultrasound (US), computed tomography (CT) and magnetic resonance imaging (MRI). With the development and introduction of contrast-enhanced ultrasound (CEUS) for analysis of intra-nodular vascularisation pattern, the sensitivity and specificity have been reported to be 90.9% and 100% for progressed HCC and 85.7% and 96.1% for early HCC, respectively [10]. The role of MRI and CT in producing reliable three-dimensional images is very important; however, the relationship between the radiographic and pathological tumour sizes is not yet well established. At this point application of tumour markers as supplementary analysis may provide useful information for making a diagnosis and monitoring of confirmed HCC [11, 12].
Protein induced by vitamin K absence/antagonist-II (PIVKA-II), also known as des-gamma carboxyprothrombin (DCP), is an abnormal form of prothrombin formed as a result of impaired or insufficient post-translational γ-carboxylation that occurs in the presence of vitamin K deficiency and leads to the loss of biological activity of the protein. Following synthesis in the liver, prothrombin, alongside the other hepatic vitamin K-dependent proteins undergoes transformation of specific glutamyl (GLU) residues into γ-carboxyl glutamyl (GLA) residues under the influence of vitamin K-dependent γ-glutamyl carboxylase in the presence of reduced vitamin K concentration (Fig. 1) [13]. Interestingly, carboxylation may not occur at all, which results in the formation of different variants of PIVKA-II with various degree of biological activity [14].
The role of PIVKA-II in HCC pathology is still not well established. It has been shown that PIVKA-II induces the malignant potential of HCC through stimulation of cell proliferation owing to a structural resemblance to hepatocyte growth factor [15–17]. Furthermore, PIVKA-II promotes angiogenesis in HCC resulting in local tissue invasion and metastases via stimulation of vascular endothelial growth factor (VEGF) and epidermal growth factor (EGF) [13, 18].
Methods and patient samples
The automated chemiluminescent microparticle immunoassay (ARCHITECT PIVKA-II 2P4 CMIA, Abbott) was validated and used for quantitation of PIVKA-II using the Abbott™ Architect iSystem 2000 analyser in the Human Nutristasis Unit at St Thomas’ Hospital, London, UK. Imprecision and recovery evaluations were performed in line with the appropriate standard operating procedures as part of the validation process. The CMIA is based on a two-step sandwich reaction of binding of anti-PIVKA-II antibodies and specific PIVKA-II epitopes with subsequent addition of chemiluminescent labels and registration of the relative light units as a quantitative representation of PIVKA-II concentration in the tested sample [1].
In order to exclude possible interference with anticoagulant therapeutic agents, high PIVKA-II results were tested for warfarin, as it is the most commonly used anticoagulant that interferes with the vitamin K cycle. Samples found to be positive for warfarin were disqualified from further analysis.
Eighty-seven samples from the Gassiott Gastroenterology Clinic (GGC, St. Thomas’ Hospital, London) and the Hepatocellular Carcinoma Clinic in the Institute of Liver Studies (King’s College Hospital, London) were analysed in three groups: high-risk patients with non-HCC pathology of the liver, high-risk patients currently undergoing HCC surveillance, and patients with diagnosed HCC (group A, B and C respectively). Group A (n=29) consisted of randomly selected patients at GGC with viral and non-viral cirrhosis, steatosis, fibrosis, hepatitis and benign lesions. Group B (n=24) represented high-risk patients with changes to the liver suggestive of possible HCC discovered in the course of US/MRI/CT investigations. Finally, group C (n=34) comprised of patients diagnosed with HCC at different stages; the diagnosis was established in the course of histological examination of liver biopsy samples.
All results for PIVKA-II concentrations in patient samples were statistically processed in IBM SPSS Statistics, Version 23. Tests of normality, association between different variables and receiver operating characteristic (ROC) curve were applied for the analysis.
Results and discussion
Using a cut-off of 49.4 mAU/mL, an elevated PIVKA-II concentration was found in just one patient from the negative control group, which represents 3.4% (Table 1). This patient was diagnosed with multiple cysts on the background of hepatitis; therefore, the result may be interpreted as both false positive (elevation of PIVKA-II due to non-malignant pathology) and true positive (in this case the patient would need to undergo more comprehensive screening).
In the positive group, PIVKA-II was elevated in 79.4% of the patients and demonstrated a broad scatter of values (19.06 mAU/mL for the lowest detected concentration and 340 485.5 mAU/mL for the highest detected concentration) owing to various sizes of the tumour masses at different stages of HCC and possibly existence of different PIVKA-II variants depending on the number of GLU residues involved in γ-carboxylation [19]. Normal PIVKA-II results in this group can be explained by the normalisation of PIVKA-II concentration after curative treatment, if performed [16].
Statistical processing of data showed no evidence of dependence of the results on age or gender (P>0.05 for all three groups). Area under the curve (AUC) in ROC analysis for PIVKA-II in the present research was 0.917 (CI 95% 0.847–0.986), which is suggestive of excellent clinical usefulness of PIVKA-II in HCC diagnosis (Fig. 2). AUC for alpha-fetoprotein (AFP) had slightly lower value (0.833 with CI 95% 0.722–0.945), which can still be classified as a fairly useful test (Fig. 3).
In this study the optimal cut-off value for PIVKA-II was identified by means of ROC and is 49.4 mAU/mL with sensitivity of 79.4% and specificity of 96.6%. Analysis of true and false-negative and -positive results revealed, that more than 83% of PIVKA-II results were truly reliable, whereas only 74.6% of AFP results demonstrated true diagnostic value (Table 2).
Unfortunately, sensitivity and specificity of AFP cannot accurately reflect its performance in the present study, as AFP results were available for only 17 patients from group A, which means that the study was possibly deprived of some potentially truly negative results. However, taking into account considerable difference between sensitivity and specificity rates for PIVKA-II and AFP (79.4 vs 96.6% and 70.6 vs 82.4% respectively), allows the conclusion that PIVKA-II displays slightly better clinical utility in HCC diagnosis. Similar results were reported in the previous studies [7, 20–24].
Limitations to the study
The major limitation to this research was the requirement to use anonymised samples, which prevented access to the full clinical history of the patients and impossibility to interpret the results in detail. Another limitation was the number of samples which could be considered to be insufficient to achieve aims of the project with adequate statistical power. A larger number of samples would have given the study more power and allowed a more precise ROC to be constructed and subsequently a more precise cut-off value to be identified.
Conclusion
In the present research PIVKA-II demonstrated high accuracy, sensitivity and specificity in HCC diagnosis. PIVKA-II has several advantages over AFP in terms of clinical utility for HCC diagnosis and prognosis: PIVKA-II is comparatively less frequently elevated in liver pathology [22], is more sensitive to small HCC tumours, correlates with HCC progression significantly better and has shorter half-life than AFP (40–72 hours against 5–7 days), which makes it more suitable for monitoring purposes [14]. Implementation of PIVKA-II as diagnostic test gathers pace in transplantation medicine, as this tumour marker, alongside Milan criteria has been used for recipient selection for living donor liver transplantation [16]. In addition, PIVKA-II concentrations can reflect the responsiveness of the liver to medical treatment (i.e. sorafenib), which cannot be achieved with AFP test. On the other hand, AFP is sensitive to radiological response following transarterial chemoembolisation, whereas PIVKA-II is not [12]. Also, PIVKA-II is affected by potentially interfering pharmacological agents (e.g. warfarin and certain antibiotics), it is dependent on vitamin K metabolism and can give false-positive results in non-HCC conditions which all has to be taken into account while interpreting the results.
Controversy over the best performance of tumour markers traces back to different assays used and various patient groups involved. Fortunately, AFP and PIVKA-II are independent of each other [16, 25]. Therefore, combination of PIVKA-II and AFP alongside AFP-L3, the fucosylated fraction of AFP, is suggested to be the best option for highly accurate laboratory diagnostic of HCC supplementary to imaging techniques. This multi-marker approach has been stated in the guidelines of The Japan Society of Hepatology and successfully used for diagnosis and management of HCC in Japan [26, 27].
Acknowledgement
ARCHITECT PIVKA-II 2P4 CMIA reagents and the graphics used in this article are courtesy of © Abbott Laboratories.
References
1. Kinukawa H, et al. characterization of an anti-PIVKA-II antibody and evaluation of a fully automated chemiluminescent immunoassay for PIVKA-II. Clin Biochem 2015; 48: 1120–1125.
2. Ha TY, et al. Expression pattern analysis of hepatocellular carcinoma tumour markers in viral hepatitis B and C patients undergoing liver transplantation and resection. Transplant Proc 2014; 46: 888–893.
3. Yano Y, et al. Clinical features of hepatitis C virus-related hepatocellular carcinoma and their association with α-fetoprotein and protein induced by vitamin K absence or antagonist-II. Liver Int 2006; 26: 789–795.
4. El-Serag HB, Rudolph KL. Hepatocellular carcinoma: epidemiology and molecular carcinogenesis. Gastroenterology 2007; 132: 2557–2576.
5. Aghemo A, Colombo M. Hepatocellular carcinoma in chronic hepatitis C: from bench to bedside. Semin Immunopathol 2012; 35: 111–120.
6. McMasters K, Vauthey J. Hepatocellular carcinoma: targeted therapy and multidisciplinary care. Springer 2011; Chapters 1–5, 8.
7. Ji J, et al. Diagnostic evaluation of des-gamma-carboxy prothrombin versus α-fetoprotein for hepatitis B virus-related hepatocellular carcinoma in China: a large-scale, multicentre study. PLoS One 2016; 11: e0153227.
8. Huang TS, et al. Diagnostic performance of alpha-fetoprotein, lens culinaris agglutinin-reactive alpha-fetoprotein, des-gamma carboxyprothrombin, and glypican-3 for the detection of hepatocellular carcinoma: a systematic review and meta-analysis protocol. Syst Rev 2013; 2: 37.
9. Song PP, et al. Controversies regarding and perspectives on clinical utility of biomarkers in hepatocellular carcinoma. World J Gastroenterol 2016; 22: 262–274.
10. Giorgio A, et al. Characterization of dysplastic nodules, early hepatocellular carcinoma and progressed hepatocellular carcinoma in cirrhosis with contrast-enhanced ultrasound. Anticancer Res 2011; 31: 3977–3982.
11. Chen H, et al. CT and MRI in target delineation in primary hepatocellular carcinoma. Cancer Radiother 2013; 17: 750–754.
12. Park H, Park JY. Clinical significance of AFP and PIVKA-II responses for monitoring treatment outcomes and predicting prognosis in patients with hepatocellular carcinoma. BioMed Research International 2013; 2013: 310427.
13. Yue P, et al. Des-γ-carboxyl prothrombin induces matrix metalloproteinase activity in hepatocellular carcinoma cells by involving the ERK1/2 MAPK signalling pathway. Eur J Cancer 2011; 47: 1115–1124.
14. Zhang YS, et al. Des-γ-carboxy prothrombin (DCP) as a potential autologous growth factor for the development of hepatocellular carcinoma. Cell Physiol Biochem 2014; 34: 903–915.
15. Suzuki K, et al. Positioning of novel tumor marker NX-PVKA-R in the diagnosis of hepatocellular carcinoma in comparison with PIVKA-II. Dokkyo Journal of Medical Sciences 2013; 40: 163–168
16. Inagaki Y, et al. Clinical and molecular insights into the hepatocellular carcinoma tumour marker des-γ-carboxyprothrombin. Liver Int 2010; 31: 22–35.
17. Jinghe X, et al. Vitamin K and hepatocellular carcinoma: the basic and clinic. World J Clin Cases 2015; 3: 757–764.
18. Fujikawa T, et al. Significance of des-gamma-carboxyprothrombin production in hepatocellular carcinom. Acta Med Okayama 2009; 63: 299–304.
19. Zakhary NI, et al. Impact of PIVKA-II in diagnosis of hepatocellular carcinoma. J Adv Res 2013; 4: 539–546.
20. Mathew S, et al. Biomarkers for virus-induced hepatocellular carcinoma (HCC). Infect Genet Evol 2014; 26: 327–339.
21. Lim TS, et al. Combined use of AFP, PIVKA-II, and AFP-L3 as tumor markers enhances diagnostic accuracy for hepatocellular carcinoma in cirrhotic patients. Scand J Gastroenterol 2015; 51: 344–353.
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The authors
Volha Klimovich*1 MSc; Kieran Voong2 MSc; Roy Sherwood3 MSc, DPhil; Dominic J Harrington2 MSc, PhD
1Clinical Biochemistry, Viapath, St Thomas’ Hospital, London, UK
2Human Nutristasis Unit, Viapath, St Thomas’ Hospital, London, UK
3Viapath, King’s College Hospital, London, UK
*Corresponding author
E-mail: klimovichvolha@gmail.com
Molecular detection and quantification of human rhinoviruses
The sequence diversity of the more than 150 currently recognized HRV genotypes poses challenges for the development of robust molecular methods that detect all genotypes with equal efficiency. Real-time reverse-transcription (RT)-PCR was compared to digital RT-PCR for quantification of HRV in clinical specimens when using type-specific and consensus primers and probes.
by Dr Jane Kuypers
Introduction
Human rhinoviruses (HRV) are small, positive-sense RNA viruses within the family Picornaviridae. Over 150 genotypes of this important human pathogen have been recognized within species HRVA, HRVB, and HRVC of the genus Enterovirus (http//:www.picornaviridae.com). HRV infections occur throughout the year and throughout the world. HRV are responsible for a high incidence and wide range of respiratory infections in all populations, including one-half to two-thirds of all common colds and many cases of otitis media and sinusitis in the upper respiratory tract. Lower tract infections include bronchiolitis, pneumonia and exacerbation of disease in children with asthma and cystic fibrosis, and in adults with chronic obstructive pulmonary disease. Cases of serious illness and even death due to HRV have been reported, especially in immunocompromised individuals, the elderly and infants [1, 2].
Laboratory detection of HRV is important for diagnosis and surveillance, especially in high risk populations. HRV are frequently detected as co-infections with other respiratory viruses and in individuals with long-term and asymptomatic shedding [3]. In addition to qualitative detection, accurate quantification of HRV RNA in clinical samples is needed for studies on the association of HRV viral load with viral transmission and with patient symptoms and outcomes. Viral-load studies of other respiratory viruses have shown that a correlation exists between quantity of virus and disease severity. HRV viral-load determinations may also be important for patient management, especially in asymptomatic patients who test positive for HRV at low levels. More importantly, accurate HRV viral-load assessments will be necessary for evaluating the performance of potential HRV antiviral drugs [4].
Detection
HRV were initially detected by growth in cell culture. Approximately 100 serotypes of HRV grown in cell culture were antigenically characterized by their reactions with various antisera. The serotypes were subsequently classified into two groups, A and B, according to their sensitivity towards antivirus agents [5] and are now included in HRV species A (80 genotypes) and B (32 genotypes) based on genetic sequencing. Cell culture is sensitive for detection of many, but not all HRV genotypes; 55 HRV that do not grow in the cell culture lines normally used in the clinical laboratory and have been detected only by molecular methods are classified in HRV species C (http//:www.picornaviridae.com).
The use of molecular methods for the detection of HRV in clinical specimens has provided more accurate information about the disease burden and epidemiology of these ubiquitous viruses. The molecular method most often used to detect HRV is real-time reverse-transcription (RT)-PCR [3]. RT-PCR assays, when accompanied by amplification of serially diluted standards of known RNA copy numbers (RT-qPCR), can be used to quantify the number of viral copies in a sample. By comparing the PCR Ct value (the PCR cycle at which fluorescence reaches a certain threshold) of a clinical specimen to the standard curve, the relative quantity of the analyte can be calculated [6].
Within the HRV genome, the region most frequently targeted for RT-PCR by clinical assays is the 5’ non-coding region (NCR), which exhibits the most sequence homology among the HRV genotypes. However, even in this region, there is a lot of sequence diversity, which makes it challenging to design a single, consensus PCR primer and probe set to amplify all HRV genotypes with equal efficiency. In order to amplify HRV genotypes with diverse sequences in the prime/probe binding regions, consensus PCR primer and probe sets have been designed with degenerate and modified bases or multiple oligonucleotides [7–10]. However, consensus RT-qPCR assays may not give accurate quantitative results for all HRV genotypes due to amplification inefficiency caused by base mismatches between the consensus primers and probe and the viral sequences [11].
Quantitation by RT-qPCR
To determine if a consensus RT-qPCR assay [7] could be used to accurately quantify all genotypes of HRV, including those with sequence differences in the primer and probe binding regions, we compared the efficiency and sensitivity of a consensus RT-qPCR assay to that of genotype-specific RT-qPCR assays [4]. In Figure 1(a), the results of RT-qPCR assays using type-specific primers and probes, which exactly match the target sequences, show standard curves indicating accurate and sensitive quantification of RNA transcripts from six specific HRV genotypes. However, RT-qPCR using a consensus HRV primer and probe set did not give accurate or sensitive quantification for some HRV genotypes, especially types A33 and A88 (Fig. 1b). RNA from HRV genotypes with base mismatches between the consensus primer and probe sequences and the specific viral sequences was inaccurately quantified using the consensus assay, most likely due to poor amplification efficiency.
Quantitation by RT-dPCR
Digital RT-PCR (RT-dPCR), which provides absolute nucleic acid quantification without the need for PCR Ct values and standard curves and is less affected by poor amplification efficiency, may perform better than RT-qPCR for quantification of HRV RNA. In dPCR, an amplification reaction, which contains fluorescent dye to measure amplification, is divided into 12?000 to 200?000 independent partitions, each ideally containing no more than one target molecule. The reaction is amplified to end point and the number of fluorescent (positive) and non-fluorescent (negative) partitions is counted. In specimens with more targets than partitions, Poisson statistics are used to calculate the average number of targets per positive partition and thus, the number of targets in the original sample [12, 13]. Compared to qPCR, dPCR is less susceptible to amplification inefficiency caused by primer/probe sequence mismatches because quantification derives from a PCR reaction that cycles to endpoint rather than from an amplification curve as in qPCR. Accurate quantification by dPCR is also not dependent on a well-calibrated standard [14]. These characteristics make dPCR especially useful for quantifying viral targets with many subtypes and high sequence diversity that leads to mismatches between targets and PCR primer and probe sequences, such as HRV.
To determine if consensus RT-dPCR would perform better than consensus RT-qPCR for quantification of HRV genotypes, we similarly tested RNA transcripts of HRV genotypes, including some with sequence variation in the consensus primer and probe binding region, by RT-dPCR using both type-specific and consensus primers and probes. In Figure 2(a), the results of RT-dPCR assays using type-specific primers and probes show good correlations between the expected number of RNA copies/reaction and the observed number. When amplified by RT-dPCR using the consensus assay (Fig. 1b), in contrast to RT-qPCR, the observed number of RNA copies/reaction was also closely correlated with the expected number for most of the HRV genotypes tested.
In a previous study [4], data from 16 HRV genotypes that represented the consensus primer and probe binding sequences of 128 genotypes indicated that, when using consensus primers and probe, RT-dPCR quantification of HRV RNA was more accurate than that of RT-qPCR for some genotypes. We found that although the consensus RT-qPCR did accurately quantify many HRV genotypes, it did not accurately quantify all genotypes of HRV due to sub-optimal amplification of genotypes with sequences that do not exactly match those of the primers and probe. Consensus RT-dPCR, however, did not overcome all sequence mismatch-induced amplification inefficiency, as evidenced by genotype A88 (Fig. 2b), which has a single mismatch near the middle of the forward primer.
Although RT-dPCR has been shown to be more accurate than RT-qPCR for quantification of HRV and may be applicable to other viruses with high sequence diversity, like HIV and HBV, it has some disadvantages for routine use in a clinical laboratory. RT-dPCR has a more limited dynamic range compared to RT-qPCR (104 for RT-qPCR compared to 108 for RT-qPCR), which would require dilution and retesting of samples with high viral loads. Running an RT-dPCR assay requires more hands-on technician time and has a lower throughput than current RT-qPCR assays. Digital PCR instruments and reagents are also currently more expensive than most qPCR systems.
Conclusion
In conclusion, dPCR was a better alternative to qPCR on RNA templates known to have significant sequence diversity that cannot be avoided during primer and probe design and should be considered the better molecular method for quantification of HRV in respiratory specimens.
References
1. Brownlee JW, Turner RB. New developments in the epidemiology and clinical spectrum of rhinovirus infections. Curr Opin Pediatr 2008: 20: 67–71.
2. Gern JE. The ABCs of rhinoviruses, wheezing, and asthma. J Virol 2010: 84(15): 7418–7426.
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4. Sedlak RH, Nguyen T, Palileo I, Jerome KR, Kuypers J. Superiority of digital RT-PCR over real-time RT-PCR for quantitation of highly divergent human rhinoviruses. J Clin Microbiol 2017; 55(2): 442–449.
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8. Granados A, Luinstra K, Chong S, Goodall E, Bahn L, Mubareka S, Smieja M, Mahony J. Use of an improved quantitative polymerase chain reaction assay to determine differences in human rhinovirus viral loads in different populations. Diagn Microbiol Infect Dis 2012: 74: 384–387.
9. Tapparel C, Cordey S, Van Belle S, Turin L, Wai-Ming L, Regamey N, Meylan P, Mühlemann K, Gobbini F, Kaiser L. New molecular detection tools adapted to emerging rhinoviruses and enterviruses. J Clin Microbiol 2009: 47(6): 1742–1749.
10. Bochkov YA, Grindle K, Vang F, Evans MD, Gern JE. Improved molecular typing for rhinovirus species A, B, and C. J Clin Microbiol 2014: 52(7): 2461–2471.
11. Hoffman NG, Cook L, Atienza EE, Limaye AP, Jerome KR. Marked variability of BK virus load measurement using quantitative real-time PCR among commonly used assays. J Clin Microbiol 2008: 46(8): 2671–80.
12. Vynck M, Trypsteen W, Thas O, Vandekerckhove L, De Spiegelaere W. The future of the polymerase chain reaction in virology. Mol Diagn Ther 2016: 20: 437–447.
13. Huggett JF, Cowen S, Foy CA. Considerations for digital PCR as an accurate molecular diagnostic tool. Clin Chem 2015: 61: 79–88.
14. Sedlak RH, Jerome KR. Viral diagnostics in the era of digital polymerase chain reaction. Diagn Microbiol Infect Dis 2013: 75(1): 1–4.
The author
Jane Kuypers PhD
Department of Laboratory Medicine, University of Washington, Seattle, WA, USA
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA
E-mail: kuypers@uw.edu
Still sceptical about the benefits of prophylactic statins?
In spite of the many advances in medical science that have resulted in a steady reduction in deaths from cardiovascular disease in most European countries, CVD remains the leading cause of mortality in Europe. Without appropriate interventions, the effects of an ageing population, together with the increasing prevalence of obesity and Type 2 diabetes, can only exacerbate the situation. Modifiable risk factors have of course been elucidated: tobacco use, a diet high in salt and sugar, excessive alcohol consumption, and a paucity of physical exercise. It is also known that statins, which reduce Low-Density-Lipoprotein cholesterol levels, are an effective prophylactic for people at risk of CVD.
However two and a half years ago the updating of the UK’s National Institute for Health and Clinical Excellence (NICE) advice to physicians on when to prescribe statins resulted in an outcry. NICE was now recommending that statin therapy should be prescribed for patients with an assessed risk of 10% of developing CVD within 10 years, whereas previous guidelines had stated a 20% risk. Incidentally the American Heart Association and American College of Cardiologists advocate prescribing prophylactic statins when the risk within 10 years is as low as 7.5%. An article and an open letter were soon published in The British Medical Journal urging NICE to rethink the advice that would result in 4.5 million more fundamentally healthy people becoming eligible to take statins. The authors claimed that the risk of side-effects from the medication would outweigh the benefits. Data from clinical trials involving over 8 000 subjects showed that new cases of diabetes were significantly higher in people taking statins than in those taking placebo. A longitudinal study of over 3 000 older men had also reported that subjects prescribed statins took significantly lower levels of physical exercise, due to either the side effects of muscle pain or to fatigue. The UK mass media fanned the flames, erroneously reporting that statins caused debilitating side-effects in 20% of patients. The impact of this media coverage was soon apparent: 200 000 people stopped taking statins, up to 6 000 of whom could be expected to suffer from an avoidable cardiovascular event within the decade.
A review in the Lancet published in September has now scrutinized and carefully interpreted the evidence available from randomized controlled clinical trials.It underlined the fact that CVD morbidity and mortality is effectively reduced by taking prophylactic statins and that side-effects are rare. Hopefully this information will be accurately disseminated by the popular press.
New screening strategies in prenatal care by the introduction of non-invasive prenatal testing for fetal aneuploidies
The identification of cell-free fetal DNA (cffDNA) in maternal plasma has led to the development of non-invasive prenatal testing (NIPT) for fetal aneuploidies risk assessment. The high accuracy of NIPT has profoundly influenced the field of prenatal care.
by F. Gerundino, Dr C. Giachini, C. Giuliani, E. Contini and Dr E. Pelo
Background
Several well-established screening approaches to estimate the personal risk for common autosomal aneuploidies such as trisomy 21 (T21, Down syndrome), trisomy 18 (T18, Edwards syndrome) and trisomy 13 (T13, Patau syndrome) are part of the standard prenatal care in many countries. These approaches are based on the combination of different parameters such as maternal age, markers in maternal serum and ultrasound findings in the first or second trimester of pregnancy. Overall, conventional screening tests show a detection rate (DR) of 80–95% with a high false positive rate (FPR) (3–5%). Pregnancies identified to be at high risk (using locally established cut-off values) are offered invasive prenatal diagnosis (IPD) to provide a definitive result. IPD, carried out using either chorionic villus sampling (CVS) or amniocentesis to obtain fetal cells, is associated with an estimated miscarriage risk of 0.5–1% [1]. Given the FPR of conventional screening protocols a not negligible number of pregnancies undergo unnecessary IPD. The identification of cell-free fetal DNA (cffDNA) in plasma of pregnant women [2] has opened new possibilities to improve non-invasive prenatal screening of common fetal aneuploidies. In the last decade, several groups developed massively parallel sequencing (MPS) – using targeted or whole genome approaches – of cell-free DNA (cfDNA) from maternal plasma to detect fetal aneuploidies [3, 4]. These approaches, referred to as non-invasive prenatal testing (NIPT) or non-invasive prenatal screening (NIPS), have been shown to outperform traditional screening protocols. According to a recent meta-analysis, the DR of NIPT was 99.2% for T21, 96.3%, 91.0% and 90.3% for T18, T13 and monosomy X, respectively; the FPR was below 1% for all of these aneuploidies [5]. Since 2011, NIPT became commercially available in the USA and China and was rapidly introduced into standard prenatal care in many countries.
Cell-free-DNA-based screening: validation of a method for fetal aneuploidies risk
The conventional first-trimester screening (FTS) is currently offered to all pregnant women by the public health system in Tuscany. Recently, we validated a NIPT method based on whole genome MPS approach [6], in order to introduce a more robust screening test within the public health system. In whole genome approach, maternal and fetal DNA fragments (called reads) are sequenced simultaneously in a single run. Sequence reads were aligned to specific chromosome locations within the human genome and the number of reads mapped to the chromosome of interest are counted. A relative increase or decrease in the number of reads respect to a predefined threshold value (Z-score) reveals a potential risk of aneuploidy for a specific chromosome. In particular, a trisomy was called when Z-score >3 (Fig. 1). MPS was performed on a total of 381 cfDNA samples isolated from maternal plasma by two steps: a first set of 186 euploid samples was analysed to generate a preliminary reference dataset (group A) and a second set of 195 samples (group B) – enriched by 69 aneuploid cases – was analysed in blind versus the reference dataset to verify the reliability of our sequencing protocol as well as the analysis method. One hundred and fifty samples from group A (80.6%) and 177 samples from group B (90.8%) gave resulted suitable (>10×106 mapped reads) for downstream data analysis. The two groups (A and B) were then merged to generate a definitive dataset (n=327), which was then used to re-analyse the whole study population. Since the fetal fraction (FF) (i.e. the proportion of fetal DNA to the total cfDNA in maternal plasma) is a parameter that strictly influences NIPT performance [7], a droplet digital PCR (ddPCR) protocol has been validated for its assessment [8]. FF quantification by ddPCR was performed in 178/381 (46.7%) samples after methylation-sensitive DNA digestion. Absolute quantification of both fetal (on digested RASSF1A) and total DNA (on TERT and undigested beta-actin/RASSF1A) was calculated as the ratio between the average copies/µL of fetal DNA and total DNA. An SRY assay was used for fetal gender assessment [6].
Results of the validation study
Considering the performance of the definitive reference dataset, all positive samples for T21 (n=43), T18 (n=6) and T13 (n=7) were correctly identified (sensitivity 99.9%). Five false positive (FP) results were observed: three for T21 (specificity 98.9%) and two for T13 (specificity 99.4%).
Z-score values of true positive (TP) cases for T21 and T13 were always higher than 4.6 and 6.6, respectively. Conversely, all Z-score values of FP cases for T21 and T13 lay within 3.0 and 4.0 (the so-called Z-score ‘grey zone’). Sex chromosome status was correctly assigned in 317/324 (97.8%) cases: 166 males, 149 females and 2 cases with monosomy X. In 3/327 (0.9%) samples fetal gender could not be assigned because of an inconclusive result in data analysis. Seven discordant cases between MPS and follow-up data were observed. The only case of false negative (FN) male has been explained by a low FF (0.3%), underling the importance of FF determination. Only two out of four cases with monosomy X were correctly identified by NIPT, while the remaining two cases were erroneously classified as male.
Discussion
NIPT is an accurate screening test without associated risk for the mother and/or the fetus and it can be performed early in pregnancies, starting from 9–10 weeks of gestation. It is suitable both in low- and high-risk pregnancies, even if the positive predictive value (PPV) of the test (the chance that the positive result is a true positive) is lower in low risk cohorts. Two large studies show that in the general population NIPT outperforms conventional screening tests for T21 with a PPV ranging from 45.5 to 80.9% versus a PPV of 3.4–4.2% [9, 10]. Pre- and post-test counselling to inform patients about benefits, risks, test failure and testing alternatives should be provided before offering cfDNA screening. Owing to a series of intrinsic limitations, NIPT cannot be considered a diagnostic tool, despite its high performance. In the management of pregnancies with a high-risk NIPT, the possibility of FP results should always be taken into account and IPD should be recommended after a NIPT-positive result. cffDNA derives from the apoptosis of the placental cytotrophoblast cells, therefore in rare cases it may not represent the genetic constitution of the fetus. FP results may arise from confined placental mosaicism (CMP) in which some or all trophoblastic cells are trisomic, whereas the fetus is normal (1–2% of first-trimester placentas) [11]. FN results are a very rare occurrence and can be explained by fetoplacental discrepancies, in which the fetus shows an abnormal karyotype but the chromosome aberration is absent in the cytotrophoblast and, therefore, in the cffDNA. Additional sources of FP results can be unanticipated finding such as maternal chromosome abnormalities (maternal mosaicisms, microdeletions and other copy-number variations) or maternal malignancy, or the presence of a vanishing twin with an early loss of a trisomic fetus.
Failure to provide a result occurs in 1.6–6.4% of NIPT. Both laboratory technical issues or low FF can cause the failure [12]. Low FF in frequently found in overweight pregnant women, in which the low FF could be due to a dilution effect of an increased blood volume or to the high turn-over of adipocytes [13]. In these cases it is not advisable to repeat the test on a new sample because the probability of a second test failure is quite high. An accurate clinical management of cases with low FF and normal maternal weight is instead recommended, because FF is lower in pregnancies with aneuploid fetuses (T18, T13, monosomy X and triploidy) compared to euploid pregnancies [14].
NIPT has rapidly spread in many countries through commercial provider, leading to a re-examination of current screening methods, and several models of implementation of NIPT have been proposed with pros and cons. Current guidelines recommend that “in countries where prenatal screening is offered as a public health service, governments and public health authorities should assume an active role to ensure the responsible introduction of NIPT” [15].
Our study represents the first experience of NIPT within the Italian public health system. Following our validation study, NIPT testing for T21, T13 and T18 has been introduced as a clinical service for all pregnant women after 10+4 week of gestation upon payment. Our regional health system has planned a pilot study of two years to evaluate the benefit-to-cost ratio of NIPT introduction into routine prenatal care to support the current screening strategy based on nuchal translucency measurement and maternal serum biomarker quantification. NIPT will be offered in an adequate context of pre- and post-test counselling as an alternative option to IPD in pregnant women with high risk after FTS and applying the national cut-off of 1:250. We expect that this strategy would lead to a significant reduction in unnecessary IPD due to FP results of FTS with a reduction in fetal losses associated to diagnostic procedures among high-risk women, allowing us to offer the best screening strategy currently available.
References
1. Tabor A, Alfirevic Z. Update on procedure-related risks for prenatal diagnosis techniques. Fetal Diagn Ther 2010; 27(1): 1–7.
2. Lo YM, Corbetta N, Chamberlain PF, Rai V, Sargent IL, Redman CW, Wainscoat JS. Presence of fetal DNA in maternal plasma and serum. Lancet 1997; 350(9076): 485–487.
3. Chiu RW, Chan KC, Gao Y, Lau VY, Zheng W, Leung TY, Foo CH, Xie B, Tsui NB, et al. Noninvasive prenatal diagnosis of fetal chromosomal aneuploidy by massively parallel genomic sequencing of DNA in maternal plasma. Proc Natl Acad Sci U S A 2008; 105(51): 20458–20463.
4. Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR. Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci U S A 2008; 105(42): 16266–16271.
5. Gil MM, Quezada MS, Revello R, Akolekar R, Nicolaides KH. Analysis of cell-free DNA in maternal blood in screening for fetal aneuploidies: updated meta-analysis. Ultrasound Obstet Gynecol 2015; 45(3): 249–266.
6. Gerundino F, Giachini C, Contini, E Benelli M, Marseglia G, Giuliani C, Marin F, Nannetti G, Lisi E, et al. Validation of a method for noninvasive prenatal testing for fetal aneuploidies risk and considerations for its introduction in the Public Health System. J Matern Fetal Neonatal Med 2017; 30(6): 710–716.
7. Palomaki GE, Kloza EM, Lambert-Messerlian GM, Haddow JE, Neveux LM, Ehrich M, van den Boom D, Bombard AT, Deciu C, et al. DNA sequencing of maternal plasma to detect Down syndrome: an international clinical validation study. Genet Med 2011; 13(11): 913–920.
8. Chan KC, Ding C, Gerovassili A, Yeung SW, Chiu RW, Leung TN, Lau TK, Chim SS, Chung GT, et al. Hypermethylated RASSF1A in maternal plasma: A universal fetal DNA marker that improves the reliability of noninvasive prenatal diagnosis. Clin Chem 2006; 52(12): 2211–2218.
9. Bianchi DW, Parker RL, Wentworth J, Madankumar R, Saffer C, Das AF, Craig JA, Chudova DI, Devers PL, et al. DNA sequencing versus standard prenatal aneuploidy screening. N Engl J Med 2014; 370(9): 799–808.
10. Norton ME, Jacobsson B, Swamy GK, Laurent LC, Ranzini AC, Brar H, Tomlinson MW, Pereira L, Spitz JL, et al. Cell-free DNA analysis for noninvasive examination of trisomy. N Engl J Med 2015; 372(17): 1589–1597.
11. Kalousek DK, Vekemans M. Confined placental mosaicism. J Med Genet 1996; 33(7): 529–533.
12. Yaron Y. The implications of non-invasive prenatal testing failures: a review of an under-discussed phenomenon. Prenat Diagn 2016; 36(5): 391–396.
13. Haghiac M, Vora NL, Basu S, Johnson KL, Presley L, Bianchi DW, Hauguel-de Mouzon S. Increased death of adipose cells, a path to release cell-free DNA into systemic circulation of obese women. Obesity 2012; 20(11): 2213–2219.
14. Rava RP, Srinivasan A, Sehnert AJ, Bianchi DW. Circulating fetal cell-free DNA fractions differ in autosomal aneuploidies and monosomy X. Clin Chem 2014; 60(1): 243–250.
15. Dondorp W, de Wert G, Bombard Y, Bianchi DW, Bergmann C, Borry P, Chitty LS, Fellmann F, Forzano F, et al. Non-invasive prenatal testing for aneuploidy and beyond: challenges of responsible innovation in prenatal screening. Summary and recommendations. Eur J Hum Genet 2015; doi: 10.1038/ejhg.2015.56.
The authors
Francesca Gerundino BS, Claudia Giachini PhD, Costanza Giuliani BS, Elisa Contini BS, Elisabetta Pelo* MD
Diagnostic Genetic Unit, Careggi University Hospital, Florence, Italy
*Corresponding author
E-mail: peloe@aou-careggi.toscana.it
Is it a urinary tract infection?
Whilst the respiratory tract is the most frequent site of infection, in the developed world the urinary tract is the second most frequent site; 50% of otherwise healthy women will experience at least one UTI. The shortness of the female urethra and its proximity to both vagina and anus in women contribute to the much greater frequency of UTIs in women compared with men. Sexually active women are most vulnerable to infection, and up to 40% of women who have a UTI will experience a repeat infection within a year. Quite apart from the extreme discomfort that uncomplicated UTIs can cause, complications such as pyelonephritis can lead to pyonephrosis, sepsis and kidney failure. Thus, diagnosed UTIs are treated with antibiotics such as trimethoprim, alone or in combination with sulfamethoxazole.
However, resistance to antibiotics and the urgent need for ‘antibiotic stewardship’ is rightfully discouraging primary healthcare providers from prescribing these drugs without a diagnosis of a bacterial infection. This is especially relevant in the case of upper respiratory tract infections, which are most frequently viral and self-limiting. But in the case of UTIs the dipstick test, and even standard lab-cultured urine, can give false negative results resulting in the withholding of necessary treatment. A recently published Belgian study based at the University of Ghent carried out quantitative PCR on urine samples for two bacterial species (and four sexually transmitted pathogens), in addition to standard urine culture, in 220 women with symptoms of UTI and 86 healthy volunteers. The tested bacterial species were Escherichia coli, present in the intestinal flora and the causative organism of over two thirds of UTIs, and Staphylococcus saprophyticus, the second most common causative organism present in the normal perineal and vaginal flora. In the symptomatic group standard culture detected uropathogens in 81% of the urine samples, but with qPCR 95.9% of these samples were found positive for E.coli and 8.6% for S. saprophyticus. There were also two samples positive for sexually transmitted pathogens. Overall 98% of the symptomatic women were found to have pathogens in their urine, compared with 10% of the control women.
So standard urine culture is not as infallible as was previously thought and dipstick tests, whilst quick and cheap, cannot be used to rule out a UTI. Because of the reliance on such tests, many women presenting with a UTI are sent home again without antibiotics being prescribed. Yes, we need stewardship, but clearly women with the typical urinary symptoms have an infection that should be treated!