The determination of autoantibodies is an important component in the diagnosis and differentiation of glomerular disease. Key analyses include antibodies against phospholipase A2 receptors (anti-PLA2R), the glomerular basement membrane (anti-GBM), neutrophil granulocyte cytoplasm (ANCA), double-stranded DNA (anti-dsDNA) and nucleosomes (ANuA). With these tests autoimmune reactions can be identified as causative factors of renal disease.
by Dr Jacqueline Gosink
Glomerulonephritis (GN) is an inflammation of the blood-filtering structures of the kidneys (glomeruli) which can lead to kidney failure if left untreated. The disease is associated with the symptom complexes nephritic syndrome and nephrotic syndrome. Nephritic syndrome is characterised by hematuria, mild to moderate proteinuria and hypertension and is observedain diseases such as post-infectious GN, lupus nephritis, rapid progressive GN and IgA nephropathy. Nephrotic syndrome combines heavy proteinuria, hypoalbuminemia, hyperlipidemia and edema and is typical of membranous GN, minimal change GN and focal segmental glomerulosclerosis.
Because of the wide range of potential causes, the diagnosis of GN can be difficult. The diagnostic process is based on clinical examination, biopsy, and laboratory tests on urine and blood. The serological analysis of specific autoantibodies allows autoimmune forms of GN to be identified and distinguished from nephropathies of other origins, for example hereditary conditions, infections, drug intoxication, electrolyte or acid-base disturbances, diabetes and hypertension.
Autoantibodies in GN may be directed against specific renal targets, such as PLA2R or the GBM, resulting in diseases that predominantly injure the kidneys. Or they may be non-organ-specific, for example ANCA, anti-dsDNA or ANuA. Non-organ-specific autoantibodies cause damage to a wide variety of organs. Thus, GN may represent just one manifestation of a complex systemic autoimmune disease, for example systemic lupus erythematosus (SLE) or ANCA-associated vasculitis (AAV).
Anti-PLA2R antibodies
Autoantibodies against PLA2R are a new and highly specific marker for primary membranous glomerulonephritis (MGN), also known as idiopathic membranous nephropathy. Primary MGN is a chronic inflammatory autoimmune disease of the glomeruli and is one of the leading causes of nephrotic syndrome in adults. It is distinguished from secondary MGN, which is triggered by an underlying disease such as a malignant tumour, an infection, drug intoxication or another autoimmune disease such as SLE. Primary MGN accounts for 70-80% of cases of MGN, while the secondary form comprises around 20-30%. Clinical differentiation of the two forms is crucial since primary MGN is treated with immunosuppressants, whereas therapy for secondary MGN focuses on the causal disease.
The immune reactions leading to primary MGN, which were first described in 2009 [1], stem from autoantibodies binding to PLA2R (transmembrane glycoproteins, [Figure 1]) on the surface of the podocytes [Figure 2]. PLA2R of type M have been identified as the major target antigen of the autoantibodies. The antigen-antibody complexes are deposited in the GBM, triggering complement activation with overproduction of collagen IV and laminin. This damages the podocytes, resulting in protein entering the primary urine. With increasing proteinuria there is a higher long-term risk of kidney failure with major morbidity and mortality, especially from thromboembolic and cardiovascular complications.
Primary MGN is diagnosed by kidney puncture followed by histological examination or electron microscopy of the tissue to detect immunoglobulin-containing deposits in the GBM. Serological determination of anti-PLA2R antibodies supports the diagnostic procedure and has the advantage of being less time-consuming and less stressful for patients. Anti-PLA2R antibody analysis is, moreover, suitable for monitoring the activity of primary MGN and the response to therapy.
Until recently there was no reliable test to detect anti-PLA2R antibodies. A new recombinant-cell anti-PLA2R indirect immunofluorescence test (IIFT) developed to address this deficit has rapidly established itself as the gold standard for the serological diagnosis of primary MGN. The assay utilizes transfected human cells expressing recombinant PLA2R as the antigenic substrate [Figure 3] to provide monospecific antibody detection [2, 3]. The sensitivity of the test for primary MGN amounts to around 50-80% depending on the characteristics of cohort individuals, for example their disease activity or therapy status. In a retrospective clinical study [2] the Anti-PLA2R IIFT demonstrated a sensitivity of 52% in a cohort of 100 patients with biopsy-proven primary MGN and a specificity of 100% with respect to control subjects. In the first prospective study [4] the sensitivity amounted to 82% in patients with biopsy-proven MGN where no secondary cause could be found. An ELISA based on purified recombinant PLA2R has also been developed. It demonstrates >98% correlation with the IIFT and is particularly useful for quantification of antibody levels in therapy monitoring.
Anti-GBM antibodies
Autoantibodies against GBM are a highly specific and sensitive marker for Goodpasture’s syndrome, a rare, but potentially fatal autoimmune disease which is characterized by rapidly progressive GN and lung haemosiderosis. Diagnosis of this disease is challenging because of the speed of progression to organ failure and the initially unspecific symptoms. Serological parameters such as anti-GBM play a crucial role in obtaining an early diagnosis.
The primary target antigen of anti-GBM antibodies is the NC1 domain of the alpha chain of type IV collagen. The antibodies target the alveolar basement membrane or the GBM. In cases without lung involvement they are detected in more than 60% of patients and in cases with lung involvement in over 90%. Clinical progression of the disease correlates with antibody concentration, with high-titre circulating anti-GBM antibodies indicating an unfavourable prognosis.
Anti-GBM antibodies can be detected serologically by IIFT using sections of primate kidney as the antigenic substrate. Inclusion of a second substrate comprising microdots of purified GBM allows results to be confirmed at a glance. The substrates are positioned side by side as BIOCHIP Mosaics in the test fields of a microscope slide [Figure 4] and incubated in parallel. Further substrates for differential diagnostics, for example HEp-2 cells, granulocytes or other microdot substrates, can also be included in the BIOCHIP Mosaics, yielding a detailed patient antibody profile following a single incubation. Serum anti-GBM antibodies can alternatively be detected or confirmed quantitatively using the Anti-GBM ELISA.
ANCA
ANCA determination is a well-established tool for serological diagnosis and differentiation of different types of AAV, which often present as a rapidly progressive GN among other symptoms. The most important ANCA parameters include antibodies against proteinase 3, which are sensitive and specific markers for Wegener’s granulomatosis, and antibodies against myeloperoxidase (MPO), which occur in microscopic polyangiitis and other forms of AAV.
The standard method for detecting ANCA is IIFT using granulocytes to identify the typical staining patterns of anti-PR3 antibodies (cytoplasmic, cANCA) and anti-MPO antibodies (perinuclear, pANCA). BIOCHIP Mosaics are particularly useful for this application as they allow different substrates to be combined and analysed in parallel [Figure 5]. Recently, several new substrates have been developed to improve the ease and reliability of ANCA analysis still further. HEp-2 cells coated with granulocytes allow immediate differentiation between ANCA and anti-nuclear antibodies, while BIOCHIPs containing microdots of purified MPO or PR3 enable monospecific antibody characterization at the same time as the ANCA screening [5, 6].
Monospecific enzyme immunoassays such as ELISA or immunoblot are used to characterize the specificity of the target antigen. A recent major advance in ANCA ELISA is the development of a novel PR3 diagnostic antigen comprising an optimized mixture of native human (hn) PR3 and designer recombinant PR3 expressed authentically in human cells (hr). An ELISA based on this combined antigen provides unsurpassed sensitivity for the detection of anti-PR3 antibodies – 14% higher than even a capture ELISA (7). The Anti-PR3-hn-hr ELISA thus enhances ANCA diagnostics and is also suitable for long-term evaluation of patients.
Anti-dsDNA and anti-nucleosome antibodies
Anti-dsDNA and ANuA are among the immunological parameters used to diagnose SLE, which counts nephritis among its many and variable manifestations. These two markers provide the highest specificity and sensitivity in the serological diagnosis of SLE.
Anti-dsDNA antibodies are found in 60-90% of patients and represent the most established marker for SLE. A recently developed ELISA provides an exceptionally high sensitivity and specificity for detection of these antibodies owing to the use of a novel coating technology based on highly adhesive nucleosomes. The unspecific reactions that typically occur with traditionally used coating materials are thus avoided, and the clear presentation of the major DNA epitopes ensures a remarkably high sensitivity. In a published clinical comparison study using a large cohort of patients with SLE and other diseases [8], the Anti-dsDNA-NcX ELISA demonstrated the highest sensitivity for SLE (60.8%), exceeding that of conventional ELISA (35.4%), Crithidia luciliae IIFT (27.4%) and even Farr-RIA (53.1%) [Figure 6].
ANuA [Figure 7] are specific for SLE and are a prognostic indicator for SLE with renal involvement. The frequency of ANuA is especially high in severe cases requiring transplantation (79%), compared to less severe lupus nephritis (18%) and SLE without nephritis (9%) [9]. The relevance of ANuA is, however, highly dependent on the assay used to detect them. If insufficiently purified nucleosomes are used in ELISA, then sera from patients with scleroderma or other diseases also frequently react, resulting in an unacceptably low specificity. The 2nd generation Anti-Nucleosome ELISA, in contrast, is based on a patented preparation of highly purified mononucleosomes, which are free of contaminating histone H1, non-histone proteins such as Scl-70, and chromatin DNA fragments. This ELISA provides an SLE specificity of close to 100% and a sensitivity of around 54%. Significantly, with this highly specific test ANuA have been shown to be present in 16-18% of SLE sera that are negative for anti-dsDNA antibodies [Table 1] [10, 11]. Thus, the determination of ANuA substantially enriches the serological diagnosis of SLE. When both ANuA and anti-dsDNA antibodies are analysed in parallel as first-line serological tests, the detection rate for SLE can be increased to 87%.
Conclusions
Recent developments in autoantibody diagnostics for nephrology include the groundbreaking anti-PLA2R IIFT for identifying primary MGN, as well as considerable improvements in the sensitivity, specificity and convenience of tests for ANCA, anti-GBM, anti-dsDNA and ANuA. These advances have boosted the ease, reliability and relevance of autoantibody testing, aiding the diagnosis of autoimmune forms of GN, especially in their early stages. This is crucial to allow the implementation of interventional therapy and prevent the nephropathy progressing to a fatal end stage.
References
1. Beck et al. N. Engl. J. Med. 2009: 361: 11.21
2. Hoxha et al. Nephrology Diagnosis Transplantation 2011: 26 (8): 2526-32.
3. Debiec et al. Nat. Rev. Nephrol. 2011: 7(9): 496-8
4. Hoxha et al. Kidney International. 2012: 82: 797-804
5. Buschtez et al. Zeitschrift für Rheumatologie 2007: Band 66: 43, 10942-10.
6. Damoiseaux et al. JIM 2009: 348: 67-73
7. Damoiseaux J. et al. Ann. Rheum. Dis. 2009; 68: 228-233.
8. Biesen et al. Lupus 2008; 17(5): 506-507.
9. Stinton et al. Lupus 2007; 15: 394-400.
10. Suer et al. J. Autoimmunity 2004: 22: 325-334.
11. Schluter et al. J. Lab Med. 2002; 26: 516-517.
The author
Jacqueline Gosink, PhD
Euroimmun AG
Luebeck, Germany
by Dr Magnus Borres Molecular allergology enables quantification of IgE antibodies to single allergen protein components at the molecular level. This helps the clinician establish the cause of allergic sensitisation, evaluate the risk for severe allergic reactions and improve patient management. New tests and technologies enable the laboratory to assist in an efficient manner.
Diagnosing cutaneous leishmaniasis histologically depends on the identification of the amastigotes, which is inconclusive and leads to cases of missed diagnosis or misdiagnosis. In this article, we describe a rapid diagnostic molecular method for Leishmania species identification and differentiation using DNA extracted from formalin-fixed paraffin-embedded (FFPE) skin tissue biopsies.
by L. Yehia and Dr I. Khalifeh
Clinical background
Cutaneous leishmaniasis is a chronic disease caused by Leishmania protozoan parasites that is on the increase in endemic and non-endemic regions because of environmental changes triggered by humans [1, 2]. It is most prevalent in the Middle East and North Africa. With changes in vector (sandfly), habitat and increased travel among populations, the incidence of leishmaniasis is showing a clear increase [3].
There are more than 20 strains of Leishmania that are pathogenic to humans [4], and these are partially responsible for its clinical diversity. The diagnosis of cutaneous leishmaniasis rests on the pathological identification of the amastigotes, which may be inconclusive [5]. This is dependent on the strain type, host response and the disease stage. Accurate microscopic diagnosis is essential to permit appropriate targeted therapy [6].
Clinically, cutaneous leishmaniasis may be asymptomatic and self-limiting. However, cases progressing to mutilating ulceration and disfiguring scarring have also been reported [7]. As the disease progresses, the number of amastigotes decreases to the point where none can be detected microscopically. The absence of amastigotes is a common problem encountered in up to 47% of cases [8]. In such instances, the diagnosis of cutaneous leishmaniasis must not be excluded [4].
Materials and methods
Skin biopsies embedded into FFPE tissue blocks were collected for 122 patients diagnosed clinically with cutaneous leishmaniasis. Cases included in the study were restricted to cutaneous lesions of patients who did not receive treatment prior to the biopsy. Cases with visceral or mucocutaneous involvement and with material insufficient for PCR or histopathological examination were excluded. Clinical information pertaining to the lesion was also collected including: number, duration, location and dermatologic appearance. In addition, the patient’s age, gender and country of residency were tabulated.
Cases were classified according to the modified Ridley’s parasitic index, a traditionally used pathological scoring system based on microscopic analysis of hematoxylin and eosin stained slides. DNA was then extracted from FFPE tissue blocks of each patient. Polymerase chain reaction (PCR) was performed using Leishmania-specific ribosomal internal transcribed spacer 1 (ITS1-PCR). Nested ITS1-PCR was performed on cases negative for conventional ITS1-PCR. ITS1-PCR amplicons were then digested with HaeIII for subsequent restriction fragment length polymorphism (RFLP) subspeciation.
Results
Of the 122 skin biopsies, microscopic evaluation of stained slides identified 54 cases (44.3%) labeled as histologically negative (with no unequivocal amastigotes detected). Of these negative cases, 9 (17%) were shave biopsies and 45 (83%) were punch biopsies.
DNA extracted from FFPE tissue blocks collected for all cases ranged from 4 to 1672 ng/μl (mean=213 ng/μl, SD=289 ng/μl). The oldest blocks were 19 years of age, whereas the newest were less than 1 year old. The quantity of the extracted DNA dating back to 1992 was 166 ng/μl (SD=128 ng/μl), whereas that for specimen from the year 2010 was 272 ng/μl (SD=161 ng/μl) indicating that a good quantity of DNA could be extracted from archival well-preserved FFPE tissues, even when they were old.
ITS1-PCR was performed on DNA extracted from all cases. Initially, and regardless of the histopathological analysis, 55 (45%) cases were positive and showed a band of between 300 and 350 base pairs indicative of Leishmania by agarose gel electrophoresis. The remaining 67 (55%) were negative (Fig. 1A, B). The negative cases were subjected to nested ITS1-PCR and 100% of these cases actually turned out to be positive for Leishmania (Fig. 1C).
Comparing the resultant ITS1-PCR bands to the DNA pattern of normal skin tissues, we identified 54 cases – that had been shown as negative by histopathology according to Ridley’s parasitic index – that amplified DNA with Leishmania-specific primers by conventional or nested ITS1-PCR, and that failed to show the normal skin profile seen in the negative controls tested. RFLP analysis identified L. tropica subspecies in all cases, identified by the presence of a 200 and 60 base pairs restriction fragments (Fig. 2) [9].
Clinical and diagnostic significance
Cutaneous leishmaniasis is a disease that is endemic in many regions of the world. With the ease of travel in the world, human and animal reservoirs of Leishmania parasites have been established in regions that previously were not known to harbour the sandfly vector because of habitat incompatibility. Thus, novel endemic areas have emerged in regions across the world. Therefore, a high index of suspicion becomes crucial for early diagnosis and control of leishmaniasis. With the advent of molecular diagnostic techniques and their high sensitivity and specificity, it has become easier to detect and control many infectious diseases, including leishmaniasis, as shown in this and other studies.
Traditionally, direct detection of parasites is performed by microscopic examination of clinical specimens or by cultivation, but either approach may be diagnostically problematic [1, 4, 10]. Cultures may take long periods, possibly weeks, for sufficient parasites to grow for species characterization. In addition, success in microscopic identification of amastigotes in stained preparations varies depending on the number of parasites present and/or the experience of the person examining the slide [11]. This is mainly due to the fact that all Leishmania species are morphologically similar and may present with a variable number of amastigotes. As the disease progresses, the number of amastigotes decreases to the point where none can be detected histopathologically.
Despite these drawbacks, microscopic identification and parasite cultivation are still the primary diagnostic tools used in most regions where leishmaniasis is endemic. However, it is stressed that accurate and rapid species identification is not possible using either technique. In the last decade, polymerase chain reaction (PCR) analysis has been successfully introduced and has been proven to be the most sensitive molecular tool for direct detection and parasite characterization of Leishmania species in clinical samples [1, 5, 12].
Accurate Leishmania species identification and subspeciation in clinical specimens is now possible by subjecting the extracted DNA to PCR, followed by enzymatic digestion to identify restriction fragments indicative of the subspecies. Such amplification using Leishmania-specific primers allows the indirect yet conclusive detection of the amastigotes, when present in a given clinical specimen. A highly sensitive method is valuable especially in chronic cases where the parasitic index is low and potentially undetectable by conventional microscopy.
Conclusion
This study successfully identified L. tropica in 54 skin biopsies from patients clinically suspected of having cutaneous leishmaniasis with negative biopsies. The importance of this result is manifested in the need for diagnostic tools that are sensitive, specific, rapid and capable of identifying all clinically significant Leishmania species from FFPE tissue blocks (Fig. 3).
Therefore, ITS1-PCR carried out on DNA extracted from FFPE tissue specimens, followed by HaeIII RFLP analysis, is a valuable method for the rapid and reliable diagnosis of cutaneous leishmaniasis. In chronic cases where the parasite load is low, or when insufficient tissue is available, nested ITS1-PCR can be performed to increase sensitivity. The advantages of this method are also highlighted with the possibility of using different biological specimens, and the ability to detect both Old World and New World leishmaniasis.
The work summarized here was first published as Yehia L. et al., 2012 [13].
References
1. Schonian G, et al. Diagn Microbiol Infect Dis 2003; 47: 349.
2. Goto H, Lindoso JA. Expert Rev Anti Infect Ther 2010; 8: 419.
3. Scarisbrick JJ, et al. Travel Med Infect Dis 2006; 4: 14.
4. Ameen M. Clin Exp Dermatol 2010; 35: 699.
5. Singh S, et al. Expert Rev Mol Diagn 2005; 5: 251.
6. Salman SM, et al. Clin Dermatol 1999; 17: 291.
7. David CV, Craft N. Dermatol Ther 2009; 22: 491.
8. Safaei A, et al. Dermatology 2002; 205: 18.
9. Kazemi-Rad E. Iran J Public Health 2008; 37: 54.
10. Farah FS, et al. Arch Dermatol 1971; 103: 467.
11. Bensoussan E, et al. J Clin Microbiol 2006; 44: 1435.
12. Schonian G, et al. Trends Parasitol 2008; 24: 135.
13. Yehia L, et al. J Cutan Pathol 2012; 39: 347–355.
The authors
Lamis Yehia, BSc
Biomedical Sciences Training Program, Case Western Reserve University in Cleveland, Ohio, USA
Ibrahim Khalifeh, MD
Department of Pathology and Laboratory Medicine, American University of Beirut, Beirut, Lebanon
E-mail: ik08@aub.edu.lb
There are many peer-reviewed papers covering autoimmunity, and it is frequently difficult for healthcare professionals to keep up with the literature. As a special service to our readers, CLI presents a few key literature abstracts from the clinical and scientific literature chosen by our editorial board as being particularly worthy of attention.
Unraveling multiple MHC gene associations with systemic lupus erythematosus: model choice indicates a role for HLA alleles and non-HLA genes in Europeans
In order to determine the association with both SNPs and classical human-leukocyte-antigen (HLA) alleles, a meta-analysis of the major-histocompatibility-complex (MHC) region in systemic lupus erythematosus (SLE) was performed. Results from six studies and well-known out-of-study control data sets were combined, providing 3701 independent SLE cases and 12 110 independent controls of European ancestry. The study used genotypes for 7199 SNPs within the MHC region and for classical HLA alleles (typed and imputed). The results from conditional analysis and model choice with the use of the Bayesian information criterion showed that the best model for SLE association includes both classical loci (HLA-DRB1*03:01, HLA-DRB1*08:01, and HLA-DQA1*01:02) and two SNPs, rs8192591 (in class III and upstream of NOTCH4) and rs2246618 (MICB in class I). The authors’ approach was to perform a stepwise search from multiple baseline models deduced from a priori evidence on HLA-DRB1 lupus-associated alleles, a stepwise regression on SNPs alone, and a stepwise regression on HLA alleles. This enabled them to identify a model that was a much better fit to the data than one identified by simple stepwise regression either on SNPs alone [Bayes factor (BF) > 50] or on classical HLA alleles alone (BF > 1,000).
Cellular targeting in autoimmunity
Many biologic agents that were first approved for the treatment of malignancies are now being actively investigated and used in a variety of autoimmune diseases such as rheumatoid arthritis (RA), antineutrophil cytoplasmic antibody (ANCA)-associated vasculitis, systemic lupus erythematosus (SLE), and Sjogren’s syndrome. The relatively recent advance of selective immune targeting has significantly changed the management of autoimmune disorders and in part can be attributed to the progress made in understanding effector cell function and their signalling pathways. This review discusses the recent FDA-approved biologic therapies that directly target immune cells as well as the most promising investigational drugs affecting immune cell function and signalling for the treatment of autoimmune disease.
Mechanisms of premature athero-sclerosis in rheumatoid arthritis and lupus
Rheumatoid arthritis (RA) and systemic lupus erythematosus (SLE), the two most common systemic autoimmune disorders, have both unique and overlapping manifestations. One feature they share is a significantly enhanced risk of atherosclerotic cardiovascular (CV) disease that significantly contributes to morbidity and mortality. The primary mechanisms that drive CV damage in these diseases remain to be fully characterized, but recent discoveries indicate that distinct inflammatory pathways and immune dysregulation characteristic of RA and SLE are likely to play prominent roles. This review focuses on analysing the major mechanisms and pathways that are potentially implicated in the acceleration of atherothrombosis and CV risk in SLE and RA, as well as in the identification of putative preventive strategies that may mitigate vascular complications in systemic autoimmunity.
The role of epigenetic mechanisms and processes in autoimmune disorders
The lack of complete concordance of autoimmune disease in identical twins suggests that non-genetic factors play a major role in determining disease susceptibility. This review considers how epigenetic mechanisms could affect the immune system and effector mechanisms in autoimmunity and/or the target organ of autoimmunity and thus affect the development of autoimmune diseases. The authors also discuss the types of stimuli that lead to epigenetic modifications and how these relate to the epidemiology of autoimmune diseases and the biological pathways operative in different autoimmune diseases. Increasing our knowledge of these epigenetic mechanisms and processes will increase the prospects for controlling or preventing autoimmune diseases in the future through the use of drugs that target the epigenetic pathways.
Coronaviruses are a group of positive sense, single-stranded RNA viruses that infect humans and animals. In a short period of time the SARS-associated coronavirus was identified and initial laboratory protocols for diagnosis of SARS were disseminated. The need for the early diagnosis of SARS is vital due to the difficulty in clinically diagnosing this infection and its rapid nosocomial transmission.
by Dr Hoon H. Sunwoo and Dr Arivazhagan Palaniyappan
Clinical background
Severe acute respiratory syndrome (SARS) is a life-threatening viral respiratory illness caused by a coronavirus known as SARS-associated coronavirus (SARS-CoV, but usually shortened to SARS). The SARS-CoV is associated with a flu-like syndrome, which may progress into pneumonia, respiratory failure, and sometimes death. It is believed that SARS-CoV originated in the Guangdong Province in southern China and the virus has subsequently spread around the world. China and its surrounding countries have witnessed the greatest numbers of SARS-related cases and death.
SARS history is short. SARS-CoV was first reported in 2002 in Asia and cases were reported until mid-year 2003. According to the World Health Organization (WHO), as of July 2003, a total of 8437 people worldwide became ill and 813 died during the SARS outbreak or epidemic. Illness was reported in more than 30 countries and on 5 continents. This new emerging disease represented the most recent threat to human health as it has been reported to be highly contagious. Infection with the SARS-CoV causes acute respiratory distress (severe breathing difficulty) and sometimes death.
SARS-CoV Diagnosis
Three major diagnosis methods are currently developed (i) viral RNA detection using quantitative reverse transcription (RT)-PCR, (ii) antibody detection using indirect fluorescence assay (IFA), and (iii) using both recombinant nucleocapsid protein (NP) and culture extract of SARS-CoV–based enzyme-linked immunosorbent assay (ELISA). ELISA based antibody detection tests with recombinant antigens are well known to offer higher specificity and reproducibility. Such tests are easy to standardize and less labour intensive than antibody detection by indirect IFA and thus avoids the requirement of growing SARS-CoV.
RT-PCR has been widely used for the rapid diagnostic of the viral genome in different clinical specimens. Early diagnosis of SARS-CoV infection, which involves viral RNA detection by RT-PCR, first targeted the polymerase (pol) 1b region of the 5’ replicase gene using different formats including one-step or two-step RT-PCR or real-time PCR assays. A comprehensive monitoring of the time periods of RT-PCR diagnosis after disease onset in different types of specimens such as tracheal and nasopharyngeal aspirates, throat swabs, nasal swabs and rectal swabs has also been studies. This study demonstrates that the peak detection rate for SARS-CoV occurred at 2 weeks after the onset of stool or rectal swab specimens and at week 4 for urine specimens [1]. It is likely that the current RT-PCR is not quite sensitive enough to detect the early diagnosis of SARS, showing that the detection rate for probable SARS was only 37.5–50%.
The presence of specific antibodies against various viral components has been a classical diagnostics method. It has been found that anti-NP antibodies in patients’ sera are detected early and with high specificity during the infection. Three different methods, Western blot, ELISA and IFA, used both native and bacterially produced SARS antigens to evaluate serum samples obtained from SARS patients, 40 patients with non-SARS pneumonia, and 38 health individuals. A report indicated that 89% of the SARS patients’ sera were found to be positive to SARS-CoV NP antigen by Western blot that had a strong ability to detect antibodies against SARS. The sensitivity and specificity was reported to be 98.5 and 100% respectively [2]. There was no cross reactivity between the N195 protein and antibodies against chicken, pig and canine coronaviruses. The Western blot assay could distinguish patients with fewer caused by other diseases from that of SARS patients, through reducing the possibility of false positives.
Our earlier study also showed that different combinations of monoclonal antibody (mAb), bispecific antibody (bsmAb), and IgY polyclonal antibody detected the SARS-CoV NP by Immunoswab assay [3] and sandwich ELISA [4] with a sensitivity of 18.5 pg/ml of recombinant SARS-CoV NP antigen in-vitro [Figure 1]. Antibodies against the NP have longer a shelf life and occur in greater abundance in SARS patients than antibodies against other viral components such as the spike protein (SP), membrane and envelope protein. This may be due to the presence of higher levels of NP, compared with other viral proteins, after SARS-CoV infection. A recombinant NP-based IgG ELISA was more sensitive than a recombinant S-protein-based IgG ELISA for diagnosis of SARS-CoV in serum [5–6], due to the highly immunogenic region of N2. It may help in explaining the present results that show less sensitivity of SP detection, compared to a previous NP detection study [4].
Recent studies demonstrate that mAbs and bsmAb could be useful reagents for the diagnosis of SARS-CoV, as well as for functional analysis of SP during infection. Further, the present study shows the development of a novel sandwich ELISA test with a potential use for the diagnosis of SARS-CoV infections based on bsmAb that recognize simultaneously the SP of SARS-CoV and the enzyme peroxidase [7] [Figure 2]. In addition to allowing the rapid diagnosis of SARS infection, the availability of diagnostic tests will help to address important questions such as the period of virus shedding during convalescence, the presence of virus in different body fluids and excreta, and the presence of virus shedding during the incubation period. Until a certain degree of standardization and quality assurance has been achieved for the SARS-CoV laboratory tests, test results must be used with utmost caution in clinical situations. It is strongly advisable to closely check on updated recommendations by the WHO and relevant national organizations regarding the availability and use of such tests.
Limitations
All tests for SARS-CoV available so far have limitations. Extreme caution is therefore necessary when management decisions are to be based on virological test results. In particular, false negative test results (due to low sensitivity, unsuitable sample type, or time of sampling, etc.) may give a false sense of security; in the worst case, they could allow persons carrying the SARS virus, and therefore capable of infecting others, to escape detection.
To aid in the better understanding of SARS, the WHO recommends that sequential samples be stored from patients with suspected or probable SARS – and also close contacts who are not ill themselves – for future use. This is particularly important for the first case(s) recognized in countries that have not previously reported SARS. Data on the clinical and contact history should also be collected in order to obtain a better understanding of the shedding pattern of the virus and the period of transmissibility. Such patient samples should be suitable for viral culture, PCR, antigen detection, immunostaining and/or serological antibody assays. The WHO also encourages each country to designate a reference laboratory for investigation and/or referral of specimens from possible SARS patients.
Future SARS outbreaks
Although the threat of SARS to public health seems to have passed, international health officials continue to remain vigilant. The WHO monitors countries throughout the world for any unusual disease activity (http://www.who.int/csr/sars/en/). Therefore, if another SARS outbreak is to occur, it should be possible to limit the spread of infection using the same measures implemented during the 2002/3 pandemic.
References
1. Chan PK, To WK, Ng KC, Lam RK, Ng TK, et al. Laboratory diagnosis of SARS. Emerg Infect Dis 2004; 10: 825–831.
2. He Q, Chong KH, Chng HH, Leung B, Ling AE, et al. Development of a Western blot assay for detection of antibodies against coronavirus causing severe acute respiratory syndrome. Clin Diagn Lab Immunol 2004; 11: 417–422.
3. Kammila S, Das D, Bhatnagar PK, Sunwoo HH, et al. A rapid point of care immunoswab assay for SARS-CoV detection. J Virol Methods 2008; 152: 77–84.
4. Palaniyappan A, Das D, Kammila S, Suresh MR, Sunwoo HH. Diagnostics of severe acute respiratory syndrome-associated coronavirus (SARS-CoV) nucleocapsid antigen using chicken immunoglobulin Y. Poult Sci 2012; 91: 636–642.
5. Rota PA, Oberste MS, Monroe SS, Nix WA, Campagnoli R, Icenogle JP. Characterization of novel coronavirus associated with severe acute respiratory syndrome. Science 2003; 300: 1394–1399.
6. Woo PC, Lau SK, Wong BH, Tsoi HW, Fung AM, et al. Differential sensitivities of severe acute respiratory syndrome (SARS) coronavirus spike polypeptide enzyme-linked immunosorbent assay (ELISA) and SARS coronavirus nucleocapsid protein ELISA for serodiagnosis of SARS coronavirus pneumonia. J Clin Microbiol 2005; 43: 3054–3058.
7. Sunwoo HH, Palaniyappan A, Ganguly A, Bhatnagar PK, et al. Quantitative and sensitive detection of the SARS-CoV spike protein using bispecific monoclonal antibody-based enzyme-linked immunoassay. J Virol Methods 2013; 187: 72–78.
The authors
Hoon H. Sunwoo* PhD and Arivazhagan Palaniyappan PhD
Faculty of Pharmacy and Pharmaceutical Sciences,
University of Alberta, Edmonton, Alberta, Canada T6G 2E
*Corresponding author
E-mail: hsunwoo@ualberta.ca
Chronic renal failure is a disease with a high and increasing prevalence. Currently about 10% of the population of Europe and North America are affected. The disease is associated with a high morbidity and mortality mainly attributed to cardiovascular diseases. In fact patients with more advanced stages of chronic renal failure have a greater risk of dying due to cardiovascular disease than of renal failure itself. Approximately 50% of these patients die from cardiovascular complications.
by Professor Berthold Hocher
Accelerated vascular calcification (VC) is but one of the important mechanisms of cardiovascular disease in dialysis patients. Under the setting of end-stage renal disease (ESRD), VC is more severe and develops in both the intima and the media of the blood vessels. VC is an active and regulated process mediated by vascular smooth muscle cells [Fig. 1], which undergo a phenotypic change to osteoblasts or chondrocytes, which, in turn, release promoters of VC and apoptosis. VC is markedly up regulated in dialysis patients, and this may be explained by the up-regulation of such promoters of VC as hyperphosphatemia, hypercalcemia, cholesterol, hyperleptinemia down-regulation of the inhibitors of VC such as matrix Gla protein, fetuin-A [1].
Fetuin-A
Fetuin-A is a 62-kilodalton glycoprotein, which belongs to the cystatin superfamily of proteins. In humans, the 349-amino acid protein, as secreted from the liver, consists of two chains: a heavy and a light chain joined by a connecting segment and linked by disulfide bonds. The N-terminus of the heavy chain consists of two cystatin domains, D1 and D2; the acidic amino acids in the D1 domain appear to account for fetuin’s ability to inhibit precipitation of calcium and phosphorus. Indeed, fetuin-A accounts for up to one-half of the in vitro capacity of the serum to prevent the precipitation of calcium and phosphorus. It is now recognised that fetuin-A can actively regulate the cell-mediated process of osteogenesis in the vessel wall, inhibits mineralisation in a concentration-dependent manner, enhances the phagocytosis of apoptotic bodies by vascular smooth muscle cells, limiting their ability to nucleate calcium phosphate. Finally, fetuin-A is an antagonist of bone morphogenetic protein-2, the promoter of VC in vascular cells.
A number of studies have demonstrated an association between serum fetuin-A levels and all-cause mortality of dialysis patients. This association of low fetuin-A levels and mortality was confirmed by clinical trial on 664 hemodialysis (HD) and 323 peritoneal dialysis (PD) patients during a median follow-up of 2.8 years. In this study, an increase in serum fetuin-A by 0.1 g per litre corresponded to a 9% lower death risk. The death predictable value of fetuin-A in this study was independent of serum C-reactive protein (CRP) levels. At the same time, in multivariate analysis of biomarkers of prediction of mortality dialysis patients where serum C-reactive protein was entered, fetuin-A lost its predictable value. The latter fact suggests further investigation of the role of fetuin-A in dialysis patients is needed to fully elucidate the pathomechanisms lowering serum fetuin-A levels in ESRD [1].
Fibroblast growth factor 23 (FGF-23)
FGF-23 is a hormone secreted by osteoblasts. It plays a role in the regulation of phosphorus and in the metabolism of vitamin D. Depletion of FGF-23 causes hyperphosphatemia, up-regulation of 1,25- dihydroxyvitamin D, ectopic calcification and early death. FGF-23 is involved in physiological maintenance of normal serum phosphate levels in the settings of variable dietary phosphorus intake. In the settings of impaired/reduced nephron mass, normal serum phosphate levels are maintained in part by reactive increase of FGF-23, which promotes excretion of phosphate via the remaining nephrons and decreases the absorption of dietary phosphorus by inhibiting the synthesis of 1,25-dihydroxyvitamin D. Depletion of FGF-23 with chronic kidney disease (CKD) progression leads to hyperphosphatemia, ectopic calcification and premature death. It was previously reported that increased serum phosphate levels and decreased 1,25-dihydroxyvitamin D levels are associated with increased mortality.
In the recent study by Gutiérrez et al., multivariable adjusted analyses showed that an increase in serum phosphate levels higher than 5.5 mg/dl and an increase of FGF-23 was associated with a 20% increase in the mortality risk, suggesting hyperphosphatemia and increased FGF-23 are sensitive biomarkers for assessment of the risk of death [reviewed in 1].
Receptor activator of NF-kB ligand–Osteoprotegerin System Osteoblasts regulate differentiation and activation of osteoclasts under conditions of normal bone turnover. Osteoblasts synthesise and secrete a protein called receptor activator of NF-κB ligand (RANKL). RANKL binds to its receptor on pre-osteoclasts and thus regulates bone turnover. Osteoprotegerin (OPG) is also secreted by osteoblasts and modulates the effects of RANKL by blocking osteoblast differentiation. These two key players are also involved in the transformation of vascular smooth muscle cells into bone formatting cells in blood vessels under the condition of chronic renal failure.
Several studies indicate a pathogenic role of OPG in the pathogenesis of cardiovascular diseases in uremic and also non-uremic patients. The OPG/RANKL system plays a key role in the pathogenesis of endothelial function. Tseng et al. suggest that an imbalance between bone formatting hormones and bone degrading hormones may play a key role in the pathogenesis of vascular calcification. High OPG might indicate a reduced degradation capacity of calcified arteries. These authors suggest that an induction of RANKL in the vessel walls might overcome this problem and thus offer even new therapeutic options for vascular calcification. However, this hypothesis needs for sure further investigations [2–4].
Vitamin D
Vitamin D is a multifunctional hormone that can affect many essential biological functions, ranging from immune regulation to mineral ion metabolism. A close association between altered activity of vitamin D and vascular calcification has been reported in various human diseases, including patients with atherosclerosis, osteoporosis and CKD. Experimental studies have shown that excessive vitamin D activities can induce vascular calcification, and such vascular pathology can be reversed by reducing vitamin D activities. The human relevance of these experimental studies is not clear, as vitamin D toxicity is relatively rare in the general population. Contrary to the relationship between vitamin D and vascular calcification, in experimental uremic models low levels of vitamin D were shown to be associated with extensive vascular calcification – a phenomenon that is very similar to the vascular pathology seen in patients with CKD. The current treatment approach of providing vitamin D analogues to patients with CKD often poses a dilemma, as studies linked vitamin D treatment to subsequent vascular calcification. In any case, a close monitoring of the vitamin D status in patients with CKD is indicated to ensure that these patients have vitamin D levels associated with the best survival likelihood [5, 6].
Osteopontin
Osteopontin (OPN) was initially identified in osteoblasts as a mineralisation-modulatory matrix protein. Recently, OPN has been studied as a multifunctional protein that is up regulated in a variety of acute and chronic inflammatory conditions, such as wound healing, fibrosis, autoimmune disease and atherosclerosis. OPN is highly expressed at sites with atherosclerotic plaques, especially those associated with macrophages and foam cells. In the context of atherosclerosis, OPN is generally regarded as a pro-inflammatory and pro-atherogenic molecule. The role of OPN in VC, which is closely related to chronic and active inflammation, is that of a negative regulator. It is an inhibitor of calcification and an active inducer of decalcification. OPN expression and its regulatory molecular mechanisms remain elusive during the process
of vascular calcification. Therefore, further research with regard to the role of OPN in diseases associated with VC is needed to identify potential OPN-related therapeutic targets [7].
References
1. Chaykovska L, Tsuprrykov O, Hocher B. Biomarkers for the prediction of mortality and morbidity in patients with renal replacement therapy. Clin Lab 2011; 57(7–8): 455–467.
2. Shin JY, Shin YG, Chung CH. Elevated serum osteoprotegerin levels are associated with vascular endothelial dysfunction in type 2 diabetes. Diabetes Care 2006; 29(7): 1664–1666.
3. Tseng W, Graham LS, Geng Y, Reddy A, Lu J, Effros RB, et al. PKA-induced receptor activator of NF-kappaB ligand (RANKL) expression in vascular cells mediates osteoclastogenesis but not matrix calcification. J Biol Chem 2010; 285(39): 29925–29931.
4. Ozkok A, Caliskan Y, Sakaci T, Erten G, Karahan G, Ozel A, et al. Osteoprotegerin/RANKL axis and progression of coronary artery calcification in hemodialysis patients. Clin J Am Soc Nephrol 2012; 7(6): 965–973.
5. Lieb W, Gona P, Larson MG, Massaro JM, Lipinska I, Keaney JF Jr, et al. Biomarkers of the osteoprotegerin pathway: clinical correlates, subclinical disease, incident cardiovascular disease, and mortality. Arterioscler Thromb Vasc Biol 2010; 30(9): 1849–1854.
6. Ellam TJ, Chico TJ. Phosphate: the new cholesterol? The role of the phosphate axis in non-uremic vascular disease. Atherosclerosis 2012; 220(2): 310–318.
7. Ketteler M, Rothe H, Krüger T, Biggar PH, Schlieper G. Mechanisms and treatment of extraosseous calcification in chronic kidney disease. Nat Rev Nephrol 2011; 7(9): 509–516.
The author
Berthold Hocher, M.D., Ph.D.
Institute of Nutritional Science, University of Potsdam,
D-14558 Nuthetal-Potsdam, Germany
E-mail: hocher@uni-potsdam.de
www.uni-potsdam.de/eem
March 2026
Prins Hendrikstraat 1
5611HH Eindhoven
The Netherlands
info@clinlabint.com
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