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Featured Articles
Scientific literature: autoimmunity
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
Morris DL et al. A J Hum Genet. 2012; doi: 10.1016/j.ajhg.2012.08.026.l
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
Rogers JL et al. Curr Allergy Asthma Rep. 2012; doi: 10.1007/s11882-012-0307-y.
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
Kahlenberg JM & Kaplan MJ. Annu Rev Med. 2012; doi: 10.1146/annurev-med-060911-090007.
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
Greer JM & McCombe PA. Biologics 2012; 6: 307–27. l
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.
Diagnosis of SARS-associated coronavirus
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
Biomarkers of vascular calcification in patients with impaired kidney function
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
Diagnosis of blood-borne parasitic infections: an overview
Methods for the diagnosis of blood-borne parasitic infections have stagnated in the last 20–30 years. However, recently, there has been a tremendous effort to focus research on the development of newer diagnostic methods focusing on serological, molecular, and proteomic approaches. This article examines the various diagnostic tools that are being used in clinical laboratories, optimized in reference laboratories and employed in mass screening programmes.
by A. Ricciardi and Dr M. Ndao
Blood-borne protozoans are the causative agents of some of the world’s most devastating and prevalent parasitic infections. This group of pathogens includes members of the Trypanosoma, Leishmania, Plasmodium, Toxoplasma, and Babesia genera. Most of these infections, with the exception of toxoplasmosis and babesiosis, have always been described as being tropical or subtropical. However, the increase in international travel as well as the arrival of new immigrants has made some of these tropical diseases realities in developed countries as well. In addition, infection via contaminated blood (transfusions and organ transplants) has become a major problem. Clearly, the transmission of blood-borne protozoans is boundless and the actual number of cases is underestimated. Quick diagnosis has always been a priority in order to determine the appropriate treatment and prevent fatalities. In addition, now more than ever, advances in diagnostics can help prevent transmission and provide active surveillance. Currently, diagnostic and reference laboratories use an array of techniques including microscopy, serological assays, and molecular assays. Here, the advantages and disadvantages of the methods will be discussed.
Toxoplasmosis
Toxoplasmosis, caused by Toxoplasma gondii, has a worldwide distribution. In immunocompetent individuals, more than 80% of primary Toxoplasma infections are asymptomatic [1]. Toxoplasmosis becomes a problem when an individual is immunocompromised or during pregnancy. Diagnosis of toxoplasmosis varies according to the immune status of the patient.
Diagnosis of immunocompetent individuals relies on serology. Early antibody responses can be detected via methods such as the dye test, immunofluorescent assay, and agglutination test whereas later IgG titres are detected by enzyme-linked immunosorbent assay (ELISA). For many years, the Sabin-Feldman dye test was the gold standard diagnostic technique due to its sensitivity and specificity. In recent years, few laboratories have continued to use this method and rather focused on newer techniques such as indirect immunofluorescent antibody tests, hemagglutination tests, capture ELISAs, and immunosorbent agglutination assays (ISAGAs). Serological assays lack the capacity to differentiate between recent and older infections; IgM levels can persist for over two years [2]. In order to determine whether an infection is recent, avidity ELISA is performed. This assay verifies IgG avidity and is based on the concept that as the immune response progresses, an immunoglobulin’s affinity for a specific antigen will increase [3].
Diagnosis of Toxoplasma infection during pregnancy is crucial in order to prevent congenital toxoplasmosis. Prenatal diagnosis involves performing real-time polymerase chain reaction (PCR) using amniotic fluid. The PCRs used often target the B1 gene of the parasite [1]. Upon delivery, PCR is performed on either the placenta or the cord blood serum in order to detect parasites. ISAGAs are also often performed. If the tests are positive, cord blood samples at one week of life are sent to a reference laboratory [1]. Follow up serology is again performed at one month and then every two to three months. There have been recent advances in the field of toxoplasmosis post-natal diagnosis. An ELISA assay that measures interferon-gamma levels upon stimulation of whole blood cells with Toxoplasma crude antigens has been developed. This method has proven to be both sensitive and specific [4].
In the case of immunocompromised patients, a quick diagnosis is essential because the infection can be fatal. Diagnosis relies on detecting parasites either by PCR or microscopy. Microscopic examination of Giemsa-stained tissues or smears is the quickest and most inexpensive method for diagnosing toxoplasmosis. However, poor sensitivity is the major pitfall of this method. PCR can also be performed on blood or cerebral spinal fluid (CSF) samples in order to detect parasite DNA. However, the degree of sensitivity attained by the PCRs is questionable and requires further investigations [1].
Leishmaniasis
Protozoans of the Leishmania genus are transmitted to humans via sand fly bites. Visceral leishmaniasis (VL), which is a lethal infection if left untreated, can also be transmitted by blood transfusions, organ transplants, and sharing of needles among intravenous drug users.
Direct parasitological methods, such as microscopy and cultures, are the gold standard methods when diagnosing VL. These methods have high specificity, but varying sensitivity. Direct detection of parasites is performed by microscopic examination of aspirates from spleen, bone marrow, or lymph nodes [5]. Using spleen samples increases sensitivity, but the procedure to obtain the aspirates risks internal bleeding. Parasite culturing from aspirates is widely used by reference laboratories.
Extensive research on the development of Leishmania serological assays has uncovered a myriad of candidate diagnostic antigens. The most promising antigens were the kinesin-related proteins. From this group, rK39 was the most tested antigen [6–8]. The rK39 antigen has been used to develop an immunochromatographic strip test (ICT)-based rapid diagnostic test which is advantageous for mass screening in endemic areas. This test requires a drop of peripheral blood and can be completed in approximately fifteen minutes [7]. Although the rK39 ICT rapid test was quite successful in Asia, it was often unable to detect Leishmania infections in African patients [5]. Additionally, rapid diagnostic tests still need standardization in order to become a regular practice in clinical laboratories.
PCR is the main molecular tool for Leishmania diagnosis due to its high sensitivity and reliability. Different PCR target sequences that are commonly used include ribosomal RNA genes, kinetoplast DNA, mini-exon derived RNA, internal transcribed spacer regions, etc., [5]. Quantitative PCR is useful because it allows for the quantification of parasites as well as species typing. Furthermore, this technique can be used to monitor treatment efficiency. Unfortunately, equipment requirements as well as the high cost limit the use of PCR for mass screening purposes in the field. The introduction of loop-mediated isothermal amplification (LAMP) could facilitate the use of molecular techniques for diagnostics. LAMP is highly specific, carried out under isothermal conditions, quick, and requires less complicated equipment (5). Moreover, reagents can be kept at room temperature, and there are no post-PCR steps. Assessment of drug treatment can also be carried out through the use of nucleic acid sequence based amplification (NASBA) which amplifies RNA sequences under isothermal conditions. Coupled to oligochromatography, NASBA can be used to monitor the progression from active disease to cure [9].
Chagas Disease (American Trypanosomiasis)
Chagas disease is the result of an infection with the blood-borne protozoan Trypanosoma cruzi. The parasite is transmitted by the triatomine bug. The second most important mode of transmission is via contaminated blood. This includes blood transfusions, organ transplants, and congenital transmission.
During the acute stage of Chagas disease, parasites can be observed in the blood. For this reason, diagnosis is carried out by direct microscopic viewing of Giemsa-stained thin and thick blood smears [10]. Parasites may also be detected through the use of hemocultures. In Chagas endemic areas, xenodiagnosis may be performed. This method involves allowing the naïve triatomine bug to take a blood-meal from the patient, and then analysing the bug for the presence of trypanosomes. It is believed that with continued research, molecular methods will eventually replace indirect diagnostic techniques such as blood cultures and xenodiagnosis [10]. However, molecular tests need to be standardized for routine clinical practice.
During the chronic stage of Chagas disease, diagnosis relies on serology; however, these tests often yield results that are difficult to interpret [10]. Commonly used, standardized serological assays include indirect immunofluorescence (IIF), indirect hemagglutination (IHA), and ELISA. IIF and IHA are commonly used due to their good sensitivity; however, their results are operator-dependent, and there is a lack of studies which analyse their reproducibility [10]. Currently, the immunoblot and radioimmunoprecipitation assays are in the process of being standardized. Both tests showed promise in early studies. A great deal of work is also being focused on the development and standardization of molecular methods such as PCR, which could be useful in monitoring chronic phase, reactivation, and treatment response.
As previously mentioned, disease transmission can also occur from mother to child, leading to congenital Chagas. Screening of neonates can be performed via direct methods, such as microscopy, or PCR using venous or cord blood samples from the newborn. These tests have very high sensitivity when performed during the first month of life [10]. Serological analysis may also be performed.
Sleeping Sickness (African Trypanosomiasis)
Trypanosoma brucei is the causative agent of African trypanosomiasis, and it is transmitted via the bite of the tsetse fly. During the first stage of the disease, parasites can be found circulating in the peripheral blood. The second stage is marked by parasites crossing the blood-brain barrier and infecting the central nervous system (CNS). The parasitic subspecies dictates geographic distribution, prognosis, and diagnosis.
T. b. gambiense causes West African trypanosomiasis, which is a slow progressing disease and is characterized by low parasite loads [11]. Definite diagnosis is carried out by microscopic observation of blood, lymph node aspirate, or CSF for the presence of parasites. In the field, the card agglutination test for trypanosomiasis (CATT/T. b. gambiense) has been widely used since its development in 1978 (12). Whole blood is used, and the assay directly detects T. b. gambiense specific antibodies. CATT/T. b. gambiense is cheap, quick, and highly sensitive. However, the test can give rise to false positives in individuals who are co-infected with malaria [12]. Although CATT/T. b. gambiense is the most sensitive, similar tests such as micro-CATT and LATEX/ T. b. gambiense can also be used. If these assays generate positive results, they need to be confirmed by microscopy or other molecular methods.
T. b. rhodesiense causes East African trypanosomiasis, which progresses quickly and is characterized by high parasite loads (11). For this subspecies, there is no diagnostic equivalent to the CATT/T. b. gambiense. However, diagnosis by microscopic observations of thick and thin smears is simple due to the elevated parasite load associated with T. b. rhodesiense.
Microscopy is the most practical technique to be used in rural areas. However, microscopy requires adequately qualified personnel in order to prevent misdiagnosis. Molecular methods would substantially improve the diagnosis of African trypanosomiasis. PCR techniques have been developed to screen the CSF of patients. The discovery of the SRA gene in T. b. rhodesiense has proven to be a breakthrough for the promotion of PCR techniques. Reactions targeting this gene have the potential to identify a single trypanosome [11]. There has also been the introduction of fluorescence in-situ hybridization in combination with peptide nucleic acid probes aimed towards ribosomal RNA. However, these tools for diagnosis are new and require further optimization. Extensive research is being focused on standardizing molecular techniques and rendering them more accessible. The use of LAMP is a step forward in improving molecular
approaches [11].
Future research needs to focus on the improvement of molecular diagnostic techniques. Currently, second stage infections are diagnosed by microscopic observation of CSF. Research is being conducted to test various cytokines and antibodies as biomarkers for CNS infection [11].
Malaria
Malaria is the most important parasitic infection in the world due to its high mortality. The causative agents, parasites of the Plasmodium genus, are transmitted by Anopheles mosquitoes. Quick diagnosis is essential in order to determine the appropriate treatment as well as to prevent further transmission.
Microscopy is the gold standard for laboratory diagnosis. This method involves detecting parasites in Giemsa-stained thick and thin blood smears. However, microscopic results are operator-dependent, thereby causing the sensitivity to vary. A great deal of effort has been focused on developing rapid diagnostic tests (RDTs) which can be used in the field. These tests can supplement microscopy, but they cannot replace it yet. Current RDTs are serology based and use three different Plasmodium antigens: Plasmodium histidine-rich protein, Plasmodium lactate dehydrogenase, or Plasmodium aldolase [13]. These tests are quick, easy to perform, and require minimal patient samples. However, they are not specific for species such as P. malariae, P. ovale, and P. knowlesi. Furthermore, false positives may be observed due to cross-reactions in patients with Schistosoma mekongi or rheumatoid factor [14]. In addition, the tests inefficiently detect P. falciparum infections from South America, as this species does not produce the common histidine-rich proteins [15].
Currently, there are no commercially available molecular assays. Although some reference and government laboratories have developed their own molecular assays, their availability is limited. LAMP is currently in the spotlight. Poon et al. developed a LAMP test which detected the target sequence of P. falciparum 18S ribosomal RNA gene [16]. They stated that the price of this test was one tenth that of a conventional PCR. Recently, LAMP was further simplified in the form of a card test. It was used in combination with DNA filter paper and melting curve analysis. This system was shown to be highly specific and sensitive [17]. Improvement of the LAMP technique should be geared towards the development of rapid diagnostic tests which could potentially be used in the field.
Babesiosis
Babesiosis is caused by parasites belonging to the Babesia genus that are spread by certain ticks commonly found in North America. The parasites infect red blood cells (RBCs), and consequently cause hemolytic anemia. The disease can be fatal in splenectomy patients, immunocompromised individuals, and the elderly. Diagnosis is complicated by the symptoms’ resemblance to other tick-borne illnesses.
The gold standard of babesiosis diagnosis relies on detecting the parasites in the patients’ RBCs. This is achieved by microscopic observation of thick and thin blood smears. Babesia infections can be easily mistaken for P. falciparum infections [18]. Additionally, false negatives are common in immunocompetent individuals whose parasitemia can be lower than 1% [18]. Samples are often sent to reference laboratories in order to confirm ambiguous results. IFFs are used to detect anti-babesial IgM and IgG [18]. They are sensitive, specific, and reliable. ELISAs and immunoblots, although not standardized, can be performed to confirm the IFF results. However, compared to IFFs, Babesia detecting ELISAs require higher concentrations of antigen and have varying sensitivity [18]. Future research on babesiosis diagnosis is aimed at developing multiplex PCR assays that will be able to detect several tick-borne infections. PCR assays have the potential to yield positive results from 100µl blood samples containing as little as three parasites; demonstrating the incredible advantage that molecular techniques could contribute to diagnosis of this parasitic disease [18].
Proteomics
Dr Momar Ndao’s laboratory focuses on the improvement and advancement of diagnosis. Through our work, we hope to encourage the development of proteomic strategies for the diagnosis of parasitic infections. Mass-spectrometry platforms are the future of proteomics, and they can be used to identify biomarkers from biological fluids. Some techniques that can be used to analyse protein expression include matrix-assisted laser desorption ionization time-of-flight mass-spectrometry (MALDI-TOF MS), surface-enhanced laser desorption ionization time-of-flight mass-spectrometry (SELDI-TOF MS), liquid chromatography combined with mass-spectrometry, isotope-coded affinity tags, and isobaric tags for relative and absolute quantification [19]. When SELDI is used, samples are directly spotted onto chemically active ProteinChip Array surfaces which can be chosen based on specific chemical and biological properties. With MALDI, samples are mixed with the matrix component prior to loading on a chip. These proteomic platforms can be useful in identifying biomarkers that are indicative of a specific pathophysiological state. Currently, members of our laboratory are using both SELDI and MALDI techniques extensively to identify biomarkers of blood borne parasites.
Summary
Quick and correct diagnosis of parasitic infections is crucial to avoid deaths and further disease transmission. Diagnostic methods include parasitological techniques, such as microscopy and culturing, serological assays, and molecular tests [Table 1]. Although several serological and molecular diagnostic tools are being tested and used by certain reference laboratories, results are always confirmed by microscopy which remains the gold standard. Many newer assays have not been standardized yet, thus, forcing diagnosticians to rely on microscopic observations. Unfortunately, the evolution of diagnosis in the field of parasitology has been slow to progress. Fortunately, in recent years, several groups have focused their research on the improvement of diagnostics. Current research emphasizes the development and optimization of molecular techniques such as PCR and LAMP. Additional work must concentrate on rendering molecular diagnostics more accessible. Although relatively new at the moment, proteomic platforms seem to be the future of diagnosis. These new techniques can identify biomarkers which can categorize susceptible individuals, distinguish between the different stages of an infection, and monitor whether treatments lead to cure. Diagnostic research has made much progression, however, there is still a lot of work to be done and improvements to be made. In order to better the diagnosis of blood-borne parasitic infections, research plus communication is the answer.
References
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The authors
Alessandra Ricciardi, BSc
National Reference Centre for Parasitology, Research Institute of the McGill University Health Center, Montreal, Canada
Momar Ndao, DVM, MSc, PhD
National Reference Centre for Parasitology at the Montreal General Hospital, Montreal, Quebec, Canada
E-mail: momar.ndao@mcgill.ca
Managing chronic disease: do clinical labs hold the key ?
Chronic diseases are placing an increasingly heavy burden on the healthcare systems of both development and emerging countries. Together with renewed prevention strategies based on systematic and coordinated approaches, clinical laboratories will have an essential role to play with the advent of new biomarkers and the development of e-health systems.
Chronic diseases are acknowledged to be one of the biggest challenges for healthcare systems. Traditionally, chronic diseases were non-communicable. Using World Health Organization (WHO) data [1], they consisted of four major groups – cardiovascular diseases, cancers, chronic respiratory diseases and diabetes, as well as some neuropsychiatric disorders and arthritis. More recently, an increase in survival rates for infectious and genetic diseases has led to expanding the definition to certain communicable diseases (such as HIV/AIDS) as well as genetic disorders like cystic fibrosis.
Attention to chronic diseases has been growing, largely due to three factors:
1. Ageing populations.
2. Early detection, or ‘secondary prevention’.
3. E-health – the possibility offered by sophisticated at-home monitoring and timely treatment.
Ageing populations
The elderly are far more susceptible to chronic disease. In the US, some 10% of the beneficiaries of Medicare, almost all with chronic disease, account for three-quarters of its budget. [2] Per capita spending is 3-10 times more for older adults with chronic diseases than those without. [3] In Europe, the EU Council has noted the “enormous burden” posed by chronic diseases and also warned that the next decade (2011-2020) will see this grow further due to an ageing population. [4]
Early detection
The early detection of chronic disease has been revolutionized by virtue of innovative and ever-faster diagnostic techniques in clinical laboratories. Clinical laboratories have, for some years, taken the lead in reducing the gap between the evolution of a chronic disease and interventional treatment, both at home and in the hospital.
In 2007, a report by the influential Milken Institute think-tank made a powerful argument to include prevention and early detection, rather than treatment alone, in the US debate on funding healthcare. The Milken report was titled ‘An Unhealthy America: The Economic Burden of Chronic Disease’. [5] It was one of the most ambitious attempts to quantify the reduction in case burden that could be achieved by such strategic reorientation: a drop by as many as 40 million cases of chronic diseases in the year 2023, in the US alone. At the time of the report’s launch, former US Surgeon General Richard Carmona noted the biggest problem with the present healthcare system was that it waited for people to get sick and then treated them at high cost.
The story is similar in Europe. Though EU-wide statistics do not yet exist, in the UK, half of hospital bed day use is accounted for by only 2.7% of all medical conditions, most of which are chronic diseases. [6] The EU Commission has called for technology-driven strategies to permit both early detection and timely monitoring of chronic disease – and do this in the context of healthy ageing.
As in the US, much European thinking about managing the burden of chronic disease involves e-Health, especially in the context of structured programmes of home care for patients. In January 2007, a major EU Commission study called “Healthy Ageing: Keystone for a Sustainable Europe” [7] approvingly highlighted a Swedish program called ‘Preventive Home Visits’ as leading to both a decrease in GP visits and lower mortality. It called for promoting and using such best-of-class practices across the EU.
E-health and clinical laboratories
All such plans essentially consist of remote acquisition of patient data using lower skilled and mobile personnel. They transfer the data in real- or near real-time for remote interpretation at a clinical laboratory, followed by consultation with a physician (e.g to modify dosage/change medicines), or to transfer the patient for intervention at a hospital.
The role of the clinical laboratory in e-Health is already advanced in telepathology. Though some telepathology efforts have aimed at remote manipulation of diagnostic equipment, the more proven approach has been to transmit images from a slide. Such systems have been in use since the mid-1990s, especially in sparsely populated areas such as parts of Canada and the north-western US, and in Norway and Sweden. France’s RESINTEL was, however, one of the first systems to establish that telepathology was at least as reliable as a physical slide examination, in a transatlantic pilot project. [8]
The largest application for telepathology has so far been in cytology. Nevertheless, microbiologists have been remotely interpreting gram stains, and hematologists have reported success with blood films.
Biomarkers: promises and challenges
The next frontier is likely to be biomarkers – pre-symptomatic signals of early disease states, detectable in blood/serum. In 2011, an article by 61 healthcare experts from Europe, the US, Brazil, Russia, India, China and some other countries called for a systemic approach to combat chronic disease, with a roadmap “for predictive, preventive, personalized and participatory (P4) medicine.” [9] The core of the proposal is to systematically identify biomarkers, which would then (progressively) be used to chart out a matrix of co-morbidities, disease severity and progression – including the critical trigger signals which predict the occurrence of abrupt transitions in the stages of a chronic disease.
The authors of the above paper cite an in-depth study on the clinical impact of telemedicine in four major chronic diseases – diabetes, asthma, heart failure and hypertension, [10] and propose that continuous monitoring of individual clinical histories and their development would be a key source of primary data, to build up a robust and extensive knowledge management infrastructure.
The role of clinical laboratories in much of the above system – from biomarker discovery to the monitoring of patients – is evident. At the moment, tests on the bulk of approved biomarkers (such as Oncotype DX and Trofile) are conducted in large reference laboratories. However, a great deal of research is also being directed at tests for use at home or at point-of-care; for example, CRP (C-reactive protein) and the hormone prolactonin are biomarkers which differentiate between bacterial and viral pneumonia in less than an hour, and reduce the use of precautionary antibiotics.
Nevertheless, there is still some way to go before biomarkers and systemic/personal approaches to medication and treatment of chronic disease become commonplace. Most barriers are regulatory [see box on this page], and are a consequence of the relative novelty of biomarkers – and their potentially sweeping impact.
In the light of this, the challenge for clinical laboratories will be to develop acceptable technical standards for the use of biomarkers, jointly with regulators and manufacturers. Clearly, given the massive challenge posed by chronic diseases in the decades ahead, any serious solution will have to involve a combination of biomarker-based personalized medicine, at-home care and clinical laboratories.
References
1. http://www.who.int/nmh/Actionplan-PC-NCD-2008.pdf
2. Berk ML, Monheit AC. The Concentration of Health Expenditures: An Update. HealthAffairs 1992; 11 (4): 145–149.
3. Fishman P, et al. Chronic Care Costs in Managed Care. Health Affairs 1997; 16 (3): 239–247.
4. http://www.consilium.europa.eu/uedocs/cms_Data/docs/pressdata/en/lsa/118282.pdf
5. http://www.milkeninstitute.org/healthreform/pdf/AnUnhealthyAmericaExecSumm.pdf
6. Chronic Disease management – a compendium of information, UK Department of Health, May 2004
7. http://ec.europa.eu/health/archive/ph_information/indicators/docs/healthy_ageing_en.pdf
8. http://pubmedcentralcanada.ca/pmcc/articles/PMC2579163/pdf/procascamc00009-0625.pdf
9. http://genomemedicine.com/content/3/7/43#B46
10. Pare G, Moqadem K, Pineau G, St-Hilaire C. Clinical effects of home telemonitoring in the context of diabetes, asthma, heart failure and hypertension: a systematic review. J Med Internet Res 2010 (12:e21).
11. http://ec.europa.eu/research/health/pdf/biomarkers-for-patient-stratification_en.pdf
12. http://www.phgfoundation.org/file/3998/