Sample collection is an important aspect of scientific work because it shapes, to a great extent, the study design and methodology, both of which may influence the outcomes of scientific research. However, often in scientific evaluations of studies which involve both field sample collection and laboratory work, only the laboratory research aspect receives serious attention, while other factors such as the socio-cultural, ecological and belief values of subjects who donate samples for laboratory studies are much less emphasised. These factors and how they play out in any particular study area are critical determinants of successful field sample collection especially in the developing countries.
by Dr Olufunmilola Ibironke, Dr Samuel Asaolu and Dr Clive Shiff
Urinary schistosomiasis is caused by a trematode worm, Schistosoma haematobium [1]. Infection with this parasite has been shown to be the commonest cause of haematuria and urogenital diseases in endemic areas. Thus, detection of haematuria in urine has been proposed as a valid indicator of schistosome infection, and has been widely adopted in many national schistosomiasis control programmes [2,3]. Diagnostic procedures in control programmes accordingly involve collection of urine samples from patients.
Most studies of urinary schistosomiasis in Nigeria and other endemic countries have targeted schoolchildren [4-8], because they represent the prime reservoir for the parasite, and children are amenable to mass chemotherapy [9]. However, studies have shown the debilitating effect of the parasite among adults in communities where it is endemic [10-13] and so this population also needs to be studied. As opposed to urine sample collection from children which is mostly done in schools, collection of urine from adults is difficult, particularly among persons who do not consider schistosomiasis as their major health problem when compared to malaria. In a school-based setting, after obtaining clearance from government health and school administrative authorities, researchers usually work with school teachers to get permission from pupils’ parents, and to educate the children involved in the study about how to follow urine sample collection instructions. However, for studies which involve adults, researchers, with the help of local health officers, would have to deal with patients directly to seek their individual involvement in the study, the acceptance of which depends on a number of the above mentioned factors.
Few studies have investigated the sociology of communities involved in such studies. We present here a study on urinary schistosomiasis in two villages in Ogun State, Nigeria, involving collection of urine samples from adults, to investigate the factors that drive their acceptance or refusal for inclusion in the study.
Methods and study sites
The study involved adults between the ages of 20 and 55 years who were mobilised to school halls in each village through the respective heads of the villages. Participants were informed of their right to accept or reject inclusion in the study. Many adults refused to come to school halls, many others who came rejected inclusion in the study. Some others accepted inclusion and collected urine sample containers but never came back while others accepted full participation. People in endemic communities show negative attitudes to urine sample collection for different reasons. To find out villagers’ attitudes to the urine sample collection process, we asked consenting participants why their friends or family refused to participate and in the process we identified some factors responsible for their attitudes. We also visited some households either to seek consent for inclusion or to understand reasons for refusing inclusion in the study.
This study was conducted in July, 2010, in Ogun State, Nigeria as a part of a study on the diagnosis of urinary schistosomiasis in six villages. For the purpose of comparison, two villages, Apojola located in Odeda Local Government Area (LGA), and Ogbere in Ijebu-east LGA, were selected. Apojola is located on Oyan Dam Reservoir. The inhabitants are all immigrant fishermen and their families, and are a mixture of Moslem Hausas and Christian Idomas. Awawa River serves Ogbere community. The inhabitants are mainly Christian Yorubas, and a mixture of farmers and Local Government Area civil servants. Ethical consideration, the data collection process, the population of each village, vegetation types and locations of each local government area have been reported previously [14].
Observations and discussion
Socio-cultural aspect
Several urinary schistosomiasis studies had been conducted in Nigeria, most of which involved urine sample collection, so there is a high level of awareness about the importance of control programmes. However, in the process of field studies there is often confusion in the minds of the participants leading to fear of exposure to strangers which was found to prevail among the villagers. Frequently researchers are mistaken for government agents visiting for revenue collections. If the researcher can work with members of the community to change these opinions it would likely improve level of cooperation for inclusion in the study. We explored this aspect in Apojola, a community located on the heavily schistosome-infested Oyan dam reservoir. We made the first attempt to recruit participants through the community leader, followed by the religious leader, a nurse and a school teacher. The number of participants recruited through the assistance of the different leaders according to age and gender are shown in Table 1a. In Table 1b, it was shown that the community leader is the most effective in helping to mobilise the villagers of both genders for urine collection.
There is also an increasing cynicism about the disease among adult patients in endemic communities. Many members of the communities who admit passing blood in the urine do not perceive it as an indication of a serious disease. They consider it as a sign of virility and puberty which is a familiar sign among adults in other villages around them. A few others who have experienced some discomfort and thought it might be a major health problem were either ashamed of their disease status or ashamed of bringing their ‘red’ urine. Past studies have noted that individuals’ perceptions on the aetiology and impact of urinary schistosomiasis differed with their levels of education and gender [13]. Lack of knowledge about the cause and effect of the disease affects patient’s turnout for sample collection and this in turn has a direct influence on field data coverage and research quality.
Apart from lack of health education on the cause of the disease, the willingness to participate in the urine sample collection process is seemingly greater among patients with some level of education than among the uneducated. We investigated how patient’s level of education impacts turnout for urine sample collection in Ogbere community. Ogbere inhabitants are a mixture of uneducated farmers, who have nought to six years of formal education, and the educated comprising teachers and Local Government Area civil servants, who have from seven to 16 years of formal education. In Table 2, data from both groups are presented for comparison to show turnout according to education level and gender.
This Table shows the percentage contributions by the Community Leader (CL), Nurse (N), Teacher (T) and Religious Leader (RL) on the total number of respondents. CL is best for mobilising males in the community (P = 0.00155). CL is also best for mobilising male and female with calculated P = 0.052 just higher than 0.05. N is best for mobilising females but this is not statistically significant.
Ecological aspect
Transmission of urinary schistosomiasis is through freshwater snails, Bulinus species, as intermediate hosts and varies with different ecological factors. In many endemic communities, the ecological factors which favour disease transmission also promote agricultural practices such as farming, cattle rearing and fishing. Therefore, transmission to humans often occurs as a result of irrigation systems for agricultural purposes or when visits are made to the rivers for washing and swimming. As such, the rate of transmission to humans varies, to a great extent, with occupation.
However, since diagnosis is by urine testing, many peasant farmers and fishermen who are thought to be the most impacted with S. haematobium because of frequent water contact may remain undiagnosed and untreated. Urine sample collection for the diagnosis of urinary schistosomiasis is preferably done between the hours of 10:00 and 14:00 for optimum egg passage [9]. These hours coincide with the time during which farmers go to farm and fishermen set nets for fish catching. This coincidence might affect turnout for sample collection and estimation of overall disease prevalence in the community.
To evaluate the impact of patient’s occupation on turnout for urine sample collection, we compared turnout of farmers and civil servants in Ogbere community. For statistical purpose, farmers, cattle rearers and fishermen are classified as farming, while students, teachers and local government workers are classified as civil servants, see Table 2. In total, there are 84 participants out of which 33 are farmers (39.2%) and 51 are civil servants (60.7%). In all, more women (79.8%) turned out for sample collection.
According to the community leader, the total adults’ population in Ogbere is 3121 and the ratio of farmers to civil servants is approximately 20:1.
Z- Distribution test was used to compare the response level between the two groups using the formula:
(see picture number 4)
where p is the difference of proportions, N1 = 149 = Educated population and N2 = 2972 = Uneducated population. At all levels of significance 0.05, 0.01 and 0.001, response from the educated civil servant population was significantly higher than response from the uneducated farmer population.
Belief structures
Christians in Apojola and Ogbere communities were relatively unhindered by religious belief regarding their willingness to come forward for education about the project and provision of their urine samples. However there was gender problem with urine collection among the Muslim families at Apojola. The Muslim families at Apojola have the culture of restricting married women within the family household compounds and forbiding male visitors of adolescent age and older from entering the compounds or visiting the women. In order to be able to collect urine samples from these Muslim women, the local community nurse and a female member of our research team were accompanied by a local female Muslim field assistant and interpreter before being allowed access to the compounds to explain the importance of the disease and purpose of the study.
Conclusion
This study attempts to find out patients‘ attitudes to scientific research especially during a field sample collection process and suggests possible reasons for rejection of inclusion in scientific research by patients. In general, this study showed that social and ecological values including educational background, occupation, religious practices and poor knowledge about the aims and objectives of the study, strongly influence turnout for urine sample collection. Therefore, such values are worth considering for a holistic understanding of the scientific study results.
References
1. Edungbola LD, Asaolu SO, Omonisi MK, Aiyedun BA. Schistosoma haematobium infection among schoolchildren in the Babana district, Kwara State, Nigeria. Afr J Med Sci 1988; 7: 187-193.
2. Koukounari A, Gabrielli AF, Toure S, Bosque-Oliva E, Zhang Y, Sellin B, Donnelly CA, Fenwick A, Webster JP. Schistosoma haematobium infection and morbidity before and after large-scale administration of praziquantel in Burkina Faso. J Infect Dis 2007; 196: 659-669.
3. Webster JP, Koukounari A, Lamberton PH, Stothard JR, Fenwick A. Evaluation and application of potential schistosome-associated morbidity markers within large-scale mass chemotherapy programmes. Parasitology 2009; 136: 1789-1799.
4. Abdel-Wahab MF, Esmat G, Ramzy I, Fouad R, Abdel-Rahman M, Yosery A, Narooz S, Strickland GT. Schistosoma haematobium infection in Egyptian schoolchildren: demonstration of both hepatic and urinary tract morbidity by ultrasonography. Trans R Soc Trop Med Hyg 1992; 86: 406-409.
5. Fenwick A, Webster JP, Bosque-Oliva E, Blair L, Fleming FM, Zhang Y, Garba A, Stothard JR, Gabrielli AF, Clements AC, Kabatereine NB, Toure S, Dembele R, Nyandindi U, Mwansa J et al. The Schistosomiasis Control Initiative (SCI): rationale, development and implementation from 2002-2008. Parasitology 2009; 136: 1719-1730.
6. French MD, Rollinson D, Basanez MG, Mgeni AF, Khamis IS, Stothard JR. School-based control of urinary schistosomiasis on Zanzibar, Tanzania: monitoring micro-haematuria with reagent strips as a rapid urological assessment. J Pediatr Urol 2007; 3: 364-368.
7. Nduka FO, Ajaero CM, Nwoke BE. Urinary schistosomiasis among school children in an endemic community in south-eastern Nigeria. Appl Parasitol 1995; 36: 34-40.
8. Okoli EI, Odaibo AB. Urinary schistosomiasis among schoolchildren in Ibadan, an urban community in south-western Nigeria. Trop Med Int Health 1999; 4: 308-315.
9. Ibironke OA, Phillips AE, Garba A, Lamine SM, Shiff C. Diagnosis of Schistosoma haematobium by detection of specific DNA fragments from filtered urine samples. Am J Trop Med Hyg 2011; 84: 998-1001.
10. Koukounari A, Webster JP, Donnelly CA, Bray BC, Naples J, Bosompem K, Shiff C. Sensitivities and specificities of diagnostic tests and infection prevalence of Schistosoma haematobium estimated from data on adults in villages northwest of Accra, Ghana. Am J Trop Med Hyg 2009; 80: 435-441.
11. Mostafa MH, Sheweita SA, O’Connor PJ. Relationship between schistosomiasis and bladder cancer. Clin Microbiol Rev 1999; 12: 97-111.
12. Mungadi IA,.Malami SA. Urinary bladder cancer and schistosomiasis in North-Western Nigeria. West Afr J Med 2007; 26: 226-229.
13. Sarkinfada F, Oyebanji AA, Sadiq IA, Ilyasu Z. Urinary schistosomiasis in the Danjarima community in Kano, Nigeria. J Infect Dev Ctries 2009; 3: 452-457.
14. Ibironke O, Koukounari A, Asaolu S, Moustaki I, Shiff C. Validation of a new test for Schistosoma haematobium based on detection of Dra1 DNA fragments in urine: evaluation through latent class analysis. PLoS Negl Trop Dis 2012; 6: e1464.
The authors
Dr Olufunmiola Ibironke*
Cell and DNA Repository
Rutgers, The State University of New Jersey
New Brunswick
New Jersey, USA
e-mail: oai5@rutgers.edu
Dr Clive Shiff
Department of Molecular Microbiology and Immunology
Johns Hopkins Bloomberg School of Public Health
Baltimore, MD, USA
e-mail: cshiff@jhsph.edu
Dr Samuel Asaolu
Department of Zoology
Obafemi Awolowo University
Ile-Ife
Nigeria
*Corresponding author
The cardiorenal syndrome (CRS) involves both kidney failure and heart failure, with the failing organ initially being either the heart or the kidney; usually one failing organ leads to the failure of the other. While use of biomarkers and imaging techniques can assess cardiovascular function, the assessment of renal injury and function is complex in CRS patients. This article discusses some of the novel markers for the assessment of renal function and injury.
Renal dysfunction is an independent and significant contributor to poor heart failure outcomes. Serum creatinine (SCr), the most frequently used marker for clinical assessment of renal dysfunction and injury, is at best a retrospective window. Glomerular filtration rate, GFR, considered the best overall measure of renal function (RF), is similarly affected by multiple variables, but novel biomarkers of renal injury and function are now available. These biomarkers also have limitations but they address gaps in the information provided from use of conventional biomarkers
A marker of renal function: cystatin-C
Cystatin C, an endogenous proteinase inhibitor of low molecular weight (13-kDa), possesses many features that make it attractive as a surrogate marker of RF and GFR. It is synthesised and released into plasma by all nucleated cells at a constant rate, is freely filtered by the glomerulus and completely reabsorbed by the proximal tubules. It can be easily measured in the serum and plasma without the need for a urine sample or complex equations. It is not affected by changes in body mass, nutrition, age or gender, making it potentially more beneficial in critically ill patients, elderly and children. It has been validated as a marker of GFR in patients with pre-existing renal dysfunction and acute kidney injury (AKI) as levels increase before SCr. In congestive heart failure (CHF) Cys-C is superior to SCr based estimates, which underestimate GFR. This appears to extend to acure decompensated heart failure (ADHF) admissions without advanced RF. Cys-C also reflects myocardial stress and damage, reflects more advanced left ventricular diastolic and right ventricular systolic dysfunction and is an independent predictor of long-term prognosis after adjusting for myocardial factors. The advantage of Cys-C over SCr appears greater and more conclusive for ruling in renal injury in affected patients. Other benefits include predicting future CV events in intermediate-risk individuals, mainly through the identification of those unlikely to develop events; it is a stronger predictor of adverse events than conventional measurement of RF and, in combination with cardiac troponin T and N-terminal–pro-brain natriuretic peptide, it improves risk stratification for CV mortality (inclusive of HF) beyond models of established risk factors.
Novel assessments of renal injury
The potential to attenuate or reverse renal injury is far less likely when renal dysfunction is already evident, at least based on current assessment methods. Several promising AKI biomarkers arenow available.
Neutrophil gelatinase-associated lipocalin
Human neutrophil gelatinase-associated lipocalin (NGAL) is a 25-kDa protein initially described to be bound to gelatinase in specific granules of the neutrophil, with recent evidence suggesting physiological activity in the kidney. It is expressed and secreted by immune cells, hepatocytes and renal tubular cells in various pathologic states. NGAL exerts bacteriostatic effects, which are explained by its ability to capture and deplete siderophores, small iron-binding molecules that are synthesised by certain bacteria as a means of iron acquisition and role in cell survival, inflammation and matrix degradation. NGAL is up regulated more than 10-fold in post ischaemic renal injury in a mouse model and secreted relatively early into the urine. Several recent studies in homogenous (adult and paediatric cardiac surgery), heterogeneous (intensive care and emergency department) and chronic kidney disease (CKD) populations have supported the use of NGAL as an important biomarker in early diagnosis and prediction of duration and severity of AKI. NGAL differentiates AKI from changes in GFR due to chronic disease progression, predicts duration of ICU stay and provides prognostic value. Specifically, a single urine level of NGAL in the emergency department differentiates AKI from normal function and from pre-renal azotaemia, and predicts poor in-patient outcome.
A recent multicentre pooled analysis of published data on 2322 critically ill children and adults with the cardiorenal syndrome revealed the surprising finding that approximately 20% of patients display early elevations in NGAL concentrations but never develop increases in serum creatinine. Importantly, this sub-group of ‘NGAL-positive creatinine-negative’ subjects encountered a substantial increase in adverse clinical outcomes, including mortality, dialysis requirement, ICU stay and overall hospital stay. Thus, early NGAL measurements can identify patients with sub-clinical AKI who have an increased risk of adverse outcomes, even in the absence of diagnostic increases in serum creatinine.
Among acute decompensated heart failure patients, high admission serum NGAL levels were associated with increased risk of worsening RF. In particular, patients with an NGAL of >140 ng/mL on admission had a 7.4-fold increased risk, with a sensitivity and specificity of 86% and 54%, respectively. NGAL values are also significantly increased and parallel the clinical severity of CHF. After a 2-year follow up, patients with baseline NGAL > 783 ng/mL had a significantly higher mortality. These findings may suggest that NGAL plays a pivotal role in the systemic adaptation to CHF. Elevated baseline serum levels in acute post-myocardial infarction and CHF correlated with clinical and neurohormonal deterioration and adverse outcomes. In a rat model of post-MI HF, NGAL/lipocalin-2 gene expression was increased in the non-ischaemic left ventricle segments, primarily located to cardiomyocytes. Strong NGAL immunostaining was found within cardiomyoctes in experimental and clinical HF. Furthermore interleukin-1β and agonists for toll-like receptors 2 and 4 were potent inducers of NGAL/lipocalin-2 in isolated neonatal cardiomyocytes supporting a role for the innate immune system in HF pathogensesis. Urinary NGAL also increases in parallel with the NYHA classes for HF and is also closely correlated with serum NGAL, Cys-C, SCr and eGFR. This suggests tubular damage may accompany renal dysfunction in CHF, which has prognostic consequences.
Evidence is continuing to accumulate and NGAL measurement appears to be of diagnostic and prognostic value. In a recent meta-analysis, NGAL levels predicted renal replacement therapy initiation and in-hospital mortality. Several recent studies showing the response of urinary levels to therapy suggest a future role for NGAL for follow up and monitoring the status and treatment of diverse renal diseases reflecting defects in the glomerular filtration barrier, proximal tubule reabsorption and distal nephrons. Thus the prospects of NGAL being used as a diagnostic tool, even beyond the realms of nephrology, are exciting but require further clinical research. The commercial availability of standardised clinical platforms for the accurate and rapid measurement of NGAL in the urine and plasma will facilitate future investigations as well as direct clinical applications.
Interleukin-18
Interleukin (IL)-18 is a proinflammatory cytokine which induces interferon-g production in T cells and natural killer cells. It is synthesised as a biologically inactive precursor, which requires cleavage into an active molecule by an intracellular cysteine protease similar to IL-1b. IL-18 is both a mediator and biomarker of ischaemic AKI. Several early studies demonstrate increases in patients with acute tubular necrosis, prerenal azotaemia, nephrotic syndrome, delayed graft function after renal transplantation, chronic renal insufficiency and urinary tract infections. In contrast nephropathy, cardiopulmonary bypass, critically ill children and kidney transplantation, urinary IL-18 rises two days earlier than SCr. Urine IL-18 increases four to six hours after cardiopulmonary bypass, peaks at over 25-fold at 12 hours, and remains elevated up to 48 hours later. IL-18 levels also predict graft recovery and need for dialysis up to three months later. There is also significant evidence that IL-18 contributes to clinical HF and other acute and chronic cardiovascular presentations. Presently there are no studies with patients with CRS.
Major concerns over IL-18 surround its discriminatory capacity and appropriate use. One concern is a spill over into the urine and its effects as a confounder, differentiating elevated cardiac as opposed to renal injury. Additionally, serum IL-18 may be increased in other disease states e.g. autoimmune disorders such as SLE, certain leukaemias, postoperative sepsis, chronic liver disease and acute coronary syndromes. On a positive note serum IL-18 levels were not different between those with and without AKI post paediatric cardiac surgery, and data suggesting its pathophysiological contribution to the renal damage observed during ischaemia/reperfusion are positive signs for its discriminatory values and causative effects in renal injury. Thus IL-18 appears to be a worthwhile addition to a biomarker panel in the assessment of AKI.
Kidney injury molecule-1
Kidney injury molecule-1 (KIM-1) is a transmembrane protein that is highly over expressed in proximal tubule cells after ischaemic or nephrotoxic AKI. Several studies have shown KIM-1 in urine and renal biopsy to be elevated from predominately ischaemic AKI and not from prerenal azotaemia, chronic renal disease, and contrast nephropathy. KIM-1 appears to play a role in the pathogenesis of tubular cell damage and repair in experimental and human kidney disease. KIM-1 is a sensitive marker for the presence of tubular damage. It is virtually undetectable in healthy kidney tissue, but tubular KIM-1 expression is strongly induced in acute and chronic kidney disease as well as transplant dysfunction, where it is significantly associated with tubulointerstitial damage and inflammation. Elevated urinary KIM-1 levels are strongly related to tubular KIM-1 expression in experimental and human renal disease, indicating that urinary KIM-1 is a very promising biomarker for the presence of tubulo-interstitial pathology and damage. Furthermore, urinary excretion of KIM-1 is an independent predictor of graft loss in renal transplant recipients, demonstrating its prognostic impact. Studies after cardiopulmonary bypass surgery have noted similar findings. KIM-1 also predicts adverse clinical outcomes in various forms of AKI. Data from non-diabetic proteinuric patients suggest that urinary excretion of KIM-1 may have the potential to guide renoprotective intervention therapy. KIM-1 could potentially provide additional prognostic data for tubular damage in CHF. Future studies will reveal whether the sensitive biomarker KIM-1 will become a therapeutic target itself. Kim-1/KIM-1 dipsticks can provide sensitive and accurate detection of Kim-1/KIM-1, thereby providing a rapid diagnostic assay for kidney damage and facilitating the rapid and early detection of kidney injury in preclinical and clinical studies. The primary limitation of KIM-1 is that time to peak is 12 to 24 hours after insult. While it may be of limited use as an early AKI biomarker, the ability to detect KIM-1 in urine makes it an attractive option, possibly in a biomarker panel togther with NGAL and IL-18.
L-FABP
The fatty acid-binding proteins (FABPs) are 15kDa cytoplasmic proteins. There are two types: heart type (H-FABP) located in the distal tubular cells and liver type (L-FABP) which is expressed in the proximal tubular cells. Both markers have been suggested as useful for the rapid detection and monitoring of renal injury. H-FABP has been tested as a marker for ischaemic injury in donor kidneys. L-FABP has been tested in progressive ESRD as well as renal injury post renal transplantation and cardiopulmonary bypass and more recently acute coronary syndromes. In patients undergoing PCI for unstable angina, urine L-FABP levels were significantly elevated after two and four hours and remained elevated for 48 hours. SCr did not change significantly during the study period. Among nondiabetic CKD patients, urine L-FABP levels correlated with urine protein and SCr levels. Notably, L-FABP levels were significantly higher in patients with mild CKD who progressed to more severe disease. Neither SCr nor urine protein differed between those same groups. H-FABP however is produced by myocardial damage and its clearance is determined by RF. The ratio of H-FABP to myoglobin after haemodialysis may be a useful marker for estimating cardiac damage and volume overload in haemodialysis. This may be an advantage for FABP in a panel of biomarkers to discrimnate background noise and cases where troponin levels can’t be interpreted. The main drawbacks are lack of evidence in the HF setting, small sample sizes in existing studies, and non-availability of a commercially usable assay. Additional longitudinal studies are needed to demonstrate the ability of L-FABP to predict AKI as well as CKD and its progression in cohorts with CKD of multiple aetiologies.
Biomarker panels
Each of these biomarkers has advantages and limitations. It will be a while yet before any of these biomarkers match serum troponin and act as a ‘standalone’ marker. However they do provide a safety mechanism initially to highlight anticipated risk, as well as additional information on likely renal pathophysiology. It may ultimately be that a panel of biomarkers is required. Candidates for inclusion are NGAL, IL-18, KIM-1, Cys C and L-FABP. The alternative may be selective use of markers when renal injury is anticipated, a strategy synonymous with acute coronary syndromes with serial cardiac enzymes, i.e.’serial renal enzymes’. Ultimately a point of care device would be ideal, and some kits are already in place. It is however clear that the learning paradigm is still ongoing. Future studies will need to validate these biomarker panels in a large heterogenous cohort.
The future of renal assessments in cardiovascular patients
Renal dysfunction is an independent and significant contributor to poor heart failure outcomes. Idiosyncracies in cardiorenal physiology and limitations of conventional diagnostic tools are factors in the poor prescribing of proven heart failure therapies in these patients. Novel biomarkers of renal injury and function are currently available. These biomarkers do have limitations but they address gaps in the information gained from conventional biomarkers i.e. improvements in injury chronology and functional accuracy. Significant limitations in how these markers are used as well as issues of availability and cost can only be addressed by further work. Future research studies should consider addressing these questions.
Reference
Abstracted with permission from Iyngkaran P et al. Cardiorenal syndrome – definition, classification and new perspective in diagnostics. Seminars in Nephrology 2012; 32: 3-17.
Maternal screening is offered to all expectant women during the first or second trimester of pregnancy. The purpose of this screening is to test for fetal abnormalities including chromosomal abnormalities such as Down’s syndrome, Trisomy 18 and neural tube defects such as spina bifida. Testing is performed by taking a blood sample from the patient’s arm which is then tested for a combination of biomarkers. Clinical results in addition to the maternal age are considered and used to calculate the risk of Down’s syndrome.
by Leah Hoencamp and Lynsey Adams
Down’s syndrome is a genetic condition and occurs when an individual inherits an extra copy of one chromosome. This means that affected people have three copies of chromosome 21, where there should be only two. The extra chromosome causes characteristic physical and intellectual features. The reasons why an extra copy of chromosome 21 causes Down’s syndrome are not known, which is why screening is so essential.
A combination of tests is used to screen for Down’s syndrome. Two types of screening are available and which is used depends on the stage of pregnancy of the patient. These stages are divided into first and second trimester.
First trimester screening includes:
• Free beta-hCG
• Pregnancy associated plasma protein (PAPP-A)
Second trimester screening includes:
• Double test AFP and hCG)
• Triple Test (AFP, hCG and uE3)
• Quadruple Test (AFP, hCG, uE3 and inhibin A)
If the results generated from this screening appear within the ‘higher risk’ category, more definitive tests are needed to confirm a diagnosis, such as amniocentesis or a chorionic villus sample. These tests provide a definitive result and involve taking samples of fluid from around the unborn baby. However, it is a highly invasive procedure and carries a small risk of miscarriage.
Internal quality control in maternal screening
Quality control (QC) is a crucial part of any clinical testing programme to ensure the accuracy and reliability of patient test results. Quality control is designed to detect, reduce and correct deficiencies in the laboratory’s internal analytical process prior to the release of patient results and to improve the quality of the results reported by the laboratory. Quality controls are manufactured to mimic a patient sample and contain one or more analytes of known concentration. They are made using a base material normally human serum, bovine serum, urine or spinal fluid. A laboratory will use quality controls to validate the patient samples. If QC results are within their target range then patient results should also be accurate. Once validated, the patient results can be used for diagnosis, prognosis and treatment planning. If QC values are outside the target range, it may indicate a number of issues including inaccurate calibration, instrument failure, operator error or reagent issues. In the field of maternal screening, the main aim is to minimise the risk of false positive and false negative results, ultimately ensuring results obtained are accurate and reliable.
In any type of screening the majority of errors take the form of false positive or false negative results. In other areas false negative results are of more concern as the patient will be perceived as healthy and will therefore not receive the required treatment. However, in prenatal screening, false positive results are also of major concern. If a patient tests positive they may have to undergo an invasive amniocentesis procedure with risk to the fetus in order to confirm if a chromosomal disorder like Downs’s syndrome is present. It is clear that such screening requires a robust and reliable quality control procedure in order to avoid potential errors.
To facilitate the increased screening for Down’s syndrome, trisomy 18 and neural tube defects, Randox has developed the only commercially available multi-analyte; tri-level control specifically designed to cover both first and second trimester prenatal screening, with the following benefits:
• The unique combination of inhibin A and PAPP-A in addition to AFP, total hCG, free B-hCG and uE3 reduce the need to purchase separate controls thus saving money
• Manufactured from 100% human serum providing a matrix similar to the patient sample while reducing cross reactivity and ultimately shifts in QC values
• Three distinct levels of control are available, accurately covering the complete clinical range. The level one control contains suitably low levels of AFP whereas the level three control contains high levels of hCG. Moreover, the uE3 levels are in line with those typically found during the first twenty weeks of pregnancy
• True third party control providing an unbiased, independent assessment of performance. Highly accurate instrument specific target values and ranges are provided for the most popular analysers used in maternal screening
• Excellent reconstituted stability of seven days at +2–8 oC
• Excellent vial-to-vial homogeneity (%CV <1 %)
• Suitable for first trimester double screen and second trimester triple and quad screens.
Internal quality control (IQC) will help ensure results are reliable. An inter-laboratory data management package such as Acusera 24.7 can be used to further ensure quality. An effective IQC and peer group reporting scheme will help improve your laboratory’s analytical performance, help meet regulatory requirements and most importantly ensure the accuracy and reliability of patient test results. Acusera 24.7 enables laboratories to monitor analytical performance, access peer group reports and compare results with other laboratories using the same quality controls, method and instrument.
External quality assessment in maternal screening
To further assess the performance of maternal screening tests, laboratories should also be involved in an external quality assessment (EQA)/proficiency testing (PT) scheme. External quality assessment (EQA) is an essential aspect of any laboratory operation. EQA measures a laboratory’s accuracy using ‘blind’ samples that are analysed as if they were patient samples. EQA provides a means of assessing the analytical performance of a laboratory compared to other laboratories utilising the same methods and instruments. Participation in an EQA scheme will help produce reliable and accurate reporting of patient results. Quality results will reduce time and labour costs, and most importantly provide accurate patient diagnosis and treatment. Such a scheme is of paramount importance during testing such as maternal screening.
Randox International Quality Assessment Scheme (RIQAS) offers a Maternal Screening Programme which is capable of monitoring all 6 parameters involved in first and second trimester screening. RIQAS is the world’s largest global EQA scheme with more than 20 000 participants in over 100 countries worldwide.
Effective screening is essential for the detection of fetal abnormalities including Down’s syndrome, trisomy 18 and spina bifida. However, equally important in this process for laboratories responsible for processing the results is quality control. Effective quality control will help reduce false positives and false negatives, thereby ensuring reliable results and improving care of the patient overall.
Abbreviations
AFP, alpha-fetoprotein; hCG, human chorionic gonadotropin; uE3, unconjugated estriol.
The authors
Leah Hoencamp BSc & Lynsey Adams BSc
Randox Laboratories
55 Diamond Road, Crumlin,
Co. Antrim, UK BT29 4QY
E-mail: marketing@randox.com
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].
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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
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