Protocols for Preparation and Mass Spectrometry Analysis of Clinical Urine Samples to Identify Candidate Biomarkers of Schistosoma-Associated Bladder Cancer.

Advances in mass spectrometry instrumentation have revolutionized analytical capability in clinical proteomics. In parallel, various sample preparation methods have been developed to try to address the inherent complexity and dynamic range of clinical samples, typically involving a combination of depletion of abundant proteins followed by extensive prefractionation. However, the depth of coverage routinely achieved in discovery proteomics experiments on peripheral fluids such as serum, still leaves something to be desired, especially if no depletion or prefractionation is done in order to increase the throughput of clinical samples. Remarkably, despite being an easily accessible, typically sterile and diagnostically rich clinical sample, urine is often overlooked and as such has received less development effort. As an ultrafiltrate of blood, urine contains proteins and protein fragments originating from all parts of the body which may have diagnostic or prognostic potential if accurately and reproducibly quantified. Here, we describe an efficient and simple method for the concentration of urine samples by methanol-chloroform precipitation and subsequent in-solution tryptic digestion prior to discovery or targeted mass spectrometry analysis. We exemplify this method by reference to the discovery of novel candidate urinary biomarkers of schistosomiasis. Importantly, the methods described here have been used to identify >1900 protein groups in human urine by label-free discovery proteomics, without requiring any prior depletion or prefractionation, making this approach amenable to high throughput clinical biomarker studies in many diseases.

Evaluation of serum biomarkers and proteinuria for the early detection of renal damage in dogs with heartworm (Dirofilaria immitis).

Glomerulonephropathy associated with Dirofilaria immitis (heartworm) is relatively frequent in infected dogs. Given the importance and the scarcity of studies focused on its prevalence and diagnosis, the objective was to determine the prevalence of proteinuria and functional indicators of glomerular filtration rate in dogs with heartworm disease and discuss its utility in the detection of renal impairment. Sera and urine from 47 infected dogs were analyzed in a reference laboratory. Urea, creatinine, plasma proteins and serum symmetric dimethylarginine (SDMA) were analyzed in sera, while the UPC ratio was performed in urine. Dogs were further evaluated for the presence/absence of microfilariae, pulmonary and systemic hypertension, and the parasite burden was assessed. The results showed that 19.1 % of dogs showed proteinuria (UPC > 0.5) and 17 % showed borderline proteinuria (UPC 0.2-0.5). Creatinine and SDMA were high (>1.8 mg/dl and ≥18 µg/dl, respectively) in 4.2 % of dogs. UPC ratio was significantly increased in dogs with high parasite burden and in dogs with microfilariemia (p < 0.05). Dogs with pulmonary hypertension showed higher increases in proteinuria as well, which was probably due to the chronicity of the infection. No significant differences were found in serum and urine values regarding systemic blood pressure. Despite the limitations of this study, proteinuria/borderline proteinuria was present in 36.2 % of dogs with heartworm disease, and this may be due to glomerular disease. Therefore, the detection of proteinuria, along with other renal biomarkers in the diagnostic protocols, could help identify kidney alterations or risk of renal damage in heartworm disease.

Chyluria with massive proteinuria: Do not reach for the biopsy gun!

We report on a patient presenting with persistent chyluria due to filariasis, whose clinical course was complicated by massive proteinuria and severe hypoalbuminemia. Treatment with dietary manipulation, antifilarials, and sclerotherapy resulted in successful reversal of the above abnormalities. It has been reported that chyluria is not associated with massive proteinuria, or that even in cases of massive proteinuria, hypoalbuminemia is not seen and implies a glomerular pathology. We argue that chyluria is always associated with proteinuria, which may be massive, and does not warrant a kidney biopsy unless proteinuria persists despite resolution of chyluria.

Evaluation of Urine Reagent Strip as a Tool for Routine Diagnosis of Maternal Urogenital Schistosomiasis at Antenatal Clinic Visit in Munyenge, South West Region, Cameroon.

Urine reagent strip used in detecting microhaematuria has been recommended in pregnancy for diagnosis of urogenital schistosomiasis (UGS) during routine antenatal care (ANC). This study evaluated its sensitivity, specificity, and predictive values in the diagnosis of maternal UGS using filtration method as a reference test. We also assessed the variation in its performance in the diagnosis of UGS using multiple-sample collection. A total of 93 pregnant women reporting for first ANC clinic visit at any of the three functional health care centres (Munyenge Integrated Health Centre, Banga Annex Health Centre, and Trans African Health Centre) were enrolled and followed up for three consecutive monthly visits. Urine samples were observed microscopically for S. haematobium egg using urine filtration and screened for microhaematuria and proteinuria using urine reagent strips. Twenty-two (23.7%) out of the 93 women were diagnosed for UGS, all of whom showed S. haematobium egg excretion during all three visits. There was a significant difference (p < 0.001) between the prevalence of S. haematobium infection and the prevalence of microhaematuria. The intensity of infection was significantly higher in microhaematuria-positive women compared with microhaematuria-negative cases. Sensitivity of reagent strip ranged from 54.5 to 59.1%, while specificity was above 98.0% (range: 98.6-100%). The measure of agreement between urine filtration and reagent strip method was substantial (0.61-0.8) irrespective of different sampling periods. Urine reagent strip is a moderately sensitive method in the detection of UGS and will most likely identify women with high egg load burden. Proper diagnosis of schistosomiasis during pregnancy is recommended.

A cross-sectional study on urogenital schistosomiasis in children; haematuria and proteinuria as diagnostic indicators in an endemic rural area of Nigeria.

BACKGROUND: Rapid and accurate diagnosis is necessary for the management of schistosomiasis in endemic areas. OBJECTIVE: To assess the burden of urogenital schistosomiasis and the diagnostic efficiency of morbidity indicators of the disease in an endemic rural community of Nigeria. METHODS: A cross-sectional school-based study was conducted. Urine samples of 487 pupils were screened microscopically for S. haematobium and tested for haematuria and proteinuria using chemical reagent strips. RESULTS: The prevalence and intensity of infection were 57.1% and 45.0 eggs/10 mL urine respectively. Prevalence of infection in male (54.1%) and female (60.3%) individuals showed no significant variation (P>0.05). However, prevalence of infection was age dependent with those in age groups 3-5 and 12-14 years having the least and highest prevalence of infection respectively (P<0.05). Microhaematuria and proteinuria varied significantly with ages of the pupils with least (14.0, 40.0%) and highest (60.0, 80.0%) prevalence recorded in age groups 3-5 and 15-19 years respectively (P<0.05). Proteinuria showed higher sensitivity (80.3%) compared to microhaematuria (73.3%). CONCLUSION: Schistosomiasis is highly endemic in the study area and the use of microhaematuria and proteinuria for mapping the infected population prior treatment could be adopted.

Performance of three rapid screening methods in the detection of Schistosoma haematobium infection in school-age children in Southeastern Nigeria.

BACKGROUND: A cross-sectional study of primary school children was conducted to evaluate and compare the performance of some rapid screening methods in the detection of Schistosoma haematobium infection in Nigeria Cement Factory (NigerCem) and Nike Lake areas of Southeastern Nigeria. METHODS: Urine samples of school children were examined for macro-haematuria and tested for micro-haematuria and proteinuria using reagent strips followed by egg microscopy. Self-reported haematuria was assessed using simple questionnaire. The performances of these rapid diagnoses singly and in combination were calculated using egg microscopy as gold standard. RESULTS: The prevalence of the infection was 26·6% in NigerCem and 5·1% in Nike Lake area, classifying these areas as moderate- and low-prevalence areas (MPA and LPA); while in the subsample used for self-reported haematuria, the prevalence was 27·2 and 4·2% in MPA and LPA, respectively. The positive predictive value (PPV) of micro-haematuria was comparable in MPA (55·26%) and LPA (57·89%). Overall PPV of macro-haematuria was 87·50% in MPA and 66·70% in LPA while in the detection of heavy infection; PPV was higher in LPA (75%) than in MPA (66·67%). In LPA and MPA, combination of micro-haematuria and proteinuria, and concomitant presence of macro-haematuria, micro-haematuria, and proteinuria had PPV of 83·33 and 63·16%, and 100 versus 66·67%, respectively. Generally, the rapid screening tests had lower negative predictive values (NPVs) in MPA than in LPA. The use of simple questionnaire increased the PPV of heavy infection in MPA (77·78%). This was further increased to 80% when self-reported haematuria was combined with micro-haematuria. CONCLUSION: The result suggests that in MPA with chronic infections, combination of self-reported haematuria and micro-haematuria may reduce the chance of missing those who should be treated.

Efficacy of Praziquantel against Schistosoma haematobium in Dulshatalo village, western Ethiopia.

BACKGROUND: Praziquantel (PZQ) is the drug of choice for treatment of all human schistosomes. It is used in population based targeted or mass deworming strategies in several countries. The effect of PZQ on S. hematobium has not been studied in Ethiopia. The objective of this study was to determine the efficacy of PZQ against S. haematobium in Dulshatalo village, western Ethiopia. METHODS: A prospective study was conducted from October to December, 2007. Urine samples from 341 residents were collected and screened for haematuria and proteinuria using urinalysis dipstick. S. haematobium eggs were detected and quantified using filtration techniques. The participants who were positive for haematuria were treated with a standard dose of PZQ (40 mg/kg). Data on pre and 24 hours post treatment symptoms were collected via questionnaire. Urine samples were also collected 7 weeks after treatment and examined to assess the cure and the egg reduction rates. RESULTS: The prevalence of S. haematobium among the study participants was 57.8% (197/341). Haematuria was detected in 234 (68.6%) of the study participants. For PZQ efficacy asessment, 152 of the treated participants were considered. The presence of S. haemetaobium eggs showed statistically significant association (p < 0.05) with haematuria and proteinuria. Seven weeks post treatment, the extent of haematuria and proteinuria decreased from 100% to 40.8% and 94.07% to 48.7%, respectively. The cure and the parasitological egg reduction rates seven weeks post treatment were 86% and 85%, respectively. Post treatment symptoms revealed a wide range of side effects including straining, abdominal pain, nausea and headache. CONCLUSIONS: There were marked cure and egg reduction rates, together with mild and short lived side effects of PZQ for treatment of S. haematobium, in this study.

Performance of microhaematuria and proteinuria as measured by urine reagent strips in estimating intensity and prevalence of Schistosoma haematobium infection in Nigeria.

OBJECTIVE: To assess if microhaematuria and proteinuria as measured by reagent strips could estimate intensity of Schistosoma haematobium (S. haematobium) infection in endemic areas and evaluate their screening performance among children in Benue State, Nigeria. METHODS: A total of 1,124 urine samples were collected, screened for microhaematuria and proteinuria using reagent strips (Combi 9) and results were compared to filtration technique, the gold standard method. RESULTS: A significant correlation was observed between microhaematuria (rho= 0.66, P<0.01), proteinuria (rho = 0.71, P<0.01) and intensity of S. haematobium eggs. Proteinuria had sensitivity of 95.7% and specificity of 67.2%, while microhaematuria had sensitivity of 64.8% and specificity of 89.6%. The proportion of false positive diagnoses was higher in proteinuria (19.2%) than microhaematuria (6.0%). CONCLUSIONS: The findings suggest that use of urine reagent strips could potentially estimate intensity of S. haematobium infection and their performance to screen urinary schistosomiasis agreed with previous observations.

Renal effects of Dirofilaria immitis in experimentally and naturally infected cats.

Canine heartworm infection has been associated with glomerular disease and proteinuria. We hypothesized that proteinuria, likely due to glomerular damage, would also be found in cats experimentally and naturally infected with Dirofilaria immitis. Two populations of cats were evaluated, including 80 that were each experimentally infected with 60 infective heartworm larvae as part of a drug safety study, and 31 that were naturally infected with D. immitis. Each had a control population with which to be compared. In the experimentally infected group, we evaluated urine from 64 cats. Ten of these cats were shown to have microalbuminuria 8 months post infection. No cat refractory to infection with larvae and no cats from the control group demonstrated microalbuminuria. All 10 microalbuminuric cats were shown to have significant proteinuria, as measured by the urine protein:creatinine ratio. There was a subtle, but significant, association between worm burden and proteinuria, and although the presence of adult heartworms was required for the development of proteinuria, both microfilaremic and amicrofilaremic cats were affected. Neither the presence of circulating heartworm antibodies and antigen nor the presence of antigenuria predicted the development of proteinuria. Both heavily infected cats (5-25 adult heartworms) and cats with worm burdens compatible with natural infections (1-4 adult heartworms) developed proteinuria, and the relative numbers of cats so affected were similar between heavily and more lightly infected cats. Naturally infected cats, for which only dipstick protein determinations were available, were shown to have a significantly greater incidence of proteinuria (90% vs 35%) than did those in an age- and gender-matched control population. Additionally, the proteinuria in heartworm-infected cats was 3- to 5-fold greater in severity. We conclude that cats infected with mature adult heartworms are at risk for developing proteinuria and that this is recognized relatively soon after infection. While heavier infections may predispose cats to developing proteinuria, this complication is seen in naturally infected cats and experimental cats with worm burdens similar to those seen in natural infections (i.e., "clinically appropriate" worm burdens). The clinical relevance of heartworm-associated proteinuria is yet to be determined.

Free light-chain proteinuria and normal renal histopathology and function in 11 dogs exposed to Lleishmania infantum, Ehrlichia canis, and Bbabesia canis.

The purpose of this retrospective study was to investigate the relationship among proteinuria consisting of immunoglobulin free light chains (FLCs), renal histopathologic findings, and routine markers of renal function in 11 dogs exposed to Leishmania infantum (n = 8), Ehrlichia canis (n = 2), and Babesia canis (n = 1). FLC proteinuria was suspected based on identification of a 22- to 27-kDa band by sodium dodecyl sulfate-agarose gel electrophoresis (SDS-AGE) and later confirmed by immunofixation electrophoresis. SDS-AGE identified an isolated band of 22-27 kDa in 8 dogs, whereas the remaining 3 had a 22- to 27-kDa band and an additional band of 67-72 kDa. The median urine protein-to-urine creatinine ratio was 0.37 (range, 0.11-2.24) and increased ratios were found in 6 dogs (54.5%) (reference value, <0.7). All dogs underwent histologic examination of renal percutaneous biopsy specimens and determination of serum creatinine and urea concentrations. Tissue samples for light microscopy were stained with hematoxylin-eosin, periodic acid-Schiff, Goldners trichrome, and methenamine silver. In the study group, the glomerular tufts, mesangium, tubulointerstitium, and vessels appeared unaffected. The median serum creatinine concentration in these 11 dogs was 1.3 mg/dL (range, 0.8-1.5 mg/dL; reference range, 0.6-1.5 mg/dL), whereas the concentration for urea was 28 mg/dL (range, 22-52 mg/dL; reference range, 20-50 mg/dL). All dogs had normal renal morphology and had normal serum creatinine and urea concentrations, suggesting that immunoglobulin FLC may be detected in the urine of dogs exposed to L. infantum, E. canis, and B. canis without any apparent structural or functional renal derangement.

Malaria in pregnancy in Hodiedah, Republic of Yemen.

In the Republic of Yemen, Plasmodium falciparum is the predominant causative agent of malaria and is associated with adverse consequences for pregnant women and their babies. The prevalence and clinical manifestations of malaria among 500 pregnant (260) and non-pregnant (240) women were compared. Clinical examinations, laboratory investigations and a structured questionnaire were used to collect data. The prevalence of malaria was higher among pregnant women (55%) than non-pregnant women (20%). Anaemia was significantly more prevalent among pregnant woman than non-pregnant women and also more prevalent in pregnant women with malaria than non-pregnant women with malaria.

Evaluation of the effectiveness of dipstick haematuria and proteinuria in screening Schistosoma haematobium infection among school children in upper Egypt.

This study aimed at evaluation of validity and reliability of dipstick haematuria and proteinuria in screening school children for Schistosoma haematobium infection. It included a random sample of 400 school children aged 6-15 years in rural area of Fayoum Governorate, upper Egypt. Urine samples of the studied children were tested parasitologically by urine filtration technique as a reference test and semiquantitatively for haematuria and proteinuria using urine reagent strips as screening tests. Results of the study revealed that haematuria was a better indicator for Schistosoma haematobium infection than proteinuria, as it was more sensitive (85.5% 73.4%, respectively), specific (94.4% 82.9%, respectively) and reliable (kappa=92% 80%, respectively). Moreover, it had stronger relationship with intensity of infection (r=0.88 & 0.67, respectively). A combination of different grades of haematuria and proteinuria did not significantly increase either sensitivity or specificity. Dipstick haematuria could be a valuable technique in screening rural Egyptian school children who are at risk of urinary schistosmiasis.

Frequency of haematuria and proteinuria among Schistosoma haematobium infected children of two communities from Liberia and Tanzania.

The frequencies of haematuria and proteinuria among children of two rural communities with different Schistosoma haematobium endemicity from Liberia and Tanzania were compared. Although the prevalence and intensity of S. haematobium infections were lower in the Tanzanian community, the frequencies of haematuria and proteinuria were significantly higher when compared to the Liberian community. The semi-quantitative dip stick tests for haematuria and proteinuria showed a comparable, good specificity (haematuria 85%, proteinuria 80%), but a community-specific sensitivity. The dip stick test for haematuria detected 85% (proteinuria 82%) of all S. haematobium infected subjects in Tanzania compared to 68% (proteinuria 57%) in Liberia. The significance of these observations in relation to S. haematobium related morbidity is discussed.