Diagnostic accuracy and applicability of a PCR system for the detection of Schistosoma mansoni DNA in human urine samples from an endemic area

Schistosomiasis caused by Schistosoma mansoni, one of the most neglected human parasitoses in Latin America and Africa, is routinely confirmed by microscopic visualization of eggs in stool. The main limitation of this diagnostic approach is its lack of sensitivity in detecting individual low worm burdens and consequently data on infection rates in low transmission settings are little reliable. According to the scientific literature, PCR assays are characterized by high sensitivity and specificity in detecting parasite DNA in biological samples. A simple and cost effective extraction method for DNA of Schistosoma mansoni from urine samples in combination with a conventional PCR assay was developed and applied in an endemic area. This urine based PCR system was tested for diagnostic accuracy among a population of a small village in an endemic area, comparing it to a reference test composed of three different parasitological techniques. The diagnostic parameters revealed a sensitivity of 100%, a specificity of 91.20%, positive and negative predictive values of 86.25% and 100%, respectively, and a test accuracy of 94.33%. Further statistical analysis showed a k index of 0.8806, indicating an excellent agreement between the reference test and the PCR system. Data obtained from the mouse model indicate the infection can be detected one week after cercariae penetration, opening a new perspective for early detection and patient management during this stage of the disease. The data indicate that this innovative PCR system provides a simple to handle and robust diagnostic tool for the detection of S. mansoni DNA from urine samples and a promising approach to overcome the diagnostic obstacles in low transmission settings. Furthermore the principals of this molecular technique, based on the examination of human urine samples may be useful for the diagnosis of other neglected tropical diseases that can be detected by trans-renal DNA.     

Long-Term Frozen Storage of Urine Samples: A Trouble to Get PCR Results in Schistosoma spp. DNA Detection?

Background

Human schistosomiasis remains a serious worldwide public health problem. At present, a sensitive and specific assay for routine diagnosis of schistosome infection is not yet available. The potential for detecting schistosome-derived DNA by PCR-based methods in human clinical samples is currently being investigated as a diagnostic tool with potential application in routine schistosomiasis diagnosis. Collection of diagnostic samples such as stool or blood is usually difficult in some populations. However, urine is a biological sample that can be collected in a non-invasive method, easy to get from people of all ages and easy in management, but as a sample for PCR diagnosis is still not widely used. This could be due to the high variability in the reported efficiency of detection as a result of the high variation in urine samples’ storage or conditions for handling and DNA preservation and extraction methods.

Methodology/Principal Findings

We evaluate different commercial DNA extraction methods from a series of long-term frozen storage human urine samples from patients with parasitological confirmed schistosomiasis in order to assess the PCR effectiveness for Schistosoma spp. detection. Patientś urine samples were frozen for 18 months up to 7 years until use. Results were compared with those obtained in PCR assays using fresh healthy human urine artificially contaminated with Schistosoma mansoni DNA and urine samples from mice experimentally infected with S. mansoni cercariae stored frozen for at least 12 months before use. PCR results in fresh human artificial urine samples using different DNA based extraction methods were much more effective than those obtained when long-term frozen human urine samples were used as the source of DNA template.

Conclusions/Significance

Long-term frozen human urine samples are probably not a good source for DNA extraction for use as a template in PCR detection of Schistosoma spp., regardless of the DNA method of extraction used.     

A Schistosoma haematobium-Specific Real-Time PCR for Diagnosis of Urogenital Schistosomiasis in Serum Samples of International Travelers and Migrants

Background

Diagnosis of urogenital schistosomiasis by microscopy and serological tests may be elusive in travelers due to low egg load and the absence of seroconversion upon arrival. There is need for a more sensitive diagnostic test. Therefore, we developed a real-time PCR targeting the Schistosoma haematobium-specific Dra1 sequence.

Methodology/Principal Findings

The PCR was evaluated on urine (n = 111), stool (n = 84) and serum samples (n = 135), and one biopsy from travelers and migrants with confirmed or suspected schistosomiasis. PCR revealed a positive result in 7/7 urine samples, 11/11 stool samples and 1/1 biopsy containing S. haematobium eggs as demonstrated by microscopy and in 22/23 serum samples from patients with a parasitological confirmed S. haematobium infection. S. haematobium DNA was additionally detected by PCR in 7 urine, 3 stool and 5 serum samples of patients suspected of having schistosomiasis without egg excretion in urine and feces. None of these suspected patients demonstrated other parasitic infections except one with Blastocystis hominis and Entamoeba cyst in a fecal sample. The PCR was negative in all stool samples containing S. mansoni eggs (n = 21) and in all serum samples of patients with a microscopically confirmed S. mansoni (n = 22), Ascaris lumbricoides (n = 1), Ancylostomidae (n = 1), Strongyloides stercoralis (n = 1) or Trichuris trichuria infection (n = 1). The PCR demonstrated a high specificity, reproducibility and analytical sensitivity (0.5 eggs per gram of feces).

Conclusion/Significance

The real-time PCR targeting the Dra1 sequence for S. haematobium-specific detection in urine, feces, and particularly serum, is a promising tool to confirm the diagnosis, also during the acute phase of urogenital schistosomiasis.     

Molecular approaches for the detection of Schistosoma mansoni: possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection

The detection of specific DNA sequences by polymerase chain reaction (PCR) has proved extremely valuable for the analysis of genetic disorders and the diagnosis of a variety of infectious disease pathogens. However, the application to the detection of Schistosoma mansoni is rare, despite a recommendation of the World Health Organization that a major focus of research on schistosomiasis should be on the development and evaluation of new strategies and tools for control of the disease. In this context, a few studies were published for the detection of the parasite in snails, monitoring of cercariae in water bodies, and diagnosis of human infection. The present minireview describes sensitive and specific PCR based systems to detect S. mansoni, indicating possible applications in the detection of snail infection, monitoring of transmission sites, and diagnosis of human infection.     

The Rapid-Heat LAMPellet Method: A Potential Diagnostic Method for Human Urogenital Schistosomiasis

Background

Urogenital schistosomiasis due to Schistosoma haematobium is a serious underestimated public health problem affecting 112 million people - particularly in sub-Saharan Africa. Microscopic examination of urine samples to detect parasite eggs still remains as definitive diagnosis. This work was focussed on developing a novel loop-mediated isothermal amplification (LAMP) assay for detection of S. haematobium DNA in human urine samples as a high-throughput, simple, accurate and affordable diagnostic tool to use in diagnosis of urogenital schistosomiasis.

Methodology/Principal Findings

A LAMP assay targeting a species specific sequence of S. haematobium ribosomal intergenic spacer was designed. The effectiveness of our LAMP was assessed in a number of patients´ urine samples with microscopy confirmed S. haematobium infection. For potentially large-scale application in field conditions, different DNA extraction methods, including a commercial kit, a modified NaOH extraction method and a rapid heating method were tested using small volumes of urine fractions (whole urine, supernatants and pellets). The heating of pellets from clinical samples was the most efficient method to obtain good-quality DNA detectable by LAMP. The detection limit of our LAMP was 1 fg/µL of S. haematobium DNA in urine samples. When testing all patients´ urine samples included in our study, diagnostic parameters for sensitivity and specificity were calculated for LAMP assay, 100% sensitivity (95% CI: 81.32%-100%) and 86.67% specificity (95% CI: 75.40%-94.05%), and also for microscopy detection of eggs in urine samples, 69.23% sensitivity (95% CI: 48.21% -85.63%) and 100% specificity (95% CI: 93.08%-100%).

Conclusions/Significance

We have developed and evaluated, for the first time, a LAMP assay for detection of S. haematobium DNA in heated pellets from patients´ urine samples using no complicated requirement procedure for DNA extraction. The procedure has been named the Rapid-Heat LAMPellet method and has the potential to be developed further as a field diagnostic tool for use in urogenital schistosomiasis-endemic areas.     

Epidemiology of Schistosomiasis and Usefulness of Indirect Diagnostic Tests in School-Age Children in Cubal,Central Angola

Introduction

Schistosomiasis remains a public health major problem and little is known in many areas, mainly in Sub-Saharan Africa

Objectives

To assess the burden and risk factors of schistosomiasis and intestinal parasitic helminthes in the children of Cubal, Angola, and to compare different diagnostic approaches for urinary schistosomiasis under field conditions.

Methods

A cross-sectional study was conducted. Urine and faeces samples of school children were microscopically studied. A random sample of children was obtained from an alphabetically arranged list of children, taking one of two children. Urine dipstick, colorimetric test and macrohaematuria were considered as indirect diagnostic methods and compared to direct urine examination. Possible risk factors for the infection were sex, age, distance to the river and previous treatment with praziquantel; the assessment was performed using Chi-square test.

Results

A total of 785 (61.18%) children showed S. haematobium eggs in urine; children living within 500 meters from the river had a higher odds for infection: Odds ratio 1.97 (1.45–2.7 CI 95%); urine dipstick showed sensitivity of 96% and specificity of 61.3%, with a positive predictive value; colorimetric test showed sensitivity of 52.5%, specificity of 74.6% and a positive predictive value of 77%. Proteinuria was present in 653 (51.1%) children, being more frequent in children with S. haematobium in urine (75.2%); 32 of 191 stool samples (16%) showed the presence of other intestinal parasites and 8 (4%) for S. haematobium.

Conclusions

Prevalence of urinary schistosomiasis in our study area is much higher than the national average, considering it as a high-risk community. Proximity to a source of water was a risk factor for the infection. Indirect tests, as urine dipstick and colorimetric test, were useful tools for diagnosis, due to ease of use and low cost. Proteinuria was a common finding, probably showing an early structural damage due to schistosomiasis in this group of children.     

Internal Amplification Control for a Cryptosporidium Diagnostic PCR: Construction and Clinical Evaluation

Various constituents in clinical specimens, particularly feces, can inhibit the PCR assay and lead to false-negative results. To ensure that negative results of a diagnostic PCR assay are true, it should be properly monitored by an inhibition control. In this study, a cloning vector harboring a modified target DNA sequence (≈375 bp) was constructed to be used as a competitive internal amplification control (IAC) for a conventional PCR assay that detects ≈550 bp of the Cryptosporidium oocyst wall protein (COWP) gene sequence in human feces. Modification of the native PCR target was carried out using a new approach comprising inverse PCR and restriction digestion techniques. IAC was included in the assay, with the estimated optimum concentration of 1 fg per reaction, as duplex PCR. When applied on fecal samples spiked with variable oocysts counts, ≈2 oocysts were theoretically enough for detection. When applied on 25 Cryptosporidium-positive fecal samples of various infection intensities, both targets were clearly detected with minimal competition noticed in 2-3 samples. Importantly, both the analytical and the diagnostic sensitivities of the PCR assay were not altered with integration of IAC into the reactions. When tried on 180 randomly collected fecal samples, 159 were Cryptosporidium-negatives. Although the native target DNA was absent, the IAC amplicon was obviously detected on gel of all the Cryptosporidium-negative samples. These results imply that running of the diagnostic PCR, inspired with the previously developed DNA extraction protocol and the constructed IAC, represents a useful tool for Cryptosporidium detection in human feces.     

Field-based evaluation of a reagent strip test for diagnosis of schistosomiasis mansoni by detecting circulating cathodic antigen (CCA) in urine in low endemic area in Ethiopia

The sensitivity, specificity, positive and negative predictive values of a reagent strip test for the diagnosis of schistosomiasis mansoni by detecting circulating cathodic antigen (CCA) in urine were evaluated using 184 stool and urine samples collected from schoolchildren living in relatively low endemic area of schistosomiasis mansoni in Ethiopia. A combined result of stool samples processed by Kato and formol-ether concentration methods was used as gold standard. The results showed that detection of CCA in urine using reagent strip test was slightly higher than the combined results of the stool techniques (65.2 % vs 42.4 %, p > 0.05) in suggesting the prevalence of the disease. The sensitivity, specificity, positive and negative predictive values of the reagent strip test were 76.9 %, 43.4 %, 50 % and 71.9 %, respectively. The result of egg counts using Kato method suggested that detection of urine CCA could be used to indicate the intensity of infection. Nevertheless, like that of stool examination, the reagent strip test was found to be less sensitive in case of light to moderate infections. About 23.1 % of the study children who were excreting the eggs of the parasite were found negative by the reagent strip test. The relative insensitivity of a reagent strip test in low intensity of infection necessitates for the development of more sensitive assay that can truly discriminate schistosome-infected from non-infected individuals.     

Spatial and temporal variation in malaria transmission in a low endemicity area in northern Tanzania

Background

Current detection or screening for malaria infection necessitates drawing blood by fingerprick or venipuncture, which poses risks and limitations for repeated measurement. This study presents PCR detection of Plasmodium falciparum in human urine and saliva samples, and illustrates this potential application in genotyping malaria infections.

Methods

Urine and saliva were obtained from 47 thick film positive and 4 negative individuals one day after collection of blood slides and filter paper blood spots. P. falciparum DNA was extracted from blood, urine and saliva, in separate groups, using the Chelex method or Qiagen DNEasy^® kit (urine and saliva only). Blood, urine and saliva extracts were subjected to PCR in separate batches. Amplicons from the various sample types were examined for MSP2 polymorphisms and restriction fragment patterns on DHFR amino acid codon 59.

Results and discussion

Malaria infections exhibited primarily low-grade parasite densities, with a geometric mean of 775 asexual parasites/μl. Regularly matching polymorphic MSP2 genotypes were found between the corresponding urine, saliva and peripheral blood amplicons of each individual, with different inter-individual polymorphic genotypes. Amplicon yields were significantly dependent on DNA extraction method, parasite density and primer set (p < 0.001). A Qiagen^® kit extraction had more than 2× higher amplicon yield than the Chelex method, for both urine and saliva. Amplicon yields were 1.6 fold higher from saliva than urine. For each unit increase in log parasite density, the probability of amplicon enhanced 1.8 fold. Highest amplicon yields were obtained from the primer set with the shortest PCR product.

Conclusion

P. falciparum infection is detectable by PCR on human urine and saliva samples. Subject to further refinement of extraction technique and amplicon yields, large-scale malaria parasite screening and epidemiological surveys could be possible without the need to collect blood and use of needles or sharps.     

Newly Established Monoclonal Antibody Diagnostic Assays for Schistosoma mansoni Direct Detection in Areas of Low Endemicity

Background

Current available methods for diagnosis of schistosomiasis mansoni lack sufficient sensitivity, which results in underreporting of infectious in areas of low endemicity.

Methodology/Principal Findings

We developed three novel diagnostic methodologies for the direct detection of schistosome infection in serum samples. These three new methods were evaluated with positive patients from a low endemicity area in southeast Brazil. The basis of the assay was the production of monoclonal antibodies against the protein backbone of heavily glycosylated Circulating Cathodic Antigen (CCA). The antibodies were also selected for having no specificity to repeating poly-Lewis x units. Assays based on the detection CCA-protein should not encounter a limitation in sensitivity due to a biological background of this particular epitope. Three diagnostic methodologies were developed and validated, (i) Immunomagnetic Separation based on improved incubation steps of non-diluted serum, (ii) Direct Enzyme-linked Immunosorbent Assay and (iii) Fluorescent Microscopy Analysis as a qualitative assay. The two quantitative assays presented high sensitivity (94% and 92%, respectively) and specificity (100%), equivalent to the analysis of 3 stool samples and 16 slides by Kato-Katz, showing promising results on the determination of cure.

Conclusions/Significance

The Immunomagnetic Separation technique showed excellent correlation with parasite burden by Cohen coefficient. The qualitative method detected 47 positive individuals out of 50 with the analysis of 3 slides. This easy-to-do method was capable of discriminating positive from negative cases, even for patients with low parasite burden.     

Monoclonal Antibody-Based Dipstick Assay: A Reliable Field Applicable Technique for Diagnosis of Schistosoma mansoni Infection Using Human Serum and Urine Samples

A field applicable diagnostic technique, the dipstick assay, was evaluated for its sensitivity and specificity in diagnosing human Schistosoma mansoni infection. A monoclonal antibody (mAb) against S. mansoni adult worm tegumental antigen (AWTA) was employed in dipstick and sandwich ELISA for detection of circulating schistosome antigen (CSA) in both serum and urine samples. Based on clinical and parasitological examinations, 60 S. mansoni-infected patients, 30 patients infected with parasites other than schistosomiasis, and 30 uninfected healthy individuals were selected. The sensitivity and specificity of dipstick assay in urine samples were 86.7% and 90.0%, respectively, compared to 90.0% sensitivity and 91.7% specificity of sandwich ELISA. In serum samples, the sensitivity and specificity were 88.3% and 91.7% for dipstick assay vs. 91.7% and 95.0% for sandwich ELISA, respectively. The diagnostic efficacy of dipstick assay in urine and serum samples was 88.3% and 90.0%, while it was 90.8% and 93.3% for sandwich ELISA, respectively. The diagnostic indices of dipstick assay and ELISA either in serum or in urine were statistically comparable (P>0.05). In conclusion, the dipstick assay offers an alternative simple, rapid, non-invasive technique in detecting CSA or complement to stool examinations especially in field studies.     

Detection of Soluble Antigen and DNA of Trypanosoma cruzi in Urine Is Independent of Renal Injury in the Guinea Pig Model

The diagnosis of Chagas disease in humans is generally limited to the detection of specific antibodies. Detection of T. cruzi antigens in urine has been reported previously, but is not used in the diagnosis. In this study, soluble T. cruzi antigens and DNA were detected in urine samples and were associated with kidney injury and systemic detection of the parasite. We used 72 guinea pigs infected with T. cruzi Y strain and 18 non-infected guinea pigs. Blood, kidney, heart and urine samples were collected during the acute phase and chronic phase. Urine samples were concentrated by ultrafiltration. Antigens were detected by Western Blot using a polyclonal antibody against trypomastigote excretory-secretory antigen (TESA). T. cruzi DNA was detected by PCR using primers 121/122 and TcZ1/TcZ2. Levels of T. cruzi DNA in blood, heart and kidney were determined by quantitative PCR. T. cruzi antigens (75 kDa, 80 kDa, 120 kDa, 150 kDa) were detected in the acute phase (67.5%) and the chronic phase (45%). Parasite DNA in urine was detected only in the acute phase (45%). Kidney injury was characterized by high levels of proteinuria, kidney injury molecule-1 (KIM-1) and urea, and some histopathological changes such as inflammation, necrosis, fibrosis and scarce parasites. The detection of antigens and DNA in urine was associated with the presence of parasite DNA in blood and heart and with high levels of parasite DNA in blood, but not with the presence of parasite in kidney or kidney injury. These results suggest that the detection of T. cruzi in urine could be improved to be a valuable method for the diagnosis of Chagas disease, particularly in congenital Chagas disease and in immunocompromised patients.     

A high-throughput assay for the detection of Tyr-phosphorylated proteins in urine of bladder cancer patients

Background

Bladder cancer has the peculiarity of shedding neoplastic cells and their components in urine representing a valuable opportunity to detect diagnostic markers. Using a semi-quantitative method we previously demonstrated that the levels of Tyr-phosphorylated proteins (TPPs) are highly increased in bladder cancer tissues and that soluble TPPs can also be detected in patient's urine samples. Although the preliminary evaluation showed very promising specificity and sensitivity, insufficient accuracy and very low throughput of the method halted the diagnostic evaluation of the new marker. To overcome this problem we developed a quantitative methodology with high sensitivity and accuracy to measure TPPs in urine.

Methods

The Immobilized Metal Affinity Chromatography (IMAC) was miniaturized in a 96 well format. Luminescence, visible and infrared fluorescence antibody-based detection methods were comparatively evaluated.

Results

Due to their low abundance we evidenced that both phosphoprotein enrichment step and very sensitive detection methods are required to detect TPPs in urine samples. To pursue high throughput, reproducibility and cost containment, which are required for bladder cancer screening programs, we coupled the pre-analytical IMAC procedure with high sensitive detection phases (infrared fluorescence or chemiluminescence) in an automated platform.

Conclusions

A high throughput method for measuring with high sensitivity TPP levels in urine samples is now available for large clinical trial for the establishment of the diagnostic and predictive power of TPPs as bladder cancer marker.

General significance

The new assay represents the first quantitative and high throughput method for the measurement of TPPs in urine.     

Trypanosoma evansi: a comparative study of four diagnostic techniques for trypanosomosis using rabbit as an experimental model

The goal of this study was to compare two parasitological diagnostic techniques, such as by Micro-Haematocrit Centrifugation Technique (MHCT) and Direct Microscopic Examination (DME) with a serological method (iELISA), and a molecular procedure PCR, in rabbits experimentally infected with Trypanosoma evansi, in order to determine their sensitivity throughout the course of disease. The parasitological methods were not able of detecting the presence of the parasite during the phases of low parasitemia, the prepatency period and the chronic phase. In contrast, PCR detected T. evansi in the prepatency and chronic phase, when increase the amount of DNA from 100 to 300 ng. 100% detection was observed with iELISA only in the chronic stage of the disease. In the acute phase, all samples were positively diagnosed using either MHCT or PCR, whereas only few samples were diagnosed by DME. Samples obtained from day 15 post infection were also detected by iELISA. The highest diagnostic register during the course of infection was achieved by the PCR technique (93.8%), followed by iELISA (71.1%), MHCT (59%) and DME (13.6%). Therefore, we recommend the use of PCR in epidemiological studies in order to implement sanitary control plans for the improvement of livestock productivity in the country.     

Galactomannan-Based and PCR-Based Assays in Bronchoalveolar Lavage to Diagnose Invasive Aspergillosis: Current Status and Future Prospects

Invasive pulmonary aspergillosis (IPA) is a life-threatening complication in patients receiving chemotherapy or undergoing allogeneic haematopoietic stem cell transplantation for acute leukemia. The existing tools to diagnose IPA lack specificity or sensitivity, or both; the search for improved diagnostic tools for IPA has focused on novel serologic and molecular methods. Aspergillus Galactomannan enzyme-linked immunosorbent assay (GM) analyses showed sensitivity rates in serum samples ranging in a wide span; testing GM in bronchoalveolar lavage (BAL) originated from the primary site of the infection seems to be more sensitive in patients with IPA. Other novel diagnostic markers to detect fungal DNA directly in clinical samples, rapidly, early, sensitively and specifically, are provided by polymerase chain reaction (PCR) based assays; higher sensitivity and specificity rates have been observed for BAL samples in IPA, even under antifungal treatment. The clinical place value of a diagnostic approach combining PCR and GM in BAL is unclear.     

Sensitive and specific target sequences selected from retrotransposons of Schistosoma japonicum for the diagnosis of schistosomiasis

Background

Schistosomiasis japonica is a serious debilitating and sometimes fatal disease. Accurate diagnostic tests play a key role in patient management and control of the disease. However, currently available diagnostic methods are not ideal, and the detection of the parasite DNA in blood samples has turned out to be one of the most promising tools for the diagnosis of schistosomiasis. In our previous investigations, a 230-bp sequence from the highly repetitive retrotransposon SjR2 was identified and it showed high sensitivity and specificity for detecting Schistosoma japonicum DNA in the sera of rabbit model and patients. Recently, 29 retrotransposons were found in S. japonicum genome by our group. The present study highlighted the key factors for selecting a new perspective sensitive target DNA sequence for the diagnosis of schistosomiasis, which can serve as example for other parasitic pathogens.

Methodology/Principal Findings

In this study, we demonstrated that the key factors based on the bioinformatic analysis for selecting target sequence are the higher genome proportion, repetitive complete copies and partial copies, and active ESTs than the others in the chromosome genome. New primers based on 25 novel retrotransposons and SjR2 were designed and their sensitivity and specificity for detecting S. japonicum DNA were compared. The results showed that a new 303-bp sequence from non-long terminal repeat (LTR) retrotransposon (SjCHGCS19) had high sensitivity and specificity. The 303-bp target sequence was amplified from the sera of rabbit model at 3 d post-infection by nested-PCR and it became negative at 17 weeks post-treatment. Furthermore, the percentage sensitivity of the nested-PCR was 97.67% in 43 serum samples of S. japonicum-infected patients.

Conclusions/Significance

Our findings highlighted the key factors based on the bioinformatic analysis for selecting target sequence from S. japonicum genome, which provide basis for establishing powerful molecular diagnostic techniques that can be used for monitoring early infection and therapy efficacy to support schistosomiasis control programs.     

Prospective study of urine cytology screening for BK polyoma virus replication in renal transplant recipients

Objective:  BK virus (BKV) may be associated with interstitial nephritis in renal transplant recipients and this can lead to irreversible chronic allograft dysfunction. Early diagnosis of BKV nephropathy determines its progress because no specific antiviral therapy exists. Urine cytology, detection of viral DNA in urine or blood and renal biopsy are the main diagnostic tools. The purpose of this study was to evaluate the use of urine cytology for diagnosis of BKV replication in renal graft recipients.
Patients and methods:  We studied 32 de novo renal transplant recipients prospectively with sequential urine samples for a period of 1 year. Thin-Prep methodology was used to prepare the slides. Cytology results were correlated with polymerase chain reaction (PCR) in urine and blood.
Results:  Decoy cells indicative of BKV infection were detected in 14 (7.3%) of the 190 urine samples derived from 11 recipients. In three cases with positive decoy cells, BK viraemia and viruria were simultaneously identified. In a further three cases, BKV active replication was confirmed in urine by both cytology and PCR.
Conclusions:  Urine cytology is an easy and rapid method of detecting decoy cells in cases where renal biopsy is not possible. However, the low incidence of detection of decoy cells in the present study, together with poor correlation with PCR results, questions its sensitivity and specificity in diagnosing BKV reactivation.     

Advances in the Diagnosis of Human Opisthorchiasis: Development of Opisthorchis viverrini Antigen Detection in Urine

Background

Many strategies to control opisthorchiasis have been employed in Thailand, but not in the other neighbouring countries. Specific control methods include mass drug administration (MDA) and health education to reduce raw fish consumption. These control efforts have greatly shifted the epidemiology of Opisthorchis viverrini (OV) infection over the last decade from presenting as densely concentrated "heavy" infections in single villages to widespread "light" OV infections distributed over wide geographical areas. Currently, the "gold standard" detection method for OV infection is formalin ethyl-acetate concentration technique (FECT), which has limited diagnostic sensitivity and diagnostic specificity for light OV infections, with OV eggs often confused with eggs of minute intestinal flukes (MIFs) in feces. In this study, we developed and evaluated the diagnostic performance of a monoclonal antibody-based enzyme-linked immunosorbent assay for the measurement of OV excretory-secretory (ES) antigens in urine (urine OV-ES assay) for the diagnosis of opisthorchiasis compared to the gold standard detection FECT method.

Methodology

We tested several methods for pre-treating urine samples prior to testing the diagnostic performance of the urine OV-ES assay. Using trichloroacetic acid (TCA) pre-treated urine, we compared detection and quantification of OV infection using the urine OV-ES assay versus FECT in OV-endemic areas in Northeastern Thailand. Receiver operating characteristic (ROC) curves were used to determine the diagnostic sensitivity and specificity of the urine OV-ES assay using TCA pre-treated urine, and to establish diagnostic positivity thresholds. The Positive Predictive Value as well as the likelihood of obtaining a positive test result (LR+) or a negative test result (LR-) were calculated for the established diagnostic positivity threshold. Diagnostic risks (Odds Ratios) were estimated using logistic regression.

Results

When urine samples were pre-treated with TCA prior to use in the urine OV-ES assay, the analytical sensitivity was significantly improved. Using TCA pre-treatment of urine, the urine OV-ES assay had a limit of detection (LoD) of 39 ng/ml compared to the LoD of 52 ng/mL reported for coprological antigen detection methods. Similarly, the urine OV-ES assay correlated significantly with intensity of OV infection as measured by FECT. The urine OV-ES assay was also able to detect 28 individuals as positive from the 63 (44.4%) individuals previously determined to be negative using FECT. The likelihood of a positive diagnosis of OV infection by urine OV-ES assay increased significantly with the intensity of OV infection as determined by FECT. With reference to FECT, the sensitivity and specificity of the urine OV-ES assay was 81% and 70%, respectively.

Conclusion

The detection of OV-infection by the urine OV-ES assay showed much greater diagnostic sensitivity and diagnostic specificity than the current "gold standard" FECT method for the detection and quantification of OV infection. Due to its ease-of-use, and noninvasive sample collection (urine), the urine OV-ES assay offers the potential to revolutionize the diagnosis of liver fluke infection and provide an effective tool for control and elimination of these tumorigenic parasites.     

Performance of a rapid immuno-chromatographic test (Schistosoma ICT IgG-IgM) for detecting Schistosoma-specific antibodies in sera of endemic and non-endemic populations

BackgroundSchistosomiasis, an acute and chronic parasitic disease caused by human pathogenic Schistosoma species, is a neglected tropical disease affecting more than 220 million people worldwide.For diagnosis of schistosomiasis, stool and urine microscopy for egg detection is still the recommended method, however sensitivity of these methods is limited. Therefore, other methods like molecular detection of DNA in stool, detection of circulating cathodic antigen in urine or circulating anodic antigen in urine and serum, as well as serological tests have gained more attention. This study examines the sensitivity and specificity of a rapid diagnostic test based on immunochromatography (Schistosoma ICT IgG-IgM, LD Bio, Lyon, France) for simultaneous detection of specific IgG and IgM antibodies in serum, against Schistosoma spp. in endemic and non-endemic populations.Methodology/Principal findingsFrozen banked serum samples from patients with confirmed schistosomiasis, patients with other helminth infections, patients with seropositive rheumatoid arthritis and healthy blood donors were used to assess the sensitivity and the specificity of the Schistosoma ICT IgG-IgM rapid diagnostic test.The test showed a sensitivity of 100% in patients with parasitologically confirmed schistosomiasis, irrespective of the species (S. mansoni, S. haematobium, S. japonicum, S. mekongi). In healthy blood donors and patients with rheumatoid factor positive rheumatoid arthritis from Europe, specificity was 100%. However, in serum samples of patients with other tissue invasive helminth infections, the test showed some cross-reactivity, resulting in a specificity of 85%.Conclusion/SignificanceWith its high sensitivity, the Schistosoma ICT IgG-IgM rapid diagnostic test is a suitable screening test for detection of Schistosoma specific antibodies, including S. mekongi. However, in populations with a high prevalence of co-infection with other tissue invasive helminths, positive results should be confirmed with other diagnostic assays due to the test’s imperfect specificity.     

An Improved PCR-RFLP Assay for Detection and Genotyping of Asymptomatic Giardia lamblia Infection in a Resource-Poor Setting

Laboratory workers, in resource-poor countries, still consider PCR detection of Giardia lamblia more costly and more time-consuming than the classical parasitological techniques. Based on 2 published primers, an in-house one-round touchdown PCR-RFLP assay was developed. The assay was validated with an internal amplification control included in reactions. Performance of the assay was assessed with DNA samples of various purities, 91 control fecal samples with various parasite load, and 472 samples of unknown results. Two cysts per reaction were enough for PCR detection by the assay with exhibited specificity (Sp) and sensitivity (Se) of 100% and 93%, respectively. Taking a published small subunit rRNA reference PCR test results (6%; 29/472) as a nominated gold standard, G. lamblia was identified in 5.9% (28/472), 5.2%, (25/472), and 3.6% (17/472) by PCR assay, RIDA^® Quick Giardia antigen detection test (R-Biopharm, Darmstadt, Germany), and iodine-stained smear microscopy, respectively. The percent agreements (kappa values) of 99.7% (0.745), 98.9% (0.900), and 97.7% (0.981) were exhibited between the assay results and that of the reference PCR, immunoassay, and microscopy, respectively. Restriction digestion of the 28 Giardia-positive samples revealed genotype A pattern in 12 and genotype B profile in 16 samples. The PCR assay with the described format and exhibited performance has a great potential to be adopted in basic clinical laboratories as a detection tool for G. lamblia especially in asymptomatic infections. This potential is increased more in particular situations where identification of the parasite genotype represents a major requirement as in epidemiological studies and infection outbreaks.