Diagnostic urinary cfDNA detected in human cystic echinococcosis.

Cystic echinococcosis (CE) is a major neglected tropical zoonotic disease caused by the tissue-dwelling larval stage of the cestode parasite Echinococcus granulosus. For individuals suspected of CE, the diagnostic standard is imaging using ultrasonography, X rays, or computed tomography. These resource-demanding and expensive procedures are rarely available in endemic rural areas where CE is most prevalent. There is a critical need for a new approach to identify CE patients so that they can be managed early in the course of their infection. This study reports on the results of a diagnostic approach that identifies E. granulosus-derived cell-free DNA (cfDNA) in the urine of CE patients. Utilizing PCR to amplify a fragment of a major tandem repeat element found in E. granulosus nuclear DNA, urine samples from all seven imaging-confirmed CE patients who harbored active liver cysts were positive. In addition, the urine samples from 2/4 patients who presented with non-viable/calcified liver cysts were also PCR positive for the repeat fragment. To our knowledge, this is the first report of using parasite cfDNA from urine to diagnose CE. This approach provides an easy to implement and cost-effective method to survey for the prevalence of E. granulosus in humans populations.

Performance of a real-time PCR approach for diagnosing Schistosoma haematobium infections of different intensity in urine samples from Zanzibar.

BACKGROUND: Efforts to control and eliminate schistosomiasis have accelerated over the past decade. As parasite burden, associated morbidity and egg excretion decrease, diagnosis with standard parasitological methods becomes harder. We assessed the robustness and performance of a real-time PCR (qPCR) approach in comparison with urine filtration microscopy and reagent strip testing for the diagnosis of Schistosoma haematobium infections of different intensities. METHODS: The robustness of DNA isolation and qPCR was validated in eight laboratories from Europe and Africa. Subsequently, 792 urine samples collected during cross-sectional surveys of the Zanzibar Elimination of Schistosomiasis Transmission (ZEST) project in 2012-2017 were examined with qPCR in 2018. Diagnostic sensitivity of the qPCR was calculated at different infection intensity categories, using urine filtration microscopy as reference test. Spearman's rank correlation between Ct-values and S. haematobium egg counts was assessed and Ct-value percentiles for infection intensity categories determined. RESULTS: S. haematobium Dra1 DNA-positive samples were identified correctly in all eight laboratories. Examination of urine samples from Zanzibar revealed Dra1 DNA in 26.8% (212/792) by qPCR, S. haematobium eggs in 13.3% (105/792) by urine filtration, and microhaematuria in 13.8% (109/792) by reagent strips. Sensitivity of the qPCR increased with augmenting egg counts: 80.6% (29/36) for counts between 1 and 4 eggs, 83.3% (15/18) for counts between 5 and 9 eggs, 100% (23/23) for counts between 10 and 49 eggs, and 96.4% (27/28) for counts of 50+ eggs. There was a significant negative correlation between Ct-values and egg counts (Spearman's rho = - 0.49, P < 0.001). Seventy-five percent of the Ct-values were ≥ 33 in the egg-negative category, < 31 in the light intensity category, and < 24 in the heavy intensity category. CONCLUSIONS: While the sensitivity of the qPCR was ~ 80% for very light intensity infections (egg counts < 10), in general, the Dra1 based qPCR assay detected twice as many S. haematobium infections compared with classical parasitological tests. The qPCR is hence a sensitive, urine-based approach for S. haematobium diagnosis that can be used for impact assessment of schistosomiasis elimination programmes, individual diagnosis, and in improved format also for verification and certification of elimination. TRIAL REGISTRATION: ISRCTN, ISRCTN48837681 . Registered 05 September 2012 - Retrospectively registered.

Detection of Schistosoma mansoni-derived DNA in human urine samples by loop-mediated isothermal amplification (LAMP).

BACKGROUND: Schistosoma mansoni is the main species causing hepatic and intestinal schistosomiasis in Sub-Saharan Africa, and it is the only species in South America. Adult stages of the parasite reside in the mesenteric venous plexus of infected hosts, and eggs are shed in feces. Collecting patient stool samples for S. mansoni diagnostic purposes is difficult in large-scale field trials. Urine samples would be an alternative approach for molecular S. mansoni detection since they have several advantages over stool samples, including better handling, management and storage. Additionally, loop-mediated isothermal amplification (LAMP) technology is a powerful molecular diagnostic tool for infectious diseases, particularly under field conditions in developing countries. The present study aimed to assess the effectiveness of our previously developed LAMP assay (SmMIT-LAMP) for S. mansoni-specific detection in clinical urine samples. METHODOLOGY/PRINCIPAL FINDINGS: The sensitivity of SmMIT-LAMP in urine was established in simulated fresh human urine samples artificially spiked with genomic DNA from S. mansoni. LAMP for 120 min instead of 60 min improved the sensitivity, reaching values of 0.01 fg/µL. A set of well-defined frozen stored human urine samples collected from Sub-Saharan immigrant patients was selected from a biobank to evaluate the diagnostic validity of SmMIT-LAMP. The set included urine samples from patients with microscopy-confirmed infections with S. mansoni, S. haematobium and other nonschistosome parasites, as well as urine samples from patients with microscopy-negative eosinophilia without a confirmed diagnosis. The SmMIT-LAMP was incubated for 60 and 120 min. A longer incubation time was shown to increase the LAMP-positive results in patient urine samples. We also tested urine samples from mice experimentally infected with S. mansoni, and LAMP-positive results were obtained from the third week after infection. A real-time LAMP assay was also performed with three individual urine samples. CONCLUSIONS/SIGNIFICANCE: The SmMIT-LAMP could effectively detect S. mansoni DNA in mouse urine samples and produced promising results for human clinical samples. The detection of S. mansoni DNA in mouse urine samples from the third week after infection indicates that early diagnosis of active S. mansoni infection is possible using urine as a source of DNA. Further studies are still needed, but our method could be used as a promising molecular tool applicable to urine samples to diagnose human intestinal schistosomiasis caused by S. mansoni.

Detection of Taenia solium DNA in the Urine of Neurocysticercosis Patients.

Neurocysticercosis (NCC), caused by Taenia solium larvae that reside in the central nervous system, results in serious public health and medical issues in many regions of the world. Current diagnosis of NCC is complex requiring both serology and costly neuroimaging of parasitic cysts in the brain. This diagnostic pipeline can be problematic in resource-constrained settings. There is an unmet need for a highly sensitive and clinically informative diagnostic test to complement the present diagnostic approaches. Here, we report that T. solium-derived cell-free DNA is readily detectable in the urine of patients with the subarachnoid and parenchymal forms of NCC, and discuss the potential utility of this approach in enhancing and refining T. solium diagnostics.

Transrenal DNA-based diagnosis of Strongyloides stercoralis (Grassi, 1879) infection: Bayesian latent class modeling of test accuracy.

For epidemiological work with soil transmitted helminths the recommended diagnostic approaches are to examine fecal samples for microscopic evidence of the parasite. In addition to several logistical and processing issues, traditional diagnostic approaches have been shown to lack the sensitivity required to reliably identify patients harboring low-level infections such as those associated with effective mass drug intervention programs. In this context, there is a need to rethink the approaches used for helminth diagnostics. Serological methods are now in use, however these tests are indirect and depend on individual immune responses, exposure patterns and the nature of the antigen. However, it has been demonstrated that cell-free DNA from pathogens and cancers can be readily detected in patient's urine which can be collected in the field, filtered in situ and processed later for analysis. In the work presented here, we employ three diagnostic procedures-stool examination, serology (NIE-ELISA) and PCR-based amplification of parasite transrenal DNA from urine-to determine their relative utility in the diagnosis of S. stercoralis infections from 359 field samples from an endemic area of Argentina. Bayesian Latent Class analysis was used to assess the relative performance of the three diagnostic procedures. The results underscore the low sensitivity of stool examination and support the idea that the use of serology combined with parasite transrenal DNA detection may be a useful strategy for sensitive and specific detection of low-level strongyloidiasis.

Diagnosis of schistosomiasis mansoni: an evaluation of existing methods and research towards single worm pair detection.

The inadequacy of current diagnostics for the detection of low worm burdens in humans means that schistosomiasis mansoni is more widespread than previously acknowledged. With the inception of mass drug treatment programmes aimed at disease elimination and the advent of human vaccine trials, the need for more sensitive diagnostics is evident. In this review, we evaluate the merits and limitations of the principal diagnostic methods, namely detection of eggs in faeces; anti-schistosome antibodies in serum; parasite-derived proteins and glycans in serum or urine; parasite DNA in blood, faeces or urine. Only in the baboon model, where actual worm burden is determined by portal perfusion, have faecal smear and circulating antigen methods been calibrated, and shown to have thresholds of detection of 10-19 worm pairs. There is scope for improvement in all the four methods of detection, e.g. the identification of single targets for host antibodies to improve the specificity of enzyme linked immunosorbent assay. Despite recent advances in the definition of the schistosome secretome, there have been no comprehensive biomarker investigations of parasite products in the urine of infected patients. Certainly, the admirable goal of eliminating schistosomiasis will not be achieved unless individuals with low worm burdens can be diagnosed.

Point of care diagnosis of multiple schistosome parasites: Species-specific DNA detection in urine by loop-mediated isothermal amplification (LAMP).

Schistosomes are easily transmitted and high chance of repeat infection, so if control strategies based on targeted mass drug administration (MDA) are to succeed it is essential to have a test that is sensitive, accurate and simple to use. It is known and regularly demonstrated that praziquantel does not always eliminate an infection so in spite of the successes of control programs a residual of the reservoir survives to re-infect snails. The issue of diagnostic sensitivity becomes more critical in the assessment of program effectiveness. While serology, such as antigen capture tests might improve sensitivity, it has been shown that the presence of species-specific DNA fragments will indicate, most effectively, the presence of active parasites. Polymerase chain reaction (PCR) can amplify and detect DNA from urine residue captured on Whatman No. 3 filter paper that is dried after filtration. Previously we have detected S. mansoni and S. haematobium parasite-specific small repeat DNA fragment from filtered urine on filter paper by PCR. In the current study, we assessed the efficacy of detection of 86 urine samples for either or both schistosome parasites by PCR and loop-mediated isothermal amplification (LAMP) that were collected from a low to moderate transmission area in Ghana. Two different DNA extraction methods, standard extraction kit and field usable LAMP-PURE kit were also evaluated by PCR and LAMP amplification. With S. haematobium LAMP amplification for both extractions showed similar sensitivity and specificity when compared with PCR amplification (100%) verified by gel electrophoresis. For S. mansoni sensitivity was highest for LAMP amplification (100%) for standard extraction than PCR and LAMP with LAMP-PURE (99% and 94%). The LAMP-PURE extraction produced false negatives, which require further investigation for this field usable extraction kit. Overall high positive and negative predictive values (90% - 100%) for both species demonstrated a highly robust approach. The LAMP approach is close to point of care use and equally sensitive and specific to detection of species-specific DNA by PCR. LAMP can be an effective means to detect low intensity infection due to its simplicity and minimal DNA extraction requirement. This will enhance the effectiveness of surveillance and MDA control programs of schistosomiasis.

LOOP-MEDIATED ISOTHERMAL AMPLIFICATION (LAMP): SENSITIVE AND RAPID DETECTION OF SCHISTOSOMA HAEMATOBIUM DNA IN URINE SAMPLES OF EGYPTIAN SUSPECTED CASES.

Schistosomiasis haemalobium is a major endemic parasitic disease in many tropical regions including Egypt. Typical infection results in haematuria, dysuria, anaemia, genital as well as urinary tract lesions, with prospect of kidney damage in complicated cases. In addition, deposited eggs in the tissue, eventually leads to squamous cell carcinoma of urinary bladder in chronically infected individuals. Microscopic detection of excreted ova in urine samples remains the gold standard diagnostic method, in spite of its inherited low sensitivity, inconsistent egg excretion and unreliable results in chronic phase of the disease. Moreover due to pre-requisite for skilled personals and pricey equipment, PCR-based technologies are of limited use especially in low-income endemic countries. So emergence of loop-mediated isothermal DNA amplification (LAMP) seemed a promising technique. Our study evaluated application of LAMP technique in detection of S. haematobium DNA in 69 urine samples of suspected patients for urogenital schistosomiasis, versus conventional urine filtration followed by microscopy ova detection method. Specificity of LAMP was tested using other parasites DNA samples that showed no cross reactivity. Furthermore our results of the calculated diagnostic parameters for sensitivity and specificity for LAMP assay were 100%, with 95% CI (88.78%-100%), and 63.16%, with 95% CI (45.99%-78.19%) respectively, moreover Positive likelihood ratio (LR+) 2.7, and Negative likelihood ratio (LR-) 0.0, which display that LAMP technique is an up-to-date simple, sensitive, diagnostic important tool that could be employed in clinical diagnosis in poorly equipped facilities, as well as in surveillance of infectious diseases. As authors knowledge, this is the first national report evaluation of LAMP technique as a promising diagnostic tool for urogenital schistosomiasis.

Impact of short-time urine freezing on the sensitivity of an established Schistosoma real-time PCR assay.

Urogenital schistosomiaisis is a serious public health problem in sub-Saharan Africa. In this study, we have updated an established real-time polymerase chain reaction (PCR) routinely used in our laboratory. Schistosoma genus-specific real-time PCR was performed on DNA isolated from 85 urine samples and pellets obtained after centrifugation without and after frozen storage. The results revealed that concentration by centrifugation of the urine samples and freezing of the samples before extracting DNA improves the sensitivity of the PCR.

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.

Diagnosis of Schistosoma haematobium by detection of specific DNA fragments from filtered urine samples.

Definitive diagnosis of Schistosoma haematobium infection in adult patients is a clinically important challenge. Chronically infected adults pass few eggs in the urine, which are often missed when current diagnostic methods are used. In the work presented here, we report on an alternative diagnostic method based on presence of the S. haematobium-specific Dra 1, 121 bp repeat fragment in human urine. A novel method of collecting the urine specimens in the field and filtering them through heavy Whatman No. 3 paper was introduced. After drying, the samples remained viable for several months at room temperature. To test the potential use of this method, 89 urine specimens from school children in Kollo District, Niger, were examined. In all, 52 of 89 (58.4%) were positive for hematuria, 4 of 89 (49.4%) were positive for eggs, and 51 of 89 (57.3%) showed parasite-specific DNA. These were compared with 60 filtered urine specimens obtained from random samples of adults from two study sites in Nigeria, one endemic and one non-endemic for S. haematobium. In the 30 patients from the endemic site, all 10 samples with detectable eggs and 7 of the 20 egg-negative samples were DNA positive. It was concluded that the urine filter paper method was sufficiently sensitive to detect low and cryptic infections, that DNA detection was more sensitive than egg detection, and that the filtration method facilitated specimen collection and transport from the field.

A new PCR-based approach for the specific amplification of DNA from different Schistosoma species applicable to human urine samples.

Currently available methods for the diagnosis of human schistosomiasis often lack enough sensitivity and specificity. Recently, several authors have developed more specific and sensitive diagnostic methods, mainly based on the polymerase chain reaction (PCR) technique. Nevertheless, these have been only applied for the diagnosis of 1 out of 4 Schistosoma species affecting man (S. mansoni). Additionally, application of specific PCR has been exclusively used for blood or faecal patients' samples. Here, we develop a new, high sensitive PCR approach that allows the genus- and species-specific amplification of the main 4 Schistosoma species causing disease in man plus S. bovis. We further successfully apply this technique for the detection of parasite DNA in easy-to-handle urine samples from patients with schistosomiasis. With these samples, we have found 94.4% sensitivity and 99.9% specificity when applying a genus-specific (Schistosoma spp.) primer pair, and 100% sensitivity and 98.9% specificity in a species-specific (S. mansoni) PCR.

Schistosoma mansoni: a diagnostic approach to detect acute schistosomiasis infection in a murine model by PCR.

Schistosomiasis represents an increasing problem in non-endemic areas, due to the growing number of immigrants and to tourists contracting this disease in "off-the-beaten-track" tourism. Acute schistosomiasis is not diagnosed early due to the lack of diagnostic tools that are sufficiently sensitive enough to detect the parasite during the first weeks of infection. We have developed a diagnostic approach based on the detection of parasite DNA by polymerase chain reaction (PCR) in urine, comparing the performance of this new approach with the two currently used schistosomiasis diagnostic tools (Kato-Katz and ELISA) and the PCR in stool samples. This comparison was done in a Schistosoma mansoni murine experimental model, which permits follow up of the parasite from the acute to the chronic stage of infection. Our results suggest that this new PCR-based approach could be useful for the detection of acute schistosomiasis in easy-to-handle clinical samples such the urine.

Diagnosis of Wuchereria bancrofti infection by the polymerase chain reaction using urine and day blood samples from amicrofilaraemic patients.

A sensitive and specific polymerase chain reaction (PCR) based on a highly repeated deoxyribonucleic acid (DNA) sequence (188 bp; SspI repeat) was tested for the detection of Wuchereria bancrofti DNA in blood and urine samples collected during the day from individuals in Coque, Recife, Brazil, an endemic area for W. bancrofti. All microfilaraemic individuals were also positive by PCR, irrespective of the samples used. The PCR system was capable of detecting W. bancrofti DNA in amicrofilaraemic individuals: c. 93% were positive by PCR when day blood samples were used and 59.7% when urine samples collected at 07:00 were used. Thus, nocturnally periodic W. bancrofti infection can be detected in blood samples collected during the day, which is convenient for large-scale screening. In addition, non-invasive urine collection provided suitable samples for PCR, which is clearly advantageous for preliminary mass diagnosis.