DNA In Urine Samples Research Articles

Mixed Leptospira infections in domestic animals from a rural community with high leptospirosis endemicity.

Leptospirosis is one of the most common zoonoses in the world which is associated with a severe febrile disease in humans causing a variety of syndromes including meningitis, interstitial nephritis, hepatitis, and sometimes death. Leptospirosis is caused by different pathogenic Leptospira species divided into almost 30 serogroups and more than 300 serovars which are carried by some animal asymptomatic chronic infections. Humans can become infected through direct contact with animal urine or indirectly by coming into contact with fresh water or mud contaminated with urine. In this research, we looked for leptospiral DNA in urine samples from dogs living in a rural, low-income and highly endemic community in the coast of Ecuador. We used molecular biology and next-generation sequencing for the detection. Our results showed evidence of two Leptospira species, L interrogans and L. santarosai, genomes in three dogs. It has been widely known that animal carriers are typically infected with a single leptospiral strain. However, recent reports, including the present one, indicate that carrier animals may be coinfected with two or more leptospiral species.

Optimizing Human Papillomavirus (HPV) Screening: Urine Sample Analysis and Associated Factors in Uzbekistan.

Objective This study assessed the accuracy of detecting Human Papillomavirus (HPV) DNA in urine samples compared to cervical samples and identified factors associated with HPV DNA positivity in Uzbekistan. Methods A total of 218 paired urine and cervical samples were collected from women in Uzbekistan. HPV DNA was detected using polymerase chain reaction (PCR) with genotyping. Sensitivity, specificity, positive predictive value (PPV), negative predictive value (NPV), and Cohen's Kappa coefficient were calculated. Univariate and multivariate analyses were conducted to identify factors associated with HPV DNA positivity. Results The study included 32.6% (71/218) positive HPV DNA by cervical samples, which demonstrated 19.7% (43/218) HPV DNA presence by urine samples. Urine HPV testing had a sensitivity of 57.7%, specificity of 98.6%, PPV of 95.3%, NPV of 82.9%, and Kappa coefficient of 60.1. To reveal factors associated with HPV DNA positivity, we set the positive 71 cases as the main group, and 147 negative samples as the control group. The results of the multivariate analysis found the association between HPV DNA positivity and included age 31-40 years (AOR=8.2), working as medical staff (AOR=7.51), condom use (AOR=0.12), having foamy (AOR=7.26) or purulent (AOR=6.84) vaginal discharge. Conclusion Urine HPV testing showed good agreement with cervical samples, although sensitivity was lower than some previous reports. Several sociodemographic and clinical factors were associated with HPV DNA positivity. Further optimization of urine collection, storage, and testing methods may improve sensitivity. Targeted screening of high-risk groups could enhance HPV prevention strategies in Uzbekistan.

Detection of Mycobacterium tuberculosis transrenal DNA in urine samples among adults in Peru

Diagnosis of pulmonary tuberculosis (TB) relies on a sputum sample, which cannot be obtained from all symptomatic individuals. Mycobacterium tuberculosis (Mtb) transrenal DNA (trDNA) has been detected in urine, an easily obtainable, noninvasive, alternative sample type. However, reported sensitivities have been variable and likely depend on collection and assay procedures and aspects of trDNA biology. We analyzed three serial urine samples from each of 75 adults with culture-confirmed pulmonary TB disease in Lima, Peru for detection of trDNA using short-fragment real-time PCR. Additionally, we examined host, urine, and sampling factors associated with detection. Overall per-sample sensitivity was 38 % (95 % Confidence Interval [CI] 30–45 %). On an individual level (i.e., any of the three samples positive), sensitivity was 73 % (95 % CI: 62–83 %). Sensitivity was highest among samples from patients with smear-positive TB, 92 % (95 % CI: 62–100 %). Specificity from a single sample from each of 10 healthy controls was 100 % (95 % CI: 69–100 %). Adjusting our assay positivity threshold increased individual-level sensitivity to 88 % (95 % CI: 78–94 %) overall without affecting the specificity. We did not find associations between Mtb trDNA detection and individual characteristics or urine sample characteristics. Overall, our results support the potential of trDNA detection for TB diagnosis.

A comparative study on the detection of Mycobacterium leprae DNA in urine samples of leprosy patients using Rlep-PCR with other conventional samples.

Mycobacterium leprae causes leprosy that is highly stigmatized and chronic infectious skin disease. Only some diagnostic tools are being used for the identification M. leprae in clinical samples, such as bacillary detection, and histopathological tests. These methods are invasive and often have low sensitivity. Currently, the PCR technique has been used as an effective tool fordetecting M. leprae DNA across different clinical samples. The current study aims to detect M. leprae DNA in urine samples of untreated and treated leprosy patients using the Rlep gene (129 bp) and compared the detection among Ridley-Jopling Classification. Clinical samples (Blood, Urine, and Slit Skin Smears (SSS)) were collected from leprosy and Non-leprosy patients. DNA extraction was performed using standard laboratory protocol and Conventional PCR was carried out for all samples using Rlep gene target and the amplicons of urine samples were sequenced by Sanger sequencing to confirm the Rlep gene target. The M. leprae DNA was successfully detected in all clinical samples across all types of leprosy among all the study groups using RLEP-PCR. Rlep gene target was able to detect the presence of M. leprae DNA in 79.17% of urine, 58.33% of blood, and 50% of SSS samples of untreated Smear-Negative leprosy patients. The statistical significant difference (p = 0.004) was observed between BI Negative (Slit Skin Smear test) and RLEP PCR positivity in urine samples of untreated leprosy group. The PCR positivity using Rlep gene target (129 bp) was highest in all clinical samples among the study groups, across all types of leprosy. Untreated tuberculoid and PNL leprosy patients showed the highest PCR positivity in urine samples, indicating its potential as a non-invasive diagnostic tool for leprosy and even for contact screening.

Abstract LB105: Sensitive urothelial cancer detection via high volume urine DNA analysis

Abstract Background: The detection of cancer-associated DNA in urine samples has significant potential in urothelial cancer (UC) detection and surveillance. Every year millions of patients with urinary symptoms undergo cystoscopy, an examination of the bladder using a flexible scope. Only 10% of these invasive examinations result in a cancer diagnosis. Cystoscopy is also used in post-operative surveillance, with many UC patients requiring life-long cystoscopies. Methods: We developed a high-volume urine DNA collection kit and laboratory platform and prospectively analyzed 433 urine samples collected from diagnostic and post-operative timepoints from 167 UC patients and 95 negative controls from January 2021 to September 2023. We used a hybridization capture assay targeting the coding regions of 21 commonly mutated genes in urothelial tumors, as well as 8 frequently copy number altered loci. Samples were sequenced using Illumina NovaSeq instruments. Mutations, copy number alterations, and chromosomal rearrangements were computationally identified. Key Findings: In the diagnostic settings our assay achieved a sensitivity of 100% for pT1+ bladder cancer, 97% for pTa bladder cancer, and 91% for upper tract urothelial cancer (UTUC) (100% for pT1+ or high grade) in an all-comers UC cohort, at a specificity of 93%. Post-operative urine DNA surveillance informed by preoperative urine mutations detected 28/30 (93%) residual cancer cases found in cystoscopy or surgery, with two pTa low grade recurrences missed. Using a secondary, highly stringent urine tumor DNA detection threshold (100% specificity for UC), we show that urine DNA testing in the diagnostic setting may allow up to 68% of UC patients to be scheduled directly to operating room procedures, reducing hospital workload and expediting care. In addition, we demonstrate the feasibility of urine DNA for detecting underlying Lynch syndrome in UC patients by identifying three UTUC patients who carried germline defects in mismatch repair genes MSH2 or MSH6, indicative of Lynch syndrome. In two of these patients our assay also found a somatic second hit and high mutation load. Conclusions and Clinical Implications: These results show promise in urine DNA based cancer diagnostics even in patients with low grade tumors. The assay is designed to offer a non-invasive and cost-effective alternative for cystoscopies. Citation Format: Jussi Nikkola, Lauri Ryyppö, Juuso Vuorinen, Heini Kallio, Hanna Selin, Pyry Jämsä, Jonne Åkerla, Tarmo Pekkarinen, Antti Kaipia, Matti Nykter, Thea Veitonmäki, Matti Annala. Sensitive urothelial cancer detection via high volume urine DNA analysis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 2 (Late-Breaking, Clinical Trial, and Invited Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(7_Suppl):Abstract nr LB105.

Development and performance of recombinase-aided amplification (RAA) assay for detecting Schistosoma haematobium DNA in urine samples

Rapid diagnosis of urogenital schistosomiasis caused by Schistosoma haematobium requires an accurate and timely assay, especially for low-intensity S. haematobium infection cases and in non-endemic areas. The mitochondrial cytochrome c oxidase 1 (cox1) gene fragment of S. haematobium was selected as detection target as this short fragment, which can be rapidly sequenced and yet possess good diagnostic resolution. A pair of primers and a fluorescent probe were designed according to the principle of recombinase-aided amplification (RAA), which was subsequently optimized and applied as an S. haematobium-specific RAA assay. Its diagnostic performance was validated for sensitivity and specificity in comparison to microscopy-based egg counting after urine filtration. The RAA assay could detect as little as 10 copies/μL of S. haematobium recombinant plasmid, and no cross-reactions were observed with S. mansoni, S. japonicum, Ancylostoma duodenale, Clonorchis sinensis, Echinococcus granulosus, or Ascaris lumbricoides. This test can be conducted at 39 °C and the whole RAA reaction can be completed within 20 min. The validation of the RAA assay showed that it had 100 % consistency with urine-egg microscopy, as it does not require an elaborate reading tool, is simple to use, and should be useful for field diagnostics and point-of-care applications.

Status of Urinary shedders of Leptospires among cattle in Tamilnadu: Implications on zoonosis.

Bovine leptospirosis causes jaundice, mastitis, infertility, abortion, and death of the animal. This research aimed to study the status of urinary shedders of pathogenic Leptospira among the cattle population and identify the infecting serogroup circulating in this region. A total of 305 blood and 305 urine samples were collected from organized farms (n = 44), individually housed animals (n = 81) and animals from the slaughterhouse (n = 180). Microscopic agglutination test was carried out to detect anti‑leptospiral antibodies. Dark‑field microscopic examination and culture of urine were done to detect and isolate the Leptospira. The isolated Leptospira were identified by cross‑agglutination test and gene sequencing. PCR and real‑time PCR were carried out to detect leptospiral genomic DNA in urine samples to detect the shedders. The anti‑leptospiral antibodies were detected in 6.2% of animals. The Leptospira genomic DNA was detected in 9.2% (28 of 305) of urine samples. Of the 28 Leptospira positive urine samples, 39.2% were from animals with clinical signs suggestive of leptospirosis and 60.8% Leptospira positive samples were from slaughterhouse animals. The Leptospira isolated were identified as Leptospira interrogans serogroup Sejroe and Hebdomadis. The present study demonstrates the need to include leptospirosis in cattle health surveillance programmes to prevent leptospirosis by vaccination, preventing renal carriage.

Detection of Quebec Polyomavirus DNA in Samples from Different Patient Groups.

Polyomaviruses infect many species, including humans. So far, 15 polyomaviruses have been described in humans, but it remains to be established whether all of these are genuine human polyomaviruses. The most recent polyomavirus to be detected in a person is Quebec polyomavirus (QPyV), which was identified in a metagenomic analysis of a stool sample from an 85-year-old hospitalized man. We used PCR to investigate the presence of QPyV DNA in urine samples from systemic lupus erythematosus (SLE) patients (67 patients; 135 samples), multiple sclerosis patients (n = 35), HIV-positive patients (n = 66) and pregnant women (n = 65). Moreover, cerebrospinal fluid from patients with suspected neurological diseases (n = 63), nasopharyngeal aspirates from patients (n = 80) with respiratory symptoms and plasma samples from HIV-positive patients (n = 65) were examined. QPyV DNA was found in urine from 11 (16.4%), 10 (15.4%) and 5 (14.3%) SLE patients, pregnant women, and multiple sclerosis patients, respectively. No QPyV DNA could be detected in the other samples. Alignment with the only available QPyV sequence in the GenBank revealed amino acid substitutions in the HI-loop of capsid protein VP1 in 6/28 of the isolates. Our results show that QPyV viruria can occur, but whether it may cause clinical symptoms in the patients remains to be determined.

Lack of HPV in pterygium with no evidence of autoinoculation and the role of cytokines in pterygium with dry eye

This study evaluated human papillomavirus’s (HPV) role in pterygium pathogenesis, its autoinoculation from genitalia to ocular surface, potential cytokines involved, and crosstalk cytokines between pterygium and dry eye (DE). This cross-sectional study enrolled 25 healthy controls (HCs) and 116 pterygium patients. Four subgroups of pterygium and DE were used in cytokine evaluations. Conjunctival and pterygium swabs and first-void urine samples (i.e., genitalia samples) were collected for HPV DNA detection using real-time polymerase chain reaction. Tear cytokines interleukin (IL)-6, IL-18, and vascular endothelial growth factor (VEGF) in tears were evaluated. No HPV DNA was detected in conjunctival or pterygium swabs. No association was found between HPV DNA in urine samples and that from conjunctival or pterygium swabs. Tear VEGF levels were significantly higher in pterygium patients than in HCs, with no markedly different levels between primary and recurrent pterygia. Tear IL-6, IL-18, and tear VEGF were significantly higher in participants with DE, regardless of pterygium status. In conclusion, HPV infection was not a pathogenic factor of pterygia. The hypothesis of HPV transmitting from the genitals to ocular surfaces was nullified. Tear VEGF was involved in both pterygia and DE, whereas tear IL-6 and IL-18 played roles only in DE.

HPyV6 and HPyV7 in urine from immunocompromised patients

BackgroundHuman polyomavirus 6 (HPyV6) and HPyV7 are two of the novel polyomaviruses that were originally detected in non-diseased skin. Serological studies have shown that these viruses are ubiquitous in the healthy adult population with seroprevalence up to 88% for HPyV6 and 72% for HPyV7. Both viruses are associated with pruritic skin eruption in immunocompromised patients, but a role with other diseases in immunoincompetent patients or malignancies has not been established.MethodsPCR was used to determine the presence of HPyV6 and HPyV7 DNA in urine samples from systemic lupus erythematosus (n = 73), multiple sclerosis (n = 50), psoriasis vulgaris (n = 15), arthritic psoriasis (n = 15) and HIV-positive patients (n = 66). In addition, urine from pregnant women (n = 47) and healthy blood donors (n = 20) was investigated.ResultsHPyV6 DNA was detected in 21 (28.8%) of the urine specimens from SLE patients, in 6 (9.1%) of the urine samples from the HIV-positive cohort, and in 19 (40.4%) samples from pregnant women. HPyV7 DNA was only found in 6 (8.2%) of the urine specimens from SLE patients and in 4 (8.5%) samples from pregnant women. No HPyV6 and HPyV7 viruria was detected in the urine samples from the other patients.ConclusionsHPyV6, and to a lesser extend HPyV7, viruria seems to be common in SLE and HIV-positive patients, and pregnant women. Whether these viruses are of clinical relevance in these patients is not known.

Detection of Streptococcus pneumoniae in Urine by Loop-Mediated Isothermal Amplification

Abstract Objective To assess the loop-mediated isothermal amplification (LAMP) to detect cell-free DNA from Streptococcus pneumoniae in urine samples from children with pneumococcal pneumonia. Methods LAMP reactions using four primers (backward inner primer, forward inner primer, B3, and F3) targeting conserved regions of the S. pneumoniae ply gene and DNA from the recombinant plasmid pTrc99A-ply were optimized for temperature (65°C) and MgSO4 concentration (8 mM) conditions. Urine samples from 71 patients with symptoms of pneumonia and from 17 healthy children were tested side by side using the isothermal methodology LAMP and the commercial urinary antigen test, BinaxNOW S. pneumoniae assay. Percentages of sensitivity, specificity, positive predictive value (PPV), negative predictive value, and positive (LR) were calculated to compare both tests. Results The specificity of the LAMP reaction was confirmed against several species of bacteria and yeast that can cause pneumonia or urine infections. The suitability of the LAMP assay was evaluated in urine samples from 71 patients and 17 healthy children. All patients (100%) with confirmed pneumococcal pneumonia were positive for the LAMP assay. Among patients with possible/probable pneumonia, 74.1% were identified as positive using the LAMP test. Notably, a higher specificity (95.4%), PPV (94.1%) and positive LR (21.7) were found compared with the urinary antigen test. Conclusion The presence of S. pneumoniae cell-free DNA in urine samples of pediatric patients can be used as a specific diagnostic biomarker for community-acquired pneumonia by using the LAMP methodology.

Colorimetric Urinalysis for On-Site Detection of Metabolic Biomarkers.

Over the past few decades, colorimetric assays have been developed for cost-effective and rapid on-site urinalysis. Most of these assays were employed for detection of biomarkers such as glucose, uric acid, ions, and albumin that are abundant in urine at micromolar to millimolar levels. In contrast, direct assaying of urinary biomarkers such as glycated proteins, low-molecular-weight reactive oxygen species, and nucleic acids that are present at significantly lower levels (nanomolar to picomolar) remain challenging due to the interferences from the urine sample matrix. State-of-the-art assays for detection of trace amounts of urinary biomarkers typically utilize time-consuming and equipment-dependent sample pretreatment or clean-up protocols prior to assaying, which limits their applicability for on-site analysis. Herein, we report a colorimetric assay for on-site detection of trace amount of generic biomarkers in urine without involving tedious sample pretreatment protocols. The detection strategy is based on monitoring the changes in optical properties of poly(3-(4-methyl-3'-thienyloxy)propyltriethylammonium bromide) upon interacting with an aptamer or a peptide nucleic acid in the presence and absence of target biomarkers of relevance for the diagnosis of metabolic complications and diabetes. As a proof of concept, this study demonstrates facile assaying of advanced glycation end products, 8-hydroxy-2'-deoxyguanosine and hepatitis B virus DNA in urine samples at clinically relevant concentrations, with limits of detection of ∼850 pM, ∼650 pM, and ∼ 1 nM, respectively. These analytes represent three distinct classes of biomarkers: (i) glycated proteins, (ii) low-molecular-weight reactive oxygen species, and (iii) nucleic acids. Hence, the proposed methodology is applicable for rapid detection of generic biomarkers in urine, without involving sophisticated equipment and skilled personnel, thereby enabling on-site urinalysis. At the end of the contribution, we discuss the opportunity to translate the homogeneous assay into a paper-based format.

Strong-LAMP Assay Based on a Strongyloides spp.-Derived Partial Sequence in the 18S rRNA as Potential Biomarker for Strongyloidiasis Diagnosis in Human Urine Samples.

Human strongyloidiasis a soil-transmitted infection caused by Strongyloides stercoralis is one of the most neglected amongst the so-called Neglected Tropical Diseases (NTDs). S. stercoralis is a nematode, which is distributed worldwide; it has been estimated that it could affect millions of people, mainly in tropical and subtropical endemic regions. The difficulties of diagnosis lead to infection rates being underreported. Asymptomatic patients have chronic infections that can lead to severe hyperinfection syndrome or disseminated strongyloidiasis in immunocompromised patients. Strongyloidiasis can easily be misdiagnosed because conventional faecal-based techniques lack of sensitivity for the morphological identification of infective larvae in faeces. None of the currently used molecular methods have used urine samples as an alternative to faecal samples for diagnosing strongyloidiasis. This study was thus aimed at comparing, for the first time, the use of a new loop-mediated isothermal amplification (LAMP) molecular assay (Strong-LAMP) to traditional methods on patients' urine samples. Twenty-four urine samples were taken from patients included in a study involving two Spanish hospitals for strongyloidiasis screening using parasitological and serological tests. Strongyloides larvae were found in 11 patients' faecal samples, thereby ascertaining that they had the disease. Other patients had high antibody titres but no larvae were found in their faeces. All urine samples were analysed by PCR and Strong-LAMP assay. No amplification occurred when using PCR. Strong-LAMP led to detecting S. stercoralis DNA in urine samples from patients having previously confirmed strongyloidiasis by parasitological tests and/or a suspicion of being infected by serological ones. The Strong-LAMP assay is a useful molecular tool for research regarding strongyloidiasis in human urine samples. After further validation, the Strong-LAMP assay could also be used for complementary and effective diagnosis of strongyloidiasis in a clinical setting.

Development of a recombinase polymerase amplification (RPA) fluorescence assay for the detection of Schistosoma haematobium

BackgroundAccurate diagnosis of urogenital schistosomiasis is vital for surveillance and control programmes. While a number of diagnostic techniques are available there is a need for simple, rapid and highly sensitive point-of-need (PON) tests in areas where infection prevalence and intensity are low. Recombinase Polymerase Amplification (RPA) is a sensitive isothermal molecular diagnostic technology that is rapid, portable and has been used at the PON for several pathogens.ResultsA real time fluorescence RPA assay (RT-ShDra1-RPA) targeting the Schistosoma haematobium Dra1 genomic repeat region was developed and was able to detect 1 fg of S. haematobium gDNA. Results were obtained within 10 minutes using a small portable battery powered tube scanner device that incubated reactions at 40 °C, whilst detecting DNA amplification and fluorescence over time. The assay’s performance was evaluated using 20 urine samples, with varying S. haematobium egg counts, from school children from Pemba Island, Zanzibar Archipelago, Tanzania. Prior to RPA analysis, samples were prepared using a quick crude field DNA extraction method, the Speed Extract Kit (Qiagen, Manchester, UK). Positive assay results were obtained from urine samples with egg counts of 1–926 eggs/10 ml, except for two samples, which had inconclusive results. These two samples had egg counts of two and three eggs/10 ml of urine.ConclusionsThe RT-ShDra1-RPA assay proved robust for S. haematobium gDNA detection and was able to amplify and detect S. haematobium DNA in urine samples from infected patients. The assay’s speed and portability, together with the use of crude sample preparation methods, could advance the rapid molecular diagnosis of urogenital schistosomiasis at the PON within endemic countries.

Epidemiology of polyparasitism with Taenia solium, schistosomes and soil-transmitted helminths in the co-endemic village of Malanga, Democratic Republic of Congo

Helminth co-infections are common in sub-Saharan Africa. However, little is known about the distribution and determinants of co-infections with Taenia solium taeniasis/cysticercosis. Building on a previous community-based study on human cysticercosis in Malanga village, we investigated co-infections with Taenia solium, soil-transmitted helminths (STHs) and Schistosoma spp and associated risk factors in a random subsample of 330 participants. Real time PCR assays were used to detect DNA of soil-transmitted helminths (STHs), T. solium and Schistosoma in stool samples and Schistosoma DNA in urine samples. Serum samples were tested for T. solium cysticercosis using the B158/B60 monoclonal antibody-based antigen ELISA. Bivariate analysis and logistic regression were applied to assess associations of single and co-infections with common risk factors (age, sex, area, hygiene) as well as pair wise associations between helminth species.Overall, 240 (72.7%) participants were infected with at least one helminth species; 128 (38.8%) harbored at least two helminth species (16.1% with STHs-Schistosoma, 14.5% with STHs-T. solium taeniasis/cysticercosis and 8.2% with Schistosoma-T. solium taeniasis/cysticercosis co-infections). No significant associations were found between Schistosoma-T. solium taeniasis/cysticercosis co-infection and any of the risk factors studied. Males (OR=2 (95%CI=1.1–5), p=0.03) and open defecation behavior (OR=3.8 (95%CI=1.1–6.5), p=0.04) were associated with higher odds of STHs-T. solium taeniasis/cysticercosis co-infection. Village districts that were found at high risk of T. solium taeniasis/cysticercosis were also at high risk of co-infection with STHs and T. solium taeniasis/cysticercosis (OR=3.2 (95%CI=1.1–7.8), p=0.03). Significant pair-wise associations were found between T. solium cysticerci and Necator americanus (OR=2.2 (95%CI=1.2–3.8), p<0.01) as well as Strongyloides stercoralis (OR=2.7 (95%CI=1.1–6.5), p=0.02).These findings show that co-infections with T. solium are common in this polyparasitic community in DRC. Our results on risk factors of helminth co-infections and specific associations between helminths may contribute to a better integration of control within programmes that target more than one NTD.

A Modified Protocol with Improved Detection Rate for Mis-Matched Donor HLA from Low Quantities of DNA in Urine Samples from Kidney Graft Recipients.

BackgroundUrine from kidney transplant recipient has proven to be a viable source for donor DNA. However, an optimized protocol would be required to determine mis-matched donor HLA specificities in view of the scarcity of DNA obtained in some cases.MethodsIn this study, fresh early morning urine specimens were obtained from 155 kidney transplant recipients with known donor HLA phenotype. DNA was extracted and typing of HLA-A, B and DRB1 loci by polymerase chain reaction-specific sequence primers was performed using tailor-made condition according to the concentration of extracted DNA.ResultsHLA typing of DNA extracted from urine revealed both recipient and donor HLA phenotypes, allowing the deduction of the unknown donor HLA and hence the degree of HLA mis-match. By adopting the modified procedures, mis-matched donor HLA phenotypes were successfully deduced in all of 35 tested urine samples at DNA quantities spanning the range of 620–24,000 ng.ConclusionsThis urine-based method offers a promising and reliable non-invasive means for the identification of mis-matched donor HLA antigens in kidney transplant recipients with unknown donor HLA phenotype or otherwise inadequate donor information.

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.

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

BackgroundUrogenital 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 FindingsA 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/SignificanceWe 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.

Molecular detection and characterization of BK and JC polyomaviruses in urine samples of renal transplant patients in Southern Brazil.

The human polyomaviruses JC (JCPyV) and BK (BKPyV) are widespread in the human population. Following the primary infection, virus reactivation may lead to nephropathy and graft rejection in renal transplant patients. This study was carried out to access the presence of BKPyV and JCPyV DNA in urine samples collected from renal transplant patients (n = 92) and healthy individuals (n = 88) in Porto Alegre, Rio Grande do Sul. The samples were submitted to a nested PCR. A significantly higher frequency (P < 0.001) of BKPyV was found in renal transplant patients (65.2%) in comparison to the control group (32.9%). JCPyV was detected equally in both groups. Phylogenetic analysis of both BKPyV and JCPyV amplicons demonstrates the presence of the BKPyV subtypes I and II, whereas for JCPyV, four different groups are found (1, 2, 3, and 4).

Interest of Human Papillomavirus DNA quantification and genotyping in paired cervical and urine samples to detect cervical lesions

Cervical cancer is caused by persistent infection with high-risk human papillomavirus (HR-HPV). Conventional human papillomavirus (HPV) testing requires cervical sampling. However, vaginal and urine self-sampling methods are more acceptable for patients and result in increased participation when they are available in screening programs. In this context, we have developed a non-invasive screening method via the detection of HPV DNA in urine samples. To compare HPV viral loads and genotypes in paired cervical and urine samples, and to assess correlation between virological and cytological results in women seeking gynecological consultation. Paired urine and cervical specimens were collected and analyzed from 230 of 245 women participating in the previously described prospective PapU study. HPV DNA detection and quantification were performed using a real-time PCR method with short fragment PCR primers. Genotyping was carried out using the INNO-LiPA HPV genotyping assay. The prevalence of HPV in the 230 paired urine and cervical smear samples was 42 and 49 %, respectively. Overall agreement for HPV positivity and negativity between the paired samples was 90 % (κ = 0.80). High HPV viral load in both cervical and urine samples was associated with cytological abnormalities. HPV-positive women were mostly infected with HR-HPV types. The agreement between high- and low-risk HPV (LR-HPV) detection in both samples was 97 % (κ = 0.95 for HR-HPV and κ = 0.97 for LR-HPV). High concordance rates for HPV-DNA quantification and high/low-risk HPV genotyping in paired urine/cervical samples suggest that urinary HPV DNA testing could be useful for cervical lesion screening.