Urine Of Infected Individuals Research Articles

ELISA for leptospiral 3-hydroxyacyl-CoA dehydrogenase in urine is a promising screening tool for acute leptospirosis.

Introduction. Leptospirosis is a zoonotic disease that is prevalent worldwide. Leptospiral 3-hydroxyacyl-CoA dehydrogenase (3-HADH) is excreted in the urine of infected individuals. However, the potential use of 3-HADH as a biomarker for the diagnosis of leptospirosis using enzyme-linked immunosorbent assay (ELISA) has not been investigated. A technique that identifies Leptospira in a patient in urine sample will be valuable in regular diagnostics and epidemic scenarios, as opposed to existing serological approaches. This study aimed to develop and evaluate an ELISA that can detect 3-HADH in the urine of patients with confirmed acute leptospirosis and to assess its potential as a screening test for leptospirosis. Methods. Laboratory confirmation of acute leptospirosis was done by flaB-nested polymerase chain reaction (PCR) of plasma samples from suspected patients. ELISA-based determination of the presence of 3-HADH in the urine of PCR-positive patients versus PCR-negative patients matched for fever date was performed by coating ELISA plates with urine supernatants and using rabbit anti-3-HADH as the primary antibody. Receiver operating characteristic curve analysis was used to determine the cutoff values for the ELISA. The diagnostic measures between the PCR-positive and PCR-negative patients were compared using the Mann-Whitney U test. Results. In total, 158 febrile patients were assessed, of whom 121 (76.6 %) were male. Of the 15 flaB-nested PCR-positive patients, 12 were in the acute phase of the febrile illness. The best cutoff was an average optical density (ODav) value of 0.2200 for febrile patients. Sensitivity and specificity were 83.33% [95 % confidence interval (CI), 51.59-97.91 %) and 83.33 % (95 % CI, 76.05-89.13 %), respectively. The ODav values for PCR-positive patients in the acute phase of the disease (≤7 days of fever) were significantly higher than those for PCR-negative patients (P<0.001, U=114.0, z=-4.946). Conclusion. Detection of 3-HADH in urine by ELISA appears to be promising for the screening of acute leptospirosis in suspected patients.

Reduction and discharge of SARS-CoV-2 RNA in Chicago-area water reclamation plants.

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) RNA is commonly excreted in the feces and urine of infected individuals and is, therefore, detected in wastewaters where infection is present in the surrounding population. Water reclamation plants (WRPs) that treat these wastewaters commonly discharge treated effluents into the surrounding environment, yet little is known about the removal or persistence of SARS-CoV-2 RNA through wastewater treatment systems and potential for eventual release into the environment. We collected 361 24-hour composite influent and effluent samples from seven WRPs in the Greater Chicago Area in Illinois. Samples were collected over a period of 21 weeks for three large WRPs (with design max flows of 1.89-2.32 billion gallons per day and serving a combined population of 4.62 million people) and 11 weeks for four smaller WRPs (with design max flows of 96.3-186 million gallons per day and serving a combined population of >0.5 million people). A total of two of the larger WRPs implemented seasonal disinfection (using UV light or chlorination/dechlorination) for 8 weeks of this sampling period. SARS-CoV-2 RNA was quantified in the influent and effluent samples by reverse-transcription quantitative PCR (RT-qPCR) of the N1 and N2 targets of the nucleocapsid (N) gene. Although SARS-CoV-2 RNA was regularly detected in influent and effluent from all WRPs, viral RNA concentrations in the effluent samples were considerably lower, with mean effluent: influent gene copy concentration ratios ranging from 1:160 to 1:2.95 between WRPs. Samples collected while disinfection was active vs. inactive did not show any significant difference in the portion of RNA persisting through the treatment process (P >.05).

Urinary Viral Shedding of COVID-19 and its Clinical Associations: A Systematic Review and Meta-analysis of Observational Studies.

To review the current literature on the presence of COVID-19 virus in the urine of infected patients and to explore the clinical features that can predict the presence of COVID-19 in urine. A systematic review of published literature between 30th December 2019 and 21st June 2020 was conducted on Pubmed, Google Scholar, Ovid, Scopus, and ISI web of science. Studies investigating urinary viral shedding of COVID-19 in infected patients were included. Two reviewers selected relative studies and performed quality assessment of individual studies. Meta-analysis was performed on the pooled case reports and cohort with a sample size of ≥ 9. Thirty-nine studies were finally included in the systematic review; 12 case reports, 26 case series, and one cohort study. Urinary samples from 533 patients were investigated. Fourteen studies reported the presence of COVID-19 in the urinary samples from 24 patients. The crude overall rate of COVID-19 detection in urinary samples was 4.5%. Considering case series and cohorts with a sample size of ≥ 9, the estimated viral shedding frequency was 1.18 % (CI 95%: 0.14 - 2.87) in the meta-analysis. Urinary viral load in most reports were lower than rectal or oropharyngeal samples. In adult patients, urinary shedding of COVID-19 was commonly detected in patients with moderate to severe disease (16 adult patients with moderate or severe disease versus two adult patients with mild disease). In children, urinary viral shedding of COVID-19 was reported in 4 children who all suffered from mild disease. Urinary viral shedding of COVID-19 was detected from day 1 to day 52 after disease onset. The pathogenicity of virus isolated from urine has been demonstrated in cell culture media in one study while another study failed to reveal replication of isolated viral RNA in cell cultures. Urinary symptoms were not attributed to urinary viral shedding. While COVID-19 is rarely detected in urine of infected individuals, infection transmission through urine still remains possible. In adult patients, infected urine is more likely in the presence of moderate or severe disease. Therefore, caution should be exerted when dealing with COVID-19 infected patients during medical interventions like endoscopy and urethral catheterization especially in symptomatic adult patients while in children caution should be exerted regardless of symptoms.

Absence of molecular evidence of Leptospira spp. in urine samples collected from rodents captured in Yucatán, México

Leptospira spp. is a spirochete bacteria, causal agent of leptospirosis, zoonotic disease endemic in Mexico that represents a serious public health and veterinary problem. Rodents are recognised as the most important reservoirs of this bacteria, which is transmitted mainly through direct or indirect contact with the Leptospira spp. excreted in the urine of infected individuals. The aim of this study was to evaluate the circulation of Leptospira spp. in urine samples of wild and synanthropic rodents from Yucatan, Mexico. Eighty-four rodents were captured in the community of Cenotillo, Yucatan. Twenty-six urine samples were collected from the bladder and were used in the total DNA extraction. The identification of Leptospira spp. was intended through the polymerase chain reaction test in its endpoint variant. No evidence of Leptospira spp. was found in the urine samples. It is necessary to use other tissues for the identification of Leptospira spp., before concluding that the rodents used in the present study are not reservoirs of this bacteria.

In vivo imaging of schistosomes to assess disease burden using positron emission tomography (PET).

BackgroundSchistosomes are chronic intravascular helminth parasites of humans causing a heavy burden of disease worldwide. Diagnosis of schistosomiasis currently requires the detection of schistosome eggs in the feces and urine of infected individuals. This method unreliably measures disease burden due to poor sensitivity and wide variances in egg shedding. In vivo imaging of schistosome parasites could potentially better assess disease burden, improve management of schistosomiasis, facilitate vaccine development, and enhance study of the parasite's biology. Schistosoma mansoni (S. mansoni) have a high metabolic demand for glucose. In this work we investigated whether the parasite burden in mice could be assessed by positron emission tomography (PET) imaging with 2-deoxy-2[18F]fluoro-D-glucose (FDG).Methodology/Principal FindingsLive adult S. mansoni worms FDG uptake in vitro increased with the number of worms. Athymic nude mice infected with S. mansoni 5–6 weeks earlier were used in the imaging studies. Fluorescence molecular tomography (FMT) imaging with Prosense 680 was first performed. Accumulation of the imaging probe in the lower abdomen correlated with the number of worms in mice with low infection burden. The total FDG uptake in the common portal vein and/or regions of elevated FDG uptake in the liver linearly correlated to the number of worms recovered from infected animals (R2 = 0.58, P<0.001, n = 40). FDG uptake showed a stronger correlation with the worm burden in mice with more than 50 worms (R2 = 0.85, P<0.001, n = 17). Cryomicrotome imaging confirmed that most of the worms in a mouse with a high infection burden were in the portal vein, but not in a mouse with a low infection burden. FDG uptake in recovered worms measured by well counting closely correlated with worm number (R2 = 0.85, P<0.001, n = 21). Infected mice showed a 32% average decrease in total FDG uptake after three days of praziquantel treatment (P = 0.12). The total FDG uptake in untreated mice increased on average by 36% over the same period (P = 0.052).ConclusionFDG PET may be useful to non-invasively quantify the worm burden in schistosomiasis-infected animals. Future investigations aiming at minimizing non-specific FDG uptake and to improve the recovery of signal from worms located in the lower abdomen will include the development of more specific radiotracers.

Mass Spectrometric Identification of Aberrantly Glycosylated Human Apolipoprotein C-III Peptides in Urine from Schistosoma mansoni-infected Individuals

Schistosomiasis is a parasitic infection caused by Schistosoma flatworms, prime examples of multicellular parasites that live in the mammalian host for many years. Glycoconjugates derived from the parasite have been shown to play an important role in many aspects of schistosomiasis, and some of them are present in the circulation of the host. The aim of this study was to identify novel glycoconjugates related to schistosomiasis in urine of Schistosoma mansoni-infected individuals using a combination of glycopeptide separation techniques and in-depth mass spectrometric analysis. Surprisingly, we characterized a heterogeneous population of novel aberrantly O-glycosylated peptides derived from the C terminus of human apolipoprotein C-III (apoC-III) in urine of S. mansoni-infected individuals that were not detected in urine of non-infected controls. The glycan composition of these glycopeptides is completely different from what has been described previously for apoC-III. Most importantly, they lack sialylation and display a high degree of fucosylation. This study exemplifies the potential of mass spectrometry for the identification and characterization of O-glycopeptides without prior knowledge of either the glycan or the peptide sequence. Furthermore, our results indicate for the first time that as a result of S. mansoni infection the glycosylation of a host protein is altered.

Targeted identification of a unique glycan epitope of Schistosoma mansoni egg antigens using a diagnostic antibody

The eggs of Schistosoma mansoni express a plethora of glycoconjugate antigens. A specific subset of these antigens can be detected in the serum or urine of infected individuals by a diagnostic sandwich ELISA using the anti-carbohydrate monoclonal antibody (mAb) 114-4D12-A [Nourel Din MS, Nibbeling R, Rotmans JP, Polderman AM, Krijger FW, Deelder AM. Quantitative determination of circulating soluble egg antigen in urine and serum of Schistosoma mansoni-infected individuals using a combined two-site enzyme-linked immunosorbent assay. Am J Trop Med Hyg 1994;50:585–94]. We used affinity chromatography to isolate the 114-4D12-binding glycoprotein subset from soluble egg antigens (SEA) of S. mansoni. SEA and the isolated SEA-subset (SEA-4D12) were subjected to reductive β-elimination and hydrazinolysis to release intact glycans and glycan fragments, respectively, from the protein backbones. The released glycans were characterised by matrix-assisted laser-desorption–ionisation time-of-flight (MALDI-TOF) mass spectrometry (MS), liquid-chromatography (LC)–MS and gas chromatography (GC)–MS linkage analysis. Glycans released by reductive β-elimination from SEA-4D12 were larger and more heavily fucosylated than glycans released from SEA. Most SEA-4D12 glycans contained a branched O-glycan core structure carrying up to 4 N-acetylhexosamines per chain which were substituted with maximum 12 fucose residues. Hydrazinolysis of SEA-4D12 resulted in the release of fucosylated antenna fragments. After 2-aminobenzamide (2AB)-labelling these fragments were subjected to 114-4D12-affinity purification. Normal phase (NP)-LC analysis of the flow-through and retained fractions indicated that the Fucα1-2Fucα1-3GalNAcβ1-4(Fucα1-2Fucα1-3)GlcNAcβ1- element forms the epitope of mAb 114-4D12. Most O-glycans from SEA-4D12 contain this structural element. Epitope-bearing N-glycans have not been found. In terms of abundance in total SEA, only a minority of all glycans possesses the epitope. This multifucosylated motif has so far only been found in schistosomes, providing a structural basis for the high specificity of the diagnostic antibody.

Immunochemical characterization and diagnostic potential of a 63-kilodalton Schistosoma antigen.

Schistosoma circulating antigens were used for the detection of active infection. Anti-S. mansoni IgG2a monoclonal antibody (MAb) designated C5C4 was generated. The target epitope of this MAb was detected in adult worms, eggs, and cercariae antigenic extracts of S. mansoni and S. haematobium, had a molecular size of 63 kD, and was not detected in Fasciola hepatica and Ascaris. In addition, a 50-kD degradation product was identified only in the urine of infected individuals. Analysis by high-performance liquid chromatography of the purified antigen demonstrated only one peak. The 63-kD antigen was characterized as a protein containing 40.4% hydrophobic, 7.5% acidic, and 8.8% basic amino acids. The C5C4 MAb was used in a Fast Dot-ELISA for rapid and simple diagnosis of human schistosomiasis. The 63-kD circulating antigen was detected in 92% of urine samples from 330 S. mansoni-infected individuals, with 16% false-positive results among 130 noninfected individuals.

Rapid detection of a Schistosoma mansoni circulating antigen excreted in urine of infected individuals by using a monoclonal antibody.

Schistosoma circulating antigens were used to indicate the infection intensity and to assess cure. An immunoglobulin G2a (IgG2a) mouse monoclonal antibody was used in a fast dot-enzyme-linked immunosorbent assay (ELISA; FDA) for rapid and simple diagnosis of schistosomiasis in the field. Seven hundred Egyptians were parasitologically examined for Schistosoma mansoni and other parasitic infections. A rectal biopsy was done as a "gold standard" for individuals showing no S. mansoni eggs in their feces. Egg counts were obtained by the Kato smear method for only 100 of 152 individuals with eggs in their feces. Specific anti-schistosome IgG antibodies were evaluated in sera by ELISA. Urine samples from the 700 individuals were tested by FDA for detection of the circulating antigen. The assay showed a sensitivity of 93% among 433 infected individuals and a specificity of 89% among 267 noninfected individuals. FDA showed the highest efficiency of antigen detection (91%) compared with the efficiency of antibody detection by ELISA (75%) and stool analysis (60%). In addition, FDA detected infected patients with 20 eggs/g of feces. Also, the sensitivity of FDA ranged from 90 to 94% among samples from patients with different clinical stages of schistosomiasis. All the assay steps can be completed within 30 min at room temperature for 96 urine samples. The monoclonal antibody identified a 74-kDa antigen in different antigenic extracts of S. mansoni and Schistosoma haematobium and in the urine of infected individuals. In addition, a 30-kDa degradation product was identified only in the urine samples. On the basis of these results, FDA should be used as a rapid tool for the sensitive and specific diagnosis of Schistosoma infection.

Detection ofLegionella DNA in human and guinea pig urine samples by the polymerase chain reaction

A detection system for Legionella DNA in urine samples based on the polymerase chain reaction (PCR) was developed and tested on infected guinea pigs and patients suffering from pneumonia. Results were compared with standard methods for diagnosis of Legionnaires' disease. A primer system was selected which amplifies a 108 bp DNA fragment of the 5S rRNA gene. The sensitivity of the PCR system was one femtogram of extracted Legionella DNA. Three methods were tested for pretreatment of urine samples. Of these, the Geneclean II kit (Bio 101, USA) gave the best results for artificially contaminated urine samples as well as those from infected guinea pigs or patients. Thirty-seven urine samples from 15 guinea pigs intraperitoneally infected with either Legionella pneumophila serogroup 1, 3 and 6 or Legionella micdadei, 26 urine samples of 21 patients suffering from pneumonia, and 30 control samples of patients with urinary tract infection (UTI) were tested. Legionella DNA was detected in 29 of the guinea pig urine samples; whereas, urinary antigen detection using EIA was positive in only 20 of the samples. PCR was also positive in the samples of 11 patients with pneumonia, 9 of which were confirmed by other microbiological methods, such as culture, direct fluorescent antibody test, urinary antigen detection and antibody testing. However, of the 30 control samples from patients with UTI, three samples yielded positive results. The results demonstrate that Legionella DNA is excreted in the urine of infected individuals and that the PCR shows a higher degree of sensitivity than EIA to the detection of soluble Legionella antigen in urine.(ABSTRACT TRUNCATED AT 250 WORDS)

Application of immunoassay methods in the serodiagnosis of human filariasis.

To be more definitive as a serodiagnostic method, an ideal immunoassay for detection of filaria-specific antibody and filarial antigens in body fluids should identify active filarial infection and differentiate between past and present infection. It should perform well in the areas of precision, reproducibility, parallelism, and sensitivity and should use reference and quality control reagents prepared with human body fluids that contain defined amounts of filarial antigen. Moreover, a sufficient quantity of reference serum in stable form should be made available to permit interlaboratory cross-standardization. Difficulty with uniform radiolabeling of filarial antigen extracts and the interference of human antibody have combined to eliminate the competitive-binding immunoassay as a useful method. Of the noncompetitive methods evaluated, the immunoradiometric assay (IRMA) and its nonisotopic counterpart, the immunoenzymetric assay (IEMA), perform best. As a factor masking diagnostically important antigenic determinants, the variable amount of human antibody in blood interfered with all assay designs tested. Better characterization of the antigen(s) circulating in the blood or excreted into the urine of infected individuals will improve assay specificity and sensitivity and will facilitate the preparation of antibody probes more specifically targeted to filarial antigens for use in both the IRMA and IEMA.

The Occurrence of Microfilariae of Onchocerca volvulus in Urine of Infected Individuals

The Occurrence of Microfilariae of Onchocerca volvulus in Urine of Infected Individuals