The urinary microbiome shows different bacterial genera in renal transplant recipients and non-transplant patients at time of acute kidney injury - a pilot study.

BACKGROUND: In the past urine was considered sterile. Through the introduction of next generation sequencing, it has become clear that a urinary microbiome exists. Acute kidney injury (AKI) represents a major threat to kidney transplant recipients. Remarkable changes in the urinary metabolome occur during AKI, which may influence the urinary microbiome. To our knowledge, this is the first study that examines the urinary microbiome in renal transplant recipients (RTX) and non-transplant recipients (nRTX) at time of AKI. METHODS: In this cross-sectional pilot-study the urinary microbiome of 21 RTX and 9 nRTX with AKI was examined. Clean catch morning urine samples were obtained from all patients on the first day of AKI diagnosis. AKI was defined according to KDIGO guidelines. Urinary microbiota and the urinary metabolome during AKI were assessed in one patient. 16S rRNA sequencing was performed. Sequences were processed using UPARSE-pipeline for operational taxonomic units (OTU) and taxon finding. RESULTS: We successfully extracted and sequenced bacterial DNA from 100% of the urine samples. All 30 patients revealed at least 106,138 reads. 319 OTU and 211 different genera were identified. The microbiotic diversity richness in the RTX group was no different from the nRTX group. Eighteen genera were solely present in nRTX and 7 in RTX. CONCLUSIONS: The urinary microbiome at time of AKI showed different bacterial genera in RTX compared to nRTX. The nRTX group exhibited no different diversity to the RTX group. Irrespective of the status of a previous renal transplantation, the urinary microbiome comprised > 210 different genera. An intraindividual change in microbiota diversity and richness was observed in one study patient during recovery from AKI.

Benchmarking urine storage and collection conditions for evaluating the female urinary microbiome.

Standardized conditions for collection, preservation and storage of urine for microbiome research have not been established. We aimed to identify the effects of the use of preservative AssayAssure® (AA), and the effects of storage time and temperatures on reproducibility of urine microbiome results. We sequenced the V3-4 segment of the 16S rRNA gene to characterize the bacterial community in the urine of a cohort of women. Each woman provided a single voided urine sample, which was divided into aliquots and stored with and without AA, at three different temperatures (room temperature [RT], 4 °C, or -20 °C), and for various time periods up to 4 days. There were significant microbiome differences in urine specimens stored with and without AA at all temperatures, but the most significant differences were observed in alpha diversity (estimated number of taxa) at RT. Specimens preserved at 4 °C and -20 °C for up to 4 days with or without AA had no significant alpha diversity differences. However, significant alpha diversity differences were observed in samples stored without AA at RT. Generally, there was greater microbiome preservation with AA than without AA at all time points and temperatures, although not all results were statistically significant. Addition of AA preservative, shorter storage times, and colder temperatures are most favorable for urinary microbiome reproducibility.

Prospective study of canine leptospirosis in shelter and stray dog populations: Identification of chronic carriers and different Leptospira species infecting dogs.

Dogs are highly susceptible to the leptospiral infection, notably stray and sheltered dogs. Unsanitary conditions often observed in dog shelters may predispose the introduction and spread of leptospires among sheltered populations, potentially increasing the chances for the inadvertent adoption of asymptomatically infected animals. The present work describes a longitudinal study using a multidisciplinary approach for the identification of chronically infected dogs and the characterization of potentially pathogenic strains circulating among stray and sheltered dog populations in São Paulo, Brazil. A total of 123 dogs from three populations were included. The initial evaluation consisted of blood and urine quantitative PCR testing (qPCR), the detection of specific antibodies by microscopic agglutination test (MAT), physical examination and hematological and serum biochemistry analyses. The qPCR-positive dogs were prospectively examined, and reevaluations also included culture from urine samples. Positive qPCR samples were subjected to 16S rRNA and secY gene phylogenetic analysis. The recovered strains were characterized by Multilocus Sequence Typing, polyclonal serogroup identification and virulence determination. Leptospiruria was detected in all populations studied (13/123), and phylogenetic analysis revealed that 10 dogs had L. interrogans infection. Three dogs (3/13) had L. santarosai infection. The secY phylogenetic analysis revealed that the L. santarosai sequences clustered separately from those obtained from other hosts. Ten leptospiruric dogs were reevaluated, and three dogs presented persistent leptospiruria, allowing culturing from two dogs. The strains were characterized as L. interrogans serogroup Canicola (virulent) and L. santarosai serogroup Sejroe (not virulent). Serum samples were retested by MAT using the DU92 and DU114 strains as antigens, and no increased seroreactivity was detected. Asymptomatic L. santarosai infection was observed in all populations studied, suggesting a possible role of dogs in the chain of transmission of this leptospiral species. The results suggest a genetic distinction between lineages of Brazilian L. santarosai maintained by dogs and other animal hosts. Our findings revealed that dogs could act as maintenance hosts for distinct pathogenic Leptospira, highlighting also that asymptomatically infected dogs can be inadvertently admitted and adopted in dog shelters, potentially increasing the risks of zoonotic transmission.

Profiling the Urinary Microbiota in Male Patients With Bladder Cancer in China.

Mounting evidence indicates that microbiome plays an important role in the development and progression of cancer. The dogma that urine in healthy individuals must be sterile has been overturned. Dysbiosis of the urinary microbiome has been revealed responsible for various urological disorders, including prostate cancer. The link between chronic inflammation, microbiome and solid tumors has been established for various neoplastic diseases. However, a detailed and comprehensive analysis of urinary microenvironment of bladder cancer has not been yet reported. We performed this study to characterize the potential urinary microbial community possibly associated with bladder cancer. Mid-stream urine was collected from 31 male patients with bladder cancer and 18 non-neoplastic controls. DNA was extracted from urine pellet samples and processed for high throughput 16S rRNA amplicon sequencing of the V4 region using Illumina MiSeq. Sequencing reads were filtered using QIIME and clustered using UPARSE. We observed increased bacterial richness (Observed Species, Chao 1 and Ace indexes; cancer vs. control; 120.0 vs. 56.0; 134.5 vs. 68.3; and 139.6 vs. 72.9, respectively), enrichment of some bacterial genera (e.g., Acinetobacter, Anaerococcus, and Sphingobacterium) and decrease of some bacterial genera (e.g., Serratia, Proteus, and Roseomonas) in cancer group when compared to non-cancer group. Significant difference in beta diversity was found between cancer and non-cancer group, among different risk level, but not among different tumor grade. Enrichment of Herbaspirillum, Porphyrobacter, and Bacteroides was observed in cancer patients with high risk of recurrence and progression, which means these genera maybe potential biomarkers for risk stratification. The PICRUSt showed that various functional pathways were enriched in cancer group, including Staphylococcus aureus infection, glycerolipid metabolism and retinol metabolism. To our knowledge, we performed the most comprehensive study to date to characterize the urinary microbiome associated with bladder cancer. A better understanding of the role of microbiome in the development and progression of bladder cancer could pave a new way for exploring new therapeutic options and biomarkers.

Prevalence of chlamydia among HIV positive and HIV negative patients in the Vhembe District as detected by real time PCR from urine samples.

BACKGROUND: Chlamydia is a bacterial infection that has long plagued humanity as the most commonly contracted STD and is caused by Chlamydia trachomatis. With the emergence of HIV/AIDS, sexually transmitted diseases have also re-emerged as a grave public health problem, particularly in developing countries. Updated Information about the relative frequencies in developing countries is sparse. This study aims at establishing the relative occurrence of chlamydia using real time PCR technique in the Vhembe District of South Africa where reports on the prevalence of chlamydia are not available. METHODS: A total of 243 Urine samples were collected from patients attending different ARV clinics in the Vhembe District and genomic DNA was purified using blood genomic DNA kit from Sigma-Aldrich. Real-Time PCR protocol targeting the 16S rRNA gene of C. trachomatis was used to confirm the presence of chlamydia among these patients. Demographic information as well as clinical data was collected as well. RESULTS: Of all the participants, 70.4% were females. The age varied from 19 to 72 years. The overall prevalence of chlamydia was 32.1%. The prevalence was significantly higher among females (39.2%) compared to males (15.5%) patients (P = 0.001) and was highest among pregnant women followed by patients who had reported any allergic reaction. Among the HIV positive patients, the prevalence was higher among those who were not taking ARV (38.1%) compared to those who were taking them (28.5%). The age group within which the highest prevalence was found was between 26-45 years. CONCLUSIONS: The present study shows a high prevalence of chlamydia among HIV and AIDS patients in the Vhembe District emphasizing the need to enhance STI control and particularly chlamydia among all young people. The particularly high prevalence of chlamydia among pregnant women is of great concern as this predisposes them to complications, while allergy migh predispose people to chlamydia infections. Further studies are needed in the general population both HIV positive and HIV negative persons to further determine the impact of these infections in the community.

A comparison of two real-time polymerase chain reaction assays using hybridization probes targeting either 16S ribosomal RNA or a subsurface lipoprotein gene for detecting leptospires in canine urine.

Leptospires are excreted in the urine of infected animals, and the prompt detection of leptospiral DNA using polymerase chain reaction (PCR) is increasingly being used. However, contradictory data has emerged concerning the diagnostic accuracy of the most popular PCR assays that target either the 16S ribosomal RNA (rrs) or the subsurface lipoprotein (LipL32) genes. In order to clarify the effect of the gene target, a novel hydrolysis probe-based, quantitative real-time PCR (qPCR) assay targeting the LipL32 gene was developed, validated, and then compared directly to the previously described rrs hydrolysis probe-based qPCR using a convenience collection of canine urine samples. The novel LipL32 qPCR assay was linear from 5.9 × 10(6) to 59 genome equivalents per reaction. Both the LipL32 and the rrs qPCR assays showed a limit of detection of 10 target copies per reaction indicating an approximately equivalent analytical sensitivity. Both assays amplified all 20 pathogenic leptospiral strains tested but did not amplify a representative collection of bacteria commonly found in voided canine urine. When the field samples were assayed, 1 and 5 out of 184 samples yielded an amplification signal in the LipL32 and rrs assays, respectively. Nevertheless, when the limit of detection was considered as the cutoff for interpreting findings, the 4 discordant cases were judged as negative. In conclusion, our study confirmed that both LipL32 and rrs are suitable targets for qPCR for the detection of leptospiral DNA in canine urine. However, the rrs target requires the mandatory use of a cutoff value in order to correctly interpret spurious amplifications.

Diagnosis of canine leptospirosis by a highly sensitive FRET-PCR targeting the lig genes.

Canine leptospirosis is underdiagnosed due to its wide spectrum of clinical presentations and the lack of a rapid and sensitive test for the accurate diagnosis of acute and chronic infections. In this study, we developed a highly sensitive and specific fluorescence resonance energy transfer (FRET)-PCR to detect common pathogenic leptospires in dogs, including Leptospira interrogans serovars Autumnalis, Canicola, Copenhageni (Icterohaemorrhagiae serogroup) and Pomona, and Leptospira kirschneri serovar Grippotyphosa. This PCR targets the lig genes, exclusively found in the pathogenic Leptospira species but not in saprophytic species (L. biflexa). A robust, high-stringency step-down real-time platform was coupled to the highly specific detection of leptospiral DNA by fluorescently labeled FRET probes. This enabled the detection of a single copy of the lig gene in a PCR containing DNA from up to 50 µL canine blood or 400 µL urine. Sensitivity determination by use of limiting serial dilutions of extracted leptospiral DNA indicated that the lig FRET-PCR we established was almost 100-fold more sensitive than the widely accepted lipL32 SYBR assay and 10-fold more sensitive than a 16S rRNA TaqMan assay. Application of this method to 207 dogs with potential leptospiral infection enabled us to diagnose three cases of canine leptospirosis characterized by low amounts of leptospiral DNA in body fluids. Detection of canine leptospirosis with the lig FRET-PCR was more sensitive with the lig FRET-PCR than with the 16S rRNA TaqMan PCR, which detected only 2 of the 3 cases, and the lipL32 SYBR PCR, which detected none of the 3 dogs with leptospirosis.

Extra-pulmonary tuberculosis: differential aspects and role of 16S-rRNA in urine.

BACKGROUND: Early diagnosis of extra-pulmonary tuberculosis (EPTB) is important for successful treatment. METHODS: All cases of EPTB diagnosed at Ramon y Cajal Hospital, Madrid, Spain, from 1997 to 2008 were analysed and compared with pulmonary tuberculosis (PTB) patients to identify differential parameters that could serve to predict the presence of EPTB at initial presentation. Different microbiological techniques were analysed, including amplification of 16S-rRNA in urine. RESULTS: During the study period, 814 cases of TB were diagnosed at our centre; 330 (40.5%) were EPTB. Concomitant PTB was detected in 45% of EPTB cases. The main clinical forms of EPTB were lymphadenitis (86, 26%), miliary TB (60, 18%), and multifocal TB (43, 13%). Variables independently associated with EPTB were human immunodeficiency virus (HIV) infection (OR 3.6, 95%CI 2.4-5.4), older age (>60 years) (OR 3.7, 95%CI 2.5-5.6) and mortality (OR 2.9, 95%CI 1.3-6.3). 16S-rRNA in urine was performed in 82 EPTB patients (25%), among whom a positive result was obtained in 70%; in the PTB group, a positive result was found in 5 of 28 patients (18%) (P <0.001). CONCLUSIONS: HIV infection and older age appear to be the main risk factors associated with EPTB. In this study, mortality was significantly higher in patients with EPTB. A positive 16S-rRNA test result in urine is a useful marker of EPTB.

In situ electrokinetic enhancement for self-assembled-monolayer-based electrochemical biosensing.

This study reports a multifunctional electrode approach which directly implements electrokinetic enhancement on a self-assembled-monolayer-based electrochemical sensor for point-of-care diagnostics. Using urinary tract infections as a model system, we demonstrate that electrokinetic enhancement, which involves in situ stirring and heating, can enhance the sensitivity of the strain specific 16S rRNA hybridization assay for 1 order of magnitude and accelerate the time-limiting incubation step with a 6-fold reduction in the incubation time. Since the same electrode platform is used for both electrochemical signal enhancement and electrochemical sensing, the multifunctional electrode approach provides a highly effective strategy toward fully integrated lab-on-a-chip systems for various biomedical applications.

Purulent pericarditis due to Streptococcus pneumoniae diagnosed by pneumococcal urinary antigen assay and 16S rDNA sequence of the pericardial fluid.

A 57-year old woman was admitted to our hospital with massive pericardial fluid. Culture of the pericardial fluid was negative, however, Binax NOW Streptococcus pneumoniae urinary antigen test was positive in pericardial fluid. 16S rDNA sequencing and PCR for lyt(A) gene of the pericardial fluid sample confirmed the microbiological diagnosis of S. pneumoniae. The patient was treated with surgical drainage and continuous intravenous infusion of penicillin G and its concentration in the serum and pericardial effusion was monitored. Incorporation of molecular methods such as antigen testing and nucleic acid sequencing would benefit the management of infectious diseases especially in culture negative cases.

Detection and identification of bacteria using in-house broad range 16S rDNA PCR amplification and genus-specific DNA hybridization probes, located within variable regions of 16S rRNA genes.

Broad range PCR amplification and genus-specific 16S ribosomal DNA hybridization was used to demonstrate that Chlamydia, Helicobacter and Mobiluncus hybridization probes, located within variable regions V3, V4, and V9 of the 16S rDNA, specifically bound to the corresponding PCR product obtained from pure cultures of the three genera. The sensitivity of the assay was determined by analysis of C. trachomatis serially diluted in urine. The detection limit was 1-10 elementary bodies using a hybridization probe derived from the variable region V3 of the 16S rRNA gene. A PCR product was furthermore formed in urine specimens not containing C. trachomatis, showing amplification of Chlamydia also in the presence of DNA from the resident urethral flora that competes for annealing sites. Analysis of a restricted number of male urine specimens using the C. trachomatis-specific probe showed complete agreement with culture and a commercially available PCR kit. Our method not only has the capacity to detect C. trachomatis in microbiologically mixed urine samples but also the potential advantage of identifying other bacterial pathogens from the same PCR product by varying the hybridization probes.