Multimode detection of ß-glycosidase and pathogenic bacteria via cation exchange assisted signal amplification.

A rapid strategy for the ß-glycosidase (ß-Gal) and Escherichia coli (E. coli) sensing is presented, which is based on selective recognition reactions of QDs using visualization/fluorescence (FL)/atomic fluorescence spectrometry (AFS)/inductively coupled plasma mass spectrometry (ICP-MS) multimode assay. CdTe QDs can selectively recognize Ag+ and Ag NPs with a cation exchange reaction (CER) where Ag+ triggers the release of Cd2+ and quenches the fluorescence signal of QDs. Taking advantage of the fact that ß-Gal can hydrolyze 4-Aminophenyl ß-D-galactopyranoside (PAPG) to produce p-aminophenol (PAP), which has the ability to reduce Ag+ to form Ag NPs. The ß-Gal can be easily detected by visualization or FL in a turn-on manner. Furthermore, combining with the selective separation of Cd2+ by filter membrane, AFS and ICP-MS with higher sensitivity were used for the determination of the enzyme. Under optimized conditions, the system limits of detections (LODs) were 0.01 U/L, 0.03 mU/L, and 0.02 mU/L using FL, AFS, and ICP-MS as the detector, respectively. The relative standard deviations (RSDs, n = 7) for 0.1 U/L ß-Gal were 2.2, 2.0, and 1.3% using FL/AFS/ICP-MS as the detector, respectively. And 0.1 U/L of ß-Gal can be discriminated from the blank solution with the naked eye. In addition, given that the ß-Gal can serve as an indicator of E. coli, we have successfully applied this strategy for the detection of E. coli with a LOD of 25 CFU/mL. Application of the method was demonstrated by analyzing human urine samples and milk samples for ultra-trace detection of E. coli. Graphical abstract The CVG-AFS/ICP-MS/visual/FL multimode ß-Gal and E.coli detection via CER.

Fast and Accurate Bacterial Species Identification in Urine Specimens Using LC-MS/MS Mass Spectrometry and Machine Learning.

Fast identification of microbial species in clinical samples is essential to provide an appropriate antibiotherapy to the patient and reduce the prescription of broad-spectrum antimicrobials leading to antibioresistances. MALDI-TOF-MS technology has become a tool of choice for microbial identification but has several drawbacks: it requires a long step of bacterial culture before analysis (≥24 h), has a low specificity and is not quantitative. We developed a new strategy for identifying bacterial species in urine using specific LC-MS/MS peptidic signatures. In the first training step, libraries of peptides are obtained on pure bacterial colonies in DDA mode, their detection in urine is then verified in DIA mode, followed by the use of machine learning classifiers (NaiveBayes, BayesNet and Hoeffding tree) to define a peptidic signature to distinguish each bacterial species from the others. Then, in the second step, this signature is monitored in unknown urine samples using targeted proteomics. This method, allowing bacterial identification in less than 4 h, has been applied to fifteen species representing 84% of all Urinary Tract Infections. More than 31,000 peptides in 190 samples were quantified by DIA and classified by machine learning to determine an 82 peptides signature and build a prediction model. This signature was validated for its use in routine using Parallel Reaction Monitoring on two different instruments. Linearity and reproducibility of the method were demonstrated as well as its accuracy on donor specimens. Within 4h and without bacterial culture, our method was able to predict the predominant bacteria infecting a sample in 97% of cases and 100% above the standard threshold. This work demonstrates the efficiency of our method for the rapid and specific identification of the bacterial species causing UTI and could be extended in the future to other biological specimens and to bacteria having specific virulence or resistance factors.

Sensitive electrochemiluminescence (ECL) immunoassays for detecting lipoarabinomannan (LAM) and ESAT-6 in urine and serum from tuberculosis patients.

BACKGROUND: Tuberculosis (TB) infection was responsible for an estimated 1.3 million deaths in 2017. Better diagnostic tools are urgently needed. We sought to determine whether accurate TB antigen detection in blood or urine has the potential to meet the WHO target product profiles for detection of active TB. MATERIALS AND METHODS: We developed Electrochemiluminescence (ECL) immunoassays for Lipoarabinomannan (LAM) and ESAT-6 detection with detection limits in the pg/ml range and used them to compare the concentrations of the two antigens in the urine and serum of 81 HIV-negative and -positive individuals with presumptive TB enrolled across diverse geographic sites. RESULTS: LAM and ESAT-6 overall sensitivities in urine were 93% and 65% respectively. LAM and ESAT-6 overall sensitivities in serum were 55% and 46% respectively. Overall specificity was ≥97% in all assays. Sensitivities were higher in HIV-positive compared to HIV-negative patients for both antigens and both sample types, with signals roughly 10-fold higher on average in urine than in serum. The two antigens showed similar concentration ranges within the same sample type and correlated. CONCLUSIONS: LAM and ESAT-6 can be detected in the urine and serum of TB patients, regardless of the HIV status and further gains in clinical sensitivity may be achievable through assay and reagent optimization. Accuracy in urine was higher with current methods and has the potential to meet the WHO accuracy target if the findings can be transferred to a point-of-care TB test.

Protein Expression Analysis by Western Blot and Protein-Protein Interactions.

Western blot analysis is widely used for detecting protein expression, analysis of protein-protein interactions, and searching for new biomarkers. Also, it is a diagnostic tool used for detection of human diseases and microorganism infections.Some Streptococcus pneumoniae proteins are important virulence factors and a few of them are diagnostic markers. Here, we describe the detection of two pneumococcal proteins, pneumolysin and PpmA, in human urine by using monoclonal and polyclonal antibodies.

Ultrafast and Ultrasensitive Naked-Eye Detection of Urease-Positive Bacteria with Plasmonic Nanosensors.

Identifying the pathogen responsible for an infection is a requirement in order to personalize antimicrobial treatments. Detecting bacterial enzymes, such as proteases, lipases, and oxidoreductases, is a winning approach for detecting pathogens at the point of care. In this Article, a new method for detecting urease-producing bacteria rapidly and at ultralow concentrations is reported. In this method, longsome bacteriological culture steps are substituted for a 10 min capture procedure with positively charged magnetic beads. The presence of urease-positive bacteria on the particles is then queried with a plasmonic signal generation step that generates blue- or red-colored nanoparticle suspensions upon addition of the enzyme substrate. These colorimetric signals, which can be easily identified by eye, are generated by the NH3-dependent assembly of gold nanoparticles in the presence of bovine serum albumin (BSA). The proposed method can detect Proteus mirabilis with a limit of detection of 101 cells mL-1, with a total assay time of 40 min, even in the presence of a large excess of urease-negative bacteria ( Pseudomonas aeruginosa). Furthermore, it does not require bulky equipment, and it can detect P. mirabilis at clinically relevant concentrations within minutes, making it suitable for detecting urease-positive pathogens at the point of care.

Silver enhanced nano-gold dot blot immunoassay for leptospirosis.

Leptospirosis is a widespread zoonotic disease and lacks in efficient diagnostic tools. In the present study, a nanogold based dot blot immunoassay was developed and evaluated for the detection of leptospirosis in human urine samples. This method was found to be rapid (<4 h) with higher sensitivity (>4.2-14.6%) than horse radish peroxidase (HRP) conjugated dot blot assay.

Needle lost in the haystack: multiple reaction monitoring fails to detect Treponema pallidum candidate protein biomarkers in plasma and urine samples from individuals with syphilis.

Background: Current syphilis diagnostic strategies are lacking a sensitive manner of directly detecting Treponema pallidum antigens. A diagnostic test that could directly detect T. pallidum antigens in individuals with syphilis would be of considerable clinical utility, especially for the diagnosis of reinfections and for post-treatment serological follow-up. Methods: In this study, 11 candidate T. pallidum biomarker proteins were chosen according to their physiochemical characteristics, T. pallidum specificity and predicted abundance. Thirty isotopically labelled proteotypic surrogate peptides (hPTPs) were synthesized and incorporated into a scheduled multiple reaction monitoring assay. Protein extracts from undepleted/unenriched plasma (N = 18) and urine (N = 4) samples from 18 individuals with syphilis in various clinical stages were tryptically digested, spiked with the hPTP mixture and analysed with a triple quadruple mass spectrometer. Results: No endogenous PTPs corresponding to the eleven candidate biomarkers were detected in any samples analysed. To estimate the Limit of Detection (LOD) of a comparably sensitive mass spectrometer (LTQ-Orbitrap), two dilution series of rabbit cultured purified T. pallidum were prepared in PBS. Polyclonal anti- T. pallidum antibodies coupled to magnetic Dynabeads were used to enrich one sample series; no LOD improvement was found compared to the unenriched series. The estimated LOD of MS instruments is 300 T. pallidum/ml in PBS. Conclusions: Biomarker protein detection likely failed due to the low (femtomoles/liter) predicted concentration of T. pallidum proteins. Alternative sample preparation strategies may improve the detectability of T. pallidum proteins in biofluids.

Candidate Treponema pallidum biomarkers uncovered in urine from individuals with syphilis using mass spectrometry.

AIM: A diagnostic test that could detect Treponema pallidum antigens in urine would facilitate the prompt diagnosis of syphilis. MATERIALS & METHODS: Urine from 54 individuals with various clinical stages of syphilis and 6 controls were pooled according to disease stage and interrogated with complementary mass spectrometry techniques to uncover potential syphilis biomarkers. RESULTS & CONCLUSION: In total, 26 unique peptides were uncovered corresponding to four unique T. pallidum proteins that have low genetic sequence similarity to other prokaryotes and human proteins. This is the first account of direct T. pallidum protein detection in human clinical samples using mass spectrometry. The implications of these findings for future diagnostic test development is discussed. Data are available via ProteomeXchange with identifier PXD009707.

Gold-copper nanoshell dot-blot immunoassay for naked-eye sensitive detection of tuberculosis specific CFP-10 antigen.

Herein, a straightforward and highly specific dot-blot immunoassay was successfully developed for the detection of Mycobacterium tuberculosis antigen (10 kDa culture filtrate protein, CFP-10) via the formation of copper nanoshell on the gold nanoparticles (AuNPs) surface. The principle of dot-blot immunoassay was based on the reduction of Cu2+ ion on the GBP-CFP10G2-AuNPs conjugates, which has gold binding and antigen binding affinities, simultaneously, favouring to appear red dot that can be observed with naked-eye. The dot intensity is proportional to the concentration of tuberculosis antigen CFP-10, which offers a detection limit of 7.6 pg/mL. The analytical performance of GBP-CFP10G2-AuNPs-copper nanoshell dot-blot was superior than that of conventional silver nanoshell. This method was successfully applied to identify the CFP-10 antigen in the clinical urine sample with high sensitivity, specificity, and minimized sample preparation steps. This method exhibits great application potential in the field of nanomedical science for highly reliable point-of-care detection of CFP-10 antigen in real samples to early diagnosis of tuberculosis.

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.

Discovery of a unique Mycobacterium tuberculosis protein through proteomic analysis of urine from patients with active tuberculosis.

Identification of pathogen-specific biomarkers present in patients' serum or urine samples can be a useful diagnostic approach. In efforts to discover Mycobacterium tuberculosis (Mtb) biomarkers we identified by mass spectroscopy a unique 21-mer Mtb peptide sequence (VVLGLTVPGGVELLPGVALPR) present in the urines of TB patients from Zimbabwe. This peptide has 100% sequence homology with the protein TBCG_03312 from the C strain of Mtb (a clinical isolate identified in New York, NY, USA) and 95% sequence homology with Mtb oxidoreductase (MRGA423_21210) from the clinical isolate MTB423 (identified in Kerala, India). Alignment of the genes coding for these proteins show an insertion point mutation relative to Rv3368c of the reference H37Rv strain, which generated a unique C-terminus with no sequence homology with any other described protein. Phylogenetic analysis utilizing public sequence data shows that the insertion mutation is apparently a rare event. However, sera from TB patients from distinct geographical areas of the world (Peru, Vietnam, and South Africa) contain antibodies that recognize a purified recombinant C-terminus of the protein, thus suggesting a wider distribution of isolates that produce this protein.

Pathogen-specific leptospiral proteins in urine of patients with febrile illness aids in differential diagnosis of leptospirosis from dengue.

Leptospirosis and dengue are two commonly seen infectious diseases of the tropics. Differential diagnosis of leptospirosis from dengue fever is often difficult due to overlapping clinical symptoms and lack of economically viable and easy-to-perform laboratory tests. The gold standard for diagnosis is the microscopic agglutination test (MAT). In this study, the diagnostic potential of screening for pathogen-specific leptospiral antigens in urine samples is presented as a non-invasive method of disease diagnosis. In a study group of 40 patients, the serum was tested for anti-leptospiral antibodies by MAT and enzyme-linked immunosorbent assay (ELISA). Urine of these patients was screened for leptospiral antigens by ELISA using specific antibodies against LipL32, LipL41, Fla1, HbpA and sphingomyelinase. Group I patients (n = 23) were classified as leptospirosis-positive based on MAT and high titres of circulating IgM-specific anti-leptospiral antibodies. All of these patients excreted all five leptospiral antigens in the urine. The 17 MAT-negative cases included six patients with pyrexia of unknown origin (PUO; Group II) and 11 confirmed dengue patients (Group III). The latter tested negative for both serum anti-leptospiral antibodies and urinary leptospiral antigens. A salient outcome of this study was highlighting the usefulness of screening for urinary leptospiral antigens in disease diagnosis, as their presence confirmed leptospiral aetiology in two PUO patients. Immunoblots of urinary antigens identified well-defined bands corresponding to LipL32, HbpA and sphingomyelinase; the significance of the 42- and 58-kDa sphingomyelinase bands is discussed.

AmpC and extended spectrum beta-lactamases production among urinary isolates from a tertiary care hospital in Lalitpur, Nepal.

BACKGROUND: Production of AmpC and extended spectrum beta-lactamases among urinary isolates has created a serious problem to the successful management of the urinary tract infection. The main purpose of this study was to determine the rates of the extended spectrum beta-lactamase (ESBL) production and AmpC beta-lactamase (ABL) production among urinary isolates. RESULTS: Among total 564 urinary isolates, 514 (91.1%) were gram negative bacilli and 50 (8.9%) were gram positive cocci. E. coli (76.1%) was the most common bacteria isolated. Staphylococcus aureus (6.7%) was the predominant gram positive bacteria isolated. 35 (6.8%) of the 514 gram negative bacilli were ESBL producers. Similarly, 14 (2.7%) of the gram negative bacilli were ABL producers. Only one isolate was ESBL and ABL co-producer. Highest rate of susceptibility of gram negative bacteria was seen toward amikacin (97.3%) followed by imipenem (94.4%). Similarly, highest rate of susceptibility among gram positive cocci was seen toward vancomycin (100%) followed by amikacin (93.5%). CONCLUSIONS: Low rates of AmpC and extended spectrum beta-lactamases production in comparison to other previous studies were reported. On the basis of the antimicrobial susceptibility patterns of the bacteria we reported in our study, amikacin, imipenem and nitrofurantoin can be used for the preliminary treatment of urinary tract infections caused by gram negative bacteria and vancomycin and amikacin for treatment of urinary tract infections caused by gram positive bacteria.

Potential of High-Affinity, Slow Off-Rate Modified Aptamer Reagents for Mycobacterium tuberculosis Proteins as Tools for Infection Models and Diagnostic Applications.

Direct pathogen detection in blood to diagnose active tuberculosis (TB) has been difficult due to low levels of circulating antigens or due to the lack of specific, high-affinity binding reagents and reliable assays with adequate sensitivity. We sought to determine whether slow off-rate modified aptamer (SOMAmer) reagents with subnanomolar affinity for Mycobacterium tuberculosis proteins (antigens 85A, 85B, 85C, GroES, GroEL2, DnaK, CFP10, KAD, CFP2, RplL, and Tpx) could be useful to diagnose tuberculosis. When incorporated into the multiplexed, array-based proteomic SOMAscan assay, limits of detection reached the subpicomolar range in 40% serum. Binding to native M. tuberculosis proteins was confirmed by using M. tuberculosis culture filtrate proteins and fractions from infected macrophages and via affinity capture assays and subsequent mass spectrometry. Comparison of serum from culture-positive pulmonary TB patients and TB suspects systematically ruled out for TB revealed small but statistically significant (P < 0.0001) differences in the median M. tuberculosis signals and in specific pathogen markers, such as antigen 85B. Samples where many M. tuberculosis aptamers produced high signals were rare exceptions. In concentrated, protein-normalized urine from TB patients and non-TB controls, the CFP10 (EsxB) SOMAmer yielded the most significant differential signals (P < 0.0276), particularly in TB patients with HIV coinfection. In conclusion, direct M. tuberculosis antigen detection proved difficult even with a sensitive method such as SOMAscan, likely due to their very low, subpicomolar abundance. The observed differences between cases and controls had limited diagnostic utility in serum and urine, but further evaluation of M. tuberculosis SOMAmers using other platforms and sample types is warranted.

Analysis of the association between Mycobacterium tuberculosis infection and Immunoglobulin A nephropathy by early secreted antigenic target 6 detection in renal biopsies: a prospective study.

OBJECTIVES: Immunoglobulin A nephropathy (IgAN) is the most frequent cause of primary renal disease, and clarifying the pathogenesis of IgAN is of great importance for its diagnosis and treatment. It is well known that Mycobacterium tuberculosis (MTB) can infect the urinary tract and result in the typical symptoms of cystitis. However, MTB can also affect the kidney more insidiously. Patients may present with glomerular disease, and sometimes with advanced renal failure. This study was to investigate the association between MTB infection and IgA nephropathy (IgAN), and the early diagnosis of MTB-mediated IgAN by means of early secreted antigenic target 6 (ESAT-6) detection in renal biopsies. METHODS: One hundred and twenty patients were divided into 3 groups: a renal tuberculosis (RTB) group, a glomerulonephritis without MTB infection (GN-TBI) group and a glomerulonephritis with MTB infection (GN+TBI) group. Morning urine samples were collected for MTB culture. Immunohistochemistry for ESAT-6 expression in renal tissues was performed. RESULTS: The incidence rate of IgAN in the GN+TBI group was 66.7%, which was significantly higher than that of the GN-TBI group. In the GN+TBI group, the ESAT-6 expression was positively associated with IgAN incidence. There was a statistical association between the positive expression of ESAT-6 and the incidence of IgAN. The sensitivity and specificity of urine MTB culture in diagnosing renal MTB infection was 23.3% and 100% respectively, while the sensitivity and specificity of ESAT-6 detection was 100% and 91.1% respectively. Compared with urine MTB culture, the sensitivity of ESAT-6 detection was significantly increased. CONCLUSION: MTB infection might be associated with the occurrence of IgAN, and ESAT-6 detection in renal tissues may be helpful for the early diagnosis of MTB-mediated IgAN.

An easy and sensitive sandwich assay for detection of Mycobacterium tuberculosis Ag85B antigen using quantum dots and gold nanorods.

Mycobacterium tuberculosis is a serious global infectious pathogen causing tuberculosis (TB). The development of an easy and sensitive method for the detection of M. tuberculosis is in urgent need due to complex and low specificity of the current assays. Herein, we present a novel method for M. tuberculosis detection based on a sandwich assay via antigen-antibody interaction using silica-coated quantum dots (SiQDs) and gold nanorods (AuNRs). A genetically engineered recombinant antibody (GBP-50B14 and SiBP-8B3) was bound to surfaces of AuNRs and SiQDs respectively, without any surface modification. The antigen-antibody interaction was revealed using M. tuberculosis-specific secretory antigen, Ag85B. Two biocomplexes showed a quenching effect in the presence of the target antigen through a sandwich assay. The assay response was in the range of 1×10-3-1×10-10µgmL-1 (R=0.969) and the limit of detection for Ag85B was 13.0pgmL-1. The Ag85B was selectively detected using three different proteins (CFP10, and BSA), and further specifically confirmed by the use of spiked samples. Compared with existing methods, a highly sensitive and selective method for Ag85B-expressing M. tuberculosis detection has been developed for better diagnosis of TB.

Emergence of vanA gene among vancomycin-resistant enterococci in a tertiary care hospital of North - East India.

BACKGROUND & OBJECTIVES: Vancomycin-resistant enterococci (VRE) have become one of the most challenging nosocomial pathogens with the rapid spread of the multi-drug resistant strain with limited therapeutic options. It is a matter of concern due to its ability to transfer vancomycin resistant gene to other organisms. The present study was undertaken to determine the emergence of vancomycin-resistant enterococci and the vanA gene among the isolates in a tertiary care hospital of North-East India. METHODS: A total of 67 consecutive enterococcal isolates from different clinical samples were collected and identified by using the standard methods. Antibiogram was done by disk diffusion method and VRE was screened by the disk diffusion and vancomycin supplement agar dilution method. The minimum inhibitory concentration (MIC) value for vancomycin was determined by E-test. The VRE isolates were analyzed by PCR for vanA gene. RESULTS: A total of 54 (81%) Enterococcus faecalis and 13 (19%) E. faecium were detected among the clinical isolates and 16 (24%) were VRE. The VRE isolates were multidrug resistant and linezolid resistance was also found to be in three. MIC range to vancomycin was 16-32 µg/ml among the VRE. The vanA gene was found in nine of 16 VRE isolates. INTERPRETATION & CONCLUSIONS: Emergence of VRE and presence of vanA in a tertiary care hospital setting in North-East India indicate toward a need for implementing infection control policies and active surveillance.

Extended-spectrum ß-lactamase (ESBL) detection directly from urine samples with the rapid isothermal amplification-based eazyplex® SuperBug CRE assay: Proof of concept.

A commercially available assay (eazyplex® SuperBug CRE) detecting the most common carbapenemase and ESBL types was evaluated directly on 50 urine samples. Eazyplex® correctly detected ESBL-encoding genes in all 30 urine samples with confirmed ESBL production (sensitivity 100%). Two specimens showed invalid and one specimen false-positive results (specificity 97.9%).

[Characteristic of virulence potential of clinical isolates of enterococci].

AIM: Determination of virulence of enterococci strains isolated from clinical material from humans on pheno- and genotype levels. MATERIALS AND METHODS: 30 strains of enterococci isolated from wound exudate, urine, newborn skin lavage were used in the study. Strain identification was carried out by multiplex PCR. Hemolytic activity was determined by dish method, gelatinase - by dissolution of gelatin column, proteolytic--by biuret method; genes coding virulence factor synthesis (gelE, sprE, cylM, cylB, cylA, cylLs, cylL1, ESP, HYL, ASA)--by using PCR. RESULTS: Clinical isolates of enterococci were assigned to E. faecalis and E. faecium species. Virulence factors on phenotype and genotype levels were detected in both species. CONCLUSION: Genetic determinants of virulence are more widespread among clinical isolates of E.faecalis species. Set of genes coding virulence factors in E. faecalis depends on biotope. Gene coding hyaluronidase synthesis is characteristic for E. faecium. A correlation between phenotypic manifestation of features and enterococci genotype was detected.

Validation of Mycobacterium tuberculosis Rv1681 protein as a diagnostic marker of active pulmonary tuberculosis.

The development of an accurate antigen detection assay for the diagnosis of active tuberculosis (TB) would represent a major clinical advance. Here, we demonstrate that the Mycobacterium tuberculosis Rv1681 protein is a biomarker for active TB with potential diagnostic utility. We initially identified, by mass spectroscopy, peptides from the Rv1681 protein in urine specimens from 4 patients with untreated active TB. Rabbit IgG anti-recombinant Rv1681 detected Rv1681 protein in lysates and culture filtrates of M. tuberculosis and immunoprecipitated it from pooled urine specimens from two TB patients. An enzyme-linked immunosorbent assay formatted with these antibodies detected Rv1681 protein in unconcentrated urine specimens from 11/25 (44%) TB patients and 1/21 (4.8%) subjects in whom TB was initially clinically suspected but then ruled out by conventional methods. Rv1681 protein was not detected in urine specimens from 10 subjects with Escherichia coli-positive urine cultures, 26 subjects with confirmed non-TB tropical diseases (11 with schistosomiasis, 5 with Chagas' disease, and 10 with cutaneous leishmaniasis), and 14 healthy subjects. These results provide strong validation of Rv1681 protein as a promising biomarker for TB diagnosis.