Variations of bacterial populations in human feces measured by fluorescent in situ hybridization with group-specific 16S rRNA-targeted oligonucleotide probes

Six 16S rRNA-targeted oligonucleotide probes were designed, validated, and used to quantify predominant groups of anaerobic bacteria in human fecal samples. A set of two probes was specific for species of the Bacteroides fragilis group and the species Bacteroides distasonis. Two others were designed to detect species of the Clostridium histolyticum and the Clostridium lituseburense groups. Another probe was designed for the genera Streptococcus and Lactococcus, and the final probe was designed for the species of the Clostridium coccoides-Eubacterium rectale group. The temperature of dissociation of each of the probes was determined. The specificities of the probes for a collection of target and reference organisms were tested by dot blot hybridization and fluorescent in situ hybridization (FISH). The new probes were used in initial FISH experiments to enumerate human fecal bacteria. The combination of the two Bacteroides-specific probes detected a mean of 5.4 x 10(10) cells per g (dry weight) of feces; the Clostridium coccoides-Eubacterium rectale group-specific probe detected a mean of 7.2 x 10(10) cells per g (dry weight) of feces. The Clostridium histolyticum, Clostridium lituseburense, and Streptococcus-Lactococcus group-specific probes detected only numbers of cells ranging from 1 x 10(7) to 7 x 10(8) per g (dry weight) of feces. Three of the newly designed probes and three additional probes were used in further FISH experiments to study the fecal flora composition of nine volunteers over a period of 8 months. The combination of probes was able to detect at least two-thirds of the fecal flora. The normal biological variations within the fecal populations of the volunteers were determined and indicated that these variations should be considered when evaluating the effects of agents modulating the flora.     

Development of a PCR assay for detection of Yersinia ruckeri in tissues of inoculated and naturally infected trout

A PCR-based method was developed for the specific detection of Yersinia ruckeri in tissues of inoculated trout and naturally infected trout. No amplification products were obtained with other yersiniae, bacterial fish pathogens, or phylogenetically related bacteria (n = 34). The sensitivity of PCR detection was 60 to 65 bacterial cells per PCR tube, which was decreased to 10 to 20 cells by hybridization with a nonradioactive probe. The PCR assay proved to be as reliable as and faster than the conventional culture method for the detection of Y. ruckeri in infected trout tissues.     

Chemiluminescent in situ hybridization for the detection of cytomegalovirus DNA

A chemiluminescent in situ hybridization assay that could combine the sensitivity of chemiluminescent substrates, the specificity of digoxigenin-labeled probes, and the spatial morphological resolution and localization of the signal of the in situ hybridization was developed for the detection of cytomegalovirus (CMV) DNA. CMV DNA in cultured CMV-infected cells and in different clinical samples (tissue sections and cellular smears) was detected using digoxigenin-labeled probes constructed in our laboratory that were immunoenzymatically visualized employing anti-digoxigenin Fab fragments labeled with alkaline phosphatase and the chemiluminescent adamantil-1,2-dioxetane phenyl phosphate substrate for alkaline phosphatase. The luminescent signal from the hybrid formation was detected, analyzed, and measured with a high performance, low light level imaging luminograph apparatus connected to an optical microscope and to a personal computer for quantitative image analysis. Increasing values of emitted photons per second per infected cell, corresponding to the presence of hybridized CMV DNA, could be found in infected cells fixed at various times after infection, following the CMV replication cycle. When the assay was performed on different clinical samples from patients with acute CMV infections, CMV DNA was detected in all positive samples tested, both in cellular samples and in frozen and paraffin-embedded tissue sections, proving specific and sensitive. The chemiluminescent in situ hybridization assay developed in this work can be a useful tool for a sensitive and specific diagnosis of viral infection and can be easily adapted to detect and study any specific gene sequence inside the cells. The assay may also be promising for an estimation and quantification of nucleic acids present in tissue samples or cellular smears and for imaging gene expression in cells.     

In vivo and in vitro detection of canine distemper virus nucleoprotein gene with digoxigenin-labelled RNA, double-stranded DNA probes and oligonucleotides by in situ hybridization

A single-stranded RNA, two double-stranded (ds) DNA probes and 10 oligonucleotides labelled with digoxigenin were comparatively evaluated for their usefulness to detect canine distemper virus (CDV) nucleoprotein RNA in in vitro infected Vero cells and in tissues of dogs with spontaneous CDV infection by in situ hybridization (ISH). In addition, results were compared to CDV nucleoprotein antigen distribution as demonstrated by immunohistochemistry. The RNA probe was derived from the virulent A75/17 strain, the DNA and oligonucleotide probes from the avirulent Onderstepoort strain of CDV. The two DNA probes were 287 and 126 base pairs long. For ISH, various factors including fixatives, proteolytic digestion, probe concentration, hybridization conditions and detection systems were compared. All probes were suitable for demonstration of CDV RNA in in vitro infected cells, regardless of the CDV strain employed. In vivo CDV nucleic acid was detected by RNA and the dsDNA probes. However, the probes varied substantially with respect to sensitivity and specificity. The CDV RNA probe was far superior in sensitivity when compared to the DNA probes. Furthermore, the shorter DNA probe displayed a higher sensitivity, indicating that length of the probe is an important parameter when selecting probes. Oligonucleotides displayed only rarely a positive signal and caused frequently hybridization signals in the nucleus, which where considered not specific for CDV. Summarized, the present study reveals that RNA probes are currently the most sensitive tool for detection of CDV RNA in tissues.     

Detection of Ralstonia solanacearum, which causes brown rot of potato, by fluorescent in situ hybridization with 23S rRNA-targeted probes

During the past few years, Ralstonia (Pseudomonas) solanacearum race 3, biovar 2, was repeatedly found in potatoes in Western Europe. To detect this bacterium in potato tissue samples, we developed a method based on fluorescent in situ hybridization (FISH). The nearly complete genes encoding 23S rRNA of five R. solanacearum strains and one Ralstonia pickettii strain were PCR amplified, sequenced, and analyzed by sequence alignment. This resulted in the construction of an unrooted tree and supported previous conclusions based on 16S rRNA sequence comparison in which R. solanacearum strains are subdivided into two clusters. Based on the alignments, two specific probes, RSOLA and RSOLB, were designed for R. solanacearum and the closely related Ralstonia syzygii and blood disease bacterium. The specificity of the probes was demonstrated by dot blot hybridization with RNA extracted from 88 bacterial strains. Probe RSOLB was successfully applied in FISH detection with pure cultures and potato tissue samples, showing a strong fluorescent signal. Unexpectedly, probe RSOLA gave a less intense signal with target cells. Potato samples are currently screened by indirect immunofluorescence (IIF). By simultaneously applying IIF and the developed specific FISH, two independent targets for identification of R. solanacearum are combined, resulting in a rapid (1-day), accurate identification of the undesired pathogen. The significance of the method was validated by detecting the pathogen in soil and water samples and root tissue of the weed host Solanum dulcamara (bittersweet) in contaminated areas.     

Chemiluminescence: a sensitive detection system in in situ hybridization

PURPOSE: To quantify, with magnetic resonance (MR) imaging, the in vivo changes in cartilage volume and thickness after physical exercise. MATERIALS AND METHODS: The patellae of eight volunteers were imaged six times at physical test by using a spoiled fat-suppressed gradient-echo sequence with an acquisition time of 4.10 minutes. The volunteers then performed 50 knee bends, and two more data sets were acquired 3-7 minutes and 8-12 minutes after exercise. The patellar cartilage volume was determined after three-dimensional reconstruction, and the thickness was assessed with a three-dimensional minimal-distance algorithm. RESULTS: Whereas repositioning had a small effect on the measurements (mean coefficient of variation, 1.4%), a statistically significant decrease in cartilage volume was observed 3-7 minutes (mean decrease, 6.0%; P < .05) and 8-12 minutes (mean decrease, 5.2%; P < .05) after exercise. The deformation was homogeneous throughout the joint surface. In one asymptomatic volunteer, a cartilage lesion became more pronounced after exercise. CONCLUSIONS: MR imaging can be used to investigate the response of articular cartilage to physical exercise in vivo. Patients or volunteers should be allowed a sufficient period of physical rest if quantitative measurements of cartilage volume and thickness are to be undertaken in longitudinal studies.     

A FISH technique for simultaneous detection of fluorescent R-band and in situ hybridization signals]

BACKGROUND: Because of serious adverse effects of centrally acting antitussive agents, is necessary to find new drugs with cough-suppressing activity. Medicinal herbs are a potential source of polysaccharides with high antitussive efficiency and on the other with minimal side effects. AIM: The study was to assess the antitussive action of mixture of a polysaccharides (RL) and polysaccharide-xylan (XY), both isolated from above-ground parts of Rudbeckie fulgida var. sullivantii. The observed activity was compared to those of narcotic and nonnarcotic antitussive substances used in clinical practice. METHODS: Cough was evoked by mechanical irritation of the airways in nonanaesthetized cats with chronic tracheal cannuly. The plant substances were administered perorally in the dose of 50 mg per kg body weight. RESULTS: Results indicate, that administration of RL induced a suppression of the followed cough parameters from both areas of airways (total fall in cough parameters by 46.6%). Administration of xylan induced the fall in the followed cough parameters with more significant influence on the laryngopharyngeal area of the airways (total fall in cough parameters 48.2%). CONCLUSION: Administration of RL and xylan did not achieve the effect of codeine, but had a more intensive antitussive effect than the peripherally acting droprozine and prenoxdiazine. (Fig. 3, Ref. 11.)     

PCR detection of DNA specific for Trichosporon species in serum of patients with disseminated trichosporonosis

Deep-seated trichosporonosis is a lethal opportunistic infection that disseminates rapidly and widely in immunocompromised patients, and early diagnosis is crucial for the treatment of this infection. We developed a novel nested-PCR assay that detects DNA specific for clinically important strains of Trichosporon in serum samples from patients with disseminated trichosporonosis. In this assay, two sets of oligonucleotide primers were derived from the sequence of 26S rRNA genes of Trichosporon asahii. The specific fragment was amplified from T. asahii and T. mucoides, but not from other microorganisms, including some other basidiomycetous fungi (Cryptococcus, Malassezia, Rhodotorula, and Sporobolomyces). Target DNA was detected by the nested PCR with as little as 5 fg of the extracted DNA of T. asahii. In a study using 11 clinical samples, the specific fragment was detected by the nested PCR in 64% (7 of 11) of sera from patients with histologically diagnosed disseminated trichosporonosis, while glucuronoxylomannan antigen was detected in only 54% (6 of 11) of the samples. Our new nested-PCR assay using serum samples can be performed repeatedly throughout the course of the disease. In addition, not only can it be used for early diagnosis of trichosporonosis, but it may also be beneficial for monitoring its progress or response to therapy.     

Simultaneous detection of X- and Y-bearing human sperm by double fluorescence in situ hybridization

Double fluorescence in situ hybridization (FISH) was used to detect sex chromosomes in decondensed human sperm nuclei. Biotinylated X chromosome specific (TRX) and digoxigenin-labeled Y chromosome specific (HRY) probes were simultaneously hybridized to sperm preparations from 12 normal healthy donors. After the hybridization, the probes were detected immunocytochemically, using two different and independent affinity systems. Ninety-six percent of the 12,636 sperm showed fluorescent labeling, of which 47.4% were haploid X and 46.8% were haploid Y. A frequency of 0.46% of XX-bearing sperm (0.28% disomic, 0.18% diploid) and 0.38% YY-bearing sperm (0.21% disomic, 0.17% diploid) was found. The overall proportions of X- and Y-bearing sperm in the ejaculates were 47.9% and 47.2%, respectively, which was not significantly different from the expected 50:50 ratio. In addition 0.21% of cells appeared to be haploid XY-bearing sperm, 0.62% were diploid XY-bearing cells, and 0.05% of cells were considered to be tetraploid cells. The application of double FISH to human sperm using X-chromosome and Y-chromosome probes has allowed a more accurate assessment of the sex chromosal complements in sperm than single FISH method and quinacrine staining for Y-bodies.     

Non-radioactive detection of K-ras mutations by nested allele specific PCR and oligonucleotide hybridization

PURPOSE: The aim of the study was to evaluate prospectively urinary alpha 1-microglobulin as a marker of proximal tubular damage following acute pyelonephritis and outflow disease of the upper urinary tract in a urological population with minimal exclusion criteria. We also measured the urinary gamma-glutamyltransferase activity, urinary albumin, urinary and serum creatinine, serum IgA and serum alpha 1-microglobulin. PATIENTS AND METHODS: We studied 483 urological patients (age: 1 to 92 years, 297 men, 186 women) excluding patients receiving nephrotoxic drugs, or suffering from type 1 diabetes or renal diseases. There were 141 patients with urinary tract infection but no fever, 36 patients with high fever of non-renal origin, 51 patients with acute pyelonephritis and 156 patients with outflow disease of the upper tract, and 99 patients were included in the reference population. RESULTS: For acute pyelonephritis, vesico-ureteral reflux, and ureteral obstruction, urinary alpha 1-microglobulin had a sensitivity of 94%, 90% and 63% respectively and a specificity of 67%, 77% and 76%. The area under the curve of the receiver operator characteristic curve was significantly (p < 0.001) higher for urinary alpha 1-microglobulin than for albumin or gamma-glutamyltransferase activity. Unexpected positive results were found in acute prostatitis. The urinary alpha 1-microglobulin was the only parameter which differentiated between acute prostatitis and pyelonephritis (p < 0.001). Creatinine clearance or age had little and gender had no influence on the urinary excretion of alpha 1-microglobulin. Urine production rate significantly increases the urinary alpha 1-microglobulin/creatinine ratio. CONCLUSION: Our results suggest that the urinary alpha 1-microglobulin/creatinine ratio is a diagnostically useful marker of tubular damage in acute pyelonephritis and vesico-ureteral reflux in the urological population. Following renal colic and chronic ureteral obstruction, a significant increase in urinary alpha 1-microglobulin excretion was observed.     

Development of internal controls for PCR detection of Bacillus anthracis

Heat shock or stress proteins (Hsp) are typically regarded as being intracellular proteins that have a range of functions including the maintenance of cellular integrity. Members of the Hsp70 family of molecules have been implicated in the processing and presentation of antigen and the cross reactivity of lymphocytes specific for pathogen-derived heat shock proteins with self Hsp70 has been suggested to be an underlying cause of certain autoimmune diseases. This study reports the presence of soluble Hsp70 in the peripheral circulation of normal individuals. Concentrations of soluble Hsp70 in females were approximately twice those in males. Circulating anti-Hsp70 antibodies were detected in all individuals assessed, but there were no differences between males and females. However, there was a significant correlation between soluble Hsp70 concentration and antibody levels in males, but not females. The physiological role for circulating heat shock proteins is intriguing, but currently unknown. These findings extend our previous observations that Hsp60 is present in the peripheral circulation and support the proposition that soluble heat shock proteins may play a regulatory role in either the prevention or protection of pathophysiological processes involving inadvertent immunorecognition or cross-recognition of heat shock proteins.     

Detection of the leghemoglobin gene on two chromosomes of Phaseolus vulgaris by in situ PCR linked-fluorescent in situ hybridization (FISH)

OBJECTIVE: To investigate the effect of phentolamine, a sympathetic blocking agent, on the spontaneous electrical activity (SEA) recorded from a locus of a myofascial trigger spot (MTrS), equivalent to a human trigger point, in rabbit skeletal muscle. DESIGN: Randomized control trial. SETTING: A university medical laboratory. PATIENTS OR OTHER PARTICIPANTS: Nine adult New Zealand rabbits. INTERVENTION: In the experimental group phentolamine mesylate (1mg/kg) was injected into the external iliac artery, followed by flushing with normal saline. The control group was treated with normal saline instead of phentolamine using the same procedure. MAIN OUTCOME MEASURES: SEA was recorded from multiple active loci of MTrSs in the biceps femoris muscle: initially SEA in the same locus was recorded before and immediately after phentolamine (or normal saline) injection; then SEA was recorded from 25 different active loci. The mean of the average integrated signal (AIS) of SEA was analyzed, comparing the effects of phentolamine and normal saline on SEA. RESULTS: In the same active locus, the AIS of SEA showed statistically a linear decay with time after phentolamine injection, with a correlation coefficient of .56 at p < .05. However, no statistical relationship could be derived for the control group data with time by using regression analysis, probably because of large variations among the rabbits and movement artifacts during the experiment. In 25 different loci in the phentolamine group, the mean of the AIS of SEA (7.92 microV) was significantly lower than that of the control group (9.89 microV) at p < .05. CONCLUSIONS: The results support the hypothesis that the autonomic nervous system is involved in the pathogenesis of myofascial trigger points. The application of the AIS as an evaluation index seems to be feasible in the quantitative measurement of SEA.     

Fluorescence in situ hybridization for the detection and monitoring of the Ph-positive clone in chronic myelogenous leukemia: comparison with metaphase banding analysis

In order to analyze the efficiency of interphase FISH for the detection and monitoring of Ph+ cells in chronic myelogenous leukemia (CML) under interferon (IFN) treatment, the following experiments were performed: (1) 98 specimens derived from 32 patients were analyzed in parallel by dual-color FISH and by conventional chromosome analysis (CCA). A 300/200 kb BCR/ABL probe was used in all tests and a smaller 35.5/39 kb probe was tested in parallel in 22 BM samples; (2) 30 BM samples were prepared by direct harvest and by 24-h culture and were analyzed in parallel; (3) PB and BM samples obtained simultaneously from 11 patients were analyzed. The cut-off point for the recognition of BCR/ABL fusion was set at 2.4%, calculated as the mean percent of false positivity in 11 controls plus 3 s.d. A very close correlation was observed (r=0.994, r2=0.988, P < 0.0001) between the percentages of Ph+ cells as assessed by CCA and by interphase FISH in 98 samples (26 at diagnosis). There was a moderate overestimation of the frequency of Ph+ cells by FISH with respect to CCA, that was more evident at low-to-medium values of Ph positivity. Seven specimens without Ph+ metaphases (17-50 cells analyzed) were shown to carry 2.5-8% interphase cells with BCR/ABL fusion. Similar percentages of BCR/ABL+ nuclei were recorded in 22 samples hybridized using the 300/200 kb and the 35.5/39 kb probe-sets (variation range: 0-5%, mean 2.3%). A very good correlation between the frequency of Ph+ interphase cells was observed when analyzing in parallel BM preparations after direct harvest and after 24-h culture. Underestimation of the percentage of BCR/ABL+ cells was noted to occur in 2/11 PB samples, compared to BM samples, the remaining nine cases showing superimposable results at either sites. We arrived at the following conclusions: (1) dual-color FISH enables an accurate detection and monitoring of the size of the Ph-positive clone in CML at diagnosis and after IFN-therapy; (2) FISH is more accurate than CCA, especially at low levels of Ph-positive cells; (3) testing of directly harvested BM samples is feasible and accurate, giving the opportunity to perform centralized FISH analysis in the context of multicentre trials; (4) the percentage of BCR/ABL+ PB cells usually, though not invariably, reflects the frequency of mutated cells in the BM.     

Universal fungus-specific primer systems and group-specific hybridization oligonucleotides for 18S rDNA

We designed two primer systems that amplify a fragment of the gene coding for the small ribosomal subunit (18S rRNA). A broadly reactive, yet fungus-specific, primer cocktail comprises two previously published primers, TR1 and TR2, which specifically amplify dermatophytes, and two newly designed primers, CA1 and AF2, which specifically amplify Candida and Aspergillus respectively. This primer cocktail amplifies a DNA fragment of approximately 578 basepairs (bp) in length (from position 838 to 1415), which contains variable, possibly species-specific regions (V5, partly V7). Another newly designed primer, UF1 (universal fungal primer 1), along with the eukaryotic primer S3 amplifies a 926-bp fragment (from position 263 to 1188) that includes the variable regions V3, V4 and V5. Both primer systems amplified DNA from Saccharomyces cerevisiae, Candida albicans, Cryptococcus neoformans, Aspergillus fumigatus, Penicillium marneffei, Fusarium oxysporum and Trichophyton mentagrophytes, but not the DNA from Prototheca zopfii, Escherichia coli or humans. The previously published oligonucleotides TR and HC, which are specific for dermatophytes and Histoplasma respectively, and the newly designed group-specific oligonucleotides, CA and AF, hybridized with T. mentagrophytes, Histoplasma capsulatum, C. albicans and A. fumigatus respectively, but not with the other six fungi or with the three controls.     

Generation of type-specific probes for the detection of single-copy human papillomavirus by a novel in situ hybridization method

The integration of human papillomavirus (HPV) DNA is associated with the pathogenesis of HPV-associated malignancies. The ability, however, of standard in situ hybridization (ISH) to detect low-copy integrated HPV DNA is limited. We describe the generation of HPV type-specific biotin-labeled DNA probes and a novel ISH method that uses the catalyzed reporter deposition (CARD) system for the detection of single-copy target HPV DNA in formalin-fixed, paraffin-embedded tissue. Consensus primers flanking the noncoding region of HPVs were used to generate biotin-labeled HPV-6b, -11, -16 and -18 probes by polymerase chain reaction (PCR). The probes were used for ISH with the novel technique of CARD to increase the sensitivity of the assay. Tissue blocks were prepared from CaSki (500-600 copies of HPV-16), SiHa (1-2 copies of HPV-16), and HeLa (10-50 copies of HPV-18) cell lines, as well as from an HPV-negative cell line, C33A, and then tested to demonstrate the sensitivity and specificity of the probes. Surgical specimens were used to show the clinical applicability of this technique. We successfully detected HPV-16 DNA in CaSki and SiHa cells but not in HeLa or C33A cells. HPV-18 DNA was detected in HeLa cells but not in CaSki, SiHa, or C33A cells. Sensitivity was increased when ISH was performed using probes with more biotin incorporation or when more cycles of signal amplification were employed, but significant nonspecific background was observed after more than two cycles of signal amplification. The probes generated in this study detected specific types of HPV in surgical specimens with much higher sensitivity than did conventional ISH. We concluded that our new method was highly sensitive and could be applied to formalin-fixed, paraffin-embedded clinical material for the detection of HPV.     

In situ hybridization with riboprobes: an overview for veterinary pathologists

Although preliminary reports indicate that fatigue is a common symptom of human immunodeficiency virus (HIV) disease, little empirical research has focused on its prevalence or characteristics among patients with acquired immunodeficiency syndrome (AIDS). We assessed the frequency of fatigue and its medical and psychological correlates, in a cross-sectional survey of ambulatory AIDS patients. Ambulatory patients with AIDS who participated in a study of quality life (N = 427) were classified into fatigue/no fatigue groups based on their responses to fatigue items on the Memorial Symptom Assessment Scale (MSAS) and the AIDS physical symptom checklist. Self-report inventories were also administered to assess psychological distress, depressive symptoms, and overall quality of life. Medical information was elicited through clinical interview and review of medical chart. Fifty-four percent of the patients endorsed both of the fatigue items from the MSAS and the AIDS physical symptom checklists, and were classified as having fatigue. Women were significantly more likely to report fatigue than men (chi square = 5.28, df = 1, P < 0.03), and patients reporting homosexual contact as their transmission risk factor were significantly less likely to report fatigue than were patients reporting injection drug use or heterosexual contact (chi square = 5.13, df = 2, P < 0.03). The presence of fatigue was significantly associated with the number of current AIDS-related physical symptoms [t(425) = 8.00, P < 0.0001], current treatment for HIV-related medical disorders (chi square = 12.51, df = 1, P < 0.0001), anemia [t(174) = -2.35, P < 0.02], and pain (chi square = 36.36, df = 1 P < 0.0001). Patients with fatigue also had significantly poorer physical functioning ability [Karnofsky: t(422) = -6.27, P < 0.0001], as well as greater degree of overall psychological distress and lower quality of life [F(5,418) = 23.79, P < 0.0001], as measured by the Brief Symptom Inventory, Beck Depression Inventory, Beck Hopelessness Scale, Functional Living Inventory for Cancer (modified for AIDS), and the MSAS Psychological Distress Subscale. Fatigue is a common symptom in ambulatory AIDS patients and is associated with significant physical and psychological morbidity.     

In situ hybridization for the detection of telomerase RNA in the progression from Barrett''s esophagus to esophageal adenocarcinoma

AIMS: To compare the value of the proximal flow convergence method and the jet area method for the determination of the severity of tricuspid regurgitation. METHODS AND RESULTS: The proximal isovelocity surface area radius and the jet area/length were measured in 71 consecutive patients with angiographically graded (grade 0/I-III) tricuspid regurgitation. Rank correlation coefficients with the angiographic grade were 0.71 (P < 0.001) for the proximal isovelocity surface area radius (aliasing border of 28 cm.s-1), 0.66 (P < 0.001) for the jet area, and 0.63 (P < 0.001) for the jet length. The proximal isovelocity surface area radius was significantly correlated with the jet area/length (correlation coefficients 0.82/0.77, P < 0.001). Correct differentiation between mild to moderate (grade I-II) and severe (grade III) tricuspid regurgitation was achieved in 62 of 71 patients (87%) by means of the proximal isovelocity surface area radius, in 61 of 71 (86%) by the jet area, and in 62 of 71 (87%) by the jet length. Grade III tricuspid regurgitation was not identified in five of 21 patients (24%) by means of the proximal isovelocity surface area radius, in six of 21 (29%) by the jet area, and in seven of 21 (33%) by the jet length. CONCLUSION: The flow convergence method and the jet area method are of similar value for the determination of the severity of tricuspid regurgitation. Both methods differentiated mild to moderate from severe tricuspid regurgitation in most patients. However, underestimation of severe tricuspid regurgitation in 20-30% of the cases represents a serious limitation of both methods.