Relevance of biofilms in the pathogenesis of Shiga-toxin-producing Escherichia coli infection.

The present study was designed to determine the relationships among biofilm formation, cellular stress and release of Shiga toxin (Stx) by three different clinical Shiga toxin-producing Escherichia coli (STEC) strains. The biofilm formation was determined using crystal violet stain in tryptic soy broth or thioglycollate medium with the addition of sugars (glucose or mannose) or hydrogen peroxide. The reactive oxygen species (ROSs) were detected by the reduction of nitro blue tetrazolium and reactive nitrogen intermediates (RNI) determined by the Griess assay. In addition, the activities of two antioxidant enzymes, superoxide dismutase (SOD) and catalase (CAT), were studied. For the cytotoxicity studies, Vero cells were cultured with Stx released of STEC biofilms. The addition of sugars in both culture mediums resulted in an increase in biofilm biomass, with a decrease in ROS and RNI production, low levels of SOD and CAT activity, and minimal cytotoxic effects. However, under stressful conditions, an important increase in the antioxidant enzyme activity and high level of Stx production were observed. The disturbance in the prooxidant-antioxidant balance and its effect on the production and release of Stx evaluated under different conditions of biofilm formation may contribute to a better understanding of the relevance of biofilms in the pathogenesis of STEC infection.

Characterization of Shiga toxin-producing Escherichia coli O130:H11 and O178:H19 isolated from dairy cows.

Shiga toxin-producing E. coli (STEC) are isolated from human patients with bloody diarrhea, hemorrhagic colitis (HC), and hemolytic uremic syndrome (HUS). In the last years, the infections with non-O157 serotypes are increasing their frequency of association with human disease. STEC produce Shiga toxin (Stx) and other virulence factors that could contribute to human pathogenesis. Cattle are the main reservoir and the transmission to humans is through the consumption of undercooked meat, non-pasteurized dairy products, and vegetables or water contaminated with feces. We have previously determined that O130:H11 and O178:H19 serotypes were the most prevalent in dairy cows from Argentina. In the present study, 37 and 25 STEC isolates from dairy cows belonging to O130:H11 and O178:H19 serotypes, respectively, were characterized regarding to their cytotoxicity on Vero cells, stx subtypes, presence of sab and typing by multiple-locus variable-number tandem repeat analysis (MLVA). All strains demonstrated a cytotoxic effect, and in O130:H11 isolates, stx2EDL933 was the predominant subtype. In O178:H19 isolates the main stx2 subtype was stx2vha. The sab gene was detected in 65 and 24% of the isolates belonging to O130:H11 and O178:H19, respectively. Only one MLVA profile was identified among the O130:H11 isolates meanwhile 10 MLVA profiles were detected among the O178:H19 isolates which were grouped in two main clusters. In conclusion, our data show that O130:H11 and O178:H19 STEC isolates encode virulence factors associated with severe human disease and both serotypes should be considered for routinely testing. Our subtyping experiments showed that isolates could be distinguished based on the stx2 subtype and the presence/absence of sab gene, and for isolates belonging to O178:H19, also when the MLVA type was considered. However, MLVA subtyping of O130:H11 isolates will require the development of more specific markers.

Differences in Shiga toxin and phage production among stx(2g)-positive STEC strains.

Shiga toxin-producing Escherichia coli (STEC) are characterized by the production of Shiga toxins (Stx) encoded by temperate bacteriophages. Stx production is linked to the induction of the phage lytic cycle. Several stx variants have been described and differentially associated with the risk of developing severe illness. The variant named stx(2g) was first identified in a STEC strain isolated from the faeces of healthy cattle. Analysis of stx(2g)-positive strains isolated from humans, animals, and environmental sources have shown that they have a close relationship. In this study, stx(2g)-positive STEC isolated from cattle were analyzed for phage and Stx production, with the aim to relate the results to differences observed in cytotoxicity. The presence of inducible phages was assessed by analyzing the bacterial growth/lysis curves and also by plaque assay. Bacterial growth curves in the absence of induction were similar for all isolates, however, notably differed among induced cultures. The two strains that clearly evidenced bacteriolysis under this condition also showed higher phage titers in plaque assays. However, only the phage plaques produced by one of these strains (FB 62) hybridized with a stx(2)-probe. Furthermore, the production of Stx was evaluated by enzyme immunoassay (EIA) and Western immunoblotting in overnight supernatants. By EIA, we detected Stx only in supernatants of FB 62, with a higher signal for induced than uninduced cultures. By immunoblotting, Stx2 could be detected after induction in all stx(2g)-positive isolates, but with lower amounts of Stx2B subunit in those supernatants where phages could not be detected. Taking into account all the results, several differences could be found among stx(2g)-positive strains. The strain with the highest cytotoxic titer showed higher levels of stx(2)-phages and toxin production by EIA, and the opposite was observed for strains that previously showed low cytotoxic titers, confirming that in stx(2g)-positive strains Stx production is phage-regulated.

Subtyping of STEC by MLVA in Argentina.

Shiga toxin-producing Escherichia coli (STEC) causes serious human illness such as hemolytic uremic syndrome (HUS). Argentina has the world's highest rate of this syndrome, which is the leading cause of acute renal failure among children. E. coli O157:H7 is the most common cause of HUS, but a substantial and growing proportion of this illness is caused by infection due to non-O157 strains. Multiple-locus variable-number tandem repeat analysis (MLVA) has become an established technique to subtype STEC. This review will address the use of routine STEC subtyping by MLVA in order to type this group of isolates and to get insight into the genetic diversity of native STEC. With regard to these objectives we modified and adapted two MLVA protocols, one exclusive for O157 and the other, a generic E. coli assay. A total of 202 STEC isolates, from different sources and corresponding to 20 serotypes, have been MLVA genotyped in our laboratory. In our experience, MLVA constitutes a very sensitive tool and enables us to perform an efficient STEC subtyping. The diversity found in many serotypes may be useful for future epidemiological studies of STEC clonality, applied to O157 as well as to non-O157 isolates.

Molecular characterization of Shiga toxin-producing Escherichia coli isolated from the environment of a dairy farm.

Environmental samples were taken from ground, cattle water troughs, and feeders from a dairy farm with different STEC prevalence between animal categories (weaning calves, rearing calves, and dairy cows). Overall, 23 % of samples were positive for stx genes, stx(2) being the most prevalent type. Isolates were analyzed by PCR monoplex to confirm generic E. coli and by two multiplex PCR to investigate the presence of stx(1), stx(2), eae, saa, ehxA, and other putative virulence genes encoded in STEC plasmids: katP, espP, subA, and stcE. The toxin genes were subtyped and the strains were serotyped. The ground and the environment of the rearing calves were the sites with the highest number of STEC-positive samples; however, cattle water troughs and the environment of cows were the places with the greater chance of finding stx(2EDL933) which is a subtype associated with serious disease in humans. Several non-O157 STEC serotypes were detected. The serotypes O8:H19; O26:H11; O26:H-; O118:H2; O141:H-; and O145:H- have been asociated with human illness. Furthermore, the emergent pathogen STEC O157:H- (stx(1)-ehxA-eae) was detected in the environment of the weaning calves. These results emphasize the risk that represents the environment as source of STEC, a potential pathogen for human and suggest the importance of developing control methods designed to prevent contaminations of food products and transmission from animal to person.

Occurrence of subtilase cytotoxin and relation with other virulence factors in verocytotoxigenic Escherichia coli isolated from food and cattle in Argentina

We investigated the presence of the gene of subtilase cytotoxin (SubAB), described in certain highly virulent verocytotoxigenic E. coli strains, in isolates from Argentina and its relation with other virulence factors. The gene subA was present in eae-negative strains mostly associated with saa, vt2 and ehxA genes.

Genetic characterization of non-O157 verocytotoxigenic Escherichia coli isolated from raw beef products using multiple-locus variable-number tandem repeat analysis.

Verocytotoxigenic Escherichia coli (VTEC) can produce serious human illness linked to the consumption of contaminated food, mainly of bovine origin. There is growing concern about non-O157 VTEC serotypes, which in some countries cause severe infections in a proportion similar to O157:H7 strains. As several epidemiological studies indicated the important role of meat as the major vehicle in the transmission of this pathogen to human consumers, our aim was to investigate the genetic diversity among non-O157:H7 VTEC isolated from raw beef products. We performed a multiple-locus variable-number tandem repeat (VNTR) analysis (MLVA), and to our knowledge, this is the first time that VTEC serotypes O8:H19, O112:H2, O113:NM, O171:NM, ONT:H7, ONT:H19, and ONT:H21 were typed by this method. MLVA typing grouped the total number of strains from this study (51) into 21 distinct genotypes, and 11 of them were unique. Several MLVA profiles were found in different serotypes, O178:H19 being the most variable. The isolates could be principally discriminated by alleles of three of seven loci studied (CVN001, CVN004, and CVN014), and on the other hand, CVN003 rendered null alleles in all the isolates. As some VNTR markers might be serotype specific, it is possible that the implementation of new VNTR loci will increase intraserotype discrimination.

Factors associated with sporadic verotoxigenic Escherichia coli infection in children with diarrhea from the Central Eastern Area of Argentina.

Verotoxigenic Escherichia coli (VTEC) are one of the most important emerging foodborne pathogens and the principal cause of hemolytic uremic syndrome (HUS). This entity has been recognized worldwide as a priority issue in the field of zoonoses and public health, and Argentina is the country with the highest incidence of HUS in children less than 5 years of age.The lack of specific treatment, combined with the high morbidity rate of VTEC infection, makes prevention the main tool for reducing the incidence of HUS. The current work aimed at assessing the factors associated with sporadic VTEC infection in children with acute diarrhea from the Central Eastern area of Argentina where the incidence rate of HUS in children under 5 is the highest worldwide. A univariate analysis was performed to identify potential factors associated with VTEC infection by calculating odds ratios (OR) with 95% confidence intervals (CI). Then, a multivariate logistic regression model was performed. Interaction and association between significant factors were checked. "Recent consumption of food prepared outside home" (OR: 2.4, 95% CI 1.05-5.7) and "recent vegetables consumption" (OR=0.4; 0.2-0.8) were identified as independent factors associated with VTEC infection. We believe that the data obtained from this study further the current knowledge about the epidemiology of VTEC infection in Argentina and could be considered when planning strategies for the prevention of the disease.

Verotoxins in bovine and meat verotoxin-producing Escherichia coli isolates: type, number of variants, and relationship to cytotoxicity.

In this study, we determined vt subtypes and evaluated verotoxicity in basal as well as induced conditions of verotoxin-producing Escherichia coli (VTEC) strains isolated from cattle and meat products. Most (87%) of the 186 isolates carried a vt(2) gene. Moreover, the vt(2) subtype, which is associated with serious disease, was present in 42% of our VTEC collection. The other vt subtypes detected were vt(1), vt(1d), vt(2vha), vt(2vhb), vt(2O118), vt(2d) (mucus activatable), and vt(2g). A total of 41 (22%) of the isolates possessed more than one vt subtype in its genome, and among them the most frequent combination was vt(1)/vt(2), but we also observed multiple combinations among vt(2) subtypes. Differences in verotoxicity titers were found among a selection of 54 isolates. Among isolates with a single vt(2) variant, those carrying the vt(2) subtype had high titers under both uninduced and induced conditions. However, the highest increase in cytotoxicity under mitomycin C treatment was detected among the strains carrying vt(2vha) or vt(2hb) variants. Notably, the isolates carrying the vt(1) subtype showed a lesser increase than that of most of the vt(2)-positive VTEC strains. Furthermore, the presence of more than one vt gene variant in the same isolate was not reflected in higher titers, and generally the titers were lower than those for strains with only one gene variant. The main observation was that both basal and induced cytotoxic effects seemed to be associated with the type and number of vt variants more than with the serotype or origin of the isolate.

Occurrence of subtilase cytotoxin and relation with other virulence factors in verocytotoxigenic Escherichia coli isolated from food and cattle in Argentina.

We investigated the presence of the gene of subtilase cytotoxin (SubAB), described in certain highly virulent verocytotoxigenic E. coli strains, in isolates from Argentina and its relation with other virulence factors. The gene subA was present in eae-negative strains mostly associated with saa, vt2 and ehxA genes.

Genetic diversity of O157:H7 and non-O157 verocytotoxigenic Escherichia coli from Argentina inferred from multiple-locus variable-number tandem repeat analysis (MLVA).

Although serotype O157:H7 has been implicated in most cases of haemolytic-uraemic syndrome (HUS), there is growing concern about non-O157 serotypes of verocytotoxigenic Escherichia coli (VTEC). Multiple-locus variable-number tandem repeat analysis (MLVA) has been focused on the specific typing of O157:H7 isolates, but recently, a generic MLVA assay for E. coli and Shigella has been developed. We performed a study of the polymorphism in 7 generic VNTR loci both in VTEC O157:H7 and non-O157 isolates from Argentina, in order to asses the ability of the method to type this group of isolates and to get insight into their genetic diversity. Sixty-four isolates from cattle, patients with diarrhoea, and contaminated food belonging to 8 different serotypes were studied. All of them could be typed by this method and revealed 41 different MLVA genotypes. The MLVA dendrogram showed 2 main clusters which corresponded to O157:H7 and non-O157, respectively. Our results confirm the suitability of this MLVA method for analyzing VTEC isolates belonging to several serotypes, both O157:H7 as well as non-O157, highlight the genetic variability of the O157:H7 serotype and the need of additional research in order to find more VNTR loci that could allow a higher discrimination among non-O157 VTEC.

Role and clinical course of verotoxigenic Escherichia coli infections in childhood acute diarrhoea in Argentina.

The aim of this study was to investigate the role and clinical course of verotoxigenic Escherichia coli (VTEC) infections in children with acute diarrhoea from Argentina, the country with the highest worldwide incidence of haemolytic uraemic syndrome (HUS). To accomplish this objective, 437 samples from children up to 6 years old with acute diarrhoea were collected and processed. More than 60 % of the children studied presented watery or mucous diarrhoea without blood, and in 25.2 % of the cases the samples contained blood. In a first screening, a multiplex PCR was performed to detect the presence of the vt(1), vt(2), eae, ehxA and saa virulence genes. The strains were then isolated and analysed to characterize their serotypes, virulence genes, antibiotic susceptibility profiles and verotoxin (VT) production. Forty-four of the 437 samples (10.1 %) were positive for VTEC virulence genes. VTEC-infected patients presented different types of diarrhoea (27.3 % belonged to the non-bloody type). Several serotypes and virulence genotypes were found. Isolates belonged to the serotypes O157 : H7, O145 : H(-), O26 : H11, O121 : H19, O111 : H2 and O118 : H2. HUS developed in 16 (36.4 %) patients positive for VTEC virulence genes. All of the VTEC isolates produced a cytopathic effect on Vero cell monolayers, confirming the ability to express VT. Despite most strains being sensitive to all of the antimicrobials studied, a positive association between clinical progression to HUS and antibiotic therapy was observed for the total number of patients studied, as well as for the VTEC(+) group. In conclusion, the data obtained in this study increase our knowledge of the role and clinical course of VTEC infection in childhood acute diarrhoea beyond bloody diarrhoea, and might be considered for the prevention, diagnosis and management of this disease. It is possible that the optimal approach for VTEC diagnosis could be using multiplex PCR to search for the presence of the vt(1), vt(2), eae and ehxA genes.

Molecular characterization of Verocytotoxigenic Escherichia coli O157: H7 isolates from Argentina by Multiple-Loci VNTR analysis (MLVA)

The aim of this work was to adapt described MLVA protocols to the molecular typing and characterization of VTEC O157:H7 isolates from Argentina. Nine VNTR loci were amplified by PCR showing diversity values from 0.49 to 0.73. Nine MLVA profiles were observed and the cluster analysis indicated both unrelated and closely related VTEC O157:H7 strains. In spite of the limited number of isolates studied, the panel of VNTR used made it possible to perform a first approach of the high genetic diversity of native strains of O157:H7 by MLVA.

Molecular characterization of Verocytotoxigenic Escherichia coli O157:H7 isolates from Argentina by Multiple-Loci VNTR Analysis (MLVA).

The aim of this work was to adapt described MLVA protocols to the molecular typing and characterization of VTEC O157:H7 isolates from Argentina. Nine VNTR loci were amplified by PCR showing diversity values from 0.49 to 0.73. Nine MLVA profiles were observed and the cluster analysis indicated both unrelated and closely related VTEC O157:H7 strains. In spite of the limited number of isolates studied, the panel of VNTR used made it possible to perform a first approach of the high genetic diversity of native strains of O157:H7 by MLVA.

Evaluation of vt2-subtyping methods for identifying vt2g in verotoxigenic Escherichia coli.

Verotoxin-producing Escherichia coli (VTEC) are important pathogens that can cause severe human disease, including haemorrhagic colitis and haemolytic-uraemic syndrome. A new variant of verotoxin, vt2g, has recently been described. It was possible to find this variant for the first time in Argentina among VTEC isolated from cattle. The present study evaluated the identification of this gene with three conventional methods used for subtyping the vt2 gene. The results show that it is possible to screen VTEC strains for the presence of vt2g without the implementation of new protocols.

Intraserotype diversity among Argentinian verocytotoxigenic Escherichia coli detected by random amplified polymorphic DNA analysis.

Most cases of diarrhoea-associated haemolytic uraemic syndrome (HUS) are caused by verocytotoxin-producing Escherichia coli (VTEC). Argentina has the highest worldwide incidence of HUS, but with a lower incidence of VTEC O157 : H7 serotype than non-Latin American countries. A large number of VTEC serotypes have been isolated from cattle and cattle-derived food products in Argentina. The aim of this work was to study intraserotype genetic diversity among these VTEC strains by random amplification of polymorphic DNA (RAPD). Strains were selected that belonged to the same serotype, but had been isolated from different sources (cattle and meat). Intraserotype genetic diversity was detected among strains belonging to O20 : H19, O113 : H21, O117 : H7, O157 : H7, O171 : H2 and O174 : H21, but only one RAPD profile corresponded to strains belonging to O91 : H21, although these isolates were from different sources.

Distribution of saa gene variants in verocytotoxigenic Escherichia coli isolated from cattle and food.

The pathogenesis of verocytotoxigenic Escherichia coli (VTEC) infection in humans is multifactorial, given that verocytotoxins are the principal virulence factor. Most strains causing serious diseases possess the eae gene that encodes the adhesin intimin, but its presence is not essential for virulence as some cases are caused by eae-negative strains. An autoagglutinating adhesin designated Saa was found in some eae-negative strains. This protein varies in size as a consequence of variation in the number of copies of a 37-aa repeat unit in the C-terminal region. Based on these findings, we designed PCR primers to amplify the region coding for these differences to detect saa gene variants present in VTEC strains isolated in Argentina from cattle and meat. The gene saa was detected in 36 (31.6%) eae-negative strains and 5 variants were found. Strains isolated from cattle possessed 4 saa variants, whereas 2 variants were present in isolates from meat. Saa variant 1 predominated (18 strains) and was distributed in strains isolated both from cattle and from meat. Our study revealed the existence of two novel saa variants, termed 4 and 5, which have a higher number of 111-bp repeats than saa genes previously studied.

Virulence genes and intimin types of Shiga-toxin-producing Escherichia coli isolated from cattle and beef products in Argentina / Genes de virulencia y tipos de intiminas de Escherichia coli productoras de toxinas Shiga aisladas de ganado bovino y carne de vacuno en Argentina

A total of 153 Shiga-toxin-producing Escherichia coli (STEC) isolates from feces of cattle and beef products (hamburgers and ground beef) in Argentina were characterized in this study. PCR showed that 22 (14%) isolates carried stx1 genes, 113 (74%) possessed stx2 genes and 18 (12%) both stx1 and stx2. Intimin (eae), enterohemolysin (ehxA), and STEC autoagglutinating adhesin (saa) virulence genes were detected in 36 (24%), 70 (46%) and in 34 (22%) of the isolates, respectively. None of 34 saa-positive isolates carried the gene eae, and 31 were ehxA-positive. Fourteen (7 of serotype O26:H11 and 4 of serotype O5:H-) isolates had intimin b1, 16 isolates possessed intimin g1 (11 of serotype O145:H- and 5 of serotype O157:H7), 5 isolates had intimin type e1 (4 of serotypes O103:H- and O103:H2), and one isolate O111:H- showed intimin type q/g2. Although the 153 STEC isolates belonged to 63 different seropathotypes, only 12 accounted for 58% of isolates. Seropathotype ONT:H- stx2 (18 isolates) was the most common, followed by O171:H2 stx2 (12 isolates), etc. The majority (84%) of STEC isolates belonged to serotypes previously found in human STEC and 56% to serotypes associated with STEC isolated from patients with hemolytic uremic syndrome (HUS). Thus, this study confirms that cattle are a major reservoir of STEC pathogenic for humans. To our knowledge, this is the first study that described the presence of saa gene in STEC of serotypes O20:H19, O39:H49, O74:H28, O79:H19, O116:H21, O120:H19, O141:H7, O141:H8, O174:H21, and ONT:H21. The serotypes O120:H19 and O185:H7 were not previously reported in bovine STEC (AU)

En este estudio hemos caracterizado un total de 153 Escherichia coli productores de toxinas Shiga (STEC) aisladas de las heces de ganado bovino y de carne picada y hamburguesas de vacuno en Argentina. Los ensayos de PCR mostraron que 22 (14%) aislamientos llevaban el gen stx1, 113 (74%) presentaban el gen stx2 y que 18 (12%) tenían ambos genes. Los genes de virulencia para la intimina (eae), la enterohemolisina (ehxA) y la adhesina autoaglutinante de STEC (saa) fueron detectados en 36 (24%), 70 (46%) y 34 (22%) de los aislamientos, respectivamente. Ninguno de los 34 aislamientos saa-positivos llevaba el gen eae, pero 31 eran ehxA-positivos. Catorce aislamientos (7 del serotipo O26:H11 y 4 del serotipo O5:H-) tenían la intimina b1, 16 poseían la intimina g1 (11 del serotipo O145:H- y 5 del serotipo O157:H7), 5 aislamientos eran positivos para la intimina tipo ε1 (4 de los serotipos O103:H- y O103:H2), y un aislamiento O111:H- mostró la intimina tipo θ/g2. Aunque los 153 aislamientos de STEC pertenecían a 63 seropatotipos, sólo 12 constituían el 58% de los aislamientos. El seropatotipo ONT:H- stx2 (18 aislamientos) fue el más común, seguido por el O171:H2 stx2 (12 aislamientos), etc. La mayoría de los aislamientos (84%) de STEC pertenecían a serotipos encontrados previamente en seres humanos y el 56% a serotipos asociados con STEC aislados de pacientes con el síndrome urémico hemolítico (HUS). Por tanto, este estudio confirma que el ganado bovino es un importante reservorio de STEC patógenos para humanos. Según nuestra información, este es el primer estudio que describe la presencia del gen saa en STEC de los serotipos O20:H19, O39:H49, O74:H28, O79:H19, O116:H21, O120:H19, O141:H7, O141:H8, O174:H21, y ONT:H21. Los serotipos O120:H19 y O185:H7 tampoco habían sido descritos previamente en STEC de origen bovino (AU)

[Enterohemorrhagic Escherichia coli and hemolytic-uremic syndrome in Argentina]. / Escherichia coli enterohemorrágica y síndrome urémico hemolítico en Argentina.

The hemolytic-uremic syndrome (HUS) is a multisystemic disorder that is characterized by the onset of acute renal failure, microangiopathic hemolytic anemia and thrombocytopenia. It is the most common cause of acute renal failure and the second cause of chronic renal failure and renal transplantation in children in Argentina. Our country has the highest incidence of HUS in the world, with approximately 420 new cases observed each year with an incidence of 12.2 cases per 100,000 children in the age group 0-5 years. Numerous etiologic factors have been associated with HUS but the infection with enterohemorrhagic Escherichia coli (EHEC) is considered the most common cause. The majority of outbreaks and sporadic cases in humans have been associated with serotype O157:H7, although other O:H serotypes have been isolated, and they are a subgroup of Verocytotoxin-producing Escherichia coli (VTEC). Cattle are the principal reservoir of VTEC. Infections in humans are a consequence of consumption of undercooked meat, raw milk and other contaminated food or water. Direct contact with animals or people infected is another source of infection.

Mixed epithelial and stromal tumors of the kidney. A report of 22 cases.

Mixed epithelial and stromal tumor of the kidney (MESTK) is a recently described subset of renal neoplasm that tends to occur in middle-aged and older women and is characterized by a distinctive histological appearance. To further characterize this lesion, we report the clinicopathological and immunohistochemical features of 22 additional cases from our institutional files. Grossly, the tumors ranged in size from 1 cm to 14 cm (mean 6.7 cm), were well circumscribed but unencapsulated, and showed a cystic cut surface. The tumors were composed of a spindle cell proliferation that resembled ovarian stroma, as well as an epithelial component lining the cystic structures, which usually consisted of flat to hobnailed cells typical of collecting-duct epithelium. Areas displaying features of Mullerian differentiation were also documented in 6 cases, including epithelium of endometrioid, tubal, clear cell and squamous cell type as well as one case showing an architecture that closely resembled Mullerian adenofibroma and adenosarcoma. Follow-up in 14 patients (average 4.4 years) showed no evidence of recurrence or metastasis. We believe these tumors represent the renal counterpart of similar mixed epithelial and stromal neoplasms occurring in the biliary tract and pancreas, which is also characterized by cystic structures lined by epithelium, admixed with ovarian-type stroma. The differential diagnosis for these tumors includes cystic nephroma and cystic partially differentiated nephroblastoma, which we believe to represent clinically and morphologically distinct entities from MESTK. In particular, the distinction from cystic nephroma in adult male patients is emphasized, and two cases of this entity are included in the study for comparison.