Desarrollo de un producto anti-toxina shiga para la prevención del síndrome urémico hemolítico / Development of a product anti-Shiga toxin for prevention of the hemolytic uremic syndrome

El síndrome urémico hemolítico (SUH) típico es una enfermedad huérfana causada por cepas de Escherichia coli productoras de toxina Shiga (Stx) y caracterizada por daño renal agudo, anemia hemolítica microangiopática y plaquetopenia. Es endémico en Argentina, el país con mayor incidencia de SUH en el mundo. Debido al rol fundamental de la Stx en su patogenia, se puede considerar que, como otras toxemias conocidas, el SUH podría ser tratado con anticuerpos. Este trabajo describe el desarrollo de un nuevo tratamiento capaz de neutralizar el efecto tóxico de distintas variantes de la Stx. El tratamiento consiste en fragmentos F(ab')2 provenientes de un antisuero equino cuya eficacia y potencia contra Stx1 y Stx2 se comprobó en diferentes modelos preclínicos. El producto mostró ser seguro en animales, presentó la farmacocinética descripta para compuestos similares y se pudo establecer una posible ventana terapéutica para su adecuada administración. En conjunto, los resultados preclínicos obtenidos validan la realización de un estudio clínico de primer uso en humanos. En dicho estudio, que se realizará en el Hospital Italiano de Buenos Aires, se analizará la seguridad y la farmacocinética del producto en voluntarios adultos sanos. Estos resultados sentarán las bases para la realización del estudio clínico fase II en pacientes pediátricos con infección por cepas de E. coli productoras de Stx.

The typical hemolytic uremic syndrome (HUS) is an orphan disease caused by Shiga toxin(Stx) -producing Escherichia coli strains and characterized by acute kidney damage, microangiopathic hemolytic anemia and low platelet count. It is endemic in Argentina, the country with the highest incidence of HUS in the world. Stx is essential for its development and therefore, HUS is considered a toxemic non-bacteremic disorder, which could be treated with antibodies. Herein we describe the development of a new treatment capable of neutralizing the toxic effect of Stx and its variants. The treatment consists of F(ab')2 fragments from an equine antiserum whose efficacy and potency against Stx1 and Stx2 were proved in different preclinical models. The product was shown to be safe in animals. Furthermore, the anti-Stx F(ab')2 pharmacokinetic was shown to be similar to that of analogous compounds and a therapeutic window for its administration was determined. Altogether, these preclinical results warrant testing in humans. The phase I clinical trial will be performed at the Hospital Italiano in Buenos Aires to evaluate the safety and pharmacokinetics of the product in healthy adult volunteers. Based on the results of this study, a phase II clinical trial will be planned in pediatric patients diagnosed with infection by Stx-producing E. coli strains.

Predictors of Success of Selective Laser Trabeculoplasty Adjusted for Intraocular Pressure Variations

PURPOSE: To investigate the efficacy, and identify predictors of success of selective laser trabeculoplasty (SLT) in open-angle glaucoma (OAG) patients after adjusting for intraocular pressure (IOP) changes in the untreated fellow eye. METHODS: This retrospective chart review included 52 eyes of 52 OAG patients who underwent SLT in one eye and were followed-up for at least 1 year after the procedure. The IOP was measured before the treatment, at 1, 2, and 3 months posttreatment, and every 3 months thereafter. To account for the possible influence of IOP fluctuations on laser outcomes, post-laser IOP values of the treated eye of each patient were also analyzed, after adjusting for IOP changes in the untreated fellow eye. Success was defined as an IOP decrease ≥20% of the pretreatment IOP. The success rate was determined based on Kaplan-Meier survival analysis and factors predictive of success were analyzed using the Cox proportional hazard model. RESULTS: The mean pretreatment IOP was 23.17 ± 6.96 mmHg. The mean IOP reduction was 5.59 ± 4.78 mmHg (29.7%) and the success rate was 65.4% at 1 year. The adjusted mean IOP reduction was 4.70 ± 4.67 mmHg (23.9%) and the adjusted success rate was 53.9%. Pretreatment IOP was associated with SLT success; the higher the pretreatment IOP, the greater the post-laser IOP reduction (p = 0.025). Age and mean deviation index did not show a significant association with SLT success (p = 0.066 and p = 0.464, respectively). CONCLUSIONS: SLT is a safe and effective alternative method of IOP reduction in OAG patients. Herein, pretreatment IOP was the only factor significantly associated with SLT success. IOP fluctuations of the untreated eye should be considered for a better understanding of the impact of treatment.

Distribution of the stx1 and stx2 genes in Escherichia coli isolated from milk cattle according to season, age, and production scale in southwestern region of Goiás, Brazil / Distribuição dos genes stx1 e stx2 em Escherichia coli isoladas de bovinos de leite de acordo com a estação, idade e escala de produção na região sudoeste de Goiás, Brasil

This study determined the distribution of stx1 and stx2 genes in Escherichia coli isolated from dairy herds with regard to animal age, season, and farm production-scale, and analyzed the phylogenetic distribution of the groups A, B1, B2, and D of 276 isolates of bovine feces Shiga toxin-producing E. coli (STEC). The stx1 profile was the most common, detected in 20.4% (202/990) of the isolates, followed by stx2 (4.54%, 45/990) and stx1+stx2 (2.92%, 29/990). The stx1 gene was detected more frequently in calves than in adult animals. In the dry season (winter), the presence of stx1+stx2 profile in cattle feces was higher than in the rainy season (summer), while no significant changes were observed between seasons for the stx1 and stx2 profiles. The most predominant phylogenetic groups in adult animals were B1, A, and D, while groups A and B1 prevailed in calves. Our data highlight the importance of identifying STEC reservoirs, since 7.5% of the tested isolates were positive for stx2, the main profile responsible for the hemolytic-uremic syndrome (HUS). Moreover, these microorganisms are adapted to survive even in hostile environments and can contaminate the food production chain, posing a significant risk to consumers of animal products.(AU)

Esse estudo determinou a distribuição dos genes stx1 e stx2 em Escherichia coli isolados de rebanhos leiteiros em relação a idade, estação e produção, e analisaram a distribuição filogenética dos grupos A, B1, B2 e D de 276 E. coli produtoras de toxina Shiga (STEC). O perfil stx1 foi mais comum, detectado em 20,4% (202/990) dos isolados, seguido de stx2 (4,54%, 45/990) e stx1+stx2 (2,92%, 29/990). O gene stx1 foi detectado mais frequentemente em bezerros que animais adultos. No período de seca (inverno), a presença do perfil stx1+stx2 nas fezes dos bovinos foi mais prevalente que no período chuvoso (verão), apesar de não haver diferença significativa entre estações para os perfis stx1 e stx2. Os grupos filogenéticos mais predominantes em animais adultos foram B1, A e D, enquanto grupos A e B2 prevaleceram em bezerros. Nossos dados enfatizam a importância de se detectar reservatórios de STEC já que 7,5% dos isolados testados foram positivos para stx2, o perfil mais prevalente em casos de síndrome hemolítica-urêmica. Ademais, esses microorganismos são adaptados à sobreviver em ambientes hostis e contaminam a cadeia alimentar, levando a risco significativo para consumidores de alimentos de origem animal.(AU)

A Case of Hyphema after Selective Laser Trabeculoplasty

PURPOSE: To report a case of hyphema after selective laser trabeculoplasty (SLT) in a patient with pseudoexfoliative glaucoma. CASE SUMMARY: A 77-year-old female was referred for elevation of intraocular pressure (IOP). Previously, she had been diagnosed with pseudoexfoliative glaucoma in the right eye and was using topical IOP-lowering agents. The best corrected visual acuity was 20/100 in the right eye and 20/40 in the left eye. IOP, measured with Goldmann applanation tonometer, was 32 mm Hg in the right eye and 20 mm Hg in the left eye. Gonioscopy revealed open-angle glaucoma with +2 trabecular meshwork pigmentation but without peripheral anterior synechiae or neovascularization. SLT was performed in the right eye. Two days later, the patient had sudden onset of blurred vision and pain in the right eye. Visual acuity was limited to light perception, and IOP was 34 mm Hg in the right eye. Slit-lamp examination revealed 1.1 mm hyphema with 4+ red blood cell count in the anterior chamber. Three weeks after the SLT, hyphema in the right eye disappeared, but IOP was measured to be 42 mm Hg. The patient underwent trabeculectomy in the right eye. CONCLUSIONS: SLT is an effective means of lowering IOP with low risk of complications. However, hyphema can rarely occur after SLT and can affect the outcome of the treatment.

Detection of E. coli O157:H7 and Shigella dysenteriae toxins in clinical samples by PCR-ELISA

Shiga toxin producing bacteria are potential causes of serious human disease such as hemorrhagic colitis, severe inflammations of ileocolonic regions of gastrointestinal tract, thrombocytopenia, septicemia, malignant disorders in urinary ducts, hemolytic uremic syndrome (HUS) Shiga toxin 1 (stx1), shiga toxin 2 (stx2), or a combination of both are responsible for most clinical symptoms of these diseases. A lot of methods have been developed so far to detect shiga toxins such as cell culture, ELISA, and RFPLA, but due to high costs and labor time in addition to low sensitivity, they have not received much attention. In this study, PCR-ELISA method was used to detect genes encoding shiga toxins 1 and 2 (stx1 and stx2). To detect stx1 and stx2 genes, two primer pairs were designed for Multiplex-PCR then PCR-ELISA. PCR products (490 and 275, respectively) were subsequently verified by sequencing. Sensitivity and specificity of PCR-ELISA method were determined by using genome serial dilution and Enterobacteria strains. PCR-ELISA method used in this study proved to be a rapid and precise approach to detect different types of shiga toxins and can be used to detect bacterial genes encoding shiga toxins.

contribution on Coliforms causing mastitis in cows with reference to serotypes and virulence factors of E. coli isolates

Escherichia coli [E. coli] is the predominant coliform species causing intramammary infections. Where in the present study, E. coll isolates were 1.8 strains [17.82%] followed by Enterobacter aerogenes 3 strains [2.97%] and Klebsiella pneumoniae one strain [0.99%] from 101 clinical mastitic milk samples of cows. Eighteen E. coli isolates were serotyped to nine different serogroups; O111:H4 [3], O127:H6 [3], O26 [2], O126 [2], O119:H6 [1], O114:H21 [1], O55:H7 [1], O44:H18 [1], O124[1] and [3] untyped. Virulence tests were performed on the 18 isolated E. coll, it was found that 15 isolates [83.3%] were serum resistant, 13 isolates [72.2%] had Congo Red binding activity, 6 isolates [33.3%] were invasive and one isolate [5.6%] had haemolytic activity. PCR was applied to detect the presence of Shiga like toxin producing E. coll [stxl and stx2 genes] on the nine different strains[one strain for each serogroup], where stxl and stx2 were found in 8 [88.9%] and 4 [44.4%] of the nine examined strains, respectively. While stxl and stx2 genes were found together in 3 strains [33.3%]. Conclusions: E. coli isolates usually posses one or more virulence factors that may help in establishment at the infection site and subsequently causing clinical bovine mastitis

Some antibiotic resistant bacteria of public health hazard isolated from raw milk sold in some Assiut City markets

A total of 50 raw milk samples were collected in this study from some Assiut City markets, Egypt, and examined for isolation of some human hazard pathogens. The percentages of the isolated pathogens were 46, 76, 78, 4 and 24% for Staph. aureus. Enterococcus faecalis, E. coli, Citrobacter freundii and Yersinia enterocolitica respectively. In vitro Antibiogram was carried out on all isolates against [8] different antimicrobial agents; moreover, ail of these isolates showed multi-drug resistance against tow or more of the tested antibiotics. The public heath hazards of the isolated pathogens were alsfrtliscussed

Prevalence and characteristics of Shiga toxin-producing Escherichia coli (STEC) from cattle in Korea between 2010 and 2011

A total of 156 Shiga-like toxin producing Escherichia coli (STEC) were isolated from fecal samples of Korean native (100/568, 18%) and Holstein dairy cattle (56/524, 11%) in Korea between September 2010 and July 2011. Fifty-two STEC isolates (33%) harbored both of shiga toxin1 (stx1) and shiga toxin2 (stx2) genes encoding enterohemolysin (EhxA) and autoagglutinating adhesion (Saa) were detected by PCR in 83 (53%) and 65 (42%) isolates, respectively. By serotyping, six STEC from native cattle and four STEC from dairy cattle were identified as O-serotypes (O26, O111, O104, and O157) that can cause human disease. Multilocus sequence typing and pulsed-field gel electrophoresis patterns highlighted the genetic diversity of the STEC strains and difference between strains collected during different years. Antimicrobial susceptibility tests showed that the multidrug resistance rate increased from 12% in 2010 to 42% in 2011. Differences between isolates collected in 2010 and 2011 may have resulted from seasonal variations or large-scale slaughtering in Korea performed to control a foot and mouth disease outbreak that occurred in early 2011. However, continuous epidemiologic studies will be needed to understand mechanisms. More public health efforts are required to minimize STEC infection transmitted via dairy products and the prevalence of these bacteria in dairy cattle.

Development of a multiplex loop-mediated isothermal amplification assay to detect shiga toxin-producing Escherichia coli in cattle

A multiplex loop-mediated isothermal amplification (mLAMP) assay was developed for simultaneous detection of the stx1 and stx2 genes and applied for detection of shiga toxin-producing Escherichia coli (STEC) in cattle farm samples. Two target genes were distinguished based on T m values of 85.03 +/- 0.54degrees C for stx1 and 87.47 +/- 0.35degrees C for stx2. The mLAMP assay was specific (100% inclusivity and exclusivity), sensitive (with a detection limit as low as 10 fg/microL), and quantifiable (R 2 = 0.9313). The efficacy and sensitivity were measured to evaluate applicability of the mLAMP assay to cattle farm samples. A total of 12 (12/253; 4.7%) and 17 (17/253; 6.7%) STEC O157, and 11 (11/236; 4.7%) non-O157 STEC strains were isolated from cattle farm samples by conventional selective culture, immunomagnetic separation, and PCR-based culture methods, respectively. The coinciding multiplex PCR and mLAMP results for the types of shiga toxin revealed the value of the mLAMP assay in terms of accuracy and rapidity for characterizing shiga toxin genes. Furthermore, the high detection rate of specific genes from enrichment broth samples indicates the potential utility of this assay as a screening method for detecting STEC in cattle farm samples.

Molecular characterization of Escherichia coli O157:H7 strains isolated from different sources and geographic regions

Escherichia (E.) coli serotype O157:H7 is a globally distributed human enteropathogen and is comprised of microorganisms with closely related genotypes. The main reservoir for this group is bovine bowels, and infection mainly occurs after ingestion of contaminated water and food. Virulence genetic markers of 28 O157:H7 strains were investigated and multilocus enzyme electrophoresis (MLEE) was used to evaluate the clonal structure. O157:H7 strains from several countries were isolated from food, human and bovine feces. According to MLEE, O157:H7 strains clustered into two main clonal groups designated A and B. Subcluster A1 included 82% of the O157:H7 strains exhibiting identical MLEE pattern. Most enterohemorrhagic E. coli (EHEC) O157:H7 strains from Brazil and Argentina were in the same MLEE subgroup. Bovine and food strains carried virulence genes associated with EHEC pathogenicity in humans.

Comparison of 180degrees and 360degrees Selective Laser Trabeculoplasty

PURPOSE: To compare the effect and safety of two regimens of Selective Laser Trabeculoplasty (SLT), SLT on 180degrees of trabecular meshwork and SLT with 100 laser spots on 360degrees of trabecular meshwork in patients with primary open-angle glaucoma and ocular hypertension. METHODS: In a retrospective clinical study, the authors compared the pressure-lowering effects of SLT in two groups of patients; group 1 (83 patients) received SLT on 180degrees, group 2 (30 patients) on 360degrees of trabecular meshwork. The clinical outcome indicators included intraocular pressure (IOP) at one day, one week, one month, two months, three months and six months after SLT, and the anterior chamber reaction at post-laser one day. RESULTS: There was no statistically significant difference in the IOP reduction between these two regimens after six months. The anterior chamber reactions in the two groups were significantly different (group 1; 0.61 +/- 0.64, group 2; 1.25 +/- 0.83, p = 0.001). The success rate of group 2 (43.3%) was not different from that of group 1 (31.3%; p = 0.23). CONCLUSIONS: SLT on 180degrees of trabecular meshwork had a similar effect compared to that of SLT on 360degrees of trabecular meshwork in terms of IOP reduction. The authors of the present study suggest that 180degrees SLT is the safest procedure with regard to success rate and complications.

Comparison of multiplex PCR with serogrouping and PCR-RFLP of fliC gene for the detection of enteropathogenic Escherichia coli (EPEC)

Enteropathogenic Escherichia coli (EPEC) comprise one of the six categories of diarrhoeagenic E. coli (DEC). EPEC is subgrouped into typical (tEPEC) and atypical (aEPEC). The identification of DEC cannot be based only on cultural and biochemical criteria, since they are indistinguishable from the non-pathogenic E. coli commonly found in human feces. Several PCR methods, with both single and multiple target genes, have been reported for detecting the different DEC pathotypes. In the present study five hundred E. coli isolates from children with diarrhea were subjected into multiplex PCR. Furthermore the strains were typed serologically with O antisera and their fliC gene was characterized by PCR-RFLP. The results obtained revealed that overall 41 (8.2 percent) isolates could be detected as EPEC by this multiplex PCR assay. Of these isolates; 27 (66 percent) were typical (escv+, bfp+) and 14 (34 percent) atypical EPEC (escv+, bfp-). None of these 41 isolates contained the Stx1 and Stx2 genes. Among 37 (90 percent) typeable strains, nine different serogroups were present. The most common serogroups were O111, followed by O86, O55 and O119 and 10 different H types were found among these isolates. The multiplex PCR assay was found to be rapid and reliable in comparison to serological test; especially when screening the large number of isolates.

Identification & characterization of Shiga toxin-producing Escherichia coli isolates from patients with diarrhoea in Iran.

Background & objectives: Verotoxigenic Escherichia coli are important serotypes of enterohaemorrhagic E. coli (EHEC) subgroup that cause attaching and effacing lesions in enterocytes by producing verotoxins or shiga-like toxins resulting in haemorrhagic colitis (HC) and haemolytic uremic syndrome (HUS). The aim of this study was to detect these serotypes specially E. coli O157:H7 in stool samples of patients with diarrhoea and identification of virulence genes (STX1, STX2, Hly and EAE) in Shahrekord-Iran area using PCR technique. Methods: Two hundred diarrhoeal stool samples of patients were collected through 2007-2008. Microbiological and biochemical examinations were done to detect the E. coli. Serological tests carried out to identify the O157 or O157:H7 serotypes. Results: Of the 58 E. coli isolates, 16 (27.6%) were detected as STX1 carrying E. coli, four (6.9%) carrying STX2, eight (13.8%) carrying both STX1 and STX2, and 12 (20.7%) were Hly carrying E. coli, but none of the isolates contained EAE gene. None of the isolates were E. coli O157 or O157:H7 serotypes. Interpretation & conclusions: Our results revealed that verotoxigenic E. coli isolates other than O157 serotype were involved in causing diarrhoea in Shahrekord-Iran.

Detection of Escherichia coli O157 and Escherichia coli O157:H7 by the immunomagnetic separation technique and stx1 and stx2 genes by multiplex PCR in slaughtered cattle in Samsun Province, Turkey

This study was conducted to investigate the presence of Escherichia (E.) coli O157 and E. coli O157:H7 and stx1 and stx2 genes on cattle carcasses and in rectal samples collected from Samsun Province of Turkey. A total of 200 samples collected from cattle carcasses and the rectal contents of 100 slaughtered cattle from two commercial abattoirs were tested using the immunomagnetic separation technique and multiplex PCR methods. E. coli O157 and E. coli O157:H7 were detected in 52 of the 200 samples (26%) tested. Of the positive samples, 49 were E. coli O157 and three were E. coli O157:H7. The E. coli O157 strain was isolated from 24 carcasses and 25 rectal samples, while E. coli O157:H7 was isolated from two carcasses and one rectal sample. Of the 49 samples positive for E. coli O157, 32 were from the rectal and carcass samples of the same animal, while two E. coli O157:H7 isolates were obtained from rectal swabs and carcasses of the same animal. The stx1 and stx2 genes were both detected in 35 E. coli O157 isolates and one E. coli O157:H7 isolate, but the stx2 gene was only detected alone in two E. coli O157 isolates. Overall, 16 carcasses tested positive for E. coli O157 and one carcass tested positive for E. coli O157:H7 based on both carcass and rectal samples. Overall, the results of this study indicate that cattle carcasses pose a potential risk to human health due to contamination by E. coli O157 and E. coli O157:H7 in the feces.

Detection of shiga-toxigenic Escherichia coli (STEC) in diarrhoeagenic stool & meat samples in Mangalore, India.

BACKGROUND & OBJECTIVE: Shiga-toxigenic Escherichia coli (STEC) are causative agents of bloody diarrhoea, haemorrhagic colitis (HC) and haemolytic uraemic syndrome (HUS). Humans acquire infections primarily through contaminated beef. In India, STEC has not been implicated as a major cause of diarrhoea. Hence, isolation of STEC from diarrhoeagenic stool samples of patients and beef samples marketed through retail outlets was attempted in Mangalore, India. METHODS: Diarrhoeagenic stool samples (n = 192) and meat samples (n = 103) were screened for STEC, using conventional culture methods and polymerase chain reaction (PCR) from December 2003 to 2006 in the department of Microbiology, Kasturba Medical College, Mangalore. All the E. coli isolates were subjected to antibiotic susceptibility testing and serotyping. RESULTS: Of the 40 eae positive E. coli isolates from meat sample, one was positive for all the STEC genes, namely stx1, stx2, rfb O157 and EHEC hlyA. This isolate belonged to O157 serogroup. Of the 110 eae positive E. coli isolated from stool samples, two were positive for EHEC hlyA and belonged to serogroup O8 and one was positive for bfp gene and found to be of O6 serogroup. Among the 192 stool enrichment broths tested, 160 were positive for eae gene, of which two were EHEC hlyA positive and one was bfp gene positive. Among the 103 meat enrichment cultures, 90 were positive for eae gene and one among them was positive for all the STEC genes. INTERPRETATION & CONCLUSION: Our results showed a low incidence of STEC and high prevalence of eae positive E. coli other than STEC in stool and meat samples. A low positivity was observed for PCR performed directly on stool and meat samples. However, PCR on enrichment cultures gave better results. Since E. coli O157 was isolated and detected by PCR in one of the meat samples, this organism may be of public health significance. A study on a large sample may provide some answer.

Split Liver Transplantation / 대한이식학회지

PURPOSE: Split liver transplantation (SLT) offers an effective way of increasing the donor pool. However, it is often difficult to perform SLT under current allocation system. We retrospectively analyzed the outcome of the patients who had undergone SLT in Seoul National University Hospital. METHODS: From the first case of SLT in Korea on November 4, 1998, 8 patients underwent SLT in our center. Three adult patients received extended right liver graft and five child patients received left lateral section graft. All liver were split by in-situ method. RESULTS: All adult patients were alive. One adult patient developed hepatic artery thrombosis one month after SLT and underwent retransplantation due to graft failure. Another patient developed biliary leakage and had to undergo operative bile duct revision. Two of child patients were died of pneumonia and hepatic failure due to HBV hepatitis, respectively. One child patient suffered from hepatic venous stricture and persistent ascites and received interventional therapy. Overall 3-year patient survival rate was 87.5% and graft survival rate was 75.0%. No primary nonfunction developed and three patients (37.5%) suffered form vascular or biliary complications. CONCLUSION: The results of SLT were similar to that of conventional deceased donor liver transplantation. Although SLT is technically difficult and increase the risk of vascular or biliary complications just like living donor liver transplantation (LDLT), its result might be acceptable and it could be a successful method to expand the donor pool if it would be performed in the center experienced in LDLT.

Aislamiento de Escherichia coli productor de toxina Shiga durante un brote de gastroenteritis en un Jardín Maternal de la Ciudad de Mar del Plata / Isolation of Shiga toxin-producing Escherichia coli strains during a gastrointestinal outbreak at a day care center in Mar del Plata City

Entre el 15 de octubre y el 8 de noviembre de 2003 ocurrió un brote de gastroenteritis en un Jardín Maternal de un Hospital de la ciudad de Mar del Plata. Catorce de un total de 80 niños (17,5%), edad promedio 23,6 ± 13,9 meses, presentaron diarrea, y un caso evolucionó a síndrome urémico hemolítico. La madre de uno de los afectados presentó diarrea simultáneamente. No se pudo establecer el origen del brote, pero probablemente la transmisión haya sido fundamentalmente persona a persona. Las prácticas habituales en el lactario del jardín maternal, y las condiciones inadecuadas de infraestructura y hábitos de higiene de la cocina del Hospital fueron señalados como factores de riesgo. En un caso se detectó Escherichia coli productor de toxina Shiga (STEC) O103:H2, y STEC O26:H11 en otro. En el niño infectado por STEC O26:H11, la excreción se extendió por un período de 37 días. La no detección de STEC en aquellos casos en los cuales el intervalo entre el inicio de los síntomas y la toma de muestra fue mayor a 6 días, enfatiza la necesidad de la recolección temprana de especímenes. Las principales conclusiones de este estudio fueron la necesidad de establecer normas óptimas de higiene, informar rápidamente la ocurrencia de casos de gastroenteritis y confirmar la negativización de la excreción del patógeno.

From October 15 to November 8, 2003, a gastrointestinal outbreak occurred at a day care center in a Hospital in Mar del Plata City. Fourteen out of 80 (17.5%) children, mean age 23.6 ± 13.9 months, and the mother of one of them had diarrhea. One case developed hemolytic uremic syndrome. No conclusive evidence of the origin of the outbreak was found, but the epidemic curve suggested person-to-person spread. The usual practices at the place where infant milk formula was prepared at the day care center, together with the inadequate infrastructure conditions and hygiene practices at the kitchen of the hospital, were considered risk factors. One case had Shiga toxin-producing Escherichia coli (STEC) O103:H2 infection and other STEC O26:H11.The duration of shedding for the child with O26:H11 infection was 37 days. In the other symptomatic children, the pathogen was not recovered from fecal samples collected 6 or more days after the onset of the illness. This emphasizes that the collection of early samples is necessary to recover STEC strains. In order to prevent and control enteric diseases in day care facilities the following measures are necessary: optimal hygiene standards, early case reporting, and exclusion of those who remain culture-positive.

Validación de una técnica de PCR múltiple para la detección de Escherichia coli productor de toxina Shiga / Validation of a multiplex PCR for detection of Shiga toxin-producing Escherichia coli

La infección por Escherichia coli productor de toxina Shiga (STEC) es causa de diarrea con o sin sangre, colitis hemorrágica y síndrome urémico hemolítico (SUH) en humanos. El objetivo de este trabajo fue validar una técnica de PCR múltiple para el diagnóstico de STEC basado en la detección de los genes stx1, stx2 y rfbO157. La validación de la técnica se realizó en dos laboratorios independientes, en forma paralela. Se determinó rango de trabajo, selectividad y robustez. Se evaluó el desempeño de la técnica al combinar distintas concentraciones de dos cepas con diferentes factores de virulencia. El rango de trabajo dependió de la cepa analizada, los valores máximos y mínimos fueron 6,6 x 107 y 1,0 x 104 UFC/50 µl. El límite de detección fue de 1,0 x 104 UFC/50 µl y el límite de corte de 1,0 x 105 UFC/50 µl. La robustez fue óptima al modificar diferentes variables. Se obtuvo 100% de inclusividad, exclusividad, precisión analítica, valor predictivo positivo y valor predictivo negativo. No se observó interferencia al combinar distintas concentraciones de los factores de virulencia blanco de la reacción. La técnica validada es una alternativa apropiada para la detección y confirmación de STEC O157 y no-O157 a partir de cultivos bacterianos.

Shiga toxin-producing Escherichia coli (STEC) cause non-bloody or bloody diarrhea, hemorrhagic colitis and hemolytic uremic syndrome (HUS) in humans. The aim of the present study was to validate a multiplex PCR for the STEC diagnosis based on the detection of stx1, stx2 and rfbO157 genes. The multiplex PCR validation was carried out in two independent laboratories in a parallel way. Work range, selectivity and robustness were established. The PCR performance was evaluated using different concentrations of two STEC strains harboring different target genes. The work range depended on the strain analyzed, the maximum and the minimum values were 6.6 x 107 and 1.0 x 104 CFU/50 µl. The detection limit was 1.0 x 104 CFU/50 µl and the cut limit 1.0 x 105 CFU/50 ml. A good robustness was observed when different variables were introduced. Inclusivity, exclusivity, positive predictivity, negative predictivity and analytical accuracy were of 100%. Interference was not shown when different concentrations of STEC strains, carrying different genes, were used. The validated technique is an appropriate alternative for detection and confirmation of STEC O157 and non-O157 strains from bacterial cultures.

Postdiarrheal Shiga toxin-mediated hemolytic uremic syndrome similar to septic shock

The inflammatory response of host endothelial cells is included in the development of vascular damage observed in enterohemorrhagic Escherichia coli (EHEC) infection, resulting in hemolytic uremic syndrome (HUS). The response to a non-conventional treatment for a group of D+ HUS (diarrhea positive HUS) patients, with clinical hemodynamic parameters of septic shock was evaluated in this prospective study (1999-2003). Twelve children 2.8 +/- 0.6 years old, with D+ HUS produced by E. coli infection with serological evidence of Shiga toxin, presenting severe unstable hemodynamic parameters and neurological dysfunction at onset, were studied. The protocol included fresh frozen plasma infusions, methylprednisolone pulses (10mg/k/day) for three consecutive days and plasma exchange for five days, starting after admission to the intensive care unit (ICU). The twelve patients with increased pediatric risk of mortality (PRISM) score: 18 +/- 2 after admission to intensive care unit (ICU), required dialysis for 17.4 +/- 4 days, mechanical ventilator assistance for 10 +/- 1 days and early inotropic drugs support for 10.5 +/- 1 days. Neurological dysfunction included generalized tonic-clonic seizures lasting for 5.4 +/- 1 days, n:8. Focal seizures were present in the remaining patients. Dilated cardiomyopathy was present in 6 children. Eight children suffered hemorrhagic colitis. Nine patients survived. Within one year of the injury, neurological sequelae, Glasgow outcome scale (GOS) 3 and 4, were present in two patients, chronic renal failure in one patient. We suggest that early introduction of this protocol could benefit D+ HUS patients with hemodynamic instability and neurological dysfunction at onset. Further studies are likely to elucidate the mechanisms involved in this early adverse clinical presentation of D+ HUS patients.

La respuesta inflamatoria de la célula endotelial se incluye en el desarrollo del daño vascular observado en la infección por Escherichia coli enterohemorrágica que deviene en Síndrome Urémico Hemolítico (SUH). Se evaluó en forma prospectiva, entre 1999 y 2003, la respuesta a un tratamiento no convencional, en doce pacientes, edad 2.8 ± 0.6 años, que desarrollaron SUH con presencia de diarrea sanguinolenta (SUH D+) y evidencia serológica de toxina Shiga, los cuales en fase inicial presentaron parámetros hemodinámicoscompatibles con shock séptico y compromiso neurológico grave. El protocolo incluyó transfusión de plasmafresco, pulsos de metilprednisolona (10mg/k/día) por tres días consecutivos y plasmaféresis por cinco días, iniciados en las primeras 48 horas. Los doce pacientes ingresaron en terapia intensiva, presentando unapuntuación de riesgo de mortalidad pediátrica (PRISM): 18 ± 2, con requerimiento de diálisis por 17.4 ± 4 días, asistencia ventilatoria mecánica por 10 ± 1días y soporte temprano con drogas inotrópicas por un período de10.5 ± 1 días. La disfunción neurológica se presentó con convulsiones tónico-clónicas generalizadas por 5.4 ±1 días en 8 pacientes y con convulsiones focalizadas en los restantes. Seis pacientes desarrollaron miocardiopatíadilatada y 8 presentaron colitis hemorrágica. Sobrevivieron a la etapa aguda de la enfermedad 9 pacientes. Alfinalizar el primer año de seguimiento, dos de ellos presentaban secuelas neurológicas (escala de seguimientode Glasgow; GOS 3 y 4 respectivamente) y uno, fallo renal crónico. La introducción temprana de este protocolo podría beneficiar a pacientes con SUH D+ con inestabilidad hemodinámica grave y disfunción neurológica al inicio. Los mecanismos involucrados en esta temprana presentación clínica adversa de SUH D+ permanecen aún sin dilucidar.

Isolation and identification of Escherichia coli O157:H7 using different detection methods and molecular determination by multiplex PCR and RAPD

Escherichia coli O157:H7 is recognized as a significant food-borne pathogen, so rapid identification is important for food hygiene management and prompt epidemiological investigations. The limited prevalence data on Shiga toxin-producing E. coli (STEC) and E. coli O157:H7 in foods and animals in Korea made an assessment of the risks difficult, and the options for management and control unclear. The prevalence of the organisms was examined by newly developed kit-E. coli O157:H7 Rapid kit. For the isolation of E. coli O157:H7, conventional culture, immunomagnetic separation, and E. coli O157:H7 Rapid kit were applied, and multiplex PCR and randomly amplified polymorphic DNA (RAPD) were performed for the molecular determination. There was high molecular relatedness among 11 Korean isolates and 17 U.S. strains at 63% level. Additionally, distinct differentiation between pig and cattle isolates was determined. It implied that RAPD had a capacity to distinguish strains with different sources, however it could not discriminate among isolates according to their differences in the degree of virulence. In antimicrobial susceptibility tests, 45.5% of isolates showed antibiotic resistance to two or more antibiotics. Unlike the isolates from other countries, domestic isolates of E. coli O157:H7 was mainly resistant to ampicillin and tetracylines. In summary, the application of E. coli O157:H7 Rapid kit may be useful to detect E. coli O157:H7 due to its sensitivity and convenience. Moreover, combinational analysis of multiplex PCR together with RAPD can aid to survey the characteristics of isolates.