Placental chemical elements concentration in small fetuses and its relationship with Doppler markers of placental function.

INTRODUCTION: In this study, we aimed at quantifying placental concentrations of 22 chemical elements in small fetuses (SGA) as compared with normally grown fetuses (AGA), and to assess the relationship with Doppler markers of placental function. METHODS: Prospective cohort study, including 71 SGA fetuses (estimated fetal weight < 10th percentile) and 96 AGA fetuses (estimated fetal weight > 10th percentile), recruited in the third trimester of gestation. The placental concentration of 22 chemical elements was determined by inductively coupled plasma optical emission spectrophotometer (ICP-OES, ICAP 6500 Duo Thermo): aluminum (Al), beryllium (Be), bismuth (Bi), calcium (Ca), cadmium (Cd), cobalt (Co), chrome (Cr), copper (Cu), magnesium (Mg), manganese (Mn), molybdenum (Mo), nickel (Ni), phosphorus (P), lead (Pb), rubidium (Rb), sulfur (S), strontium (Sr), titanium (Ti), thallium (Tl), antimony (Sb), selenium (Se), and zinc (Zn). Placental function was assessed by measuring the following fetal-maternal parameters: Uterine artery Pulsatility Index (UtA PI), Umbilical artery Pulsatility Index (UA PI) and Middle Cerebral artery Pulsatility Index (MCA PI). The association between the chemical elements concentration and study group and the association with Doppler measures were evaluated. RESULTS: SGA was associated with significantly (p < 0.05) lower concentrations of Al (AGA 21.14 vs SGA 0.51 mg/kg), Cr (AGA 0.17 vs SGA 0.12 mg/kg), Cu (AGA 0.89 vs SGA 0.81 mg/kg), Mg (AGA 0.007 vs SGA 0.006 g/100g), Mn (AGA 0.60 vs SGA 0.47 mg/kg), Rb (AGA 1.68 vs SGA 1.47 mg/kg), Se (AGA 0.02 vs SGA 0.01 mg/kg), Ti (AGA 0.75 vs SGA 0.05 mg/kg) and Zn (AGA 9.04 vs SGA 8.22 mg/kg). Lower placental concentrations of Al, Cr, Mn, Se, Ti were associated with abnormal UtA, UA and MCA Doppler. DISCUSSION: Lower placental concentrations of Al, Cr, Cu, Mn, Rb, Se, Ti and Zn are associated with SGA fetuses and abnormal fetal-maternal Doppler results. Additional studies are required to further understand how chemical elements affect fetal growth and potentially find strategies to prevent SGA.

Dynamics of crAssphage as a human source tracking marker in potentially faecally polluted environments.

Recent studies have shown that crAssphage is abundant in human faecal samples worldwide. It has thus been postulated as a potential microbial source tracking (MST) marker to detect human faecal pollution in water. However, an effective implementation of crAssphage in water management strategies will depend on an understanding of its environmental dynamics. In this work, the abundance and temporal distribution of crAssphage was analysed in the effluent of wastewater treatment plants using different sewage treatments, and in two rivers (water and sediments) that differ in pollution impact and flow regime. Additionally, the influence of environmental conditions (temperature and rainfall) on the removal of the marker was studied along a river section, and natural inactivation was assessed by a mesocosms approach. Molecular and culture-based tools were used to compare crAssphage abundance and dynamics with those of bacteria and bacteriophages currently applied as global indicators (E. coli, somatic coliphages, Bacteroides GA17 bacteriophages, and the human-associated MST markers HF183 and HMBif). CrAssphage concentrations in sewage effluent and river samples were similar to those of HF183 and HMBif and higher than other general and/or culture-based indicators (by 2-3 orders of magnitude). Measurement of crAssphage abundance revealed no temporal variability in the effluent, although rainfall events affected the dynamics, possibly through the mobilisation of sediments, where the marker was detected in high concentrations, and an increase in diffuse and point pollution. Another factor affecting crAssphage inactivation was temperature. Its persistence was longer compared with other bacterial markers analysed by qPCR but lower than culturable markers. The results of this study support the use of crAssphage as a human source tracking marker of faecal pollution in water, since it has similar abundances to other molecular human MST markers, yet with a longer persistence in the environment. Nevertheless, its use in combination with infectious bacteriophages is probably advisable.

Genetic risk assessment of thrombophilia in patients with adverse obstetric outcomes.

OBJECTIVES: To investigate the incidence of inherited thrombophilias in patients with adverse obstetric outcomes and to compare detection rates of thrombophilias between standard blood tests and a novel genetic test. METHODS: This is a case-control prospective study performed in Hospital Sant Joan de Déu in Barcelona, Spain. Cases had a history of intrauterine growth restriction requiring delivery before 34 weeks gestation, placental abruption before 34 weeks gestation, or severe preeclampsia. Controls had at least two normal, spontaneously conceived pregnancies at term, without complications or no underlying medical disease. At least 3 months after delivery, all case and control women underwent blood collection for standard blood tests for thrombophilias and saliva collection for the genetic test, which enables the diagnosis of 12 hereditary thrombophilias by analyzing genetic variants affecting different points of the blood coagulation cascade. RESULTS: The study included 33 cases and 41 controls. There were no statistically significant differences between cases and controls in the standard blood tests for thrombophilias in plasma or the TiC test for genetic variables. One clinical-genetic model was generated using variables with the lowest P values: ABO, body mass index, C_rs5985, C_rs6025, and protein S. This model exhibited good prediction capacity, with an area under the curve of almost 0.7 (P <0.05), sensitivity of almost 67%, and specificity of 70%. CONCLUSION: Although some association may exist between hypercoagulability and pregnancy outcomes, no significant direct correlation was observed between adverse obstetric outcomes and inherited thrombophilias when analyzed using either standard blood tests or the genetic test. Future studies with a larger sample size are required to create a clinical-genetic model that better discriminates women with a history of adverse pregnancy outcomes and an increased risk of poor outcomes in subsequent pregnancies.

Bluephage: A rapid method for the detection of somatic coliphages used as indicators of fecal pollution in water.

The use of somatic coliphages as indicators of fecal and viral pollution in water and food has great potential due to the reliability, reproducibility, speed and cost effectiveness of methods for their detection. Indeed, several countries already use this approach in their water management policies. Although standardized protocols for somatic coliphage detection are available, user-friendly commercial kits would facilitate their routine implementation in laboratories. The new method presented here allows detection of up to 1 somatic coliphage in under 3.5 h, well within one working day. The method is based on a modified Escherichia coli strain with knocked-out uidB and uidC genes, which encode the transport of glucuronic acid inside cells, and overexpressing uidA, which encodes the enzyme ß-glucuronidase. The enzyme accumulated in the bacterial cells only has contact with its substrate after cell lysis, such as that caused by phages, since the strain cannot internalize the substrate. When the enzyme is released into the medium, which contains a chromogen analogous to glucuronic acid, it produces a change of color from yellow to dark blue. This microbiological method for the determination of fecal pollution via the detection of culturable microorganisms can be applied to diverse sample types and volumes for qualitative (presence/absence) and quantitative analysis and is the fastest reported to date.

Spread of mcr-1-carrying Enterobacteriaceae in sewage water from Spain.

Objectives: The mobile colistin resistance gene mcr-1 has been identified worldwide in human and animal sources, while its occurrence in the environment is still largely unknown. The aim of this study was to investigate the presence of mcr-1 -harbouring Enterobacteriaceae in water samples obtained from rivers and waste water treatment plants in the area of Barcelona, Spain. Methods: The presence of mcr-1 was detected by PCR. Bacterial identification was performed via MALDI-TOF MS. Resistance to colistin was determined by a broth dilution method. The epidemiological relationship between the positive isolates was assessed with PFGE and ST was determined by MLST. Plasmid characterization was performed by transformation experiments, antimicrobial susceptibility testing and incompatibility group PCR. Results: Thirty MDR isolates bearing mcr-1 , 29 Escherichia coli (ST632 and ST479) and 1 Klebsiella pneumoniae (ST526), were identified in sewage from two different waste water treatment plants, whereas the gene was not found in river water. All isolates, including the K. pneumoniae , harboured bla CTX-M-55 and bla TEM-1 . mcr-1 was in all cases associated with an IncI2 plasmid, which only conferred resistance to colistin. mcr-1 was harboured by two predominant E. coli clones that were found in both waste water treatment plants. Conclusions: This study showed a high occurrence of mcr-1 in the sewage of Barcelona, mainly due to the dissemination of two E. coli pulsotypes that are circulating in the population. The presence of mcr-1 in the environment is a cause for concern, and suggests high prevalence of mcr-1 in the community.

Evaluation of the Ex-PRESS(®) P-50 implant under scleral flap in combined cataract and glaucoma surgery.

AIM: To evaluate the efficacy and safety of glaucoma drainage device Ex-PRESS(®) P-50 for combined cataract surgery and glaucoma. METHODS: Patients having cataract and open angle glaucoma or patients with open advanced glaucoma which needed two or more antiglaucoma medications were included. Combined cataract surgery and glaucoma with Ex-PRESS(®) P-50 model placed under scleral flap was performed. RESULTS: Out of 40 eyes of 40 patients (55% male and 45% female) completed the study during one-year follow-up. The mean of age was 76.6±11.02y. The intraocular pressure (IOP) decreased significantly during the 12-month follow-up from 23.5 mm Hg to 16.8 mm Hg (Wilcoxon signed ranks test, P<0.001). A 59.5% of patients did not need any topical treatment, 10.8% of them needed one active principle, 27% needed two active principles, and 2.7% of them needed three active principles for successful IOP control (<21 mm Hg). CONCLUSION: Combined surgery of phacoemulsification with ExPRESS(®) P-50 lowers IOP from the preoperative baseline and reduces significantly the number of antiglaucoma active principles for IOP control after the operation.

Premio al mejor póster presentado en el XXXVI Congreso, celebrado en Valencia. Importancia del equipo multidisciplinar en la eficacia de la instilación de mitomicina C posoperatoria inmediata / No disponible

El tumor vesical es una patología con alta prevalencia, a causa de su tendencia a la recidiva, un porcentaje alto de pacientes tiene recidiva tumoral en 1-2 años. Se ha demostrado que la instilación precoz de mitomicina C, tras la resección transuretral, disminuye las recidivas; pero la eficacia de este tratamiento está condicionada por el tiempo que pasa desde la resección del tumor y la administración de Mitomicina C, que no ha de ser superior a las 24 horas y con preferencia en las primeras 6 horas

No disponible

ExPRESS Shunt Surgery for Glaucoma with Post-Traumatic Aniridia.

A 76-year-old male presented with post-traumatic aphakia and aniridia in the right eye. The ocular pressure was 24 mmHg despite treatment with mixed eyedrops of timolol 0.5% and dorzolamide, brimonidine 0.1%, and Latananoprost 0.005%. The glaucomatous cup excavation was 0.8. Because it was his only eye, we informed him of the possibility of the Ex-PRESS implant as a possible prevention measure of possible postoperative hypotony. Seidel, hypotony, and choroidal detachment were not observed in the postoperative period. In the first two postoperative months, the pressure remained at 14 mmHg. The ocular pressure reached 20 mmHg at three months, so he was treated with mixed eyedrops of timolol 0.5% and brimonidine 0.1%. The ocular pressure decreased to 12 mmHg and remained at that level at the 10-month follow-up. The absence of serious postoperative complications suggest that this procedure may be an alternative in selected situations, such as for our patient.

[Cystic tumors in the retrorectal space. Do these two cases have something in common?]. / Tumoraciones quísticas del espacio retrorrectal. ¿Tienen algo en común estos dos casos?

Cystic lesions in the retrorectal or presacral space present a broad differential diagnosis. Rare but typical lesions at this site are those related to embryonic development, which are the most frequent presacral congenital lesions in adults. Amongst these tumors, epidermoid cysts and cystic hamartomas are the most common lesions. Cystic masses, which are asymptomatic in approximately 50% of the cases at diagnosis, may show complications such as infection or malignant degeneration. Initial diagnosis is based on imaging techniques although definite lesion characterization, essential due to their malignancy risk, is given by the pathological analysis of the surgical piece.

[Evaluation of conjunctival epithelium of filtering blebs by impression cytology]. / Estudio del epitelio conjuntival de las ampollas filtrantes mediante citología de impresión.

PURPOSE: To study the ocular surface in filtering blebs using impression cytology, comparing the bleb side and areas outside the bleb edges. METHODS: Twelve filtering blebs of 8 patients were included: 4 cases of trabeculectomy without mitomycin C (MMC), 6 cases of trabeculectomy with MMC, and 2 cases of non-penetrating glaucoma surgery. Impression cytology specimens were taken from filtering blebs as well as outside the bleb area. A classification scale from 0 to 3 was used to describe the distribution of epithelial cells and the density of goblet cells. Grade 0 indicated cohesive epithelial cells and abundant goblet cells; and the grade 3 indicated loss of epithelial cohesion and absence of goblet cells. RESULTS: The mean grade of cytology in filtering blebs was 2.4 ± 0.9, and in the outside bleb area of 0.8 ± 0.3 (P<.001). These differences were independent of the use of MMC (P=.48). The large majority (83%) of filtering blebs showed a decrease in epithelial cohesion and absence of goblet cells. Outside the bleb area, 100% of the cases had cohesive epithelial cells with different grades of goblet cells. CONCLUSION: The conjunctival epithelium overlying the filtering blebs showed significant changes that consisted of increased intercellular spaces and loss of goblet cells. These increased intercellular spaces could explain the trans-epithelial pathway of aqueous humor. The least amount of mucin due to loss of goblet cells could contribute to increase the risk of infection in filtering blebs.

The relationship between floppy eyelid syndrome and obstructive sleep apnoea.

PURPOSE: To determine the prevalence of eyelid hyperlaxity and floppy eyelid syndrome (FES) in obstructive sleep apnoea (OSA), and the presence of OSA in FES. PARTICIPANTS: One-hundred and fourteen patients who had been consecutively admitted for OSA evaluation and 45 patients with FES in which sleep studies were recorded. METHODS: Subjects underwent eyelid laxity measurement, slit-lamp examination and polysomnography. RESULTS: Eighty-nine patients were diagnosed of OSA. Fourteen patients with OSA had FES (16%) and 54/89 (60.67%) had eyelid hyperlaxity. Two of the 25 non-OSA patients had FES (8%) and 8 of 25 (32%) had eyelid hyperlaxity. There was a significantly higher incidence of eyelid hyperlaxity in OSA than in non-OSA patients (p=0.004). Thirty-eight of the 45 patients with FES were diagnosed of OSA (85%) and 65% had severe OSA. CONCLUSIONS: OSA might be an independent risk factor for eyelid hyperlaxity and severe OSA is common in patients with FES.

Pterygium surgery by means of conjunctival autograft: long term follow-up.

PURPOSE: To demonstrate the long term of follow-up of the recurrence rate after conjunctival autograft for pterygium surgery. METHODS: A total of 112 patients operated for pterygium with conjunctival autografts and with more than one year follow-up were registered. Patients were called for voluntary examinations of their respective ocular surface statuses. Completing the study was only possible in 44 cases. Seven had bilateral pterygium. (N=51 operated pterygium). RESULTS: The mean follow-up time was 49.06 months. In 29.45% of the cases, attachment was performed using non-absorbable sutures (nylon 10/0), while fibrin glue was used in 70.55% of the cases. Six cases of recurrence (11.76%) were found. The recurrence rate between primary and recurrent pterygium, and between sutures and fibrin glue proved to be not statistically significant; p>0.05. There were no significant differences between women and men with respect to recurrence. The median of age in the recurrence group was 40 years old, in contrast to 55 in the non-recurrence group; p=0.01517. All of these recurrences were associated with patients of Hispanic origins (from Latin America); p=0.001506. CONCLUSIONS: After a long follow-up period after autograft pterygium surgery, there were no statistically significant differences in recurrence rates for the application of sutures as opposed to fibrin glue; similarly, there were no statistically significant differences between the use of autograft in primary and recurrent pterygium. The greatest risk factors for recurrence were young age and Hispanic ethnicity.

Evaluación mediante navegación intraoperatoria de la laxitud del ligamento cruzado anterior en su reconstrucción anatómica monofascicular / Evaluation of intraoperative anterior cruciate ligament laxity using a navigation system in the anatomical single bundle reconstruction

Objetivo. La técnica de reconstrucción anatómica del ligamento cruzado anterior (LCA) persigue, reproduciendo la orientación nativa de sus fibras, alcanzar una mejor estabilidad rotacional en la rodilla. El objetivo principal del presente trabajo es la evaluación cuantitativa intraoperatoria, mediante el uso del sistema de navegación de Orthopilot®, de la laxitud anteroposterior y rotacional de la rodilla antes y después de una ligamentoplastia anatómica. Material y método. Descripción de la técnica de navegación y estudio transversal sobre una cohorte de 20 pacientes intervenidos en nuestro centro por rotura primaria crónica del LCA desde enero de 2010 hasta mayo de 2011. Con la ayuda del navegador se definió la posición exacta de los túneles en base a referencias anatómicas intraarticulares y se realizaron pruebas de estabilidad, tanto en el plano sagital como en el axial. Resultados. Nuestra técnica de reconstrucción anatómica del LCA situó el túnel tibial a una distancia media de 16,8±4,92 mm del ligamento cruzado posterior y a un 44,1%±4,35% de la anchura total del platillo tibial. La distancia media del centro del túnel femoral a la cortical posterior del cóndilo lateral fue de 7,89±2,78 mm. Intraoperatoriamente y antes de la reconstrucción, los valores medios (±DE) de traslación anteroposterior, rotación interna y rotación externa de la tibia a 30° fueron de 15,5mm (±5,11); 19° (±3,62) y 19,65° (±3,26) respectivamente. Tras la reconstrucción dichos valores disminuyeron a 5,6mm (±1,72); 12,17° (±3,76) y 16,9° (±4,42). Conclusiones. El empleo de sistemas de navegación como apoyo al cirujano permite sistematizar la posición de los túneles óseos y estandarizar el procedimiento en relación a la reconstrucción deseada. La reconstrucción del LCA según la técnica descrita, mejora la estabilidad anteroposterior y rotacional monoplanar respecto al estado preoperatorio pudiendo restablecer los valores de laxitud a los considerados como fisiológicos acorde al conocimiento científico actual (AU)

Purpose. The anatomical anterior cruciate ligament (ACL) reconstruction attempts to, by reproducing the natural orientation of its fibres, achieve a better rotational stability of the knee. The aim of this paper is to quantify the anteroposterior and rotational laxity of the knee before and after an anatomic ligamentoplasty using the Orthopilot® navigation system as a supporting tool. Matherial and method. We describe the distinctive steps of Orthopilot® navigation as well as conducting a retrospective cross-sectional study on a cohort of 20 patients operated in our hospital for chronic primary ACL rupture from january 2010 to may 2011. The precise location of the tunnels was defined with the help of the navigator and the intra-articular landmarks and stability tests were performed in both the sagittal and axial planes. Results. In our technique for anatomical ACL reconstruction placed the tibial tunnel at a mean distance of 16.8±4.92mm from the posterior cruciate ligament in a position that represented 44.1%±4.35 of the total width of the tibial plateau. The average distance from the centre of the femoral tunnel to the posterior cortex of the lateral condyle was 7.89±2.78mm. Intra-operatively and before ACL reconstruction, the mean (±SD) anteroposterior movement, internal rotation and external rotation of the tibia at 30° position were 15.5mm (±5.11), 19° (±3.62) and 19.65° (±3.26), respectively. After reconstruction these values decreased to 5.6mm (±1.72°), 12.17° (±3.76) and 16.9° (±4.42), respectively. Conclusions. The use of navigation systems supporting the surgery allows the systematic positioning of bone tunnels and standardises the procedures for the desired reconstruction. ACL reconstruction using the technique described, improves the anteroposterior and rotational stability compared to preoperative status, to a stability state that could be considered physiological according to current scientific knowledge (AU)

Pterygium surgery by means of conjunctival autograft: long term follow-up / Cirurgia de pterígio por meio de transplante autólogo de conjuntiva: acompanhamento a longo prazo

PURPOSE: To demonstrate the long term of follow-up of the recurrence rate after conjunctival autograft for pterygium surgery. METHODS: A total of 112 patients operated for pterygium with conjunctival autografts and with more than one year follow-up were registered. Patients were called for voluntary examinations of their respective ocular surface statuses. Completing the study was only possible in 44 cases. Seven had bilateral pterygium. (N=51 operated pterygium). RESULTS: The mean follow-up time was 49.06 months. In 29.45% of the cases, attachment was performed using non-absorbable sutures (nylon 10/0), while fibrin glue was used in 70.55% of the cases. Six cases of recurrence (11.76%) were found. The recurrence rate between primary and recurrent pterygium, and between sutures and fibrin glue proved to be not statistically significant; p>0.05. There were no significant differences between women and men with respect to recurrence. The median of age in the recurrence group was 40 years old, in contrast to 55 in the non-recurrence group; p=0.01517. All of these recurrences were associated with patients of Hispanic origins (from Latin America); p=0.001506. CONCLUSIONS: After a long follow-up period after autograft pterygium surgery, there were no statistically significant differences in recurrence rates for the application of sutures as opposed to fibrin glue; similarly, there were no statistically significant differences between the use of autograft in primary and recurrent pterygium. The greatest risk factors for recurrence were young age and Hispanic ethnicity.

OBJETIVO: Demonstrar a taxa de recorrência em cirurgia do pterígio com transplante autólogo de conjuntiva após um acompanhamento a longo prazo. MÉTODOS: Cento e doze pacientes operados de pterígio e transplante autólogo de conjuntiva, com mais de um ano de acompanhamento foram registrados. Os pacientes foram chamados para um exame voluntário de estado da superfície ocular. O estudo completo só foi possível em 44 pacientes. Sete deles tinha tido pterígio bilateral. (N=51). RESULTADOS: A média de acompanhamento a partir do momento da cirurgia até o exame foi de 49,06 meses. A sutura não-absorvível (nylon 10/0) foi usada em 29,45% dos casos, enquanto que a cola biológica de fibrina foi usada em 70,55%. A recidiva do pterígio foi detectada apenas em 6 casos (11,76%). As recidivas entre pacientes operados de pterígio primário e recorrente, e entre o uso de suturas ou cola de fibrina não foram significativos (p>0,05). Também não houve diferença significativa na recorrência com relação ao sexo. A idade média no grupo de recidiva foi de 40 anos e no grupo sem recidiva de 55 anos (p=0,01517). Todas essas recorrências ocorreram em pacientes de origem hispânica (América latina) (p=0,001506). CONCLUSÕES: A longo prazo da remoção cirúrgica de pterígio utilizando um transplante autólogo de conjuntiva, não há diferença estatisticamente significativa com o uso de sutura ou cola de fibrina. Também não há diferenças na recidiva, se o transplante autólogo de conjuntiva utilizado for em casos primários ou recorrentes de pterígio. O maior fator de risco para a recorrência parece estar entre os jovens e pacientes de etnia hispânica.

[Evaluation of intraoperative anterior cruciate ligament laxity using a navigation system in the anatomical single bundle reconstruction]. / Evaluación mediante navegación intraoperatoria de la laxitud del ligamento cruzado anterior en su reconstrucción anatómica monofascicular.

PURPOSE: The anatomical anterior cruciate ligament (ACL) reconstruction attempts to, by reproducing the natural orientation of its fibres, achieve a better rotational stability of the knee. The aim of this paper is to quantify the anteroposterior and rotational laxity of the knee before and after an anatomic ligamentoplasty using the Orthopilot(®) navigation system as a supporting tool. MATHERIAL AND METHOD: We describe the distinctive steps of Orthopilot(®) navigation as well as conducting a retrospective cross-sectional study on a cohort of 20 patients operated in our hospital for chronic primary ACL rupture from january 2010 to may 2011. The precise location of the tunnels was defined with the help of the navigator and the intra-articular landmarks and stability tests were performed in both the sagittal and axial planes. RESULTS: In our technique for anatomical ACL reconstruction placed the tibial tunnel at a mean distance of 16.8±4.92 mm from the posterior cruciate ligament in a position that represented 44.1%±4.35 of the total width of the tibial plateau. The average distance from the centre of the femoral tunnel to the posterior cortex of the lateral condyle was 7.89±2.78 mm. Intra-operatively and before ACL reconstruction, the mean (±SD) anteroposterior movement, internal rotation and external rotation of the tibia at 30° position were 15.5 mm (±5.11), 19° (±3.62) and 19.65° (±3.26), respectively. After reconstruction these values decreased to 5.6 mm (±1.72°), 12.17° (±3.76) and 16.9° (±4.42), respectively. CONCLUSIONS: The use of navigation systems supporting the surgery allows the systematic positioning of bone tunnels and standardises the procedures for the desired reconstruction. ACL reconstruction using the technique described, improves the anteroposterior and rotational stability compared to preoperative status, to a stability state that could be considered physiological according to current scientific knowledge.

Quantification of Shiga toxin 2-encoding bacteriophages, by real-time PCR and correlation with phage infectivity.

AIMS: To evaluate a qPCR-based protocol for the enumeration of Shiga toxin (Stx) 2 phages and to compare the results of qPCR with the number of infective Stx phage particles. METHODS AND RESULTS: An approach based on qPCR was applied to count Stx phages in five phage lysates of known titre. The number of viral particles from each phage lysate was determined by electron microscopy using latex spheres. The infectivity of the Stx phages was evaluated onto three bacterial host strains, by double agar layer assay and plaque blot hybridization. The number of phage particles detected by electron microscopy correlates with the number calculated by qPCR in all the phages assayed. The number of infectious phages was from 1 to 3 log10 units below the numbers obtained by qPCR and electron microscopy. CONCLUSIONS: The approach allows accurate quantification of Stx phages with a high recovery. The number of infectious phages is always below the number of phage particles detected by qPCR. SIGNIFICANCE AND IMPACT OF THE STUDY: The qPCR method is a good approach to enumerate Stx phages. However, these results should be carefully considered when related to the number of infectious phages for each lysate that could be applied in real samples, because values of infectious particles are always below the number of Stx phages detected by qPCR.