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Introduction: The detection of cytomegalovirus (CMV) DNA by real time polymerase chain reaction (rt-PCR) in dried blood spots collected routinely for metabolic screening has been assessed for the retrospective diagnosis of congenital CMV (cCMV) infection in many studies, but not in Spain. The aim of this study is to analyze the diagnostic accuracy of this technique in our hospital., Methods: A cross-sectional retrospective observational study was conducted including all patients born between January, 2007 and September, 2012 with confirmed cCMV infection. The assessment of CMV DNA was made by using rt-PCR in dried blood spots of these patients., Results: Fourteen patients were included: 4/14 were symptomatic and 4/14 had sequelae. The detection of CMV DNA by rt-PCR was positive in only 7 patients. A statistically significant relationship between low viral load at birth and negative rt-PCR in dried blood spots was demonstrated., Conclusions: Despite the low number of patients included, our data highlight an important amount of false negative results in the DNA CMV detection by rt-PCR in these samples for the retrospective diagnosis of cCMV infection, especially in cases with low viral load at birth., (Copyright © 2013 Elsevier España, S.L.U. y Sociedad Española de Enfermedades Infecciosas y Microbiología Clínica. All rights reserved.)

Introduction: Cytomegalovirus (CMV) is the main virus causing congenital and postnatal infections in the pediatric population. The aim of this study is to evaluate the usefulness of a quantitative real-time PCR in the diagnosis of these infections using urine as a single sample., Patients and Methods: We studied all the urine samples of newborns (< 7 days) with suspected congenital infection, and urine of patients with suspected postnatal infection (urine negative at birth). Urines were simultaneously studied by cell culture, qualitative PCR (PCRc), and quantitative real-time PCR (PCRq)., Results: We analyzed 332 urine samples (270 to rule out congenital infection and 62 postnatal infections). Of the first, 22 were positive in the PCRq, 19 in the PCRc, and 17 in the culture. PCRq had a sensitivity of 100%, on comparing the culture with the rest of the techniques. Using the PCRq as a reference method, culture had a sensitivity of 77.2%, and PCRc 86.3%. In cases of postnatal infection, PCRq detected 16 positive urines, the PCRq 12, and the cell culture 10. The urines showed viral loads ranging from 2,178 to 116,641 copies/ml., Conclusions: The genomic amplification technique PCRq in real time was more sensitive than the other techniques evaluated. This technique should be considered as a reference (gold standard), leaving the cell culture as a second diagnostic level. The low cost and the automation of PCRq would enable the screening for CMV infection in large neonatal and postnatal populations., (Copyright © 2013 Asociación Española de Pediatría. Published by Elsevier Espana. All rights reserved.)

Background: To compare the capacity of conventional centrifugation and spontaneous sedimentation to detect the presence of cytomegalovirus (CMV) in urine samples., Material and Methods: We studied urine samples from 45 renal transplant recipients. After decontamination half of each sample was centrifuged (1,500 rpm for 10 minutes) and the other half was allowed to sediment at room temperature for 30 minutes. From the supernatant of each of these 250 microliters was inoculated in a shell-vial (MRC-5). Cultures were incubated for 48 hours at 37 degrees C and stained by an indirect immunofluorescence assay. The samples considered totally toxic were re-inoculated after dilution 1:1 with maintainance medium., Results: Of 845 urine samples analyzed, 743 (88%) were negative and 102 (12%) positive. 66 (7.8%) were considered toxic: 35 (4.1%) partially toxic and 31 (3.6%) totally toxic. Of the centrifuged urine samples 86.2% were positive against 98% of the sedimented samples (p = 0.004). Of the 31 samples considered totally toxic, 17 (2.2%) corresponded with negative urine samples and 14 (13.7%) with positive samples. Off these, 12 (85.7%) were detected in centrifuged samples and 2 (14.3%) in sedimented samples (p = 0.001)., Conclusions: The percentage of toxicity in the urine samples was low (7.8%) which does not seem to suggest the need for systematic dilution of all samples. The sedimentation process enabled us to detect a greater number of urine samples positive for CMV with a lower rate of toxicity of the monolayers. The use of this process would reduce the number of samples requiring reinoculation.

Objective: To prospectively and comparatively study the usefulness of urine (viruria) and blood (antigenemia pp65 and culture) (viremia) for the diagnosis of cytomegalovirus (CMV) infection in renal transplant (RT) recipients., Material and Methods: All RT recipients at our hospital were studied from January 1995 to December 1996. After decontamination, urine specimens were inoculated into two MRC-5 cell line vials. Polymorphonuclear cells were extracted from peripheral blood by sedimentation in saline dextran and were used for antigenemia pp65 test and for culture in shell-vial., Results: A total of 1,335 specimens from 43 patients were studied. CMV was recovered from 110 out of the 913 (12%) urine specimens and from 101 out of the 422 (23.9%) blood specimens (antigenemia and/or viremia). CMV detection was first obtained by a positive blood test in 23 patients (88.4%), whereas the urine specimen was the first positive test in only three (11.6%) patients (p = 0.0001). A positive result in blood preceded a positive result in urine by a mean of 10.3 days (range: 2-73 days). Antigenemia and viremia were simultaneously positive in 61.5% of patients. In three patients a positive antigenemia preceded viremia by 14 days. In seven patients (26.9%) only the shell-vial culture was positive. Culture preceded antigenemia by a mean of 7.6 days. In the 26 patients, the time elapsed until the first positive blood specimen for CMV was 37.3 days (range: 11-88 days)., Conclusion: According to the results obtained we believe that blood (antigenemia pp65 and/or viremia) should be considered as the only really useful specimen for the diagnosis of infection/disease caused by CMV in RT recipients. The urine specimen lacks a diagnostic and clinical usefulness and therefore should not be used in these patients.

Background: The presence of pyuria and the role of mixed culture in the diagnosis of asymptomatic bacteriuria in pregnant women have been evaluated, Methods: One hundred and sixty four pregnant women without any symptomatology have been studied using two cultures of mid-stream urine samples and pyuria quantification. In addition culture of bladder urine has been carried out in 17 of these patients (12 with pure cultures and 5 with mixed cultures)., Results: 110 samples were culture negative without pyuria; 7 were pure cultures with pyuria; 19 pure culture without pyuria and the remaining 28 patients yielded mixed culture with or without pyuria in the first culture. Twenty of these 28 mixed cultures were negative in the second culture. A estimated frequency of asymptomatic bacteriuria in pregnancy was 16% and pyuria was only found in 27% of pregnant women with asymptomatic bacteriuria., Conclusion: The pyuria is not a useful marker for the diagnosis of asymptomatic bacteriuria in pregnancy.

To assess the toxicity of urine samples on human fibroblast cell monolayer using Shell-vial technique for cytomegalovirus antigen detection. A total of 227 urine samples were processed, inoculated on Shell-vial simultaneously, being either diluted (1:1) and non diluted in transport media. After 24-48 hours incubation, the fibroblast cells monolayer was stained using fluorescein-marked monoclonal anti-cytomegalovirus antibodies. Cytomegalovirus was detected in 9.6% of samples inoculated with diluted urine, and in 5.2% of samples inoculated with non-diluted urine. Destruction of monolayer cells was seen in 13.2 and 22.4, respectively. With Shell-vial technique for detecting cytomegalovirus in urine samples, a high number of positive results was obtained using diluted urine. Also a significant minor destruction of cells monolayer was observed with the same inoculum.