Analysis of human cytomegalovirus DNA in urines of newborns and infants by means of a new ultrarapid real-time PCR-system (original) (raw)
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The Open Microbiology Journal
Background: Human cytomegalovirus (HCMV) is a common opportunistic pathogen that causes serious complications in immunosuppressed patients and infected newborns. In this study, PCR-ELISA was optimized for semi-quantitative detection of infection in clinical specimens and simultaneous genotyping of glycoprotein B for 4 major genotypes, due to its significance. Method: During DIG-labeling PCR, a pair of primers amplifies a fragment of variable region of the glycoprotein B encoding sequence. Under optimized conditions, labeled Target amplicons hybridize to biotinated specific probes and are detected in an ELISA system. Results: PCR-ELISA system showed specific performance with detection limit of approximately 100 copies of CMV DNA. The linear correlation was observed between the PCR-ELISA results (OD) and logarithmic scale of CMV (r=0.979). Repeatability of PCR-ELISA detection system for intra-assay and inter-assay was evaluated for negative and positive samples. In optimized condition...
2020
Congenital cytomegalovirus (CMV) infection is the most common infectious cause of birth defects. It may cause both, immediate and long term health problems in infants. These include variety of symptoms, such as hearing loss, microcephaly, jaundice, hepatosplenomegaly and seizures. In more severe cases CMV infection can cause the death of an unborn baby and loss of pregnancy. Despite being one of the most extensively studied vertically transmitted infections recently, the adverse effects of vertically transmitted CMV infection are still not well presented to the general public, resulting in a low awareness among potential expectant mothers in Bosnia and Herzegovina. This study aims to elucidate the sensitivity of urine samples for CMV detection in infants as well as to reflect the importance of quantitative real-time PCR (qRT-PCR) in diagnostics of CMV infection in infants. qRT-PCR was used in this study as a technique for the screening of CMV DNA in a cohort of patients based in Sar...
Journal of Clinical Virology, 2012
Background: Cytomegalovirus (CMV) infection is the most common cause of congenital infection. Whereas CMV PCR has replaced viral culture and antigen detection in immunocompromised patients because of higher sensitivity, viral culture of neonatal urine is still referred to as the gold standard in the diagnosis of congenital CMV infection. Objective: To compare real-time CMV PCR with shell vial culture on urine in the diagnosis of congenital CMV, in a multicenter design. Study design: A series of neonatal urines (n = 340), received for congenital CMV diagnostics and routinely assessed with shell vial CMV culture, was retrospectively tested by real-time CMV PCR. Results: The proportion of newborns found to be congenitally infected by real-time CMV PCR was 8.2% (28/340, 95%CI 5.6-11.8%), and 7.4% (25/340, 95%CI 4.9-10.8%) by rapid culture. When considering rapid culture as reference, real-time PCR was highly sensitive (100%), whereas sensitivity of rapid culture was 89.3% when considering real-time PCR as reference. Conclusions: Our results, supported by analytical and clinical data on CMV DNA detection in neonatal urine, suggest enhanced sensitivity of recent PCR techniques when compared to viral culture. There is considerable rationale to favor real-time CMV PCR as a gold standard in the diagnosis of congenital CMV infection. A large-scale study combining both laboratory and clinical data is required to determine the exact time frame for sampling of neonatal urine when using real-time PCR.
Diagnostic Microbiology and Infectious Disease, 2020
To evaluate the potential of dried blood spots (DBS) as a congenital cytomegalovirus (cCMV) testing specimen, the laboratory diagnostic accuracy of polymerase chain reaction (PCR) on DBS was compared to viral urine cultures from neonates suspected for cCMV. Two different extraction methods (EasyMAG, bioMérieux versus Qiagen) and 2 real-time PCR protocols (in-house versus Argene) were compared. We were able to collect both DBS and urine samples in 6 Belgian neonatal units from 276 neonates suspected for cCMV registered in CMVREG (an online neonatal registry system). Forty-eight neonates (17.4%) were positive by viral culture in urine. Laboratory diagnostic accuracy parameters of DBS-PCR were both extraction method and PCR protocol dependent. Not all DBS-CMV-PCR methods successfully detected urine-culture-positive neonates born after first-trimester seroconversions. Interestingly, however, all urine-culture-positive neonates having clinical signs of cCMV did consistently score positive.
Archives of Disease in Childhood - Fetal and Neonatal Edition, 2003
Objectives: To define the incidence of congenital cytomegalovirus (CMV) infection in a defined population in Israel as diagnosed by urine polymerase chain reaction (PCR), and to assess the utility of this method for screening for congenital CMV infection. Design: A convenient sample of urine specimens from asymptomatic newborns were subjected to CMV PCR. Positive results were validated by urine tube culture and by determination of serum CMV IgM antibodies. Maternal CMV IgG was determined in a representative sample of mothers. Newborns with positive urine specimens underwent full clinical evaluation. Epidemiological characteristics of the mothers were extracted from the medical records. Settings: Two medical centres in Israel with different population characteristics. Patients: A total of 2000 newborns (1000 in each medical centre). Main outcome measure: Presence of CMV DNA in the urine. Results: Despite significant epidemiological differences between the populations in the two hospitals, the CMV seroprevalence was similar, 80.5% and 85%. Fourteen of the 2000 newborns screened (0.7%) were PCR positive. Urine culture was positive in nine of 10 specimens; IgM was positive in only two of 13 newborns with positive PCR. Eleven newborns underwent full or partial evaluation, and only one (9%) was symptomatic. Conclusions: The incidence of congenital CMV infection in the study population was 0.7%; over 90% were asymptomatic. Urinary CMV PCR is a reliable, rapid, and convenient method, and thus may serve as a screening tool for the detection of congenital CMV infection.
Saliva pools for screening of human cytomegalovirus using real-time PCR
European Journal of Pediatrics
Human cytomegalovirus (HCMV) is the leading congenital infection agent in the world. The importance of screening this infection has been debated, as 10–15% of the asymptomatic newborns with HCMV at birth will present late sequelae. The aim of this study was to test the feasibility of using saliva pools from newborns in a screening program for congenital HCMV infection, in two Portuguese hospitals. The screening was based on the use of pools of 10 saliva samples for detection of viral DNA by real-time PCR. Whenever there was a positive pool, the samples were tested individually, and for each positive sample the result was confirmed with a urine sample collected in the first 2 weeks of life. The study involved 1492 newborns. One hundred and fifty pools were screened, with 14 positive results in saliva, but only 10 were confirmed in urine samples, giving a prevalence of congenital HCMV infection in both hospitals of 0.67% (CI95% 0.36 to 1.23%). Conclusion: The overall prevalence of con...
2010
PCR has been commonly used for genomic viral diagnosis for its sensitivity and accuracy. It showed a higher sensitivity when compared to virus isolation in tissue culture and also in antigenemia detection. Definitely, DNA samples are critical factor in PCR validity. Out of 84 serum samples subjected to this study and extracted by Wizard® DNA purification mini kit, 27 samples (32.1%) were positive PCR. While, 15 samples (17.9%) only out of the same population study (84) were positive PCR for HCMV DNA in native serum samples (without DNA extraction). This result was confirmed the importance of DNA extraction from serum samples for detection of HCMV which, subsequently lead to more sensitive diagnostic tool of an ongoing HCMV infection.
Journal of Clinical Virology, 2006
There are no studies on the detection of cytomegalovirus (CMV) DNA by molecular methods in the saliva of newborn infants in large scale screening programs. Objectives: To evaluate the usefulness of saliva as a sample for the neonatal screening of congenital CMV infection as compared to urine when processed by a PCR. Study Design: Saliva and urine samples were obtained during the first week of life. Both samples were attempted to be obtained from the first 2816 neonates. Subsequently, only saliva was obtained from other 1623 infants. Urine and saliva were processed by DNA-PCR. Confirmation of positive results was done by PCR and virus isolation by 3 weeks after birth. Results: A urine sample was not obtainable from 893/2816 (31.7%) infants. Both saliva and urine samples were obtained from the remaining 1923 infants. Of these, 28 (1.45%) were CMV-infected. There was 99.7% agreement between the results with both samples. CMV excretion was similar when PCR was applied to urine (1.3%) or to saliva (1.2%) samples. Among the subsequent 1623 infants for whom only a saliva sample was planned for screening, 16 (0.98%) were CMV-infected. Conclusions: Saliva samples are as useful as urine for the identification of CMV-DNA in large use for screening programs.
Journal of clinical microbiology, 2000
Cytomegalovirus (CMV) is the most common cause of congenital infection in the developed world. We have designed and evaluated an assay that includes an internal control for amplification and detection of CMV DNA in amniotic fluid and neonatal urine samples. We present data on the use of this assay in the diagnosis of congenital CMV infection. A total of 145 amniotic and fetal fluid samples were examined by this assay; 83 were from healthy pregnant women and 62 were from women who were being investigated because of concerns over the pregnancy (diagnostic group). CMV DNA was detected in three amniotic fluid samples from the diagnostic group but was not detected in any samples taken from healthy pregnant women. Thirty-nine urine samples were obtained from 19 neonates with suspected congenital infection; CMV DNA was detected in urine from 6 of these patients. The assay provides useful information about CMV infection in the fetus and the neonate; when used in conjunction with other diagn...
Journal of Clinical Virology, 2004
Background: PCR detection of human cytomegalovirus (HCMV) DNA in amniotic fluid (AF) is the most sensitive tool for diagnosis of fetal infection, but has sub-optimal sensitivity. It has been suggested that inhibition by AF reduces the sensitivity of this assay, however this assumption has never been thoroughly studied. Several PCR assays have been shown to improve sensitivity, but comparative studies are insufficient to choose the optimal approach. Objectives: To assess the effect of AF inhibition on PCR sensitivity and to determine the most sensitive assay for diagnosing fetal infection. Study design: Plasmid containing HCMV DNA was tested by PCR, in water and in non-infected AF, to assess the inhibitory effect of AF. Twenty-three AF-infected samples were tested by various PCR protocols. AF supernatant, with or without DNA extraction, and AF cells, were assayed by single-round and semi-nested PCR. Viral load was measured in the supernatant by a commercial quantitative PCR kit. Results: The plasmid model demonstrated that single-round PCR was 2000-fold less sensitive in AF compared with water. Semi-nested PCR was only 10-fold less sensitive. Single-round PCR was 30% sensitive in HCMV-infected AF supernatants, and detected viral loads higher than 2.3 × 10 6 viral copies/ml. Extraction of DNA from the supernatant increased the sensitivity of this assay to 89% and the detection limit to 5.2 × 10 4 copies/ml. Semi-nested PCR performed on supernatant, with and without DNA extraction, was 96% and 100% sensitive, respectively, with a detection threshold of 3.8 × 10 3 copies/ml. Single-round and semi-nested PCR were 89% and 100% sensitive, respectively, in cells. The commercial quantitative PCR assay was 100% sensitive. Conclusions: AF supernatant is inhibitory to PCR. The two most sensitive assays were semi-nested PCR performed on DNA extracted from the supernatant and the commercial quantitative PCR kit. Of these two, the latter is standardized, non-labor-intensive, and allows minimal opportunity for contamination, thereby making it the preferred method for diagnosis.