Saliva pools for screening of human cytomegalovirus using real-time PCR

Abstract 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 congenital HCMV infection in both hospitals was 0.67%. The use of saliva pools proved to be effective for the screening of this congenital infection, allowing timely screening and confirmation in a large population, with associated cost reduction. What is Known:• Newborn screening for HCMV is desirable.• Saliva is a good and practical sample. What is New:• The feasibility of using saliva pools for a large-scale screening.• The cost reduction of this strategy.

Download Full-text

Maternal Fc-mediated non-neutralizing antibody responses correlate with protection against congenital human cytomegalovirus infection

10.1101/2021.12.05.21267312 ◽

2021 ◽

Author(s):

Eleanor C. Semmes ◽

Itzayana G. Miller ◽

Jennifer A. Jenks ◽

Courtney E. Wimberly ◽

Stella J. Berendam ◽

...

Keyword(s):

Cord Blood ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Passive Immunization ◽

Congenital Infection ◽

Neutralizing Antibody ◽

Antibody Responses ◽

High Avidity ◽

Igg Binding ◽

Congenital Hcmv Infection

AbstractHuman cytomegalovirus (HCMV) is the most common congenital infection and a leading cause of stillbirth, neurodevelopmental impairment, and pediatric hearing loss worldwide. Development of a maternal vaccine or therapeutic to prevent congenital infection has been hindered by limited knowledge of the immune responses that protect against placental HCMV transmission in maternal primary and nonprimary infection. To identify protective antibody responses, we measured anti-HCMV IgG binding and anti-viral functions in maternal and cord blood sera from HCMV transmitting (n=41) and non- transmitting (n=40) mother-infant dyads identified via a large U.S.-based public cord blood bank. In a predefined immune correlate analysis, maternal monocyte-mediated antibody-dependent cellular phagocytosis (ADCP) and high avidity IgG binding to HCMV envelope glycoproteins were associated with decreased risk of congenital HCMV infection. Moreover, HCMV-specific IgG engagement of FcγRI and FcγRIIA, which mediate non-neutralizing antibody responses, was enhanced in non-transmitting mother-infant dyads and strongly correlated with ADCP. These findings suggest that Fc effector functions including ADCP protect against placental HCMV transmission. Taken together, our data indicate that future active and passive immunization strategies to prevent congenital HCMV infection should target Fc-mediated non-neutralizing antibody responses.

Download Full-text

Animal models of human cytomegalovirus congenital infection

Microbiology Australia ◽

10.1071/ma15068 ◽

2015 ◽

Vol 36 (4) ◽

pp. 196

Author(s):

Helen Farrell

Keyword(s):

Animal Models ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Phylogenetic Analyses ◽

Congenital Infection ◽

Host Responses ◽

Laboratory Animals ◽

Natural Hosts ◽

Congenital Hcmv Infection ◽

Species Specific

Human cytomegalovirus (HCMV) infection is highly species-specific, which means that it is unable to productively infect laboratory animals. Despite this caveat, studies of animal CMV counterparts in their natural hosts have revealed significant correlations with observed neuropathological effects of congenital HCMV infection and have improved our understanding of host responses to vaccination. The biological relatedness between human and animal CMVs has been confirmed by phylogenetic analyses; the conservation of ‘core' genes that are essential for virus replication as well as genes that contribute similar mechanisms for virus persistence in their respective host species. The common animal models of HCMV congenital infection include Rhesus CMV (RhCMV), guinea-pig CMV (GPCMV) and mouse CMV (MCMV). Whilst animal models of CMV do not fully recapitulate HCMV infection, they each offer specific advantages in understanding HCMV congenital/perinatal infection (summarised in Table 1).

Download Full-text

Sequence analysis of human cytomegalovirus US28 gene in low-passage clinical isolates from children and AIDS patients.

Acta Biochimica Polonica ◽

10.18388/abp.2011_2270 ◽

2011 ◽

Vol 58 (2) ◽

Cited By ~ 2

Author(s):

Rong He ◽

Chang Xia ◽

Qiang Ruan ◽

Ying Qi ◽

Yan-Ping Ma ◽

...

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

Congenital Infection ◽

Opportunistic Pathogen ◽

Clinical Isolates ◽

Aids Patients ◽

C Terminus ◽

Congenital Hcmv Infection ◽

Low Passage ◽

Tissue Tropisms

Human cytomegalovirus (HCMV) is often a dangerous opportunistic pathogen that causes significant morbidity and mortality in newborn children and immunocompromised patients. The different symptoms and tissue tropisms of HCMV infection may result from genetic polymorphism. This study investigated the sequence variability of the HCMV US28 ORF, which shows sequence homology to the G protein-coupled receptor. HCMV isolated from suspected pediatric cases and isolates from AIDS patients were compared in order to examine the possible associations between polymorphisms and pathogenesis. Seventy children with suspected congenital HCMV infection, who suffered from jaundice (47), megacolon (10), and microcephaly (13), and 17 AIDS patients, were studied. Mutation was prevalent among the sequences of US28, with a focus on the two ends of US28. The important functional groups of US28 are highly conserved. An unrooted tree showed that all sequences from suspected congenitally infected infants and AIDS patients were divided into three groups. Comparison showed that most of the sequences (12/17) from pediatric patients were included in the first group (G1), whereas most of the sequences (11/17) from AIDS patients were included in the third group (G3). The specific high mutation sites in US28 from children were located at the C terminus of the protein, whereas those from AIDS patients were located at the N terminus. We demonstrated the existence of polymorphisms among the US28 genes of clinical isolates of HCMV from infants with suspected congenital infection. Comparison of US28 sequences from AIDS patients with those from children showed that both sequences have their own specific high mutation points.

Download Full-text

Prevalence of human cytomegalovirus congenital infection in Portuguese newborns

Eurosurveillance ◽

10.2807/ese.14.09.19135-en ◽

2009 ◽

Vol 14 (9) ◽

Author(s):

P Paixão ◽

S Almeida ◽

P Gouveia ◽

L Vilarinho ◽

R Vaz Osório

Keyword(s):

Human Cytomegalovirus ◽

Hcmv Infection ◽

National Level ◽

Congenital Infection ◽

Dna Amplification ◽

Dna Extraction Method ◽

Wide Range ◽

Congenital Hcmv Infection ◽

National Prevalence ◽

Guthrie Cards

Human cytomegalovirus (HCMV) is considered the most frequent cause of congenital infection, occurring in 0.2 to 2.2% of all live births. Since this is a wide range of prevalences observed in different studies, it would be desirable to investigate the prevalence of this infection at national level. The aim of this study was the evaluation of the national prevalence of HCMV congenital infection. We analysed a total of 3,600 Guthrie cards collected from Portuguese newborns during a period of 14 months (August 2003 to September 2004). The cards covered all regions of Portugal and were proportional to the number of births in each region. A heat DNA extraction method was used, followed by DNA amplification by nested PCR. Sensitivity and specificity of this method were evaluated as 93% and 100%, respectively, using 28 cards from HCMV-positive and 280 cards from HCMV-negative children. The national prevalence of congenital HCMV was determined as 1.05% (95% confidence interval: 0.748-1.446). This is the first study of the prevalence of HCMV congenital infection at national level in Portugal. It suggests that Portugal may have one of the highest prevalences of congenital HCMV infection in Europe.

Download Full-text

Detecting human cytomegalovirus drug resistant mutations and monitoring the emergence of resistant strains using real-time PCR

Journal of Clinical Virology ◽

10.1016/j.jcv.2014.07.008 ◽

2014 ◽

Vol 61 (2) ◽

pp. 270-274 ◽

Cited By ~ 5

Author(s):

Pavlina Volfova ◽

Martina Lengerova ◽

Jana Lochmanova ◽

Dana Dvorakova ◽

Dita Ricna ◽

...

Keyword(s):

Real Time ◽

Human Cytomegalovirus ◽

Real Time Pcr ◽

Drug Resistant ◽

Resistant Strains

Download Full-text

Development and evaluation of duplex TaqMan real-time PCR assay for detection and differentiation of wide-type and MGF505-2R gene-deleted African swine fever viruses

10.21203/rs.3.rs-59710/v2 ◽

2020 ◽

Author(s):

zhenhua Guo ◽

Kunpeng Li ◽

Songlin Qiao ◽

Xinxin Chen ◽

Ruiguang Deng ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Large Scale ◽

Limit Of Detection ◽

African Swine Fever ◽

Economic Losses ◽

Clinical Samples ◽

Diagnosis Method ◽

Pcr Method ◽

And Control

Abstract Background: African swine fever (ASF) is the most important disease to the pigs and cause serious economic losses to the countries with large-scale swine production. Vaccines are recognized as the most useful tool to prevent and control ASF virus (ASFV) infection. Currently, the MGF505 and MGF360 gene-deleted ASFVs or combined with CD2v deletion were confirmed to be the most promising vaccine candidates. Thus, it is essential to develop a diagnosis method to discriminate wide-type strain from the vaccines used.Results: In this study, we established a duplex TaqMan real-time PCR based on the B646L gene and MGF505-2R gene. The sequence alignment showed that the targeted regions of primers and probes are highly conserved in the genotype II ASFVs. The duplex real-time assay can specifically detect B646L and MGF505-2R gene single or simultaneously without cross-reaction with other porcine viruses tested. The limit of detection was 5.8 copies and 3.0 copies for the standard plasmids containing B646L and MGF505-2R genes, respectively. Clinical samples were tested in parallel by duplex real-time PCR and a commercial ASFV detection kit. The detection results of these two assays against B646L gene were well consistent.Conclusion: We successfully developed and evaluated a duplex TaqMan real-time PCR method which can effectively distinguish the wide type and MGF505 gene-deleted ASFVs. It would be a useful tool for the clinical diagnosis and control of ASF.

Download Full-text

The Light and Shadow of Rapid Serological Tests for SARS-CoV-2 Infection: Results from a Study in a Large Emergency Department

International Journal of Environmental Research and Public Health ◽

10.3390/ijerph17186493 ◽

2020 ◽

Vol 17 (18) ◽

pp. 6493

Author(s):

Daniela Loconsole ◽

Francesca Centrone ◽

Caterina Morcavallo ◽

Silvia Campanella ◽

Anna Sallustio ◽

...

Keyword(s):

Emergency Department ◽

Real Time ◽

Real Time Pcr ◽

Respiratory Symptoms ◽

Large Scale ◽

Serological Test ◽

Serological Tests ◽

Serological Assay ◽

Infected People ◽

Screening Programs

A critical point in the management of the SARS-CoV-2 pandemic is the need to promptly identify the greatest number of infected people and to implement strict public health measures. In this study, the performance of a rapid serological test in a clinical setting was evaluated. Samples from 819 consecutive patients (with or without respiratory symptoms) admitted to a large Emergency Department were tested between 23 March and 21 April 2020. Patient samples were tested in a real-time PCR assay and a serological assay. In total, 148/819 patients (18.1%) tested positive for SARS-CoV-2 by real-time PCR. The serological test revealed that 70/819 patients (8.5%) had anti-SARS-CoV-2 IgM and/or IgG. The prevalence of anti-SARS-CoV-2 antibodies was significantly higher in patients with respiratory symptoms lasting for >7 days than in those with respiratory symptoms lasting for 0–7 days (p < 0.001). The serological assay had an overall sensitivity of 35.1% and an overall specificity of 97.3%. A high negative predictive value (96.7%) was reported for patients without respiratory symptoms. The results confirm that rapid serological assays alone are not sufficient for diagnosis of SARS-CoV-2 infection but can be incorporated into large-scale screening programs during periods in which the virus circulation is low.

Download Full-text

Novel Real-Time Monitoring System for Human Cytomegalovirus- Infected Cells In Vitro That Uses a Green Fluorescent Protein-PML-Expressing Cell Line

Antimicrobial Agents and Chemotherapy ◽

10.1128/aac.01641-05 ◽

2006 ◽

Vol 50 (8) ◽

pp. 2806-2813 ◽

Cited By ~ 10

Author(s):

T. Ueno ◽

Y. Eizuru ◽

H. Katano ◽

T. Kurata ◽

T. Sata ◽

...

Keyword(s):

Green Fluorescent Protein ◽

Cell Line ◽

Real Time ◽

Human Cytomegalovirus ◽

Hcmv Infection ◽

Fluorescent Protein ◽

Clinical Isolates ◽

Pml Bodies ◽

Infected Cells ◽

Green Fluorescent

ABSTRACT Promyelocytic leukemia (PML) bodies are discrete nuclear foci that are intimately associated with many DNA viruses. In human cytomegalovirus (HCMV) infection, the IE1 (for “immediate-early 1”) protein has a marked effect on PML bodies via de-SUMOylation of PML protein. Here, we report a novel real-time monitoring system for HCMV-infected cells using a newly established cell line (SE/15) that stably expresses green fluorescent protein (GFP)-PML protein. In SE/15 cells, HCMV infection causes specific and efficient dispersion of GFP-PML bodies in an IE1-dependent manner, allowing the infected cells to be monitored by fluorescence microscopy without immunostaining. Since a specific change in the detergent solubility of GFP-PML occurs upon infection, the infected cells can be quantified by GFP fluorescence measurement after extraction. With this assay, the inhibitory effects of heparin and neutralizing antibodies were determined in small-scale cultures, indicating its usefulness for screening inhibitory reagents for laboratory virus strains. Furthermore, we established a sensitive imaging assay by counting the number of nuclei containing dispersed GFP-PML, which is applicable for titration of slow-growing clinical isolates. In all strains tested, the virus titers estimated by the GFP-PML imaging assay were well correlated with the plaque-forming cell numbers determined in human embryonic lung cells. Coculture of SE/15 cells and HCMV-infected fibroblasts permitted a rapid and reliable method for estimating the 50% inhibitory concentration values of drugs for clinical isolates in susceptibility testing. Taken together, these results demonstrate the development of a rapid, sensitive, quantitative, and specific detection system for HCMV-infected cells involving a simple procedure that can be used for titration of low-titer clinical isolates.

Download Full-text

Different Real-Time PCR Formats Compared for the Quantitative Detection of Human Cytomegalovirus DNA

Clinical Chemistry ◽

10.1093/clinchem/45.11.1932 ◽

1999 ◽

Vol 45 (11) ◽

pp. 1932-1937 ◽

Cited By ~ 65

Author(s):

Andreas Nitsche ◽

Nina Steuer ◽

Christian Andreas Schmidt ◽

Olfert Landt ◽

Wolfgang Siegert

Keyword(s):

Real Time ◽

Human Cytomegalovirus ◽

Real Time Pcr ◽

Detection System ◽

Quantitative Detection ◽

Fluorescence Signal ◽

Hybridization Probe ◽

Sequence Detection ◽

Hybridization Probes ◽

Assay Time

Abstract Background: The aim of this study was to compare the ABI PRISM 7700 Sequence Detection System and the LightCycler to develop a quantitative real-time PCR assay for the detection of human cytomegalovirus (HCMV) DNA suitable for routine hospital application. Methods: We used one exonuclease probe and five different hybridization probe sets as sequence-specific fluorescence detection formats. For the exonuclease assay and two hybridization probe sets, reproducibility and the detection limit were determined. To keep the total assay time to a minimum, we gradually shortened individual reaction steps on both instruments. Results: The exonuclease assay can be interchangeably performed on the 7700 and the LightCycler. No change of reaction conditions is required, except for the addition of bovine serum albumin to the LightCycler reaction. The shortest possible total assay time is 80 min for the ABI PRISM 7700 Sequence Detection System and 20 min for the LightCycler. When the LightCycler is used, the exonuclease probe can be replaced by a set of hybridization probes. All assays presented here detected HCMV DNA in a linear range from 101 to 107 HCMV genome equivalents/assay (r >0.995) with low intraassay (<5%) and interassay (<10%) variation. Conclusions: The ABI PRISM 7700 Sequence Detection System as well as the LightCycler are useful instruments for rapid and precise online PCR detection. Moreover, the two principles of fluorescence signal production allow HCMV quantification with the same accuracy.

Download Full-text

Real-time PCR versus shell vial culture on urine of patients with suspected congenital cytomegalovirus infection

Future Virology ◽

10.2217/fvl-2019-0078 ◽

2019 ◽

Vol 14 (9) ◽

pp. 585-591

Author(s):

Luana Coltella ◽

Stefania Ranno ◽

Giuseppe Pizzichemi ◽

Livia Piccioni ◽

Stefano Chiavelli ◽

...

Keyword(s):

Real Time ◽

Real Time Pcr ◽

Congenital Infection ◽

Urine Samples ◽

Congenital Cytomegalovirus ◽

Shell Vial ◽

Congenital Cmv Infection ◽

Polymerase Chain ◽

Improve Patient Management ◽

Improve Patient

Aims: Cytomegalovirus (CMV) is the most common cause of congenital infection. Aim of this study is to support quantitative real-time polymerase chain reaction (PCR) versus shell vials culture for CMV screening in urine samples. Patients & methods: A retrospective study was conducted on 255 urine samples belonging to patients admitted to Bambino Gesù Pediatric Hospital, Rome, Italy, with suspected congenital CMV infection. Results & conclusion: Quantitative real-time PCR resulted more standardized, faster, less operator-dependent, less laborious and most of all cost saving and more sensitive than shell vial culture. Since a negative result for CMV in urine means no congenital infection, a more sensitive tool for detection of CMV DNA is essential to improve patient management and to reduce healthcare costs associated to a late diagnosis.

Download Full-text