Torque teno virus viremia and QuantiFERON®-CMV assay in prediction of cytomegalovirus reactivation in R+ kidney transplant recipients.

Introduction: Cytomegalovirus (CMV) is the most frequent infectious complication following solid organ transplantation. Torque teno viruses (TTV) viremia has been proposed as a biomarker of functional immunity in the management of kidney transplant recipients (KTR). The QuantiFERON®-CMV (QF-CMV) is a commercially available assay that allows the assessment of CD8+ T-cell responses in routine diagnostic laboratories. Methods: In a prospective national multicenter cohort of 64 CMV-seropositive (R+) KTR, we analyzed the value of TTV load and the two markers of the QF-CMV assay [QF-Ag (CMV-specific T-cell responses) and QF-Mg (overall T-cell responses)], alone and in combination, in prediction of CMV reactivation (≥3 log10 IU/ ml) in the first post-transplant year. We compared previously published cut-offs and specific cut-offs optimized from ROC curves for our population. Results: Using the conventional cut-off (3.45 log10 copies/ml), TTV load at D0 [inclusion visit on the day of transplantation before induction (D0)], or at M1 (1-month post-transplant visit) perform better in predicting CMV viremia control than CMV reactivation. Survival analyses suggest a better performance of our optimized TTV cut-offs (3.78 log10 copies/ml at D0 and 4.23 log10 copies/ml at M1) for risk stratification of CMV reactivation in our R+ KTR cohort. The QF-CMV (QF-Ag = 0.2 IU/ml, and QF-Mg = 0.5 IU/ml) also appears to better predict CMV viremia control than CMV reactivation. Moreover, survival analyses suggest that the QF-Mg would perform better than the QF-Ag in stratifying the risk of CMV reactivation. The use of our optimized QF-Mg cut-off (1.27 IU/ml) at M1 further improved risk stratification of CMV reactivation. Using conventional cut-offs, the combination of TTV load and QF-Ag or TTV load and QF-Mg did not improve prediction of CMV viremia control compared to separate analysis of each marker but resulted in an increase of positive predictive values. The use of our cut-offs slightly improved risk prediction of CMV reactivation. Conclusion: The combination of TTV load and QF-Ag or TTV load and QF-Mg could be useful in stratifying the risk of CMV reactivation in R+ KTR during the first post-transplant year and thereby have an impact on the duration of prophylaxis in these patients. Clinical trial registration: ClinicalTrials.gov registry, identifier NCT02064699.

Performance Evaluation of the VIDAS(®) Measles IgG Assay and Its Diagnostic Value for Measuring IgG Antibody Avidity in Measles Virus Infection.

The objective of this study is primarily to compare the performance of the VIDAS(®) Measles immunoglobulin (Ig)G assay to that of two other serological assays using an immunoassay technique, Enzygnost(®) Anti-measles Virus/IgG (Siemens) and Measles IgG CAPTURE EIA(®) (Microimmune). The sensitivity and the agreement of the VIDAS(®) Measles IgG assay compared to the Enzygnost(®) Anti-measles Virus/IgG assay and the Measles IgG CAPTURE EIA(®) assay are 100%, 97.2% and 99.0%, 98.4%, respectively. The very low number of negative sera for IgG antibodies does not allow calculation of specificity. As a secondary objective, we have evaluated the ability of the VIDAS(®) Measles IgG assay to measure anti-measles virus IgG antibody avidity with the help of the VIDAS(®) CMV IgG Avidity reagent, using 76 sera from subjects with measles and 238 other sera. Different groups of populations were analyzed. In the primary infection measles group, the mean IgG avidity index was 0.16 (range of 0.07 to 0.93) compared to 0.79 (range of 0.25 to 1) in the serum group positive for IgG antibodies and negative for IgM. These data allow to define a weak anti-measles virus IgG antibody avidity as an avidity index (AI) < 0.3 and a strong avidity as an AI > 0.6. The VIDAS(®) Measles IgG assay has a performance equivalent to that of other available products. Its use, individual and quick, is well adapted to testing for anti-measles immunity in exposed subjects.

Study and interest of cellular load in respiratory samples for the optimization of molecular virological diagnosis in clinical practice.

BACKGROUND: Respiratory viral diagnosis of upper respiratory tract infections has largely developed through multiplex molecular techniques. Although the sensitivity of different types of upper respiratory tract samples seems to be correlated to the number of sampled cells, this link remains largely unexplored. METHODS: Our study included 800 upper respiratory tract specimens of which 400 negative and 400 positive for viral detection in multiplex PCR. All samples were selected and matched for age in these 2 groups. For the positive group, samples were selected for the detected viral species. RESULTS: Among the factors influencing the cellularity were the type of sample (p < 0.0001); patient age (p < 0.001); viral positive or negative nature of the sample (p = 0.002); and, for the positive samples, the number of viral targets detected (0.004 < p < 0.049) and viral species. CONCLUSION: The cellular load of upper respiratory samples is multifactorial and occurs for many in the sensitivity of molecular detection. However it was not possible to determine a minimum cellularity threshold allowing molecular viral detection. The differences according to the type of virus remain to be studied on a larger scale.

Development of duplex real-time PCR for detection of two DNA respiratory viruses.

A method was developed for the detection and quantitation of HAdV (human adenovirus) and HBoV (human bocavirus) based on a duplex real-time PCR, the AB PCR, using a Smartcycler instrument. A control real-time PCR was carried out on albumin DNA to standardise the non-homogenous respiratory samples. No cross-reactivity was observed with viruses or bacteria that could be found in the respiratory tract. The diagnosis rate using the AB PCR on clinical samples was 10.7%: 3.4% for HBoV detection, 6.9% for HAdV detection and 0.3% double detection HBoV-HAdV. The clinical and epidemiological characteristics of the HAdV- and HBoV-infected patients were evaluated. In the HAdV-positive group and the HBoV-positive group the samples were classified according to the severity of the disease. The HAdV viral load did not appear to be linked to the severity of the disease. Conversely, the difference between the two HBoV groups, severe and non-severe, was significant statistically when the comparison was based on the viral load (P=0.006) or after adjustment of the viral load to the number of cells in the samples (P=0.02).

Detection of human bocavirus in hospitalised children.

AIM: The objectives of this study are to assess the frequency of human bocavirus (HBoV) infection in hospitalised children and to study the clinical symptoms associated with the detection of HBoV. METHODS: Two groups of hospitalised children were included in this study: group 1 consisted of 1946 children hospitalised from 1st September 2004 to 30th May 2005, and group 2 consisted of 448 children hospitalised from 1st November 2003 to 30th March 2004. The respiratory specimens were tested by polymerase chain reaction. RESULTS: In the first group, HBoV was detected by polymerise chain reaction in 11/828 (1.3%) of nasal specimens that tested negative for other respiratory viruses. One child tested positive for HBoV in both a nasal aspirate and stool sample. In the second group, nasal specimens were tested for all respiratory viruses, including HBoV. The presence of HBoV infection was detected in seven children (1.6%). Detection of a mixed viral population was observed in four of these children. The main symptoms in children infected with HBoV were rhinitis (50%), cough (45%), dyspnoea (28%), wheezing (28%), fever (23%) and diarrhoea (22%). The final clinical diagnoses were bronchiolitis (seven children), rhinopharyngitis (five children), the exacerbation of asthma (two children) and pneumonia (one child). Moreover, four children have associated gastroenteritis. CONCLUSION: These results contribute to the interest in the HBoV detection in children. HBoV detection in hospitalised children with or without any other respiratory virus detection was essentially associated with lower respiratory tract infection and in a lower score with upper respiratory tract infection and gastroenteritis.

Human (non-severe acute respiratory syndrome) coronavirus infections in hospitalised children in France.

AIM: This study has two objectives: to study the clinical symptoms associated with the detection of the four human coronaviruses (HCoVs), 229E, OC43, NL63 and HKU1 types, in the respiratory specimens sampled from hospitalised children in France between September 2004 and May 2005; and to develop a multiplex reverse transcription polymerase chain reaction (RT-PCR) assay allowing for the simultaneous detection of the four HCoVs. METHODS: 1002 respiratory specimens were tested for HCoVs. The clinical and epidemiological data were compared on the basis of the type HCoV infection. RESULTS: A hundred coronaviruses, 33 NL63, 2229E, 27 OC43 and 38 HKU1, were detected in 97 (9.8%) of 1002 samples negative in routine tests. The clinical and epidemiological characteristics of the study children were compared in three groups, 24 OC43-, 27 NL63- and 34 HKU1-infected children. HCoVs were identified mainly in children with upper and lower respiratory tract infections (50.5% vs. 29.4%). The significant difference in clinical presentation between the three coronavirus groups was the very low association between lower respiratory tract illness and HKU1 detection. CONCLUSIONS: HCoV detection in hospitalised children without any other respiratory virus detection was associated with upper and a significant rate of lower respiratory tract illness. The four types of HCoVs were detected, and new types NL63 and HKU1 represented a substantial portion of detection. The multiplex RT-PCR enabled a sensitive one-time detection and the characterisation of all of the known HCoV types with the exception severe acute respiratory syndrome-coronavirus.

[Techniques used for the diagnostic of upper and lower respiratory tract viral infections]. / Moyens diagnostiques des infections virales respiratoires aiguës hautes et basses.

Techniques used for the diagnostic of upper and lower respiratory tract viral infections Techniques used for the diagnostic of upper and lower respiratory tract viral infections include conventional procedures and the new molecular tools. Serology has no interest for diagnosis. Antigen detection can be carried out by simple, easy and cheap tools. They are available at hospital and in community practice as well for the detection of influenza virus, RSV, hMPV, parainfluenza virus and adenovirus. To look for viruses which are non detectable by antigen detection assays (rhinovirus, coronavirus, bocavirus, polyomavirus...) or in severe or complicated respiratory lower infections, the use of molecular tools is highly indicated.

Inter- and intra-variant genetic heterogeneity of human coronavirus OC43 strains in France.

Human coronavirus OC43 (HCoV-OC43) causes acute, self-limited respiratory infections. A close relationship between bovine coronaviruses (BCoVs) and HCoV-OC43 has recently been demonstrated. This study includes seven clinical, non-cell culture-adapted, contemporary HCoV-OC43 strains detected in France in 2003. By using RT-PCR and clonal sequencing of the S1 gene of HCoV-OC43, the inter-variant heterogeneity of the HCoV-OC43 circulating strains was studied and the intra-variant diversity was assessed by investigation of a quasispecies cloud. This paper brings to the forefront a high genetic diversity of circulating HCoV-OC43 variants. Genetically different groups are defined among the variants described in this study. One of these variants holds characteristics of an outlier and presents a deletion of 12 nt, also found in BCoV strains. Moreover, the presence of HCoV-OC43 as a quasispecies cloud in vivo during an acute respiratory-tract illness was discovered. It has also been revealed that quasispecies-cloud sizes are similar for the two viral populations tested.

Comparison of multiplex PCR assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness.

The performances of four multiplex PCR (m-PCR) were compared to direct immunofluorescence assay (DFA) and HuH7 cell culture for the detection of viruses in 263 children admitted to hospital with an acute respiratory illness. One hundred fifty (57.6%) nasal aspirates were found DFA-positive; 188 (72.3%) were found positive by both DFA and HuH7 cell culture, and 242 (92%) were PCR-positive. The m-PCR detected 124 viruses which were not found by conventional methods: 68 rhinovirus, 17 human metapneumovirus, 15 respiratory syncytial virus (RSV), 8 parainfluenza virus (PIV), 5 coronavirus 229E, 3 OC43 and 3 NL63, 4 enterovirus, 2 influenza virus B and C virus. The m-PCR were more sensitive, had the advantages of a shorter delay in specific diagnosis, and a lower cost than DFA and culture. Using these m-PCR, the prevalence of each virus was compared between in-patient and out-patient groups of children attending the emergency unit of the hospital. Nasal aspirates from 411 (91.5%) children were found positive by the PCRs. RSV, rhinovirus, and influenza virus were the most frequent viruses detected in this population, representing 43.6%, 31.8%, and 8.8% of the virus found, respectively, followed by human metapneumovirus (4.4%), coronavirus (3.4%), parainfluenza virus (3.2%), adenovirus (2.3%), and enterovirus (2.1%). RSVs were detected more significantly in the in-patient group than in the out-patient group, and influenza viruses were detected more frequently in the out-patient group than in the in-patient group. Moreover, the use of m-PCR pointed out the frequency of rhinovirus and mixed viral detections in these patients. In conclusion, according to the requirements of speed and low cost of the methods, and to achieve the highest rate of detection of respiratory viruses, the combined use of DFA and m-PCR is today likely to be the best way to improve diagnosis of respiratory illnesses in children.

Detection of the new human coronavirus HKU1: a report of 6 cases.

BACKGROUND: Human coronavirus HKU1 (HCoV-HKU1), a new group 2 coronavirus, was first characterized in 2005 from 2 adults with pneumonia in Hong Kong, China. To the best of our knowledge, there is no other report to date about the detection of this new virus. We report a molecular method allowing for the detection of HCoV-HKU1 and also report the clinical presentation of 6 infected patients. METHODS: We screened 141 specimens (135 nasal samples and 6 stool samples) received in February and March 2005 in our laboratory and obtained from 135 hospitalized patients (61.5% of whom were <5 years old and 34.1% of whom were >20 years old) for HCoV-HKU1. RESULTS: HCoV-HKU1 was detected in 6 (4.4%) of the 135 nasal specimens and in 2 (33.3%) of the 6 stool samples; the positive samples were obtained from 6 patients (5 children and 1 adult). The clinical presentation of these 6 patients was as follows: 3 were admitted to the hospital for acute enteric disease resulting in severe dehydration associated with upper respiratory symptoms; 1 had fever, otitis, and febrile seizure; 1 had a sample obtained to investigate failure to thrive; and 1 had a sample obtained for exploration of X-linked agammaglobulinemia and hyperleucocytosis. CONCLUSION: HCoV-HKU1 can be detected in respiratory and stool samples from children and adults in a part of the world other than Hong Kong. Our results suggest that HCoV-HKU1 could be associated with respiratory and enteric diseases, and its detection can be related to a persistent asymptomatic infection in patients with poor underlying conditions.

Human coronavirus NL63, France.

The human coronavirus NL63 (HCoV-NL63) was first identified in The Netherlands, and its circulation in France has not been investigated. We studied HCoV-NL63 infection in hospitalized children diagnosed with respiratory tract infections. From November 2002 to April 2003, we evaluated 300 respiratory specimens for HCoV-NL63. Of the 300 samples, 28 (9.3%) were positive for HCoV-NL63. The highest prevalence was found in February (18%). The main symptoms were fever (61%), rhinitis (39%), bronchiolitis (39%), digestive problems (33%), otitis (28%), pharyngitis (22%), and conjunctivitis (17%). A fragment of the spike protein gene was sequenced to determine the variety of circulating HCoV-NL63. Phylogenetic analysis indicated that strains with different genetic markers cocirculate in France.

Replication of respiratory viruses, particularly influenza virus, rhinovirus, and coronavirus in HuH7 hepatocarcinoma cell line.

Detection of viral antigens and isolation methods has long been used for the diagnosis of respiratory virus infections. The objective was to determine the ability of HuH7 cells to support the replication of prototype and wild strains of respiratory viruses. The cell culture-adapted strains of influenza viruses A and B, parainfluenza viruses 1-4, respiratory syncytial viruses A and B, both strains of the human metapneumoviruses, numerous rhinoviruses, most of the adenoviruses, coronaviruses 229E and OC43, and a number of enteroviruses (poliovirus type 3, coxsackie virus B1, echovirus type 30) replicate in HuH7. The kinetic study of the replication of influenza A and B viruses showed that there were infected cells in HuH7 and MDCK lines as early as 24 hr post-infection. However, the replication of influenza A and B viruses was more rapid and intense on MDCK cells than on HuH7 cells. During the three winters of 1999-2000, 2000-2001, and 2001-2002, of the 1,226 (23.3%) direct fluorescent assay-positive nasal aspirates from children admitted to hospital, 788 were positive for respiratory syncytial virus, 228 for influenza virus, 133 for parainfluenza virus, and 77 for adenovirus. Of the 4,032 direct fluorescent assay-negative nasal aspirates, 571 virus isolates were identified by using HuH7 cell culture; 272 rhinoviruses, 100 influenza viruses A and B, 85 enteroviruses, 40 adenoviruses, 35 coronaviruses, 31 parainfluenza viruses, and 10 respiratory syncytial viruses. Interestingly, 100/328 (30.5%) influenza viruses A and B, 40/189 (21.1%) adenoviruses, and 31/164 (19%) parainfluenza viruses type 1-3, not detected by direct fluorescent assay, were identified by isolation in HuH7 cell culture.

Development of a PCR-and hybridization-based assay (PCR Adenovirus Consensus) for the detection and the species identification of adenoviruses in respiratory specimens.

BACKGROUND: Antigen detection assays and viral isolation techniques are routinely used to detect adenoviruses (Ad) associated with respiratory infections, and the value of the polymerase chain reaction (PCR) has recently been assessed. OBJECTIVES: This paper describes a PCR-hybridization-immunoenzymatic assay (PCR Adenovirus consensus) used to detect Ad and identify Ad species in respiratory specimens. RESULTS: On seven representative serotypes Ad 12, Ad 3, Ad 7, Ad 11, Ad 1, Ad 8, Ad 4, the mean genome equivalents per ml and the mean 50% infectious doses per ml were 10(6.3)and 10(4), respectively. Using 362 nasal aspirates from children, Ad were detected by immunofluorescence (IF) and culture in 97 cases (27%), by the PCR-Ad hexon method in 107 cases (29.5%) and by the PCR Adenovirus Consensus method in 113 cases (31.2%); 13 samples were found positive by both PCR and negative by the IF and culture methods; five samples were only positive according to the PCR Adenovirus Consensus) method. The sensitivity, specificity, predictive positive value and predictive negative value of the PCR Adenovirus Consensus method were 97.9%, 93.2%, 84%, 99.1%, respectively. The method identified the species (sp) from 91 positive amplicons: 1 Ad sp A, 44 Ad sp B, 42 Ad sp C, 3 Ad sp E, and 1 Ad sp F; 85 isolates were identified by IF or the neutralisation in culture, and 86 by a PCR-RE digestion method. The PCR Adenovirus Consensus detected six positive samples that were negative according to the IF and culture methods, and it identified the precise species of nine IF-positive and culture-negative nasal aspirates. CONCLUSION: The PCR Adenovirus Consensus technique is more efficient than the classical IF or culture techniques for the detection of Ad in respiratory samples. An internal control is included to validate the screening results, and specific probes are used to identify the Ad species.

Natural polymorphism of cytomegalovirus DNA polymerase lies in two nonconserved regions located between domains delta-C and II and between domains III and I.

We described the natural polymorphism of cytomegalovirus DNA polymerase in 42 unrelated isolates susceptible to ganciclovir, foscarnet, and cidofovir. All variations, including an eight-amino-acid deletion, were located between domains delta-C and II and between domains III and I, suggesting that these specific residues are not involved in enzymatic functions.

An outbreak of coronavirus OC43 respiratory infection in Normandy, France.

The 2 groups of human coronaviruses (HCoVs) represented by the prototype strains HCoV 229E and HCoV OC43 are mostly known as viruses responsible for common cold syndrome. HCoVs are difficult to detect, and epidemiological data are rare. From October 2000 through April 2001, we tested 1803 respiratory samples for HCoV by reverse-transcriptase polymerase chain reaction. From 8 February through 27 March 2001, HCoV OC43 was detected in samples obtained from 30 (6%) of 501 patients. The other viruses detected were respiratory syncytial virus (6.1%), parainfluenza virus 3 (1%), influenza virus A (7.8%), influenza virus B (7.2%), rhinovirus (6.4%), enterovirus (1%), and adenovirus (2%). Infection with HCoV OC43 was detected in patients of all age groups. The following clinical symptoms were noted: fever (in 59.8% of patients), general symptoms (in 30%), digestive problems (in 56.8%), rhinitis (in 36.6%), pharyngitis (in 30%), laryngitis (in 3.3%), otitis (in 13.3%), bronchitis (in 16.6%), bronchiolitis (in 10%), and pneumonia (in 6.6%). This study shows that an outbreak of HCoV OC43 respiratory infection was responsible for the lower respiratory tract symptoms observed in nearly one-third of patients identified by active surveillance for coronavirus infection.