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Development of a chemiluminescence competitive PCR for the detection and quantification of parvovirus B19 DNA using a microplate luminometer.

BACKGROUND: Quantitative PCR of viral nucleic acids can be useful clinically in diagnosis, risk assessment, and monitoring of antiviral therapy. We wished to develop a chemiluminescence competitive PCR (cPCR) for parvovirus B19. METHODS: Parvovirus DNA target sequences and competitor sequences were coamplified and directly labeled. Amplified products were then separately hybridized by specific biotin-labeled probes, captured onto streptavidin-coated ELISA microplates, and detected immunoenzymatically using chemiluminescent substrates of peroxidase. Chemiluminescent signals were quantitatively analyzed by a microplate luminometer and were correlated to the amounts of amplified products. RESULTS: Luminol-based systems displayed constant emission but had a higher detection limit (100-1000 genome copies) than the acridan-based system (20 genome copies). The detection limit of chemiluminescent substrates was lower (20 genome copies) than colorimetric substrates (50 genome copies). In chemiluminescence cPCR, the titration curves showed linear correlation above 100 target genome copies. Chemiluminescence cPCR was positive in six serum samples from patients with parvovirus infections and negative in six control sera. CONCLUSIONS: The chemiluminescence cPCR appears to be a sensitive and specific method for the quantitative detection of viral DNAs.

DNA, Viral↗

Parvovirus B19 infections.

Infections caused by human parvovirus B19 can result in a wide spectrum of manifestations, which are usually influenced by the patient's immunologic and hematologic status. In the normal host, parvovirus infection can be asymptomatic or can result in erythema infectiosum or arthropathy. Patients with underlying hematologic and immunologic disorders who become infected with this virus are at risk for aplastic anemia. Hydrops fetalis and fetal death are complications of intrauterine parvovirus B19 infection.

Erythema Infectiosum↗

Presumed parvovirus B19-associated retinal pigment epitheliopathy.

PURPOSE: To describe a patient with a white dot syndrome associated with acute erythema infectiosum. METHODS: A patient with a clinical history of erythema infectiosum and multifocal punctate white lesions at the level of the retinal pigment epithelium was followed up for an 8-month interval. Serum was tested for immunoglobulin M (IgM) and IgG antibodies to parvovirus B19 at the time of the initial evaluation and during convalescence using an indirect immunofluorescence antibody technique. Serial photographs and fluorescein angiograms were obtained. RESULTS: IgM and IgG antibodies to parvovirus were detected in the serum at the time of initial evaluation; IgM antibodies had disappeared but IgG antibodies persisted in serum obtained at 1-month follow-up. Fundus evaluation revealed clinical disappearance of some lesions, with increased pigmentation of others over the course of follow-up. CONCLUSION: The authors have identified an adult patient who presented with a white dot syndrome associated with acute erythema infectiosum documented by serologically proved parvovirus B19 infection.

Acute Disease↗

Spontaneous resolution of nonimmune hydrops fetalis secondary to human parvovirus B19 infection.

Many instances of nonimmune hydrops fetalis ascribed to human parvovirus B19 have been reported. The leading proposed pathophysiologic mechanism of hydrops in affected fetuses is viral invasion of red blood cell progenitors, causing a profound reticulocytopenic fetal anemia. Although the natural history of fetal parvovirus infection remains to be elucidated fully, there have been recent reports of funipuncuture and intrauterine blood transfusions to diagnose and manage this problem. We report two pregnancies in which parvovirus-related hydrops fetalis was observed to resolve without intervention, followed by uncomplicated vaginal deliveries of healthy infants. These observations emphasize the need for further investigation before recommending routine fetal blood transfusion in affected cases.

Adolescent↗

[Fetal erythropoiesis and parvovirus B19].

Target cells for the human parvovirus B19 include erythroid progenitors located in the bone marrow in adults and in the liver in fetuses of 12 to 30 weeks gestational age. The main manifestations of fetal parvovirus B19 infection seem to be related to lysis of the erythroid progenitors which causes non immune hydrops fetalis with severe anemia, congestive heart failure, generalized edema and death. The exact incidence of human parvovirus B19 infection during pregnancy remains unclear.

Anemia↗

[Acute vasculitis and arthritis caused by Parvovirus B 19 infection].

A 35-year old woman was admitted with fever, polyarthritis and severe vasculitis. The symptoms resolved spontaneously within a week. Two weeks later, her seven year old son developed erythema on arms and legs and the typical "slapped cheek syndrome" characteristic of erythema infectiosum. Both cases were due to acute infection with Parvovirus B 19 as demonstrated by the presence of IgM-antibodies to Parvovirus B 19. The cases are described in order to draw attention to the possibility of Parvovirus B 19 infection as the etiological agent for acute vasculitis in adults.

Acute Disease↗

[Obstructive respiratory tract diseases in children and Parvovirus B19 infections].

Acute respiratory diseases (ARD) due to parvovirus B 19 infection can be observed relative frequently in children. In 21 children (infants, toddlers and school children) we have seen acute or prolonged obstructive bronchitis/bronchiolitis (15 infants), acute subglottic laryngitis (3 toddlers) and acute asthmatic attacks (3 children of school age) in connection with parvovirus B 19 infection. Other respiratory viruses (adeno-, influenza, parainfluenza and RS-virus) could be excluded as agents causing the ARD. We suggest that parvovirus B 19 can provoke ARD with obstructive ventilatory disturbances of the upper or lower airways in children with a specific endogenous predisposition (small or unstable bronchial walls, or bronchial or tracheal mucosal hyperreactivity).

Airway Obstruction↗

[Parvovirus B19 infection as the cause of hepatitis and neutrophil granulocytosis in a 20-year old woman].

A case of Parvovirus B19 infection (erythema infectiosum) in a 20 year old woman is presented. The patient presented with fever, arthritis in one knee, neutrophil granulocytosis and biochemical evidence of hepatitis. Serological evidence of Parvovirus B19 infection was found as the only explanation of the clinical picture. Hepatitis was due to Parvovirus B19 infection as there was no serological evidence of EBV or CMV reactivation. Neutrophil granulocytosis and thrombocytosis were found and were probably due to an active bone marrow in the recovery phase of bone marrow aplasia.

Adult↗

[Cutaneous parvovirus infections: "gloves and socks" syndrome].

INTRODUCTION: Among the viral purpuric cutaneous manifestations, papular-purpuric "gloves and socks" eruptions have been lately described. The responsibility of parvovirus B19 infection has been suspected. OBSERVATION: We report two pediatric cases of papular-purpuric eruption of the extremities with a seroconversion for the parvovirus B19. One patient, a twelve-year old girl, also had cellulitic plaques. DISCUSSION: Parvovirus B19 is known for causing various cutaneous manifestations, among which erythema infectiosum as the most classical. A papular-purpuric "gloves and socks" syndrome may be individualised. Previously described in adults, this type of eruption may be prevalent in childhood.

Child↗

Human B19 parvovirus infection in an obstetric population. A prospective study determining fetal outcome.

In a prospective study of 1,967 pregnant women who were routinely screened for recent human B19 parvovirus infection, 64 (3.3%) were identified as being IgM positive. No adverse effects were documented by ultrasound in any of the fetuses. The outcome of pregnancy was favorable in 95.1% of these women, with no evidence of hydrops fetalis or any congenital abnormalities. Two neonates (3.4%) were small for gestational age, and there was one abortion. Samples of blood obtained from 20 neonates born to women with evidence of recent infection were B19 parvovirus IgM negative. Recent infection with human B19 parvovirus in pregnancy constitutes a low risk for the development of adverse fetal effects; hence, routine antenatal screening is not warranted.

Antibodies, Viral↗

B19 parvovirus-induced anemia in a normal child. Initial bone marrow erythroid hyperplasia and response to intravenous immunoglobulin.

PURPOSE: Human B19 parvovirus infection may cause severe erythroid hypoplasia in patients with an underlying hemolytic anemia. We report a case of severe parvovirus-induced anemia with initial marrow erythroid hyperplasia in a child with no underlying hematologic disorder. CONCLUSIONS: The patient's rapid hemoglobin recovery after treatment with i.v. immunoglobulin further supports this form of therapy for children with parvovirus-induced anemia.

Anemia↗

[Human parvovirus B19-induced aplastic crisis in an elderly man with intestinal hemorrhage].

An elderly man with an intestinal hemorrhage from bowel diverticulosis developed human parvovirus B19-induced aplastic crisis. A 71-year-old man noticed occasional tarry stools and at the same time showed fever, arthralgia and severe anemia. Blood counts revealed Hgb 5.3g/dl, reticulocytes 0%, and WBC 1,900/microliters. Bone marrow examination showed hypocellular marrow with rare erythroid precursors (4.8%). A few giant proerythroblasts were found in the bone marrow smears. A diagnosis of parvovirus B19 infection was made because of detection of B19-specific IgM and IgG antibodies. Parvovirus B19 infection should be carefully checked for in patients with hemorrhage as well as those with hemolysis.

Acute Disease↗

Use of digoxigenin-labelled probes for the detection of B19 parvovirus DNA in batches of blood products.

Being thermostable and non lipid-enveloped, the B19 parvovirus cannot be destroyed by the chemical and physical treatments presently used to inactivate lipid-enveloped viruses such as HIV, hepatitis C and B viruses in plasma derivatives. A polymerase chain reaction assay was used to detect B19 parvovirus DNA. Amplified products were detected through a non-radioactive technology based on the use of a digoxigenin-labelled probe. B19 parvovirus DNA was detected in 20% of the studied batches of factor coagulation concentrates and in none of the albumin batches.

Blood↗

[Infection with parvovirus B19 in pregnant women].

The viral infection caused by Parvovirus B19 which occurs at pregnant women may be reason of many different kinds of complications during pregnancy. Until this time it is not known the frequency of the Parvovirus infections at pregnant women in Poland. We have based our studies on a group of 78 pregnant women with symptoms of a abortion, a premature imminent labour, premature labor and intrauterine death of foetus. In 10 cases (12.8%) we have confirmed a presence of antibodies IgM class antiparvovirus B19 at patients serum. It seems that the Parvovirus infection is one of most often reasons of unsuccessful pregnant.

Abortion, Spontaneous↗

[Parvovirus B19 infection. New physiopathological approaches].

Knowledge of the pathophysiological mechanisms governing Parvovirus B19 infection has been modified with the recent discovery of its cellular receptor. The cell lines susceptible to infection can be identified on the basis of the nature and distribution of the virus in the human organism. The Parvovirus B19 can undergo two types of cycles within the host cell. The first is a complete cycle within the infected cell leading to cell death by lysis. In the second, the cycle aborts and cell death is caused by an association of immunological phenomena and the accumulation of viral proteins toxic to the cell. It is not known why complete cycles are restricted to a single erythrocyte line. These new data not only give a more precise explanation of the classical clinical expression of the disease, but also suggest possible mechanisms for the pathogenic power of the Parvovirus B19, some of which have already been proposed on the basis of clinical observations.

AIDS-Related Opportunistic Infections↗

Infections caused by parvovirus B19.

Parvovirus B19 was discovered in 1974 by Cossart et al; is a single stranded unenveloped DNA virus, which virion is isometric, uniform and has icosagedral symmetry. B19 infection has been found in all countries, it is almost certainly world-wide in distribution. Infections occurs most frequently in late winter, spring and early summer months and are transmitted by respiratory route. Erythema infectiosum is the most common manifestation of human parvovirus B19 infection, is most commonly acquired between 4 and 10 years of age and at least 60% of adults are seropositive. Erythema Infectiosum is characterized by three stages of rash that involves the face and may also involves trunk and extremities. In adult patients, particularly women, arthralgia or arthritis have been associated with up to 80% of Erythema Infectiosum casually starts in the small joints of the hand. Maternal parvovirus B19 infection with or without rash, can affect fetus. Transient aplastic crisis can be caused by HPV B19 in patient who have increased rate of RBC destruction or loss. Others diseases or symptoms complexes may be associated with B19 infection in the coming years as this virus and its infections continues being an interesting field of investigation.

Adolescent↗

Autonomous parvovirus transduction of a gene under control of tissue-specific or inducible promoters.

Several classes of viruses are in use, or are being developed, as gene therapy vectors. Viruses with small genomes containing few essential genes have the advantage of requiring only simple complementation systems to allow packaging of foreign DNA, substituted for the entire viral coding sequences. Retroviruses and the dependent parvovirus AAV (adeno-associated virus) have been used in this way, and both possess an efficient integration mechanism which should allow long-term expression of transduced genes. In some situations, however, long-term persistence may be undesirable and there is a need for small, non-integrating viral vectors. Autonomous parvoviruses, such as LuIII, have potential as such vectors for short-term expression of therapeutic genes. We previously described recombinants of LuIII that transduced reporter genes, expressed using the viral constitutive promoter, P4. We have now generated several recombinants containing regulated promoters. A virus including a liver-specific enhancer directed 10- to 20-fold preferential expression of the luciferase reporter in transduced human hepatoma (HepG2) versus HeLa cells. In additional LuIII recombinants, the luciferase reporter was linked with chimeric promoters containing binding sequences for either the yeast GAL4 protein or the bacterial tetracycline repressor. Luciferase expression was strongly activated when these viruses were used to infect cells containing a cognate trans-activator (GAL4 or tTA, a tetracycline repressor fusion with VP16 of herpes simplex), introduced by transfection. The response to tTA could be abolished, or reduced in a graded manner, by exposure of the infected cells to tetracycline. Further results suggested that an increase in basal expression, apparently mediated by the viral left terminal inverted repeat, could be minimized by interposing polyadenylation signals between this sequence and the promoter. These results confirm that appropriate transcriptional regulation can be achieved for genes transduced by an autonomous parvovirus vector. Such vectors therefore show promise for the delivery of therapeutic genes in situations requiring cell-specific, short-term expression, eg in targeting suicide genes for ablation of cancer cells.

Cell Line↗

Various associations of human parvovirus B19 infection.

A variety of diseases encountered in association with human parvovirus B19 infection seen at Paediatric clinic during 6 months is presented and their relation to parvovirus B19 is discussed. We conclude that investigation of parvovirus B19 in variable diseases by using the newly developed methods of molecular biology will enlighten many etiopathogenetic mechanisms.

Antibodies, Viral↗