Modification of CMV DNA detection from dried blood spots for diagnosing congenital CMV infection.

BACKGROUND: Detection of viral DNA in dried blood spots using the Guthrie card (DBS test) is a reliable and practical method of diagnosing congenital cytomegalovirus (CMV) infection. The test lends itself to epidemiological studies to establish the prevalence of the infection, but also to neonatal screening for secondary prevention of sequelae. These applications would be facilitated if it were possible to use smaller samples and do the test on pools of individual cases. OBJECTIVE: To ascertain whether doing the test on smaller, pooled samples still accurately identifies neonates with congenital CMV infection. STUDY DESIGN: We tested DBS from: (A) 39 laboratory reference cases; (B) 156 neonates suspected of having congenital CMV infection; (C) 119 children examined for the retrospective diagnosis of congenital CMV; (D) mock specimens prepared with known amounts of viral DNA. RESULTS: The test using only one third of the usual amount of dried blood was 100% sensitive and specific compared to the standard DBS test (A) and to viral isolation (A and B). Pools of three single cases gave the same results as viral isolation (B) and the small-sample test (B and C). All the versions of the test gave a detection limit of 400 copies/ml. CONCLUSIONS: The modified procedure can accurately diagnose congenital CMV infection. It achieves savings in both the patient material and the costs of testing.     

CMV DNA detection in dried blood spots for diagnosing congenital CMV infection in Japan

Human cytomegalovirus (CMV) is a leading congenital infectious agent in developed countries. In the past, the incidence of congenital infection has been rather low in Japan because a high seroprevalence of CMV present in young women. However, this seroprevalence has been decreasing in recent years, so that the incidence of congenital CMV infection in Japanese neonates may increase and approach the level seen in other developed countries. The method was used for detecting CMV DNA reported by Barbi et al. [Barbi et al. (1996): Clin Diagn Virol 6:27-32] using a dried blood spot on filter paper, to diagnose congenital CMV infection in Japanese neonates. This method is effective and less laborious than virus isolation both for epidemiological studies and for identifying asymptomatic infected babies. Japanese neonates (1,176) were examined; two of who were asymptomatic were found to be infected.     

PCR detection of cytomegalovirus DNA in serum as a diagnostic test for congenital cytomegalovirus infection.

PCR detected cytomegalovirus (CMV) DNA in the serum of 18 of 18 infants with symptomatic congenital CMV infection, 1 of 2 infants with asymptomatic congenital CMV infection, and 0 of 32 controls. Serum CMV PCR provided a rapid, sensitive, and specific method for diagnosis of congenital CMV infection in infants who were symptomatic at birth.     

DNA analysis of cystic fibrosis in Brazil by direct PCR amplification from Guthrie cards

A 3 bp deletion of condon 508 (phenylalanine) of the cystic fibrosis (CF) gene constitutes the mutation of most CF chromosomes. The frequency of this mutation (referred to as ΔF508), varies considerably between populations, ranging form 26% of the CF mutations in Turkey to 88% in Denmark. To determine the frequency of the ΔF508 mutation in Brazilian Caucasoid CF patients, we used direct polymerase chain reacion (PCR) amplification of DNA obtained from dried blood spots on Guthrie cards, followed by ethidium bromide staining of gels. Although the overall frequency of the ΔF508 mutation was 47% of 380 CF chromosomes from Brazilian Caucasoids born in five different states, significant inter-state differences were observed, ranging from a ΔF508 frequency of 27% to 53%. While our method could be used to screen patients and their relatives for carrier testing and prenatal diagnosis, the efficacy of screening only for the ΔF508 mutation would be low, and would vary from state to state. Screening for a panel of local mutations will be needed to increase the mutation detection rate and optimize genetic counseling. © 1993 Wiley-Liss, Inc.     

Cytomegalovirus (CMV): specific lymphokine production in congenital CMV infection.

The studies reported here were designed to examine cytomegalovirus (CMV)-specific lymphokine production in infants with congenital CMV infection to elucidate the role of the efferent limb of the immune response to this virus. Mononuclear cells (MNC) from adult control donors and congenitally infected infants were stimulated with mitogen (PHA) or antigens (CMV, mumps), and the supernatants were assayed for production of migration inhibitory factor (MIF) and interferon (IFN). Concordance was observed between lymphocyte proliferative responses to CMV antigen and production of MIF in both control donors and congenitally infected infants. PHA-stimulated cultures from both controls and patients resulted in immune-specific IFN-gamma production in all cases. CMV-stimulated MNC from controls produced IFN when lymphocyte proliferation responses to the antigen were present, whereas those with absent proliferation did not produce IFN. CMV-induced IFN was detected in several patients who had reduced lymphocyte proliferative responses to CMV. The predominant species of IFN stimulated by CMV was neutralized by anti-IFN-gamma, suggesting that CMV induced primarily immune-specific IFN-gamma. In some cases, IFN activity was also reduced to a lesser degree by anti-IFN-alpha, suggesting either the presence of IFN-alpha or of cross-reacting antigenic determinants shared by the two species of IFN. Mumps viral antigen induced primarily IFN-alpha regardless of the immunological status of the donor. We conclude that lymphokine production in congenital CMV reflects the state of activation and/or proliferation of CMV-specific T helper cells rather than an intrinsic defect in the efferent limb of the immune response.     

Review. Hyaluronan: a powerful tissue engineering tool

Hyaluronan (HA) is a versatile molecular tool with considerable potential for tissue engineering applications. The inclusion of HA has created biocompatible biomaterials and engineered tissues that can be crosslinked or degraded controllably and can facilitate angiogenesis, osteointegration, and cell phenotype preservation. The utility of HA in tissue engineering has been broadened further by the recently identified HA synthases, which can be manipulated to stimulate the endogenous production of HA by cells seeded within biomaterial scaffolds. Overall, HA shows great promise in the development of engineered tissues and biomaterials for a variety of biomedical needs including orthopedic, cardiovascular, pharmacologic, and oncologic applications.     

Enzyme-immunoassay: a powerful analytical tool

This paper reviews various aspects of enzyme-immunoassay (EIA). Firstly it summarizes the principles of tests in which use is made of labelled antigen or antibody. Since these constitute essential reagents in EIA they are discussed next. Many assay principles call for a bound/free separation. The various methods to accomplish this are therefore briefly discussed. Very important are the characteristics of EIA: specificity, sensitivity, precision and practicability, the latter including reagent stability, performance requirements, assay times and automation potential. Finally, fields of application are listed and some more recent developments are mentioned.     

DNA hybridization assay for congenital cytomegalovirus infection.

An in vitro DNA hybridization assay was used to test 281 newborns for congenital infection with cytomegalovirus. The assay utilized an abbreviated method for DNA preparation and a dot blot assay that provided good sensitivity (100%) and specificity (98.9%) when compared with standard tissue culture, yet substantially reduced the total time of analysis. This assay would be a useful adjunct to tissue culture to diagnose newborns with congenital infection with cytomegalovirus.     

Modified latex agglutination test for antibodies to Toxoplasma gondii in eluates from Guthrie cards.

AIMS: To determine whether the Eiken particle agglutination test could be modified to make it sufficiently sensitive to screen blood samples collected on Guthrie cards for the presence of antibodies to Toxoplasma gondii; to evaluate the specificity of the modified system; and to compare seroepidemiological data on the prevalence of T gondii in pregnant women. METHODS: Simulated dried blood spots were prepared from sera from pregnant women booking for antenatal care. Eluates from the simulated dried blood spot cards and sera were tested in parallel using the modified test (1 in 5 dilution of latex) and the standard assay (neat latex particles) and endpoints determined. Guthrie card eluates, from neonates in three Thames regions, were then tested using the modified test. RESULTS: The modified test produced a 4.21-fold increase in antibody titre in 85 sera when tested in parallel with the standard test. Eluates of 168/170 from simulated dried blood spots derived from seropositive patients gave a positive result in the modified test. The two eluates which gave a negative result were derived from patients with an equivocal titre of 1/16 in the standard serum test. Of the eluates derived from serum negative patients all 103 were negative at a dilution of 1 in 4 in the modified test. The seroprevalence of antibodies to T gondii in pregnancy was 21.8% using the standard test. A similar value of 20.5% was obtained when dried blood spots from neonates in a similar region of London were tested by the modified test. CONCLUSIONS: The modified Eiken Toxo-reagent test is sensitive, simple, and economic for screening large numbers of dried blood spots. The procedure could be easily semiautomated and the technique applied to the mass screening of neonatal blood samples collected on Guthrie cards to determine the seroprevalence of T gondii in pregnant women.     

Cytomegalovirus in urine: detection of viral DNA by sandwich hybridization.

A cytomegalovirus (CMV)-specific sandwich hybridization test was constructed by using two adjacent BamHI DNA fragments of CMV DNA as reagents. The fragments were cloned into two different vectors. One of the recombinants was attached to the filter, and the other was the labeled probe. When present in the sample, CMV DNA mediated labeling of the filter by hybridizing to both the filter-bound DNA and the probe. The sandwich hybridization test was applied for the detection of CMV DNA from urine. DNA was released from virus by 2% Sarkosyl, concentrated by 2-butanol extraction and isopropanol precipitation, denatured, and finally subjected to the sandwich hybridization test. As a result, 70 to 90% of the original viral DNA could be recovered and demonstrated by the quantitative hybridization reaction. Urine could be stored at room temperature in Sarkosyl for at least 2 days without affecting the detectability of CMV. The clinical applicability of the test was evaluated by studying urine samples from four infants excreting CMV. Sandwich hybridization demonstrated the presence of CMV DNA in all of the specimens. These contained originally 10(5) to 10(8) CMV DNA molecules per ml.     

Metabolic flux analysis: a powerful tool for monitoring tissue function.

In recent years, metabolic flux analysis has been widely used in bioprocess engineering to monitor cell viability and improve strain activity. Metabolic flux analysis refers to a methodology for investigating cellular metabolism whereby intracellular fluxes are calculated using a stoichiometric model for the major intracellular reactions and applying mass balances around intracellular metabolites. A powerful feature of this methodology is its ability to consider cellular biochemistry in terms of reaction networks. By considering the stoichiometry of biochemical reactions, it is possible to estimate the degree of engagement of each pathway participating in overall cellular activity, and hence obtain a comprehensive view of a cell s metabolic state. Given the potential impact of cellular energy metabolism on the function of engineered tissues, such comprehensive analysis of metabolic activity can be an extremely useful tool for tissue engineers. Estimates of intracellular fluxes under various environmental conditions could be used to optimize function in vivo as well as culture conditions in vitro. In this review, we provide a brief theoretical background of metabolic flux analysis and summarize the most widely used experimental approaches to obtain flux data. This review is intended as an overview of the field and as a starting point for tissue engineers wishing to learn about and eventually employ this methodology.     

Dynamic clamp: a powerful tool in cardiac electrophysiology

Under chronic hypoxia, tumour cells undergo adaptive changes involving hypoxia-inducible factors (HIFs). Here we report that ion currents mediated by Ca²⁺-activated K⁺ (K_Ca) channels in human melanoma IGR1 cells are increased by chronic hypoxia (3% O₂), as well as by hypoxia mimetics. This increase involves the HIF system as confirmed by overexpression of HIF-1α or the von Hippel-Lindau tumour suppressor gene. Under normoxic conditions the K_Ca channels in IGR1 cells showed pharmacological characteristics of intermediate conductance K_Ca subtype IK channels, whereas the subtype SK2 channels were up-regulated under hypoxia, shown with pharmacological tools and with mRNA analysis. Hypoxia increased cell proliferation, but the K_Ca channel blockers apamin and charybdotoxin slowed down cell growth, particularly under hypoxic conditions. Similar results were obtained for the cell line IGR39 and for acutely isolated cells from a biopsy of a melanoma metastasis. Thus, up-regulation of K_Ca channels may be a novel mechanism by which HIFs can contribute to the malignant phenotype of human tumour cells.     

Cytomegalovirus DNA detection in sera from patients with active cytomegalovirus infections.

Using a specific and sensitive polymerase chain reaction method, we detected reliably the presence of human cytomegalovirus (HCMV) DNA directly in serum samples collected at an early stage of HCMV infection, even before immunoglobulin M (IgM) antibodies were measurable. HCMV DNA was detected in serum from all patients with active HCMV infection; in 91% of these patients, HCMV DNA was found in the acute-phase serum. In 13 of 44 patients, HCMV DNA was found in serum before HCMV-specific IgM. For four kidney transplant recipients, the occurrence of HCMV DNA in serum, virus isolation from urine and leukocytes, and HCMV IgG and IgM serology were determined. We found a correlation between HCMV DNA in serum and positive virus isolation from leukocytes. In three of five congenitally infected infants, HCMV DNA and HCMV IgM were detected in the same sample. Two other infants were HCMV DNA positive, although no HCMV IgM antibodies were measurable. HCMV was found in urine from these infants either by virus isolation or with the polymerase chain reaction. Serum from one of the 22 healthy HCMV-seropositive blood donors was HCMV polymerase chain reaction positive.     

Diagnosis of congenital cytomegalovirus infection by detection of viral DNA in urine pools

Human cytomegalovirus (HCMV) is the most frequent cause of congenital infection. Diagnosis of this infection is important because 5-17% of asymptomatic infected babies will develop late sequelae and should be followed closely. Most of these children will remain undetected, since screening of all newborns by viral culture is too expensive. The aim of this study was to demonstrate that pool testing could be used to detect HCMV congenital infection in newborns. For this purpose, a nested-PCR technique was tested in urine pools. In phase 1, urine specimens were tested alone by nested-PCR and compared with viral culture, followed by cross experiments to test the reliability of detecting one positive specimen in a 20 samples in a urine pool. In phase 2, this pool method was applied to all urine specimens from children received in the virology laboratory of the Centro Hospitalar Cova da Beira for diagnosis of HCMV infection, between January 2002 and March 2003. In phase 1, 74 urine specimens were tested simultaneously by shell-vial culture and nested-PCR; 17 were positive and the remaining 57 negative by both methods. The negative specimens were divided into three pools and each pool was tested alone and crossed with each of the positive specimens by nested-PCR. Although the three pools were negative when tested alone, all 51 crossed results were positive. In phase 2, 15 out of the 180 urine samples tested positive by shell-vial culture and were detected by this pool method. These results suggest that urine pools can be used to detect HCMV positive urines in children, with similar sensitivity and specificity when compared with the standard method, but with a substantial labour reduction. This significant reduction in labour and consequently in cost per test, opens the possibility of applying PCR to urine pools for screening the HCMV congenital infection in newborns.     

Cytomegalovirus infection in a volunteer blood donor population.

Among 223 volunteer blood donors who were studied for evidence of cytomegalovirus (CMV) infection, 58 percent had complement-fixing antibody and 59 percent had indirect hemagglutinating antibody to CMV. No virus was isolated from any donor's washed leukocytes or leukocyte-rich plasma in fibroblast monolayer culture. In seven asymptomatic donors (3 percent), CMV was recovered from urine cultures obtained at the time of blood donation. However, at the time of reexamination, viruria was no longer present and serum antibody titers had not changed. In the three patients studied who received blood from three of the cytomegaloviruric donors, serological evidence of CMV infection developed (fourfold or greater indirect hemagglutinating antibody rise), and one recipient also developed cytomegaloviruria; no illnesses was associated with these infections. Further study is needed to establish that the detection of viruria in donors may identify potentially infective blood.     

Genetically manipulated mice: a powerful tool with unsuspected caveats

Although genetic manipulations in mice have provided a powerful tool for investigating gene function in vivo , recent studies have uncovered a number of developmental phenomena that complicate the attribution of phenotype to the specific genetic change. A more realistic approach has been to modulate gene expression and function in a temporal and tissue-specific manner. The most common of these methods, the Cre LoxP and tetracycline response systems, are surveyed here and their recently identified shortcomings discussed, along with a less well known system based on the E. coli lac operon and modified for use in mammals. The potential for further complications in interpretation due to hitherto unexpected epigenetic effects involving transfer of RNA or protein in oocytes or sperm is also explored. Given these problems we reiterate the necessity for the use of completely reversible methods that will allow each experimental group of animals to act as their own control. Using these methods with a number of specific modifications to eliminate non-specific effects from random insertion sites and inducer molecules, the full potential of genetic manipulation studies should be realized.