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M Lappalainen

Publications and source records attributed to M Lappalainen.

At least 19 recordsLinked to original sources
PubMed2006

Real-time PCR targeting a 529-bp repeat element for diagnosis of toxoplasmosis.

Sensitive and rapid detection of infection with Toxoplasma gondii in transplanted immunocompromised patients is crucial for a good prognosis. Two DNA fragments are used currently for detecting T. gondii infection by PCR, i.e., the B1 gene and a 529-bp repeat element that exists in 200-300 copies/genome. This study investigated whether targeting the 529-bp repeat element gives better sensitivity and accuracy than can be obtained when targeting the B1 gene (35 copies) when concentrations of T. gondii DNA are low. The results demonstrated that detection of the 529-bp repeat element increased diagnostic sensitivity and accuracy. Addition of an internal amplification control did not affect the PCR performance and was useful in order to monitor PCR inhibition by non-specific DNA in the LightCycler instrument. The real-time PCR was used successfully in a clinical context to monitor parasitaemia in the blood of a transplant recipient suffering from toxoplasmosis.

Animals↗
PubMed2005

Quantitative PCR in the diagnosis of CMV infection and in the monitoring of viral load during the antiviral treatment in renal transplant patients.

Cytomegalovirus (CMV) infection is a significant problem in transplantation. In this study, a quantitative PCR test was compared with the CMVpp65 antigenemia assay not only in the diagnosis CMV infections but especially in the monitoring of viral loads during ganciclovir treatment of CMV disease in individual renal transplant patients. Altogether 342 blood specimens were obtained from 116 patients. Blood specimens were used for Cobas Amplicor Monitor plasma PCR and for the pp65 assay. Also shell vial culture was performed. The patients with a positive pp65 finding were monitored for CMV weekly during ganciclovir treatment and/or until the antigenemia subsided. CMV was detected in 31/116 (27%) patients, of whom 14 (12%) developed CMV disease and were treated with ganciclovir. CMV was found by shell vial culture in 13/14 cases, but by PCR and pp65 test in all 14 patients. CMV was detected in 156 (45%) samples; by PCR in 121/156 (range 344-103,000 copies/ml) and by pp65 test in 138/156 (range 1-1,000 positive cells/50,000 leukocytes) and by culture in 59/156 (38%) only. The peak viral loads were significantly (P<0.0001) higher in CMV disease than in untreated infections (19,650 vs. 379 copies/ml, and 100 vs. 5pp65 positive cells). In the monitoring of individual patients, the time-related CMV-DNAemia and pp65 antigenemia correlated well during the treatment of CMV disease. In conclusion, Cobas Amplicor Monitor plasma PCR and CMVpp65 antigen assays can be equally used in the diagnosis CMV infection and in the monitoring of viral load during antiviral treatment.

Antigens, Viral↗
PubMed2004

Delayed maturation of IgG avidity in congenital toxoplasmosis.

The aim of this comparative study was to investigate the clinical usefulness of the measurement of Toxoplasma gondii IgG avidity in the postnatal diagnosis of congenital toxoplasmosis. IgG avidity values in serum samples from infants with congenital infection were compared with those in samples from uninfected infants, all born to mothers with toxoplasmosis acquired during gestation. This analysis revealed that IgG avidity values soon after birth reflected maternal values in the large majority of the samples. Low or borderline IgG avidity values were systematically found in the cohort of congenitally infected subjects. After birth, IgG avidity values slowly increased over time for up to 2 years in congenitally infected subjects. On the contrary, IgG avidity values in the uninfected infants remained stable over time. The presence of low IgG avidity in a newborn can be considered a marker of maternal seroconversion in the second or third trimester of gestation and, as a consequence, an indicator of risk for congenital toxoplasmosis. An IgG avidity assay can be easily carried out with antibodies eluted from dried blood spots (Guthrie cards), providing an opportunity to retrospectively evaluate the risk of congenital infection in special clinical circumstances, for example when suspicion of congenital infection arises during late infancy.

Aging↗
PubMed2004

CARD15 frameshift mutation in patients with CROHN disease is associated with immune dysregulation.

BACKGROUND: Mutations in the caspase-activating recruitment domain 15 (CARD15) gene are associated with Crohn disease (CD). CARD15 is an intracellular receptor for bacterial lipopolysaccharides (LPS). LPS-induced activation of transfectants containing the frameshift mutation (1007fs) of CARD15 is impaired. The aim of this study was to investigate whether the presence of CARD15 1007fs affects activation of CD patients' own cells. Patients (4 homozygotes, 6 heterozygotes, and 6 wild-type) were matched according to clinical picture and medication. METHODS: Immune inflammatory status was evaluated by measuring monocyte HLA-DR and CD11b densities and the proportion of CD14dimCD16+ monocytes, and was found to be comparable in the three groups. Blood mononuclear cells were cultured overnight in serum-free medium alone, or the medium supplemented with LPS (0.1-10.0 ng/mL), a combination of IFN-gamma (100 IU/mL) and granulocyte-macrophage colony stimulating factor (GM-CSF) (5 ng/mL), or both. TNF and IL-10 levels in the culture supernatant were determined. RESULTS: LPS 0.1 or 1.0 ng/mL alone did not increase TNF levels. IFN-gamma/GM-CSF induced TNF release, and co-culture with LPS 1.0 or 10.0 ng/mL was strongly synergistic. CARD15 1007fs mutation was linked in a gene-dose-dependent manner to low TNF release induced by IFN-gamma/GM-CSF (P value for linear trend = 0.001). The degree of synergism in co-culture was normal or high, suggesting that 1007fs did not depress responses to LPS. IL-10 levels were not related to CARD15 1007fs. CONCLUSIONS: In CD patients, CARD15 1007fs is associated in a gene-dose-dependent manner to low mononuclear cell TNF release by IFN-gamma/GM-CSF but does not impair TNF release by LPS. This type of immune dysregulation may influence susceptibility to and/or phenotype of CD.

Adult↗
PubMed2004

Rapid and sensitive diagnosis of Toxoplasma gondii infections by PCR.

DNA was extracted with a modified Qiagen DNA Mini Kit method from 20 clinical samples and was amplified by PCR using specific primers for the T. gondii B1 gene. T. gondii was detected correctly in 18 of the 20 clinical samples in < 5 h, with a detection limit of two parasites/sample. The results were in good agreement with those obtained by a more complicated and time-consuming procedure involving two-step nested PCR and either liquid hybridisation or colorimetric detection using internal probes.

Amniotic Fluid↗
PubMed2003

Targeting of biotinylated compounds to its target tissue using a low-density lipoprotein receptor-avidin fusion protein.

The very high binding affinity of avidin to biotin is one of the highest to occur in nature. We constructed a fusion protein composed of avidin and the endocytotic LDL receptor in order to target biotinylated molecules to cells of the desired tissues. In addition to the native avidin, charge-mutated and nonglycosylated avidins were utilized as part of the fusion proteins, in order to modify its properties. All of the fusion protein versions retained the biotin-binding capacity. Although the specificity was not increased, however, fusion proteins composed of natural avidin and nonglycosylated avidin bound most efficiently to the biotinylated ligands. Fluorescence microscopy and atomic force microscopy studies revealed the expression of the fusion protein on cell membranes, and demonstrated specific and high-affinity binding of biotin to the low-density lipoprotein receptor (LDLR)-avidin fusion protein in vitro. Additionally, systemically administered biotinylated ligand targeted with high specificity the intracerebral tumors of rats that were expressing fusion protein after the virus-mediated gene transfer. These results suggest that local gene transfer of the fusion protein to target tissues may offer a novel tool for the delivery of biotinylated molecules in vitro and in vivo for therapeutic and imaging purposes.

Animals↗
PubMed2003

CARD15/NOD2 gene variants are associated with familially occurring and complicated forms of Crohn's disease.

BACKGROUND: Variants of the caspase activating recruitment domain 15/nucleotide oligomerisation domain 2 (CARD15/NOD2) gene have been associated with susceptibility to Crohn's disease (CD). AIM: Our aim was to evaluate the allele frequencies of the CARD15 variants R702W, G908R, and 1007fs in Finnish inflammatory bowel disease (IBD) patients and to search for possible associations between CARD15 variants and occurrence of familial forms of IBD or complicated forms of CD. PATIENTS AND METHODS: We investigated 198 sporadic CD patients, 46 probands with familial CD, 27 CD probands from mixed IBD families, 99 unrelated patients with ulcerative colitis (UC), and 300 control individuals for the occurrence of the CARD15 gene variants R702W, G908R, and 1007fs. RESULTS: In CD patients, the allele frequencies for the rare variants of these polymorphisms were 3.3%, 0.6%, and 4.8% (total 8.7%), and the corresponding frequencies in healthy controls were 1.8%, 0%, and 1.7% (total 3.5%) (8.7% v 3.5%; p<0.01). In UC patients allele frequencies were comparable with those in controls. The frequency of the 1007fs polymorphism variant allele was significantly higher among all CD patients than in controls (4.8% v 1.7%; p<0.01) but there was no significant difference in allele frequencies between the CD and UC groups. The 1007fs allele frequency was higher in familial CD than in non-familial cases with CD (10.9% v 3.5%; p<0.01). There were no significant differences in the allele frequencies of the R702W and G908R polymorphisms between CD patients, UC patients, and controls. We found that 15.5% of CD patients, 9.1% of UC patients, and 6.7% of controls carried at least one of the CARD15 variants. In CD patients carrying at least one of the three NOD2 variants, the ileum was affected more often than in non-carrier CD patients (90% v 73%; p<0.05), they had stricturing or penetrating disease more often than non-carriers (88% v 56%; p<0.01), and they had an increased need for bowel surgery. CONCLUSIONS: The frequency of NOD2 gene variants was lower in genetically homogenous Finns than in other populations. The 1007fs variant was associated with CD. The occurrence of CARD15 variants predicted ileal location as well as stricturing and penetrating forms of CD.

Adolescent↗
PubMed2002

CMV infection is usually associated with concurrent HHV-6 and HHV-7 antigenemia in liver transplant patients.

Human herpesvirus 6 and 7 (HHV-6, HHV-7) have been recently reported in liver transplant patients. HHV-6 may cause fever, neurological disorders and hepatitis. The clinical significance of HHV-7 is less clear. HHV-6 and -7 are closely related to cytomegalovirus (CMV), and interactions between the viruses have also been suggested. In this study, we investigated the post transplant HHV-6 and -7 antigenemia was in relation to symptomatic CMV disease after liver transplantation. Consecutive 34 adult liver allograft recipients transplanted during 1999-2000 were included in the study. CMV infections were diagnosed by the frequent monitoring of pp65-antigenemia and by viral cultures. HHV-6 and -7 were demonstrated, by using immunoperoxidase staining and monoclonal antibodies against the virus specific antigens, in the mononuclear cells from the same blood specimens which were obtained for CMV pp65 monitoring. Altogether 322 blood specimens were analyzed. CMV disease was diagnosed in 12 (35%) patients during the first 3 months (first pp65 positive specimen mean 25 days, range 8-61 days) after transplantation. Concurrent HHV-6 antigenemia was detected in 10/12 (mean 14 days, range 6-22 days) and HHV-7 antigenemia in 9/12 patients (mean 25 days, range 10-89 days) after transplantation. HHV-6 usually appeared slightly before CMV. All CMV infections were successfully treated with ganciclovir and the CMV-antigenemia subsided. HHV-6 and -7 antigenemia also responded to the antiviral treatment, but more slowly than CMV. In conclusion, CMV infection was usually associated with HHV-6 and -7 antigenemia in liver transplant patients. The results support the suggestion that CMV, HHV-6 and -7 may have interactions. The clinical symptoms of CMV infection, may also be linked with HHV-6 or -7.

Adult↗
PubMed2001

Complement C4 deficiency and HLA homozygosity in patients with frequent intraoral herpes simplex virus type 1 infections.

Three consecutive patients with no apparent immunodeficiency who had frequent intraoral herpes simplex type 1 recurrences, a rare complication of herpes simplex virus infection, were found to have a total deficiency of either the A or B isotype of the complement component C4 and to be homozygous for the studied HLA antigens. A combination of HLA homozygosity, which may lead to impaired T cell recognition of viral peptides, and deficiency in the classical complement pathway, which can compromise virus neutralization, may predispose to severe and frequent herpes simplex virus infections.

Adult↗
PubMed2001

Real-time PCR for detection and quantitation of hepatitis B virus DNA.

A sensitive and reproducible real-time PCR assay based on TaqMan technology was developed for the detection and quantitation of hepatitis B virus (HBV) DNA in serum, and compared with an "in-house" qualitative PCR assay. HBV DNA was measured in 125 serum samples from 76 hepatitis B patients, consisting of 22 patients with an acute infection, 20 patients with a previous history of hepatitis B infection, and 34 patients with a chronic hepatitis B. Four patients with a chronic infection were treated with either an IFN-alpha monotherapy or a combination of IFN-alpha and lamivudine. Twenty-nine sera from healthy individuals and non-hepatitis B patients served as negative controls. The assay was validated by using a 10-fold dilution series of the World Virological Quality Control (VQC) sample containing 3.73 x 10(7) genome equivalents per ml. The detection limit for the real-time PCR was 3.73 x 10(2) genome equivalents per ml (geq/ml), while it was 3.73 x 10(3) geq/ml for the in-house PCR. The real-time PCR assay had an 8-logarithm dynamic range spanning from 10(2) to 10(10) geq/ml. In clinical serum samples, the real-time PCR and the in-house PCR detected HBV DNA in 81% (101/125) and 66% (83/125) of samples, respectively. HBV DNA was not detected among the negative controls by either of these assays. In conclusion, real-time PCR is a sensitive, specific, and a reproducible approach for the detection and quantitation of HBV DNA in clinical serum samples, useful also for monitoring the efficacy of antiviral treatment.

Acute Disease↗
PubMed2001

Molecular epidemiology of viral pathogens and tracing of transmission routes: hepatitis-, calici- and hantaviruses.

BACKGROUND: The need to rapidly identify new therapeutic drugs and vaccines for clinically important viral infections has resulted in intensive study of the molecular properties of viruses. Modern molecular techniques have provided tools for tracing infections and studying the evolution of viruses. OBJECTIVE STUDY AND DESIGN: Two examples illustrating how modern molecular techniques can be used in clinical virology and molecular epidemiology (hepatitis and caliciviruses), and one example documenting their importance in basic research (hantaviruses) will be discussed. RESULTS AND CONCLUSIONS: Water- and food-borne outbreaks caused by the faeco-orally spread hepatitis A virus (HAV) are common in areas lacking proper sanitation, but they are possible also in countries with low seroprevalence. In water epidemics, the sequence comparisons between the virus from patients and from water have been used successfully. Hepatitis B virus variants are clinically important and challenge the diagnostic tests and prophylactic measures. Some hepatitis C (HCV) genotypes appear to be associated with more severe pathology and others respond better to antiviral treatment. Nosocomial and occupational infections are not rare, and the source can be identified by phylogenetic analysis of nucleotide sequences obtained from the infected individuals. The overwhelming role of Norwalk-like caliciviruses (NLV) in adult diarrhoea and especially in food- and water-borne epidemics has become apparent during the last decade. Methods are under development for detecting these viruses, not only from patient samples and water, but also from other environmental samples (e.g. foodstuff and surface swabs). The analysis of the genetic variation and evolution of the Old World hantaviruses in their carrier rodents has shown that the extent of genetic diversity correlates with geographical distance. As a rule, phylogenetic relationships of hantaviruses resemble those of their rodent hosts, suggesting virus-host co-evolution. Exceptional host-switch events allow a study on still radiating hantavirus species. There is suggestive evidence that natural reassortant hantaviruses are involved in human infection.

Animals↗
PubMed2001

Comparison of plasma polymerase chain reaction and pp65-antigenemia assay in the quantification of cytomegalovirus in liver and kidney transplant patients.

BACKGROUND: Cytomegalovirus (CMV) is a significant problem in transplantation. The antiviral treatment is based on the clinical symptoms and the rapid laboratory diagnosis. Although polymerase chain reaction (PCR) methods have already been widely used, the clinical correlation of the findings is not clear. OBJECTIVE: The objective of this study was to investigate the usefulness of a quantitative plasma PCR test and compare it with the pp65-antigenemia test in the detection of clinically significant CMV infections in liver and kidney transplant patients. STUDY DESIGN: The clinical material consisted of 253 consecutive blood samples was tested using a quantitative polymerase chain reaction test, Cobas Amplicor CMV Monitor (Roche) and pp65 antigenemia assay. Plasma was used for PCR and leucocytes were used for the antigenemia test. RESULTS: CMV was detected in 89 out of 253 blood samples by one or both methods. PCR detected 78 (range 274-165000 copies/ml) and pp65 antigenemia test 79 (range 1-1500 positive cells/50000) of the positive findings. The sensitivity and specificity of PCR test was 86 and 94%, respectively. The PCR detected all clinically significant CMV infections (>10 positive cells in pp65 test) and infections which required antiviral treatment. In addition, the correlation between the two tests was almost linear. CONCLUSIONS: The quantitative PCR appears to be a suitable alternative to diagnose and monitor CMV infections in transplant patients.

Antiviral Agents↗
PubMed2001

Polymerase chain reaction for diagnosis of cerebral toxoplasmosis in cerebrospinal fluid in HIV-positive patients.

It is important but sometimes difficult to establish a diagnosis of toxoplasma encephalitis (TE) in an HIV-positive immunodeficient patient. The most promising non-invasive method is polymerase chain reaction (PCR) for Toxoplasma gondii in cerebrospinal fluid (CSF). In a retrospective study PCR was used to analyse CSF for the presence of T. gondii DNA in 5 HIV-infected patients with a clinical suspicion of TE (group 1), 8 patients with other HIV-associated symptoms (group 2) and 7 other patients with neurological disorders (group 3). PCR was positive in 2/4 patients with a final diagnosis of TE and negative in all remaining patients in all 3 groups. The 2 patients with positive PCR had a fulminant course and experienced treatment failure. The albumin index was elevated in 4/5 patients in group 1, of whom 3/4 had a final diagnosis of TE, with suspected TE in 1. This small study confirms earlier data indicating that the PCR test has a low sensitivity but a high specificity.

AIDS-Related Opportunistic Infections↗
PubMed2001

Factors predicting interferon treatment response in patients with chronic hepatitis c: late viral clearance does not preclude a sustained response.

OBJECTIVES: Because of the suboptimal efficacy, cost, and adverse effects of interferon in chronic hepatitis C (HCV), predictors have been sought to detect patients with a good treatment response. Also, markers for determining a poor response early in the course of therapy, such as the lack of early viral clearance, have been proposed. METHODS: Ninety-seven patients with chronic hepatitis C were enrolled to receive leukocyte alpha-interferon according to a stepped-care management protocol. The final virological treatment response was evaluated in 74 patients after a 6-month post-treatment follow-up. The relationship between pretreatment and during-treatment variables and the long-term response was assessed. RESULTS: Non-1 viral genotype, higher pretreatment ALT levels, and lower gamma-glutamyl transferase (GGT)/ALT ratios and GGT as well as younger age were significantly associated with a sustained response; a trend was also detected for lower serum ferritin levels. Normalization of ALT by 3 months was also a significant predictor of a long-term response. Of the 27 patients carrying the HCV genotype 3a, seven (26%) were still HCV RNA positive at 6 months. Of these patients, however, five (19%) still achieved a sustained virological response after treatment for up to 12 months. CONCLUSIONS: In contrast to some previous reports, our results suggest that a late viral clearance after 6 months of interferon monotherapy may not preclude a favorable long-term response after a 12-month treatment, especially in patients carrying a non-1 HCV genotype. A low pretreatment GGT/ALT ratio is a predictor of a good treatment response.

Adolescent↗
PubMed2001

Focal sialadenitis in patients with ankylosing spondylitis and spondyloarthropathy: a comparison with patients with rheumatoid arthritis or mixed connective tissue disease.

OBJECTIVES: To investigate the occurrence of and risk factors for focal sialadenitis in patients with rheumatoid arthritis (RA), mixed connective tissue disease (MCTD), ankylosing spondylitis (AS), and spondyloarthropathy (SpA). METHODS: A total of 85 patients (25 with RA, 19 with MCTD, 19 with AS, 22 with SpA) participated in the study. Each patient filled out a questionnaire for eye and oral symptoms and for the use of medication, and was interviewed; other tests included Schirmer's test, laboratory tests, collection of unstimulated and stimulated whole saliva, and minor salivary gland biopsy. A focus score of > or =1 was regarded as an indicator of focal sialadenitis. RESULTS: Focal sialadenitis was observed in 68% (57/84) of all patients. It affected 80% (20/25) of the patients with RA, 94% (17/18) of those with MCTD, 58% (11/19) of those with AS, and 41% (9/22) of those with SpA (chi(2) test, p=0.0013). Salivary secretion correlated negatively with the focus scores-that is, severity of focal sialadenitis. Patients with focal sialadenitis had both decreased salivary secretion and decreased tear secretion significantly more often than did patients without (chi(2) test, p=0.0074 and p=0.048 respectively). Patients with positive rheumatoid factor (RF), antinuclear antibodies (ANA), or SSA or SSB antibodies had sialadenitis significantly more often than did patients with negative antibodies. In the subgroup of patients with AS or SpA, no associations were found between focal sialadenitis and the presence of these antibodies. CONCLUSION: In addition to patients with RA or MCTD, focal sialadenitis also affects a very high proportion of patients with AS or SpA. Focus scores are significantly higher in patients with RA or MCTD than in those with AS or SpA. A significant association exists between focal sialadenitis and RF, ANA, SSA and SSB. However, in the subgroup of patients with AS or SpA, no associations were found between focal sialadenitis and serological markers or clinical symptoms.

Adult↗
PubMed2000

HHV-6 reactivation is often associated with CMV infection in liver transplant patients.

Human herpesvirus 6 (HHV-6) infection has been recently reported in liver transplant patients. HHV-6 is closely related to cytomegalo-virus (CMV), and some interaction between the viruses has been suggested. In this study, the post-transplant HHV-6 antigenemia was investigated in relation to symptomatic CMV infections in adult liver transplant patients. CMV infections were diagnosed by the pp65 antigenemia test and by viral cultures. HHV-6 infections were demonstrated by the HHV-6 antigenemia test and by serology. Significant symptomatic CMV infection was diagnosed in 42 of 75 patients during the first 6 months after transplantation. All CMV infections were successfully treated with ganciclovir. Concurrent HHV-6 antigenemia was detected in 21 (50%) of 42 patients with CMV infection. All HHV-6 infections were reactivations. HHV-6 also responded to the antiviral treatment, but with less clear effect. In conclusion, HHV-6 reactivation is often associated with CMV infection in liver transplant patients. The results support the suggestion that CMV and HHV-6 may have interactions.

Adult↗
PubMed2000

Leukocyte interferon-alpha in the treatment of chronic hepatitis C in Finland.

BACKGROUND: To evaluate the efficacy of leukocyte interferon in previously untreated patients with chronic hepatitis C, 97 patients were enrolled in a prospective study in Finland with a stepped-care management protocol. METHODS: The treatment was initiated with 3 million units of interferon-alpha subcutaneously three times a week. At 3 months, if the serum alanine aminotransferase was still abnormal, the dose was doubled. If serum hepatitis C virus (HCV) RNA had turned negative at 6 months, the treatment was stopped; if it was still positive, treatment was continued for up to 12 months. All patients were followed up after treatment for 6 months. Altogether, 74 patients completed the treatment and follow-up periods. RESULTS: Of all the originally enrolled patients 36% (35 of 97) achieved sustained virologic response, defined as HCV RNA negativity 6 months after the end of treatment. The commonest HCV genotype among these patients was 3a, and as many as 52% of such patients achieved sustained virologic response. Thirty-two per cent of the patients had HCV genotype 1a, 1b, or a mixture of these; a sustained response was achieved in only 6% of such patients but in 50% of patients with a non-1 genotype. Adverse effects caused treatment cessation for 10% of the patients and IFN dose reduction for 20%. CONCLUSIONS: Monotherapy with human leukocyte interferon resulted in sustained virologic response in 36% of patients with chronic hepatitis C. In those infected with a HCV genotype other than 1, the sustained virologic response rate was 50%.

Adult↗