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Biomedical subjects

Per Olcén

Publications and source records attributed to Per Olcén.

At least 19 recordsLinked to original sources
PubMed2006

Total variation in the penA gene of Neisseria meningitidis: correlation between susceptibility to beta-lactam antibiotics and penA gene heterogeneity.

In recent decades, the prevalence of Neisseria meningitidis isolates with reduced susceptibility to penicillins has increased. The intermediate resistance to penicillin (Pen(i)) for most strains is due mainly to mosaic structures in the penA gene, encoding penicillin-binding protein 2. In this study, susceptibility to beta-lactam antibiotics was determined for 60 Swedish clinical N. meningitidis isolates and 19 reference strains. The penA gene was sequenced and compared to 237 penA sequences from GenBank in order to explore the total identified variation of penA. The divergent mosaic alleles differed by 3% to 24% compared to those of the designated wild-type penA gene. By studying the final 1,143 to 1,149 bp of penA in a sequence alignment, 130 sequence variants were identified. In a 402-bp alignment of the most variable regions, 84 variants were recognized. Good correlation between elevated MICs and the presence of penA mosaic structures was found especially for penicillin G and ampicillin. The Pen(i) isolates comprised an MIC of >0.094 microg/ml for penicillin G and an MIC of >0.064 microg/ml for ampicillin. Ampicillin was the best antibiotic for precise categorization as Pen(s) or Pen(i). In comparison with the wild-type penA sequence, two specific Pen(i) sites were altered in all except two mosaic penA sequences, which were published in GenBank and no MICs of the corresponding isolates were described. In conclusion, monitoring the relationship between penA sequences and MICs to penicillins is crucial for developing fast and objective methods for susceptibility determination. By studying the penA gene, genotypical determination of susceptibility in culture-negative cases can also be accomplished.

Amino Acid Sequence↗
PubMed2006

Etiologic diagnosis of adult bacterial pneumonia by culture and PCR applied to respiratory tract samples.

Respiratory culture and multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae, and Chlamydophila pneumoniae were applied to sputum, nasopharyngeal swabs, and nasopharyngeal aspirates from 235 adult patients with community-acquired pneumonia and 113 controls. Both culture and multiplex PCR performed well with the different samples and appear to be useful as diagnostic tools.

Adolescent↗
PubMed2005

Should healthy children be vaccinated against influenza? A consensus report of the Summits of Independent European Vaccination Experts.

Influenza is often regarded as an illness of the elderly portion of the population because most of the excess mortality associated with influenza epidemics occurs in that age group. However, evidence derived from a large number of clinical studies carried out in different countries and various settings has clearly demonstrated that the burden of influenza is also substantial in children. The attack rates of influenza during annual epidemics are consistently highest in children, and young children are hospitalized for influenza-related illnesses at rates comparable to those for adults with high-risk conditions. Especially among children younger than 3 years of age, influenza frequently predisposes the patient to bacterial complications such as acute otitis media. Children also serve as the main transmitters of influenza in the community. A safe and effective vaccine against influenza has been available for decades, but the vaccine is rarely used even for children with high-risk conditions. Despite several existing problems related to influenza vaccination of children, the current evidence indicates that the advantages of vaccinating young children would clearly outweigh the disadvantages. Considering the total burden of influenza in children, children younger than 3 years of age should be regarded as a high-risk group for influenza, analogously with the age-based definition of high risk among persons 65 years of age or older. Annual influenza vaccination should be recommended to all children from 6 months to 3 years of age.

Age Factors↗
PubMed2005

Sequence constancies and variations in genes encoding three new meningococcal vaccine candidate antigens.

By the strategy "reverse vaccinology" a number of new antigens have been identified in Neisseria meningitidis, which are potential candidates for a highly needed broad-spectrum meningococcal vaccine. In the present study we examined the prevalence, sequence constancies and variations of the genes encoding three of these new antigens designated, genome-derived neisserial antigen (GNA) 1870, GNA1946 and GNA2132. All three genes were present in all N. meningitidis isolates tested. Concerning gna1870, three major variants of the gene sequences and deduced amino acid sequences were identified and 56% of the deduced amino acids were conserved in all isolates. In gna1946, 98% of the deduced amino acids were conserved and in gna2132, 54% of the deduced amino acids were conserved. Based on gene prevalence and conservation, all three antigens are promising candidates for an effective meningococcal vaccine against all N. meningitidis irrespective of serogroup.

Antigens, Bacterial↗
PubMed2005

Design of a multiplex PCR for Streptococcus pneumoniae, Haemophilus influenzae, Mycoplasma pneumoniae and Chlamydophila pneumoniae to be used on sputum samples.

A multiplex PCR (mPCR) was developed for simultaneous detection of specific genes for Streptococcus pneumoniae (lytA), Mycoplasma pneumoniae (P1), Chlamydophila pneumoniae (ompA), and Haemophilus influenzae (16S rRNA, with verification PCR for P6). When the protocol was tested on 257 bacterial strains belonging to 37 different species, no false negatives and only one false positive were noted. One Streptococcus mitis out of thirty was positive for lytA. In a pilot application study of 81 sputum samples from different patients with suspected lower respiratory tract infection (LRTI), mPCR identified S. pneumoniae in 25 samples, H. influenzae in 29, M. pneumoniae in 3, and C. pneumoniae in 1. All samples culture positive for S. pneumoniae (n=15) and H. influenzae (n=15) were mPCR positive for the same bacteria. In a pilot control study with nasopharyngeal swabs and aspirates from 10 healthy adults, both culture and mPCR were negative. No PCR inhibition was found in any of the mPCR-negative sputum or nasopharyngeal samples. Whether all samples identified as positive by mPCR are truly positive in an aetiological perspective regarding LRTI remains to be evaluated in a well-defined patient material. In conclusion, the mPCR appears to be a promising tool in the aetiological diagnostics of LRTI.

Adolescent↗
PubMed2005

Interlaboratory comparison of PCR-based identification and genogrouping of Neisseria meningitidis.

Twenty clinical samples (18 cerebrospinal fluid samples and 2 articular fluid samples) were sent to 11 meningococcus reference centers located in 11 different countries. Ten of these laboratories are participating in the EU-MenNet program (a European Union-funded program) and are members of the European Monitoring Group on Meningococci. The remaining laboratory was located in Burkina Faso. Neisseria meningitidis was sought by detecting several meningococcus-specific genes (crgA, ctrA, 16S rRNA, and porA). The PCR-based nonculture method for the detection of N. meningitidis gave similar results between participants with a mean sensitivity and specificity of 89.7 and 92.7%, respectively. Most of the laboratories also performed genogrouping assays (siaD and mynB/sacC). The performance of genogrouping was more variable between laboratories, with a mean sensitivity of 72.7%. Genogroup B gave the best correlation between participants, as all laboratories routinely perform this PCR. The results for genogroups A and W135 were less similar between the eight participating laboratories that performed these PCRs.

Adolescent↗
PubMed2004

Neisseria meningitidis undergoes PilC phase variation and PilE sequence variation during invasive disease.

Neisseria meningitidis colonizes the upper respiratory tract (URT), enters the blood stream, and reaches the cerebrospinal fluid (CSF). In the present study, we show that bacteria isolated from the URT adhere better to human epithelial cells, compared with bacteria from blood or CSF, which suggests that important changes of virulence-associated proteins take place during bacterial dissemination. Phase variation in the pilus adhesin PilC and sequence variation in the pilus subunit PilE occurred among strains from 1 patient. Changes were not found in the invasion-associated opacity proteins or in lipooligosaccharides. PilC was frequently expressed in serogroup B strains and in URT strains but was often switched off in other serogroups and in CSF strains. Strains lacking PilC showed impaired adhesion to epithelial cells. These data argue that N. meningitidis undergoes PilC phase variation and PilE sequence variation during invasive disease.

Amino Acid Sequence↗
PubMed2004

Molecular typing of Neisseria gonorrhoeae isolates by pyrosequencing of highly polymorphic segments of the porB gene.

For prevention and control of gonorrhea, an objective, highly discriminating, and reproducible molecular epidemiological characterization of Neisseria gonorrhoeae is essential. In the present study, in pursuance of providing such qualities, pyrosequencing technology, a fast real-time DNA sequence analysis, was applied to six short, highly polymorphic porB gene segments, with subsequent genetic variant (genovar) determination of the bacterial isolates. The sequencing templates were obtained by real-time PCR amplification, which also included fluorescence melting curve analysis of the entire porB gene in order to determine the genogroup (porB1a or porB1b allele) prior to pyrosequencing analysis. The PSQ 96 MA system used allowed rapid (in approximately 1.5 h) determination of 96 sequences of 20 to 65 correct nucleotides each. The results were reproducible and mostly in concordance with the results of conventional Sanger dideoxy sequencing, with the exception of shorter read lengths and some uncertainty in determining the correct number of identical nucleotides in homopolymeric segments. The number of sequence variants identified in each of the six highly polymorphic segments of the porB1a and porB1b alleles (encoding surface-exposed amino acid loops of the mature PorB protein) ranged from 5 to 11 and from 8 to 39, respectively. Among porB1a isolates (n = 22) and porB1b isolates (n = 65), 22 and 64 unique genovars, respectively, were identified. All isolates were typeable. The present results provide evidence of a high discriminatory ability, practically the same as that for sequencing of the entire porB gene. In conclusion, the fast and high-throughput pyrosequencing technology can be used for molecular epidemiological characterization of N. gonorrhoeae.

Alleles↗
PubMed2004

Comparison of two urinary antigen tests for establishment of pneumococcal etiology of adult community-acquired pneumonia.

The Binax NOW immunochromatographic test (ICT) detecting the pneumococcal C polysaccharide and a serotype-specific latex agglutination (LA) test detecting 23 pneumococcal capsular antigens were evaluated for establishing pneumococcal etiology in community-acquired pneumonia (CAP) by use of nonconcentrated urine. ICT was considered to be strongly positive for result lines at least as intense as the control line and weakly positive for less intense result lines. When 215 adult CAP patients were tested, strong ICT, weak ICT, and LA positivity were found in 28, 24, and 16 patients, respectively; of these patients, 13 (46%), 6 (25%), and 13 (81%), respectively, had pneumococcal bacteremia and 27 (96%), 17 (71%), and 15 (94%), respectively, had Streptococcus pneumoniae isolated from blood, sputum, and/or nasopharynx. Among 108 controls tested, 2 (1.9%) were weakly ICT positive. When weak positivity was considered negative, the sensitivity of ICT decreased from 79% (19 of 24) to 54% (13 of 24), while the specificity increased from 83% (158 of 191) to 92% (176 of 191); no controls were false positive. The sensitivity and specificity of LA were 54% (13 of 24) and 98% (188 of 191), respectively. Eight of nine LA serotypes corresponded to culture serotypes. In conclusion, using nonconcentrated urine and dividing ICT-positive results into strongly and weakly positive results is a suitable way of performing ICT. While weak ICT positivity should be interpreted with caution, strong ICT positivity and LA positivity should be considered supportive of pneumococcal etiology in adult CAP. As such, these assays might have implications for antibiotic use in CAP. LA has promising potential for pneumococcal serotyping, although further evaluation is required.

Adult↗
PubMed2003

Multiple group-specific sequencing primers for reliable and rapid DNA sequencing.

Pyrosequencing technology is a bioluminometric DNA sequencing method that employs a cascade of four enzymes to deliver sequence signals. To date this technology has been limited to the sequencing of short stretches of DNA. As an improvement to this technique, we have introduced a bacterial group-specific, multiple sequencing primer approach that circumvents sequencing of less informative semi-conservative regions of the 16S rRNA gene. This new approach is suitable for challenging templates, improving sequence data quality, avoiding sequencing of non-specific amplification products, lessening sequencing time, and moreover, this strategy should open the way for many new applications in the future. The group-specific, multiple sequencing primers can be applied in the Sanger dideoxy sequencing method as well. In addition, we have improved the chemistry of the Pyrosequencing system enabling sequencing of longer stretches of DNA, which allows numerous new applications.

Bacteria↗
PubMed2003

PCR of cerebrospinal fluid for diagnosis of bacterial meningitis during meningococcal epidemics; an example from Sudan.

Meningococcal disease is feared due to its rapid progression and high case fatality rate, especially in the African meningitis belt, where epidemics of meningococcal meningitis appear cyclically. Culture, direct microscopy and antigen detection are the basic methods for diagnosis and species identification of bacterial meningitis. These methods are known to have limitations, especially in developing countries. The aim of the present study was to document the application of PCR technology for the diagnosis of bacterial meningitis in cerebrospinal fluid (CSF) samples (n = 52) collected during epidemics in Sudan. In the application of PCR for detection of the causative agent of bacterial meningitis (based on the 16S rRNA gene), bacterial DNA was identified in 49 samples. Common bacterial species causing bacterial meningitis could be detected in 31 of the CSF samples (27 meningococci), while 18 contained DNA, mainly from normally contaminating bacteria. A specific PCR for group A meningococci (based on the sacC gene) was positive in 27 of the CSF samples. The results show that PCR technology is a sharp-edged tool for confirmation of a diagnosis of meningococcal meningitis and for obtaining a direct genogrouping of group A meningococci in CSF. It is important to stress the use of direct and specific PCRs to avoid interference by contaminating bacteria, a great problem in samples from areas in the meningitis belt.

Cerebrospinal Fluid↗
PubMed2003

Genotyping of Chlamydia trachomatis would improve contact tracing.

BACKGROUND: The reported number of Chlamydia trachomatis genital infections has increased 15% annually since 1997 in Sweden. Inaccurate partner notification might be one reason. GOAL: The goals were to determine if genotyping of C trachomatis would improve partner notification and to study the duration of infection. STUDY DESIGN: Sexual networks were constructed. C trachomatis isolates from 231 individuals attending the Orebro STD clinic during 1 year were typed by sequencing of the omp1 gene. RESULTS: All individuals were traced and diagnoses were established in 30 of 161 networks. More than one genotype was seen in seven networks. The mean duration of C trachomatis infection in each network was calculated to be 23 weeks. CONCLUSION: Genotyping could be a useful tool in partner notification when there are discrepant or uncommon genotypes. Limited clinic catchment areas create information difficulties that obstruct accurate contact tracing.

Adolescent↗
PubMed2003

Molecular characterisation of group A Neisseria meningitidis isolated in Sudan 1985-2001.

A total of 33 group A Neisseria meningitidis (Mc) isolates, collected in Sudan between 1985 and 2001, were studied in order to describe the changes over time in a country within the meningitis belt of Africa. The isolates were characterised by traditional phenotypic methods (serogrouping, serotyping, serosubtyping and antibiogram) and molecular techniques (genosubtyping, pulsed-field gel electrophoresis [PFGE] with restriction endonucleases SpeI and NheI, and multilocus sequence typing [MLST]). Three clones of group A Mc were identified: one before 1988 (sulphadiazine sensitive, serotype 4, genosubtype P1.7,13-1,35-1, sequence type 4 [ST-4]); another during and after the 1988 epidemic (sulphadiazine resistant, serotype 4, genosubtype P1.20,9,35-1, ST-5); and a third causing the 1999 epidemic (sulphadiazine resistant, serotype 4, genosubtype P1.20,9,35-1, ST-7). The first clone showed major differences compared to the other two. The second and third clones had many similarities with differences in only a single gene (pgm) in the MLST (47 of the 450 bp) but significant other differences according to the PFGE patterns. Within the clones, genosubtyping and MLST gave identical information (except one base substitution in the aroE gene in one isolate). However, the PFGE patterns showed changes over time within the clones, where SpeI revealed somewhat more diversity than NheI.

Anti-Infective Agents↗
PubMed2003

Comparison of serologic and genetic porB-based typing of Neisseria gonorrhoeae: consequences for future characterization.

Due to temporal changes in the epidemiology of gonorrhea, a precise characterization of Neisseria gonorrhoeae is essential. In the present study genetic heterogeneity in the porB genes of N. gonorrhoeae was examined, and serovar determination was compared to porB gene sequencing. Among 108 N. gonorrhoeae isolates, phylogenetic analysis of the entire porB alleles (924 to 993 bp) identified 87 unique sequences. By analyzing only the four to six most heterogeneous porB gene regions (174 to 363 bp), 86 out of these 87 genetic variants were identified. Consequently, analysis of shorter highly variable regions of the porB gene generates high-level discriminatory ability as well as fast, objective, reproducible, and portable data for epidemiological characterization of N. gonorrhoeae. Regarding putative antigenic epitopes of PorB for Genetic Systems monoclonal antibodies (MAbs), some of the previous findings were confirmed, but new findings were also observed. For several of the MAbs, however, the precise amino acid residues of PorB critical for single-MAb reactivity were difficult to identify. In addition, repeated serovar determination of 108 N. gonorrhoeae isolates revealed discrepancies for 34 isolates, mostly due to nonreproducible reactivity with single MAbs. Thus, the prospects of a genetic typing system with congruent translation of the serovar determination seem to be limited. In conclusion, analysis of short highly variable regions of the porB gene could form the basis for a fast molecular epidemiological tool for the examination of emergence and transmission of N. gonorrhoeae strains within the community.

Bacterial Typing Techniques↗
PubMed2003

Neisseria meningitidis serogroup W-135 isolated from healthy carriers and patients in Sudan after the Hajj in 2000.

The first epidemic in the world of meningococcal disease due to serogroup W-135 was reported during the Hajj in 2000, with subsequent spread. The aims of the present study were to investigate whether the Hajj 2000 Neisseria meningitidis serogroup W-135 had also been carried to Sudan in the eastern part of the African meningitis belt, by examining healthy Sudanese pilgrims (Hajj 2000) and members of their families, and whether the strain was causing meningitis. The phenotypic character of W-135 meningococci from Sudanese carriers (n = 5) and patients (n = 2) 1 y later was similar to W-135 strains associated with Hajj 2000. The present study, using the combination of the 2 molecular techniques; sequencing of the porA gene for variable regions (VR1, VR2 and VR3) and pulsed-field gel electrophoresis of the entire genome (using SpeI and NheI), shows that the Hajj 2000 serogroup W-135 clone (P1.5,2,36-2 of the ET-37 complex) most probably was introduced into Sudan, by pilgrims returning from the Hajj 2000. This strain has not been diagnosed before in Sudan. Close epidemiological surveillance is required to identify a possible new emerging meningitis epidemic.

Adolescent↗