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PubMed2005

Fluoroquinolone-resistant Neisseria gonorrhoeae: the inevitable epidemic.

The worldwide incidence of fluoroquinolone-resistant Neisseria gonorrhoeae has increased dramatically in the last few years. Single doses of fluoroquinolones can no longer be used to treat N gonorrhoeae infections acquired in the Far East, parts of the Middle East, the Pacific Islands, and parts of Western Europe and the United States. Although California and Hawaii account for most of the current United States cases, the increased incidence of FQR in some high-risk groups independent of geography heralds an imminent spread of drug-resistant strains throughout the rest of the population. The use of molecular tests has revolutionized the diagnostic field in STIs. The main limitation of their application in N gonorrhoeae testing has been the loss of culture specimens that allow antimicrobial sensitivity testing. New molecular methods have made it possible to detect antimicrobial resistance without the use of live organisms. These tests hold the promise of improving epidemiologic tracking of N gonorrhoeae drug resistance, leading to better patient management at the local level. The loss of fluoroquinolones limits available oral regimens to a single CDC-recommended antibiotic, cefixime. Oral, inexpensive, single-dose alternatives are needed to ensure continued therapeutic success.

Anti-Bacterial Agents↗

PubMed2002

Applications of selective neutrality tests to molecular ecology.

This paper reviews how statistical tests of neutrality have been used to address questions in molecular ecology are reviewed. The work consists of four major parts: a brief review of the current status of the neutral theory; a review of several particularly interesting examples of how statistical tests of neutrality have led to insight into ecological problems; a brief discussion of the pitfalls of assuming a strictly neutral model if it is false; and a discussion of some of the opportunities and problems that molecular ecologists face when using neutrality tests to study natural selection.

Animals↗

PubMedDate not supplied

Detection of hepatitis B virus DNA directly in human serum by a simplified molecular hybridization test: comparison to HBeAg/anti-HBe status in HBsAg carriers.

A simple, direct molecular hybridization test was employed to detect hepatitis B virus (HBV) DNA sequences in human serum. In 61 HBsAg carriers, many with HBV-related diseases (chronic persistent hepatitis, chronic active hepatitis, or posthepatitic cirrhosis), 28/28 (100%) who were HBeAg+ and 16/32 (50%) who were anti-HBe+ had HBV DNA sequences in their serum. Among 22 South African black patients with hepatocellular carcinoma, 7 (32%) had detectable HBV DNA in their serum but at reduced levels when compared to HBsAg carriers without hepatocellular carcinoma, suggesting that viral replication is suppressed or inactive in many hepatocellular carcinoma patients. Hybridization analysis also distinguished carriers with high, moderate, or low amounts of HBV DNA in serum. Ten to 20% of HBsAg+/HBeAg+ carriers showed high serum levels of HBV DNA but, surprisingly, a similar percentage of HBsAg+/anti-HBe+ carriers also showed relatively high serum levels of HBV DNA. Five patients who had undergone immunosuppression therapy and most of whom were on chronic hemodialysis had very high serum levels of HBV DNA, in the range observed during acute HBV infection. By epidemiologic analysis, two of these individuals were implicated in transmission of hepatitis to other hemodialysis patients, paramedical personnel, or intimate family contacts. Serum HBV DNA hybridization analysis identifies carriers with high serum levels of HBV irrespective of HBeAg/anti-HBe status and may define individuals with potentially high risk of transmitting infection to their immediate contacts.

Antibodies, Viral↗

PubMed2006

[A census of medical genetics services in Argentina].

OBJECTIVE: To describe the medical genetics services in Argentina, in order to help organize a network to coordinate those services, decrease inequity in access to them, and promote the comprehensive development of medical genetics in the country. METHODS: In April-August 2004 a survey was conducted of centers providing medical genetics services in Argentina, looking at their human resources, diagnostic procedures, the services that patients use, and funding. The survey covered service centers that conduct genetic assessment, cytogenetic diagnostic testing, molecular biology testing, and biochemical testing to detect birth defects or genetic disorders. Centers that were devoted exclusively to research were not surveyed. RESULTS: A total of 134 centers fulfilled the selection criteria for the survey; 84 of them were private services, and the other 50 were publicly managed services, at universities and hospitals. The 50 public facilities had a total of 67 physicians who had studied clinical genetics, plus 133 non-medical laboratory professionals. A majority of the 50 centers performed clinical and cytogenetic diagnoses, with a smaller number performing molecular, biochemical, and prenatal diagnoses. More than 70% of the centers and human resources were located in an area that includes the city of Buenos Aires, the province of Buenos Aires, and the nearby north-central provinces of Córdoba and Santa Fe. The 50 public centers were found mainly in large cities; 10 of the country's 23 provinces had no public services. CONCLUSIONS: There is a great geographic disparity in the availability of resources as well as wide variability in the diagnostic procedures that are provided in the different service centers. The unequal distribution of the facilities is an organizational problem that will require the application of policies to guarantee minimum services for people in all regions of the country, as well as access to more complex diagnostic procedures.

Adult↗

PubMed1995

Molecular analysis by pulsed-field gel electrophoresis of penicillin-resistant Streptococcus pneumoniae from Toulouse, France.

A sample of 28 penicillin-resistant Streptococcus pneumoniae strains isolated between 1991 and 1993 in a large hospital in Toulouse, France, was characterized by pulsed-field gel electrophoresis of genomic DNA. Also included were 6 penicillin-susceptible clinical isolates from Toulouse and 12 penicillin-resistant strains from different parts of the world. The restriction endonucleases ApaI and SmaI were used to digest intact chromosomes, and the fragments were resolved by field-inversion gel electrophoresis. Seven major pattern types could be recognized among the penicillin-resistant isolates from Toulouse. Nine of these isolates could be assigned to two clones that were also found in Spain and were associated with serotypes 6B and 9V. A third clone was isolated in South Africa and in Spain and contained serotype 23F isolates. The profiles obtained by field-inversion gel electrophoresis suggested that 15 of the 16 penicillin-resistant serogroup 23 isolates from Toulouse belonged to the same Spanish 23F clone. The molecular test profiles of penicillin-susceptible strains differed from those of resistant strains of the same serotype except those of 9V strains. These data underline the importance of the geographic spread of resistant clones from Spain in the emergence of penicillin-resistant pneumococci in France.

DNA, Bacterial↗

PubMed2003

Diagnosis of hereditary neuropathies in adult patients.

This paper reviews the clinical diagnostic approach to hereditary neuropathies in adults by analysing: elements that point to a neuropathy of inherited origin, different modalities of presentation, laboratory and instrumental diagnostic tests, including molecular tests, symptoms and signs of involvement of other organs. Different phenotypes may be identified according to: disease course; involvement of motor, sensory, autonomic fibres; site of lesion (neuropathy versus neuronopathy); calibre of involved fibres (small-fibre versus large-fibre neuropathy); presence of distinctive symptoms (neuropathic pain); involvement of other organs or apparatus. Charcot-Marie-Tooth disease, Familial Amyloid Polyneuropathy, Hereditary Sensory and Autonomic Neuropathy, Fabry disease, Tangier disease, Porphyric Neuropathies, Refsum disease, Hereditary Neuropathy with liability to Pressure Palsies, Hereditary Neuralgic Amyotrophy, and other rare disorders involving the peripheral nervous system are reviewed.

Acute Disease↗

PubMed1998

Comparison of PCR-heteroduplex characterization by automated DNA sequencing and line probe assay for the detection of rifampicin resistance in Mycobacterium tuberculosis isolates from KwaZulu-Natal, South Africa.

Progress in understanding the basis of resistance to rifampicin (RifR) has allowed molecular tests for the detection of drug-resistant tuberculosis to be developed. One hundred thirteen strains of Mycobacterium tuberculosis isolated from patients with multidrug resistant tuberculosis (MDR-TB) were investigated for genotypic analysis of RifR by polymerase chain reaction-heteroduplex formation (PCR-HDF) and characterization of mutations by automated DNA sequencing of the rpoB gene. A subset of isolates (22) representative of different mutations as confirmed by sequence analysis were also evaluated by the Line Probe Assay (LiPA). In 106 of the RifR strains, 24 mutations within an 81-bp region of the rpoB gene affecting 13 amino acids were observed. Most isolates (7/8) harboring Leu533 --> Pro codon mutation required minimum inhibitory concentrations (MICs) of < or = 8 microg/ml. There was geographic variation in the frequency of occurrence of particular rpoB mutations, with the Ser531 --> Leu/Trp codon mutation found in 59/113 of isolates. Although there are certain limitations in the use of both the rapid PCR-HDF diagnostic assay and the LiPA for the detection of rifampicin susceptibility of M. tuberculosis, these provide important and convenient tools for identifying and managing patients with MDR-TB.

Antibiotics, Antitubercular↗

PubMed1998

Factor V Leiden, activated protein C resistance, and retinal vein occlusion.

BACKGROUND: Resistance to activated protein C (APC resistance) is a thrombophilic abnormality characterized by a normal plasma level of protein C and an inherited defect in the coagulative response. This condition is believed to be caused by a point mutation in factor V, the so-called factor V Leiden, and is inherited as an autosomal dominant trait. PURPOSE: A case-control study was carried out to evaluate the prevalence of APC resistance and factor V Leiden in patients with retinal vein occlusion (RVO) and in control subjects. METHODS: Eighty-four consecutive RVO patients and 70 controls were tested for APC resistance with a commercial assay (Chromogenix). The first 30 patients and 47 controls were also studied for factor V Leiden. In addition, a repeat APC-resistance test was performed in 40 RVO patients and in 9 controls with a second-generation assay done to compare the reliability and reproducibility of the tests. RESULTS: Results of testing for APC resistance with the first-generation assay revealed positive results in 38 (45%) of the study patients and 6 (9%) of the controls. The difference in frequencies of APC resistance in patients and controls was statistically significant (P < 0.0001). In the patients tested for factor V Leiden, one (3%) was a heterozygous carrier of the Arg506GIn mutation and one (2%) of the controls was a heterozygous carrier. No homozygous individuals were identified in either the study or the control groups. The difference in frequencies of factor V Leiden in study patients and controls was not statistically significant (P = 1). The repeat APC-resistance assay using factor V-deficient plasma in 40 RVO patients and 9 controls did not show any significant difference between study patients and controls or an association between APC resistance and the determination of the factor V Leiden mutant. CONCLUSION: The first-generation commercial assay for APC resistance is not a useful screening test. The molecular test for factor V Leiden is the only definitive method. Furthermore, no significant association was found between factor V Leiden and retinal vein occlusion. Accordingly, routine testing for the presence of the factor V Leiden mutant is not advisable for patients with retinal vein occlusion.

Activated Protein C Resistance↗

PubMed2006

Two methods of self-sampling compared to clinician sampling to detect reproductive tract infections in Gugulethu, South Africa.

OBJECTIVES: To assess the validity, feasibility, and acceptability of 2 methods of self-sampling compared to clinician sampling during a speculum examination. GOAL: To improve screening for reproductive tract infections (RTIs) in resource-poor settings. STUDY DESIGN: In a public clinic in Cape Town, 450 women underwent a speculum examination and were randomized to self-sample with either a tampon or vaginal swabs. All specimens were tested for the same pathogens using the same diagnostic tests. RESULTS: Self-sampling resulted in satisfactory validity for N gonorrhoeae, C trachomatis, bacterial vaginosis, and Candida species (tampons and swabs) and high-risk human papillomavirus (swabs only) when tested with molecular tests or microscopy, but not for T vaginalis by culture. Self-sampling was feasible and acceptable, but some women preferred speculum examinations, which allow the clinician to view the vagina and cervix. CONCLUSIONS: Although self-sampling should not replace speculum examinations in all circumstances, it should be explored further as an RTI screening strategy.

Adolescent↗

PubMed2006

Multiple mutations in or adjacent to the conserved penicillin-binding protein motifs of the penicillin-binding protein 1A confer amoxicillin resistance to Helicobacter pylori.

BACKGROUND: Amoxicillin-based therapies are highly effective for the treatment of Helicobacter pylori infections, but the efficacy may decrease as the incidence of amoxicillin resistance is increasing. So far, the molecular mechanism underlying stable amoxicillin resistance has only been identified for a few naturally occurring amoxicillin-resistant (Amx) H. pylori isolates, and is mediated by mutations in penicillin-binding protein 1A (PBP1A). In this study the molecular mechanism underlying amoxicillin resistance of seven additional Amx H. pylori isolates has been established. METHODS: H. pylori strain 26695 (minimal inhibitory concentration (MIC) 0.125 mg/l) was naturally transformed with total DNA and pbp1A polymerase chain reaction (PCR) products from the seven Amx H. pylori isolates, and the MIC of amoxicillin and pbp1A gene sequence of the obtained Amx transformants were determined. RESULTS: Replacement of the wild-type pbp1A gene of H. pylori reference strain 26695 by the pbp1A gene of the Amx H. pylori isolates resulted in an increased MIC (0.5-1.0 mg/l). Sequence analysis of the smallest PBP1A fragments able to transfer the resistance indicated that several amino acid substitutions in or adjacent to the second (SKN402-404) and third (KTG555-557) conserved penicillin-binding protein motifs (PBP-motifs) mediate amoxicillin resistance in H. pylori. This was confirmed by site-directed mutagenesis using oligonucleotides that contained defined mutations in or adjacent to these PBP-motifs. CONCLUSION: In naturally occurring Amx H. pylori isolates, amoxicillin resistance is mediated by various mutational changes located in or adjacent to the second and third PBP-motifs of the PBP1A. Although we cannot exclude the role of the other genes in amoxicillin resistance, it is likely that multiple mutational changes in the PBP1A gene are the predominant cause of amoxicillin resistance in H. pylori. The findings of this study currently preclude the rapid detection of amoxicillin resistance in H. pylori by molecular tests.

Amoxicillin↗

PubMed2003

Identification of mycobacterial species by PCR sequencing of quinolone resistance-determining regions of DNA gyrase genes.

The determination of the amino acid sequence of quinolone resistance-determining regions (QRDRs) in the A and B subunits of DNA gyrase is the molecular test for the detection of fluoroquinolone resistance in mycobacteria. We looked to see if the assignment of mycobacterial species could be obtained simultaneously by analysis of the corresponding nucleotide sequences. PCR sequencing of gyrA and gyrB QRDRs was performed for 133 reference and clinical strains of 21 mycobacterial species commonly isolated in clinical laboratories. Nucleotide sequences of gyrA and gyrB QRDRs were species specific, regardless of fluoroquinolone susceptibility.

Anti-Infective Agents↗

PubMed1999

[Modern methods in the diagnosis of tuberculosis: PCR--polymerase chain reaction].

The diagnosis of tuberculosis depends on many factors but essentially on the detection of mycobacteria, their identification to the species level, and their susceptibility to major antituberculous drugs. The classic tests (direct examination-Ziehl-Neelsen, conventional culture and antibiotic susceptibility tests) are combined with modern tests (the molecular tests such as genomic amplification /PCR/ direct from clinical samples). By Polymerase Chain Reaction (PCR) over a billion identical bacteria could be multiplied from a single DNA molecule. These modern tests shortened diagnostics duration from several weeks to one or two days. The sensitivity of PCR test was 97% for Z-N positive, 68% for Z-N negative samples while specificity was 99% either for Z-N positive or negative samples.

DNA, Bacterial↗

PubMed2003

[Diagnosis of hereditary neuropathies in adult-subjects].

The clinical approach to hereditary neuropathies of adulthood has become very complex. The following issues are reviewed: elements that point to a neuropathy of inherited origin, different modalities of presentation, laboratory and instrumental diagnostic tests, including molecular tests, symptoms and signs of involvement of other organs. It is useful to identify different phenotypes according to: disease course; involvement of motor, sensory, autonomic fibres; site of lesion (neuropathy versus neuronopathy); calibre of involved fibres (small-fibre versus large-fibre neuropathy); presence of distinctive symptoms (neuropathic pain); involvement of other organs or apparatus. Charcot-Marie-Tooth (CMT) disease, familial amyloid polyneuropathy (FAP), hereditary sensory and autonomic neuropathy, Fabry disease, Tangier disease, porphyric neuropathies, Refsum disease, hereditary neuropathy with liability to pressure palsies (HNPP), hereditary neuralgic amyotrophy and other rare disorders involving the peripheral nervous system are reviewed.

Adult↗

PubMed2003

Detection of Group A beta-hemolytic Streptococcus employing three different detection methods: culture, rapid antigen detecting test, and molecular assay.

In order to study the prevalence of Group A beta-hemolytic Streptococcus (GABHS) pharyngotonsillitis in our pediatric population and to compare different sampling methods of GABHS detection, oropharyngeal swabs from 50 children with acute pharyngotonsillitis, between 1 and 12 years old, were used simultaneously for culture, molecular assay and rapid GABHS antigen detection tests. All children were clinically examined at the Division of Pediatric Otorhinolaryngology of the Federal University of São Paulo. Diagnostic criteria were based on signs and symptoms, including sore throat, fever and oropharyngeal purulent secretion. Children that had been treated with antibiotics were excluded. Overall, combining the three methods, the prevalence of GABHS was 34%. GABHS was diagnosed in 30% of the bacterial cultures, in 25% of the samples tested with the molecular nucleic acid hybridization method and in 26% of the cases tested with the rapid antigen detection test. There was no significant difference between these three methods.

Bacterial Typing Techniques↗

PubMed2003

Quality assurance in human molecular genetics testing: status and recommendations.

OBJECTIVE: The role of genetic testing has expanded with rapidly developing technology and completion of the International Human Genome Project. Development of universally acceptable quality control methods and quality assurance standards trails technology. The principle that high-quality genetic testing is important for public health motivated the Centers for Disease Control and Prevention to formulate ways for improving quality assurance of human molecular genetics testing. PARTICIPANTS: Twenty-eight panelists were chosen based on expertise in molecular genetics testing and knowledge of quality assurance practices. Representatives of professional organizations, industries, and federal agencies participated in one or more of 3 panel meetings. Consensus recommendations were developed by the 15 panelists in the third meeting. EVIDENCE: Evidence was derived from experts' opinion during 3 panel meetings. Data compiled through laboratory visits and literature review were used as reference information. Need for this project was derived from the Final Report of the Task Force on Genetic Testing, produced by the National Institutes of Health and the Department of Energy in 1997, and the Summary Report of the Subcommittee Meeting on Genetics of the Clinical Laboratory Improvement Act Advisory Committee in 1997. CONSENSUS PROCESS: Research and development needs were identified using a participatory visioning approach. A modified nominal group process was used to reach consensus. CONCLUSIONS: Five core consensus recommendations were made: research for developing positive samples for quality assurance purposes, performance evaluation programs supplementing those in existence, establishment and support of laboratory-oriented consortia, establishment of a laboratory-focused database, and support of molecular genetics training programs.

Centers for Disease Control and Prevention, U.S.↗

PubMed2002

Practical application of molecular genetic testing as an aid to the surgical pathologic diagnosis of sarcomas: a prospective study.

The strong correlation of specific reciprocal translocations with individual tumor types and the demonstration that polymerase chain reaction (PCR)-based methods can detect translocations in tissue samples have stimulated interest in the role of molecular genetic testing in diagnostic surgical pathology. To evaluate the clinical utility of PCR-based molecular analysis of soft tissue neoplasms in routine surgical pathology, 131 consecutive soft tissue tumors submitted for molecular genetic testing at a tertiary care teaching hospital were prospectively analyzed over a 36-month period. RT-PCR was used to test tumor RNA for fusion transcripts characteristic of malignant round cell tumors (including Ewing sarcoma/primitive neuroectodermal tumor, desmoplastic small round cell tumor, and alveolar rhabdomyosarcoma), spindle cell tumors (including synovial sarcoma and congenital fibrosarcoma), and fatty tumors (myxoid liposarcoma). DNA sequence analysis was used to confirm the identity of all PCR products, and the PCR results were compared with the histopathologic diagnosis. We found that sufficient RNA for RT-PCR-based testing was recovered from 96% of the 131 cases and the percentage of tumors that tested positive for the associated characteristic fusion transcript was in general agreement with those reported in the literature. DNA sequence analysis of PCR products identified both variant transcripts and spurious PCR products, underscoring the value of product confirmation steps when testing formalin-fixed, paraffin-embedded tissue. Only in rare cases did testing yield a genetic result that was discordant with the histopathologic diagnosis. We conclude that PCR-based testing is a useful adjunct for the diagnosis of malignant small round cell tumors, spindle cell tumors, and other miscellaneous neoplasms in routine surgical pathology practice.

Base Sequence↗

PubMed2004

Quality assessment in cytogenetic and molecular genetic testing: the experience of the Italian Project on Standardisation and Quality Assurance.

The first Italian national trial of external quality assessment in genetic testing was organised within the framework of the "Italian National Project for Standardisation and Quality Assurance of Genetic Tests". Sixty-eight Public Health Service laboratories volunteered for the trial, which involved molecular genetic tests (cystic fibrosis, beta-thalassaemia, familial adenomatous polyposis coli and fragile-X syndrome) and cytogenetic tests (prenatal and postnatal, the latter included cancer cytogenetics). The response rate was high (88.2%). The level of analytical accuracy was good, i.e., the percentage of laboratories that correctly genotyped all samples was 89.3% for cystic fibrosis, 90.9% for beta-thalassaemia, 100% for familial adenomatous polyposis coli (despite two laboratories did not complete the analysis because the amount of DNA was considered insufficient), and 90.5% for fragile-X syndrome. Written reports differed widely and were judged "inadequate" in over 50% of cases. Most laboratories from the present study already have experience in previous European external quality assessments for at least one genetic test; this can explain the higher analytical accuracy in the Italian external quality assessment with respect to quality control programmes in other countries. Collaborative networks are strongly suggested to improve the quality of the reports.

Cystic Fibrosis↗

PubMed2001

Molecular resistance testing of Helicobacter pylori in gastric biopsies.

OBJECTIVE: To evaluate simultaneous diagnosis of infection and molecular resistance testing of Helicobacter pylori. METHODS: Gastric biopsies were obtained from 26 rapid urease-positive and 51 rapid urease-negative test kits used to diagnose H pylori infection. Following glass bead-assisted DNA isolation, amplification of H pylori 16S ribosomal DNA (rDNA), glmM, and 23S rDNA target genes was performed. RESULTS: Helicobacter pylori DNA was successfully amplified from 100% (26/26) of urease-positive and 3.9% (2/51) of urease-negative gastric biopsies. Subsequent restriction enzyme-mediated digestion of 23S rDNA amplification products revealed that 17% (4/24) of urease-positive and H pylori DNA-positive biopsy specimens contained point mutations (A2142G or A2143G) associated with clarithromycin resistance. Helicobacter pylori DNA from gastric biopsies was successfully amplified 8 weeks following rapid urease testing. CONCLUSION: Helicobacter pylori genotyping may be used to detect macrolide-resistant H pylori in individuals prior to initiation of therapy or in patients refractory to anti-H pylori therapy. Two urease-negative specimens yielded Helicobacter DNA distinct from that of H pylori and indicated the need for further investigations of Helicobacter species present in the human stomach.

Anti-Bacterial Agents↗