Conventional methods versus 16S ribosomal DNA sequencing for identification of nontuberculous mycobacteria: cost analysis

The clinical profile of nontuberculous mycobacteria (NTM) has been raised by the human immunodeficiency virus and AIDS pandemic. Different laboratory techniques, often molecular based, are available to facilitate the rapid and accurate identification of NTM. The expense of these advanced techniques has been questioned. At the National Reference Center for Mycobacteriology and the Health Sciences Center, University of Manitoba, in Winnipeg, Canada, we performed a direct cost analysis of laboratory techniques for commercial DNA probe-negative (Gen-Probe, Inc., San Diego, Calif.), difficult-to-identify NTM. We compared the costs associated with conventional phenotypic methodology (biochemical testing, pigment production, growth, and colony characteristics) and genotypic methodology (16S ribosomal DNA [rDNA] sequence-based identification). We revealed a higher cost per sample with conventional methods, and this cost varied with organism characteristics: $80.93 for slowly growing, biochemically active NTM; $173.23 for slowly growing, biochemically inert NTM; and $129.40 for rapidly growing NTM. The cost per sample using 16S rDNA sequencing was $47.91 irrespective of organism characteristics, less than one-third of the expense associated with phenotypic identification of biochemically inert, slow growers. Starting with a pure culture, the turnaround time to species identification is 1 to 2 days for 16S rDNA sequencing compared to 2 to 6 weeks for biochemical testing. The accuracy of results comparing both methodologies is briefly discussed. 16S rDNA sequencing provides a cost-effective alternative in the identification of clinically relevant forms of probe-negative NTM. This concept is not only useful in mycobacteriology but also is highly applicable in other areas of clinical microbiology.     

Hypergonadotrophinaemia with reduced uterine and ovarian size in women born small-for-gestational-age

BACKGROUND: Fetal growth restraint has been associated with FSH hypersecretion in early infancy and in early post-menarche, and with reduced uterine and ovarian size in adolescence. It is unknown whether these reproductive anomalies persist, respectively, into late infancy and into the reproductive age range. METHODS: We report follow-up findings in two age groups of girls. A cohort of infants [n=26; n=10 born appropriate-for-gestational-age (AGA) and n=16 born small-for-gestational-age (SGA)], who had been studied at the age of approximately 4 months, was assessed again at the age of 12 months. A cohort of teenagers (n=28), who had been studied at the age of approximately 14 years, was assessed again at the age of approximately 18 years; this group was complemented by a transversal cohort of similar age (n=19) for a total of 47 young women (n=27 AGA; n=20 SGA). In infants, only serum FSH was measured; adolescents underwent endocrine-metabolic screening, ultrasound assessment of uterine-ovarian size, and evaluation of body composition by dual X-ray absorptiometry. RESULTS: Serum FSH levels were higher in SGA than AGA infant girls at 4 and 12 months, and higher in SGA than AGA adolescents at 14 and 18 years (all P<0.01). Longitudinal ultrasound assessments disclosed a late-adolescent increment of uterine size that was less obvious in SGA than AGA girls. In contrast, ovarian volume remained stable in both subgroups. Compilation of longitudinal and transversal results at 18 years of age corroborated the persistent reduction in the uterine size of SGA girls (by approximately 20%; P<0.005) and in their ovarian volume (by approximately 40%; P<0.0001); moreover, SGA girls displayed not only a persistent elevation of FSH (by approximately 50%; P<0.001), but also a rise of LH and fasting insulin, as well as an excess of abdominal fat (all P<0.01). CONCLUSIONS: The gynaecology of young women born SGA was found to be characterized by hypergonadotrophinaemia and by a reduced uterine and ovarian size.     

Deletion of decay-accelerating factor (CD55) exacerbates autoimmune disease development in MRL/lpr mice

Decay-accelerating factor (DAF, CD55) is a glycosylphosphatidylinositol-anchored membrane protein that restricts complement activation on autologous cells. It is also a ligand for CD97, an activation-associated lymphocyte antigen with seven transmembrane domains. It is widely expressed on cells of both the hematopoietic and nonhematopoietic lineages. Although deficiency of DAF on human erythrocytes is associated with the hemolytic anemia syndrome paroxysmal nocturnal hemoglobinuria, the in vivo biology of DAF is still poorly understood. We addressed the in vivo function of DAF in a knockout mouse model and describe here that deletion of DAF exacerbates autoimmune disease development in MRL/lpr mice, a model for human systemic lupus erythematosus. Compared to DAF-sufficient littermate controls, DAF-deficient female MRL/lpr mice developed exacerbated lymphadenopathy and splenomegaly, higher serum anti-chromatin autoantibody levels, and aggravated dermatitis. Consistent with the phenotype of aggravated dermatitis in DAF-deficient mice, Northern and Western blots and immunofluorescence studies showed DAF to be expressed abundantly in the mouse skin, suggesting that it may play a particularly important role in this tissue. Histology and immunostaining demonstrated inflammatory infiltrate and focal C3 deposition in early skin lesions, mostly along the dermal-epidermal junction. These results reveal a protective function of DAF in the development of a systemic autoimmune syndrome and suggest that dysfunction or down-regulation of DAF may contribute to autoimmune disease pathogenesis and manifestation.     

Comparative evaluation of the new version of the INNO-LiPA Mycobacteria and genotype Mycobacterium assays for identification of Mycobacterium species from MB/BacT liquid cultures artificially inoculated with Mycobacterial strains

The performance of two DNA line probe assays, a new version of INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium) and the GenoType Mycobacterium (Hain Diagnostika, Nehren, Germany), were evaluated for identification of mycobacterial species isolated from liquid cultures. Both tests are based on a PCR technique and designed for simultaneous identification of different mycobacterial species by reverse hybridization and line probe technology. The INNO-LiPA Mycobacteria v2 targeting the 16S-23S rRNA gene spacer region was developed for the simultaneous identification of 16 different mycobacterial species. The GenoType Mycobacterium, which targets the 23S rRNA gene, allows simultaneous identification of 13 mycobacterial species. Both tests were evaluated on 110 mycobacterial strains belonging to 22 different mycobacterial species (20 reference strains, 83 clinical strains, and 4 Mycobacterium kansasii strains isolated from tap water) that were previously inoculated into MB/BacT bottles. The sensitivity of both methods, defined as the number of positive results obtained with the Mycobacterium genus probe together with an interpretable result on the number of samples tested was 110 of 110 (100%) for INNO-LiPA and 102 of 110 (92.7%) for GenoType. For samples with interpretable results, INNO-LiPA was able to correctly identify 109 of 110 samples (99.1%), whereas the GenoType correctly identified 100 of 102 samples (98.0%). Both tests were easy to perform, rapid, and reliable when applied to mycobacterial identification directly from MB/BacT bottles.     

Galanin receptors in the post-mortem human brain. Regional distribution of I-galanin binding sites using the method of in vitro receptor autoradiography

The distribution of putative receptors for the peptide galanin was studied in the normal post-mortem human brain by using ¹²⁵I-galanin (0.5 nM) in combination with in vitro receptor autoradiography. Specific binding of ¹²⁵I-galanin was found in a large number of brain areas throughout the neuraxis. Highest binding densities occurred in the basal forebrain and hypothalamus, while the basal ganglia, major parts of the thalamus and the tectum were found to be poor in binding sites. All cortical areas harboured ¹²⁵I-galanin binding, and in the visual cortex a laminated pattern was present. In the hippocampus, ¹²⁵I-galanin binding occurred in layer 2 of the entorhinal cortex, in the uncus and in the hippocampal-amygdala area. In the brain-stem, ¹²⁵I-galanin binding was found in serotoninergic and noradrenergic cellgroups as well as in the reticular formation and in the parabrachial nuclei. Galanin receptors may, thus, mediate the response of galanin in numerous structures in the human brain.     

Stability of lyophilised specimens for the molecular detection of viral DNA/RNA.

BACKGROUND: The range of nucleic acid-based technologies for the molecular detection of pathogens has grown rapidly in recent years. The influx of new testing methods into the clinical laboratory, demands for evaluation and standardisation of methods, interpretation of results and evaluation of laboratory performance have highlighted the need for internal and External Quality Assessment (EQA) systems more than ever before. External Quality Assessment panels demand reproducible, stable specimens of consistent form, suitable for transportation. OBJECTIVES: To determine the stability of freeze-dried viral specimens in terms of molecular detection. STUDY DESIGN: When EQA specimens are prepared, they undergo long-term storage and testing as part of the quality control (QC) process. The frequency and nature of testing is dependent on the resources and methodologies available at the time. A range of virus preparations used for EQA was monitored over a period of months to years in a retrospective study; the available quality monitoring data for the five viruses, including storage temperature and method of detection were analysed. RESULTS: The nucleic acid (DNA or RNA) of the freeze-dried viruses included in the study was readily detectable over a long period of time. Quantitative analysis indicated that detectable concentrations of nucleic acid post-freeze drying were similarly maintained. Storage temperature was an important factor in the stability of HCV, but other viruses were unaffected by storage at different temperatures. CONCLUSIONS: In summary, the molecular detection of nucleic acid (DNA or RNA) in freeze-dried specimens of HSV1, HSV2, HBV, HCV and HIV is possible even after prolonged storage, in some cases at a range of temperatures. Freeze drying allows large-scale production of viral specimens of high quality for EQA, which are stable in varying storage and shipment conditions. Furthermore, detection of each virus was possible with a range of commonly used molecular diagnostic methods.     

Patient follow-up is a major problem at genetics clinics

Children with genetic diseases must be followed for long periods of time to seek new findings. Other patients require further check-ups and studies to be diagnosed. Some patients never return for medical care after the first consultation, which may have serious consequences. We reviewed 400 medical charts of patients with genetic disease to analyze overall attendance to the genetics clinic, investigate some of the causes of failure to seek medical advice, and determine the differences between those first seen as outpatients or as inpatients. The mean follow-up period was 8.3 months (range 0-79), and the average number of visits was 2.8 (range 1-16). Forty eight percent of the cases first seen as inpatients were evaluated only once and 14% twice; while 22 and 21% of the 300 cases first seen as outpatients attended once and twice, respectively (P = 0.0). Appointment keeping was apparently not affected by the presence or absence of diagnosis. Overall, 97 patients were discharged, 7 died, 55 continued on follow-up, 62 attended other hospital services-but not genetics-and 179 were completely lost to follow-up. Diagnosed patients were counseled more frequently than undiagnosed patients (62 vs. 5%); and 71% of the diagnosed patients first seen as outpatients but only 36% of undiagnosed cases first seen as inpatients were counseled, differences between these two groups were significant (P = 0.005). We conclude that keeping the patient with genetic disease on follow-up is a difficult task. New educational strategies must be planned to improve this worrisome situation.