Characterization of Mycobacterium tuberculosis complex DNAs from Egyptian mummies by spoligotyping

Bone and soft tissue samples from 85 ancient Egyptian mummies were analyzed for the presence of ancient Mycobacterium tuberculosis complex DNA (aDNA) and further characterized by spoligotyping. The specimens were obtained from individuals from different tomb complexes in Thebes West, Upper Egypt, which were used for upper social class burials between the Middle Kingdom (since ca. 2050 BC) and the Late Period (until ca. 500 BC). A total of 25 samples provided a specific positive signal for the amplification of a 123-bp fragment of the repetitive element IS6110, indicating the presence of M. tuberculosis DNA. Further PCR-based tests for the identification of subspecies failed due to lack of specific amplification products in the historic tissue samples. Of these 25 positive specimens, 12 could be successfully characterized by spoligotyping. The spoligotyping signatures were compared to those in an international database. They all show either an M. tuberculosis or an M. africanum pattern, but none revealed an M. bovis-specific pattern. The results from a Middle Kingdom tomb (used exclusively between ca. 2050 and 1650 BC) suggest that these samples bear an M. africanum-type specific spoligotyping signature. The samples from later periods provided patterns typical for M. tuberculosis. This study clearly demonstrates that spoligotyping can be applied to historic tissue samples. In addition, our results do not support the theory that M. tuberculosis originated from the M. bovis type but, rather, suggest that human M. tuberculosis may have originated from a precursor complex probably related to M. africanum.     

Microbial exposure of rural school children, as assessed by levels of N-acetyl-muramic acid in mattress dust, and its association with respiratory health

BACKGROUND: Endotoxin exposure has been shown to be associated with a decreased prevalence of atopic sensitization and symptoms. Yet endotoxin represents only a part of the indoor microbial exposure. Muramic acid, a constituent of peptidoglycan, is present in gram-negative and gram-positive bacteria in the environment and may therefore serve as an additional marker of microbial exposure. OBJECTIVE: To study the factors determining the level of indoor exposure to muramic acid/peptidoglycan, as well as its potential association with respiratory health. METHODS: In 553 farm and nonfarm school children from Austria, Switzerland, and Germany, mattress dust muramic acid concentrations were determined, and health was assessed by using IgE measurements and questionnaire information. RESULTS: The muramic acid concentration was found to be significantly higher in dust from farm children's mattresses than in dust from nonfarm children's mattresses (157 vs 131 ng/mg). Children with higher mattress dust muramic acid concentrations had a significantly lower prevalence of wheezing (odds ratio of highest vs lowest tertile of muramic acid concentration, 0.3; 95% CI, 0.1-0.9), regardless of farming status and endotoxin exposure. The association for asthma was similar, and no association was found with atopic sensitization. CONCLUSION: Next to endotoxin, muramic acid provides us with an independent marker of microbial exposure. Unlike endotoxin, muramic acid was inversely associated with wheezing rather than with atopic sensitization.     

Molecular characterization and determination of the coding capacity of the genome of equine herpesvirus type 2 between the genome coordinates 0.235 and 0.258 (theEcoRI DNA fragment N; 4.2 kbp)

The complete DNA nucleotide sequence of theEcoRI DNA fragment N (0.235 to 0.258 viral map units) of equine herpes virus type 2 (EHV-2) strain T400/3 was determined. This DNA fragment comprises 4237 bp with a base composition of 55.23% G+C and 44.77% A+T. Nineteen open reading frames (ORFs) of 50-287 amino acid (aa) residues were detected. ORF number 10 is located between the nucleotide position 2220 and 2756 coding for a protein of 179 amino acid residues. This protein shows significant homology to the cytokine synthesis inhibitory factor (CSIF; interleukin 10) of human (76.4%) and mouse (68.5%), and to the Epstein-Barr virus (EBV) protein BCRF1 (70.6%). The existence of an interleukin 10 (IL-10) analogous gene within the genome of the EHV-2 was confirmed by screening the genome of nine EHV-2 strains using specific oligonucleotide primers corresponding to the 5 and 3 region of this particular gene by polymerase chain reaction. In all experiments an 870 bp DNA product was amplified. The specifity of the amplified DNA fragments obtained from individual EHV-2 strains was confirmed by DNA-DNA hybridization experiments. The DNA sequence analysis of the amplified DNA products of the EHV-2 strain LK was carried out. This analysis revealed the identity of the corresponding IL-10 gene (540 bp) of this strain to the IL-10 gene of EHV-2 strain T400/3. The presented data indicate that the EHV-2 genome harbors a viral interleukin 10-like gene. This is further evidence that the IL-10 gene can be present in the genomes of members of the Herpesviridae family.     

Modafinil in the treatment of idiopathic hypersomnia without long sleep time—a randomized,double‐blind,placebo‐controlled study

In 2010 the European Medicines Agency withdrew the indication of modafinil for the treatment of obstructive sleep apnea, shift work sleep disorder and for idiopathic hypersomnia (IH). In uncontrolled studies, modafinil has been reported to be efficacious in the treatment of sleep disorders. We therefore performed a randomized, placebo‐controlled study with the aim of proving the efficacy of modafinil treatment in these patients. Drug‐free IH patients without long sleep according to ICSD2 criteria, age >18 years and disease duration >2 years were included. After a washout phase, patients at baseline received placebo or 100 mg modafinil in the morning and at noon over 3 weeks, followed by 1 week without medication. At each visit the Epworth Sleepiness Scale (ESS) and Clinical Global Impression (CGI) rating scale were performed. At baseline and on days 8 and 21 four Maintenance of Wakefulness Tests (MWTs)/day or per day were performed. Patients kept a sleep–wake diary throughout the study. Between 2009 and 2011 three sleep centres recruited 33 participants. Compared to placebo, modafinil decreased sleepiness significantly and improved mean sleep latency in the MWT non‐significantly. The CGI improved significantly from baseline to the last visit on treatment. The most frequent adverse events were headaches and gastrointestinal disorders; skin and psychiatric reactions were not reported. The number of reported naps and duration of daytime sleepiness decreased significantly. Total sleep time of nocturnal sleep was slightly reduced. The sleep diaries showed increases in feeling refreshed in the morning; the diurnal diaries showed significant improvement of performance and of exhaustion. Modafinil is an effective and safe medication in the treatment of IH. Adverse events are mild to moderate.     

DSC perfusion-based collateral imaging and quantitative T2 mapping to assess regional recruitment of leptomeningeal collaterals and microstructural cortical tissue damage in unilateral steno-occlusive vasculopathy

Leptomeningeal collateral supply is considered pivotal in steno-occlusive vasculopathy to prevent chronic microstructural ischaemic tissue damage. The aim of this study was to assess the alleged protective role of leptomeningeal collaterals in patients with unilateral high-grade steno-occlusive vasculopathy using quantitative (q)T2 mapping and perfusion-weighted imaging (PWI)-based collateral abundance. High-resolution qT2 was used to estimate microstructural damage of the segmented normal-appearing cortex. Volumetric abundance of collaterals was assessed based on PWI source data. The ratio relative cerebral blood flow/relative cerebral blood volume (rCBF/rCBV) as a surrogate of relative cerebral perfusion pressure (rCPP) was used to investigate the intravascular hemodynamic competency of pial collateral vessels and the hemodynamic state of brain parenchyma. Within the dependent vascular territory with increased cortical qT2 values (P = 0.0001) compared to the contralateral side, parenchymal rCPP was decreased (P = 0.0001) and correlated negatively with increase of qT2 (P  < 0.05). Furthermore, volumetric abundance of adjacent leptomeningeal collaterals was significantly increased (P < 0.01) and negatively correlated with changes of parenchymal rCPP (P = 0.01). Microstructural cortical damage is closely related to restrictions of antegrade blood flow despite increased pial collateral vessel abundance. Therefore, increased leptomeningeal collateral supply cannot necessarily be regarded as a sign of effective compensation in patients with high-grade steno-occlusive vasculopathy.     

Value of combinations of pancreatic function tests to predict mild or moderate chronic pancreatitis

Pancreatic function tests share an insufficient accuracy concerning the detection of mild or moderate forms of chronic pancreatitis. It was evaluated here whether by combination of different assays the prediction or exclusion of chronic pancreatitis could be improved. METHODS: 62 patients with chronic abdominal pain and suspected chronic pancreatitis underwent an endoscopic retrograde pancreaticography. The duct alterations were classified according to the Cambridge criteria. In all individuals the pancreolauryl test in serum (PLT-S) and urine (PLT-U) was performed and elastase 1-immunoreactivity (E) as well as chymotrypsin (Chy) activity in stool were measured. Sensitivities, specificities, receiver-operator-curves as well as cut-off points at optimal accuracies were calculated for each single assay and all test combinations. Cut offs were optimized by a mathematical model to achieve highest accuracies. RESULTS: In 30 patients the pancreatic duct was normal and in 32 patients alterations of the duct system were found. These were classified as mild in 10 patients, as moderate in 8 patients and as severe in 14 patients. In those with mild and moderate disease all pancreatic function tests showed sensitivities/specificities of 60-65% and 65-70%, respectively. Only in severe chronic pancreatitis elastase was superior to the other tests. Combinations of function tests did not lead to improved accuracy. After mathematical optimization the accuracy (sensitivity 80%, specificity 80%) was best for the combination of PLT-S (cut off 4.7 micrograms/ml) and E (cut off 500 micrograms/g). Both parameters had to be below these newly defined cut offs to diagnose chronic pancreatitis. CONCLUSIONS: The accuracy of pancreatic function tests may be improved by use of altered cut offs and a combination of serum pancreolauryl test and elastase. These newly defined cut offs will have to be evaluated in a much larger study.