Ijad Madisch

Publications

• 3.76

  Impact points

  Unique sequence features of the Adenovirus 31 complete genomic sequence are conserved in clinical isolates.

  Soeren Hofmayer, Ijad Madisch, Sebastian Darr, Fabienne Rehren, Albert Heim

  BMC genomics. 11/2009; 10(1):557.

  ABSTRACT: BACKGROUND: Human adenoviruses (HAdV) are causing a broad spectrum of diseases. One of the most severe forms of adenovirus infection is a disseminated disease resulting in significant morbidity and mortality. Several reports in recent years have identified HAdV-31 from species A (HAdV-A31)... [more] ABSTRACT: BACKGROUND: Human adenoviruses (HAdV) are causing a broad spectrum of diseases. One of the most severe forms of adenovirus infection is a disseminated disease resulting in significant morbidity and mortality. Several reports in recent years have identified HAdV-31 from species A (HAdV-A31) as a cause of disseminated disease in children following haematopoetic stem cell transplantation (hSCT) and liver transplantation. We sequenced and analyzed the complete genome of the HAdV-A31 prototype strain to uncover unique sequence motifs associated with its high virulence. Moreover, we sequenced coding regions known to be essential for tropism and virulence (early transcription units E1A, E3, E4, the fiber knob and the penton base) of HAdV-A31 clinical isolates from patients with disseminated disease. RESULTS: The genome size of HAdV-A31 is 33763 base pairs (bp) in length with a GC content of 46.36%. Nucleotide alignment to the closely related HAdV-A12 revealed an overall homology of 84.2%. The genome organization into early, intermediate and late regions is similar to HAdV-A12. Sequence analysis of the prototype strain showed unique sequence features such as an immunoglobulin-like domain in the species A specific gene product E3 CR1 beta and a potentially integrin binding RGD motif in the C-terminal region of the protein IX. These features were conserved in all analyzed clinical isolates. Overall, amino acid sequences of clinical isolates were highly conserved compared to the prototype (99.2 to 100%), but a synonymous/non synonymous ratio (S/N) of 2.36 in E3 CR1-beta suggested positive selection. CONCLUSIONS: Unique sequence features of HAdV-A31 may enhance its ability to escape the host's immune surveillance and may facilitate a promiscuous tropism for various tissues. Moderate evolution of clinical isolates did not indicate the emergence of new HAdV-A31 subtypes in the recent years.

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• 3.26

  Impact points

  Phylogeny and primary structure analysis of fiber shafts of all human adenovirus types for rational design of adenoviral gene therapy vectors.

  Sebastian Darr, Ijad Madisch, Sören Hofmayer, Fabienne Rehren, Albert Heim

  The Journal of general virology. 09/2009;

  The fiber shaft of human adenoviruses (HAdV) is essential for bringing the penton base in proximity to the secondary cellular receptor. Fiber shaft sequences of all 53 HAdV types were studied. Phylogeny of the fiber shaft revealed clustering corresponding to the HAdV species concept. An intraspecies... [more] The fiber shaft of human adenoviruses (HAdV) is essential for bringing the penton base in proximity to the secondary cellular receptor. Fiber shaft sequences of all 53 HAdV types were studied. Phylogeny of the fiber shaft revealed clustering corresponding to the HAdV species concept. An intraspecies recombination hot spot was found at the shaft/knob boundary, a highly conserved sequence stretch. For example, HAdV-D20 clustered with -D23 in the fiber shaft but with HAdV-D47 in the fiber knob. Although all shafts exhibited the typical pseudorepeats, sequence divergence was found to be as high as 92% interspecies and 54% intraspecies. In contrast to a previous study, a flexibility motif (KXGGLXFD/N) was found in eight HAdV-D types whereas the putative heparan sulfate binding site (KKTK) was only found in species HAdV-C. Our results suggest that pseudotyping of gene therapy vectors at the shaft/knob-boundary is feasible but flexibility data of shafts should be considered.

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• Phylogeny and primary structure analysis of fiber shafts of all human adenovirus types for rational design of adenoviral gene therapy vectors.

  Darr S, Madisch I, Hofmayer S, Rehren F, Heim A.

  J Gen Virol. 08/2009;
• 4.64

  Impact points

  Towards regenerating a human thumb in situ.

  Christian Weinand, Rajiv Gupta, Eli Weinberg, Ijad Madisch, Craig Neville, Jesse B Jupiter, Joseph Vacanti

  Tissue engineering. Part A. 03/2009;

  Regenerative technology promises to alleviate the problem of limited donor supply for bone or organ transplants. Most expensive and time consuming is cell expansion in laboratories. We propose a method of magnetically enriched osteoprogenitor stem cells, dispersed in self-assembling hydrogels and ap... [more] Regenerative technology promises to alleviate the problem of limited donor supply for bone or organ transplants. Most expensive and time consuming is cell expansion in laboratories. We propose a method of magnetically enriched osteoprogenitor stem cells, dispersed in self-assembling hydrogels and applied onto new ultra-high resolution, jet-based 3D printing of living human bone in a single-step for in-situ bone regeneration. Human mesenchymal stem-cells (hBMSCs) were enriched with CD 117+ osteoprogenitor cells, dispersed in different collagen I, RAD 16I hydrogels mixes and applied onto 3-dimensional printed (3DP) beta-TCP/PLGA scaffolds, printed from ultra-high-resolution volumetric CT (VCT) images of a human thumb. Constructs were directly implanted subcutaneously into nude mice for 6 weeks. In-vivo radiographic VCT scanning and histological evaluations were performed at 1, 2, 4 and 6 weeks, expression of bone-specific genes and biomechanical compression-testing at 6 weeks endpoint. Time dependant accumulation of bone-like extracellular matrix was most evident in CD 117+ hBMSCs using collagen I/RAD 16I hydrogel mix. This was shown histologically by Toluidine blue, von Kossa and alkaline-phosphatase staining, paralleled by increased radiological densities within implants approximating that of human bone, and confirmed by high expression of bone-specific osteonectin and biomechanical stiffness at 6 weeks. Human origin of newly formed tissue was established by expression of human GAPDH using RT-PCR. Statistical analysis confirmed high correlations between biomechanical stiffness, radiological densities and bone-markers. Bone tissue can be successfully regenerated in-situ using a single-step procedure with constructs comprised of RAD 16I /collagen I hydrogel, CD 117+ enriched hBMSCs and porous beta-TCP/PLGA scaffolds.
• 4.41

  Impact points

  Evidence of molecular evolution driven by recombination events influencing tropism in a novel human adenovirus that causes epidemic keratoconjunctivitis.

  Michael P Walsh, Ashish Chintakuntlawar, Christopher M Robinson, Ijad Madisch, Balázs Harrach, Nolan R Hudson, David Schnurr, Albert Heim, James Chodosh, Donald Seto, Morris S Jones

  PloS one. 02/2009; 4(6):e5635.

  In 2005, a human adenovirus strain (formerly known as HAdV-D22/H8 but renamed here HAdV-D53) was isolated from an outbreak of epidemic keratoconjunctititis (EKC), a disease that is usually caused by HAdV-D8, -D19, or -D37, not HAdV-D22. To date, a complete change of tropism compared to the prototype... [more] In 2005, a human adenovirus strain (formerly known as HAdV-D22/H8 but renamed here HAdV-D53) was isolated from an outbreak of epidemic keratoconjunctititis (EKC), a disease that is usually caused by HAdV-D8, -D19, or -D37, not HAdV-D22. To date, a complete change of tropism compared to the prototype has never been observed, although apparent recombinant strains of other viruses from species Human adenovirus D (HAdV-D) have been described. The complete genome of HAdV-D53 was sequenced to elucidate recombination events that lead to the emergence of a viable and highly virulent virus with a modified tropism. Bioinformatic and phylogenetic analyses of this genome demonstrate that this adenovirus is a recombinant of HAdV-D8 (including the fiber gene encoding the primary cellular receptor binding site), HAdV-D22, (the epsilon determinant of the hexon gene), HAdV-D37 (including the penton base gene encoding the secondary cellular receptor binding site), and at least one unknown or unsequenced HAdV-D strain. Bootscanning analysis of the complete genomic sequence of this novel adenovirus, which we have re-named HAdV-D53, indicated at least five recombination events between the aforementioned adenoviruses. Intrahexon recombination sites perfectly framed the epsilon neutralization determinant that was almost identical to the HAdV-D22 prototype. Additional bootscan analysis of all HAdV-D hexon genes revealed recombinations in identical locations in several other adenoviruses. In addition, HAdV-D53 but not HAdV-D22 induced corneal inflammation in a mouse model. Serological analysis confirmed previous results and demonstrated that HAdV-D53 has a neutralization profile representative of the epsilon determinant of its hexon (HAdV-D22) and the fiber (HAdV-D8) proteins. Our recombinant hexon sequence is almost identical to the hexon sequences of the HAdV-D strain causing EKC outbreaks in Japan, suggesting that HAdV-D53 is pandemic as an emerging EKC agent. This documents the first genomic, bioinformatic, and biological descriptions of the molecular evolution events engendering an emerging pathogenic adenovirus.

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• 6.34

  Impact points

  Distal radius in adolescent girls with anorexia nervosa: trabecular structure analysis with high-resolution flat-panel volume CT.

  Miriam A Bredella, Madhusmita Misra, Karen K Miller, Ijad Madisch, Ammar Sarwar, Arnold Cheung, Anne Klibanski, Rajiv Gupta

  Radiology. 12/2008; 249(3):938-46.

  PURPOSE: To examine trabecular microarchitecture with high-resolution flat-panel volume computed tomography (CT) and bone mineral density (BMD) with dual-energy x-ray absorptiometry (DXA) in adolescent girls with anorexia nervosa (AN) and to compare these results with those in normal-weight control ... [more] PURPOSE: To examine trabecular microarchitecture with high-resolution flat-panel volume computed tomography (CT) and bone mineral density (BMD) with dual-energy x-ray absorptiometry (DXA) in adolescent girls with anorexia nervosa (AN) and to compare these results with those in normal-weight control subjects. MATERIALS AND METHODS: The study was approved by the institutional review board and complied with HIPAA guidelines. Informed consent was obtained. Twenty adolescent girls, 10 with mild AN (mean age, 15.9 years; range, 13-18 years) and 10 age- and sex-matched normal-weight control subjects (mean age, 15.9 years; range, 12-18 years) underwent flat-panel volume CT of distal radius to determine apparent trabecular bone volume fraction (BV/TV), apparent trabecular number (TbN), apparent trabecular thickness (TbTh), and apparent trabecular separation (TbSp). All subjects underwent DXA of spine, hip, and whole body to determine BMD and body composition. The means and standard deviations (SDs) of structure parameters were calculated for AN and control groups. Groups were compared (Student t test). Linear regression analysis was performed. RESULTS: AN subjects compared with control subjects, respectively, showed significantly lower mean values for BV/TV (0.37% +/- 0.05 [SD] vs 0.46% +/- 0.03, P = .0002) and TbTh (0.31 mm +/- 0.03 vs 0.39 mm +/- 0.03, P < .0001) and higher mean values for TbSp (0.54 mm +/- 0.13 vs 0.44 mm +/- 0.04, P = .02). TbN was lower in AN subjects than in control subjects, but the difference was not significant (1.17 mm(-3) +/- 0.15 vs 1.22 mm(-3) +/- 0.07, P = .43). There was no significant difference in BMD between AN and control subjects. BMD parameters showed positive correlation with BV/TV and TbTh in the control group (r = 0.55-0.84, P = .05-.01) but not in AN patients. CONCLUSION: Flat-panel volume CT is effective in evaluation of trabecular structure in adolescent girls with AN and demonstrates that bone structure is abnormal in these patients compared with that in normal-weight control subjects despite normal BMD. SUPPLEMENTAL MATERIAL: http://radiology.rsnajnls.org/cgi/content/full/249/3/938/DC1.
• 8.20

  Impact points

  Antiviral activity of cidofovir and ribavirin against the new human adenovirus subtype 14a that is associated with severe pneumonia.

  Sebastian Darr, Ijad Madisch, Albert Heim

  Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 10/2008; 47(5):731-2.

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• Unique sequence features of the Adenovirus 31 prototype complete genomic sequence are conserved in clinical isolates from cases of disseminated infection

  S. Hofmayer, I. Madisch, A. Heim

  60. DGHM, Dresden; 09/2008
• 3.59

  Impact points

  Intrinsic respiratory gating in small-animal CT.

  Soenke H Bartling, Julien Dinkel, Wolfram Stiller, Michael Grasruck, Ijad Madisch, Hans-Ulrich Kauczor, Wolfhard Semmler, Rajiv Gupta, Fabian Kiessling

  European radiology. 08/2008; 18(7):1375-84.

  Gating in small-animal CT imaging can compensate artefacts caused by physiological motion during scanning. However, all published gating approaches for small animals rely on additional hardware to derive the gating signals. In contrast, in this study a novel method of intrinsic respiratory gating of... [more] Gating in small-animal CT imaging can compensate artefacts caused by physiological motion during scanning. However, all published gating approaches for small animals rely on additional hardware to derive the gating signals. In contrast, in this study a novel method of intrinsic respiratory gating of rodents was developed and tested for mice (n=5), rats (n=5) and rabbits (n=2) in a flat-panel cone-beam CT system. In a consensus read image quality was compared with that of non-gated and retrospective extrinsically gated scans performed using a pneumatic cushion. In comparison to non-gated images, image quality improved significantly using intrinsic and extrinsic gating. Delineation of diaphragm and lung structure improved in all animals. Image quality of intrinsically gated CT was judged to be equivalent to extrinsically gated ones. Additionally 4D datasets were calculated using both gating methods. Values for expiratory, inspiratory and tidal lung volumes determined with the two gating methods were comparable and correlated well with values known from the literature. We could show that intrinsic respiratory gating in rodents makes additional gating hardware and preparatory efforts superfluous. This method improves image quality and allows derivation of functional data. Therefore it bears the potential to find wide applications in small-animal CT imaging.

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• Unique sequence features of the Adenovirus 31 prototype complete genomic sequence are conserved in clinical isolates from cases of disseminated infection

  Sören Hofmayer, Ijad Madisch, Fabienne Rehren, Albert Heim

  01/2008
• 5.15

  Impact points

  Phylogenetic analysis and structural predictions of human adenovirus penton proteins as a basis for tissue-specific adenovirus vector design.

  Ijad Madisch, Soeren Hofmayer, Christian Moritz, Alexander Grintzalis, Jens Hainmueller, Patricia Pring-Akerblom, Albert Heim

  Journal of virology. 09/2007; 81(15):8270-81.

  The penton base is a major capsid protein of human adenoviruses (HAdV) which forms the vertices of the capsid and interacts with hexon and fiber protein. Two hypervariable loops of the penton are exposed on the capsid surface. Sequences of these and 300 adjacent amino acid residues of all 51 HAdV an... [more] The penton base is a major capsid protein of human adenoviruses (HAdV) which forms the vertices of the capsid and interacts with hexon and fiber protein. Two hypervariable loops of the penton are exposed on the capsid surface. Sequences of these and 300 adjacent amino acid residues of all 51 HAdV and closely related simian adenoviruses were studied. Adjacent sequences and predicted overall secondary structure were conserved. Phylogenetic analysis revealed clustering corresponding to the HAdV species and recombination events in the origin of HAdV prototypes. All HAdV except serotypes 40 and 41 of species F exhibited an integrin binding RGD motif in the second loop. The lengths of the loops (HVR1 and RGD loops) varied significantly between HAdV species with the longest RGD loop observed in species C and the longest HVR1 in species B. Long loops may permit the insertion of motifs that modify tissue tropism. Genetic analysis of HAdV prime strain p17'H30, a neutralization variant of HAdV-D17, indicated the significance of nonhexon neutralization epitopes for HAdV immune escape. Fourteen highly conserved motifs of the penton base were analyzed by site-directed mutagenesis of HAdV-D8 and tested for sustained induction of early cytopathic effects. Thus, three new motifs essential for penton base function were identified additionally to the RGD site, which interacts with a secondary cellular receptor responsible for internalization. Therefore, our penton primary structure data and secondary structure modeling in combination with the recently published fiber knob sequences may permit the rational design of tissue-specific adenoviral vectors.

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• 1.29

  Impact points

  Human shaped thumb bone tissue engineered by hydrogel-beta-tricalciumphosphate/poly-epsilon-caprolactone scaffolds and magnetically sorted stem cells.

  Christian Weinand, Rajiv Gupta, Eli Weinberg, Ijad Madisch, Jesse B Jupiter, Joseph P Vacanti

  Annals of plastic surgery. 08/2007; 59(1):46-52; discussion 52.

  Traumatic amputation of a thumb with bone loss leaves a patient in severe disability. Reconstructive procedures are restricted by limited shape and have the disadvantage of severe donor-site morbidity. To overcome these limitations, we used a tissue engineering approach to create a distal thumb bone... [more] Traumatic amputation of a thumb with bone loss leaves a patient in severe disability. Reconstructive procedures are restricted by limited shape and have the disadvantage of severe donor-site morbidity. To overcome these limitations, we used a tissue engineering approach to create a distal thumb bone phalanx, combining magnetically sorted 133+ human mesenchymal stem cells (hMSCs) suspended in successful tested hydrogels for bone formation and porous 3-dimensionally printed scaffolds (3DP) in the shape of a distal thumb bone phalanx. Collagen I and fibrin glue hydrogels with suspended hMSCs were first histologically evaluated in vitro for bone formation after 6 weeks.Then 3DP scaffolds, made from a mix of osteoinductive and -conductive beta-tricalciumphosphate (beta-TCP) and poly-epsilon-caprolactone (PCL), with hydrogels and suspended hMSCs, were implanted into nude mice subcutaneously for 15 weeks. Histologic evaluation, high-resolution volumetric CT (VCT) scanning, and biomechanical testing confirmed formation of bonelike tissue. Both hydrogels with CD 133+ hMSCs on 3DP scaffolds supported bone formation. Collagen I resulted in radiologically better bone formation. Bone tissue can be successfully tissue engineered with CD 133+ hMSCs, collagen I hydrogels, and porous 3DP beta-TCP/PCL scaffolds.

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• 4.16

  Impact points

  Extent of circulation of incorrectly labeled adenovirus 50 and 51 prototype preparations.

  Ijad Madisch, Albert Heim

  Journal of clinical microbiology. 07/2007; 45(6):2092; discussion 2092.
• 2.29

  Impact points

  Comparison of hydrogels in the in vivo formation of tissue-engineered bone using mesenchymal stem cells and beta-tricalcium phosphate.

  Christian Weinand, Rajiv Gupta, Albert Y Huang, Eli Weinberg, Ijad Madisch, Rameez A Qudsi, Craig M Neville, Irina Pomerantseva, Joseph P Vacanti

  Tissue engineering. 05/2007; 13(4):757-65.

  Availability of grafts and morbidity at the donor site limit autologous transplantation in patients requiring bone reconstruction. A tissue-engineering approach can overcome these limitations by producing bone-like tissue of custom shape and size from isolated cells. Several hydrogels facilitate ost... [more] Availability of grafts and morbidity at the donor site limit autologous transplantation in patients requiring bone reconstruction. A tissue-engineering approach can overcome these limitations by producing bone-like tissue of custom shape and size from isolated cells. Several hydrogels facilitate osteogenesis on porous scaffolds; however, the relative suitability of various hydrogels has not been rigorously assessed. Fibrin glue, alginate, and collagen I hydrogels were mixed with swine bone marrow-derived differentiated mesenchymal stem cells (MSCs), applied to 3-dimensionally printed porous beta-tricalcium phosphate (beta-TCP) scaffolds and implanted subcutaneously in nude mice. Although noninvasive assessment of osteogenesis in 3 dimensions is desirable for monitoring new bone formation in vivo, correlations with traditional histological and mechanical testing need to be established. High-resolution volumetric computed tomography (VCT) scanning, histological examination, biomechanical compression testing, and osteonectin (ON) expression were performed on excised scaffolds after 1, 2, 4, and 6 weeks of subcutaneous implantation in mice. Statistical correlation analyses were performed between radiological density, stiffness, and ON expression. Use of collagen I as a hydrogel carrier produced superior bone formation at 6 weeks, as demonstrated using VCT scanning with densities similar to native bone and the highest compression values. Continued contribution of the seeded MSCs was demonstrated using swine-specific messenger ribonucleic acid probes. Radiological density values correlated closely with the results of histological and biomechanical testing and ON expression. High-resolution VCT is a promising method for monitoring osteogenesis.

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• Molecular Phylogeny and bioinformatical analysis as basis for a typing system of human Adenoviruses and for the design of tissue specific adenoviral Vectors

  Ijad Madisch

  01/2007

  Degree: Ph.D.

  Supervisor: Albert R. Heim

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• Phylogenetic analysis and structural predictions of human adenovirus penton proteins: Basis for tissue specific adenoviral vector design.

  Madisch, Hofmayer, Moritz, Grintzalis, Akerblom, Heim A.

  Adenovirus: Basic research and application meeting, Ulm; 01/2007

• Phylogenetic analysis and structural predictions of human adenovirus penton proteins: Basis for tissue specific adenoviral vector design.

  Madisch, Hofmayer, Moritz, Grintzalis, Akerblom, Heim A.

  Adenovirus: Basic research and application meeting, Ulm; 01/2007
• 3.61

  Impact points

  A rapid quantitative PCR-based assay for testing antiviral agents against human adenoviruses demonstrates type specific differences in ribavirin activity.

  Rüdiger Stock, Gabi Harste, Ijad Madisch, Albert Heim

  Antiviral research. 11/2006; 72(1):34-41.

  Human adenovirus (HAdV) infections are increasingly frequent and potentially fatal as a disseminated disease in highly immunocompromised patients. Determining the in vitro sensitivity of HAdV to antiviral agents is not an easy task because HAdV CPE reduction assays are difficult to interpret and may... [more] Human adenovirus (HAdV) infections are increasingly frequent and potentially fatal as a disseminated disease in highly immunocompromised patients. Determining the in vitro sensitivity of HAdV to antiviral agents is not an easy task because HAdV CPE reduction assays are difficult to interpret and may take more than 1 week. We developed a phenotypic assay for testing the antiviral activity during the first round of replication using HAdV DNA concentration as an objective readout within 30 h. After evaluating the assay with cidofovir, we focused on determining the antiviral of ribavirin against different HAdV serotypes because clinical response of HAdV infections towards ribavirin treatment varied considerably. Several HAdV prototypes (1, 2, 5, 11, 31, 34, 48) associated with disseminated infections and clinical isolates were tested. Predominating HAdV of species C were more sensitive to ribavirin (HAdV-2 and -5: EC(50)<10 microM, EC(99) 111 and 104 microM, respectively) than HAdV of other species, for example HAdV-31 (EC(50) 56 microM, EC(99)>500 microM). Differential ribavirin sensitivity of HAdV types may contribute to the variable outcome of ribavirin therapy. Rapid screening of antiviral agents with the rapid qPCR-based assay against a multitude of HAdV serotypes may also facilitate development of future antiviral agents.

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• 8.20

  Impact points

  An outbreak of epidemic keratoconjunctivitis caused by a new intermediate adenovirus 22/H8 identified by molecular typing.

  Ilka Engelmann, Ijad Madisch, Heidi Pommer, Albert Heim

  Clinical infectious diseases : an official publication of the Infectious Diseases Society of America. 11/2006; 43(7):e64-6.

  In a 4-week period, 12 patients contracted adenoviral keratoconjunctivitis. Eight of these patients had visited the same ophthalmologist's practice before onset of symptoms. Adenovirus was detected in swab specimens obtained from 9 patients. Sequence-based typing of 2 isolates revealed type 22/H... [more] In a 4-week period, 12 patients contracted adenoviral keratoconjunctivitis. Eight of these patients had visited the same ophthalmologist's practice before onset of symptoms. Adenovirus was detected in swab specimens obtained from 9 patients. Sequence-based typing of 2 isolates revealed type 22/H8. This is, to our knowledge, the first report of a keratoconjunctivitis outbreak caused by an intermediate adenovirus type 22/H8.

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• 2.47

  Impact points

  Molecular identification of adenovirus sequences: a rapid scheme for early typing of human adenoviruses in diagnostic samples of immunocompetent and immunodeficient patients.

  Ijad Madisch, Roman Wölfel, Gabi Harste, Heidi Pommer, Albert Heim

  Journal of medical virology. 10/2006; 78(9):1210-7.

  Precise typing of human adenoviruses (HAdV) is fundamental for epidemiology and the detection of infection chains. As only few of the 51 adenovirus types are associated with life- threatening disseminated diseases in immunodeficient patients, detection of one of these types may have prognostic value... [more] Precise typing of human adenoviruses (HAdV) is fundamental for epidemiology and the detection of infection chains. As only few of the 51 adenovirus types are associated with life- threatening disseminated diseases in immunodeficient patients, detection of one of these types may have prognostic value and lead to immediate therapeutic intervention. A recently published molecular typing scheme consisting of two steps (sequencing of a generic PCR product closely adjacent to loop 1 of the main neutralization determinant epsilon, and for species HAdV-B, -C, and -D the sequencing of loop 2 [Madisch et al., 2005]) was applied to 119 clinical samples. HAdV DNA was typed unequivocally even in cases of culture negative samples, for example in immunodeficient patients before HAdV causes high virus loads and disseminated disease. Direct typing results demonstrated the predominance of HAdV-1, -2, -5, and -31 in immunodeficient patients suggesting the significance of the persistence of these viruses for the pathogenesis of disseminated disease. In contrast, HAdV-3 predominated in immunocompetent patients and cocirculation of four subtypes was demonstrated. Typing of samples from a conjunctivitis outbreak in multiple military barracks demonstrated various HAdV types (2, 4, 8, 19) and not the suspected unique adenovirus etiology. This suggests that our molecular typing scheme will be also useful for epidemiological investigations. In conclusion, our two-step molecular typing system will permit the precise and rapid typing of clinical HAdV isolates and even of HAdV DNA in clinical samples without the need of time-consuming virus isolation prior to typing.

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