Diagnosis of human metapneumovirus and rhinovirus in patients with respiratory tract infections by an internally controlled multiplex real-time RNA PCR.

BACKGROUND: Adequate laboratory diagnosis of human rhinoviruses (hRV) and human metapneumoviruses (h MPV) requires molecular methods as viral culture lacks sensitivity. However, setting up individual PCRs for all respiratory viruses is not practical so preferentially multiplex PCRs are used. OBJECTIVES: To develop for routine diagnosis a rapid real-time PCR assay for detection of hRV and h MPV including an internal control in a single tube multiplex reaction using probes carrying different fluorophores to discriminate targets. STUDY DESIGN: The multiplex real-time RNA PCR was optimized to include the internal control virus and a total of 358 respiratory samples from 239 patients taken over a one-year period were analyzed by the multiplex assay. RESULTS: The multiplex assay with co-amplification of the internal control was as sensitive and specific as the individual assays. Application of this assay on clinical samples from 239 patients in a one-year period resulted in an incidence of hRV and h MPV of 41/239 (17.1%) and 6/239 (2.5%), respectively. Inhibition, defined as poor internal control amplification, was detected in 8 (2.2%) samples. Culture was performed on these samples and only four hRV were detected. CONCLUSIONS: This real-time PCR method enables sensitive diagnosis of these two respiratory pathogens with the potential to expand the assay as part of a full molecular respiratory viral screen.

A generic sequencing based typing approach for the identification of HLA-A diversity.

Sequencing Based Typing (SBT) is a generic approach for the identification of HLA-A polymorphism. This approach includes the high resolution typing of the HLA-A broad reacting groups, HLA-A subtypes and will identify new alleles directly. The SBT approach described here uses a locus specific amplification of DNA from exon 1 to exon 5. The resulting 2,022 bp PCR product serves as a template for the subsequent sequencing reactions. Amplification is followed by direct sequencing of exons 2, 3 and 4 in both orientations with fluorescently labeled primers to define all polymorphic positions leading to a high resolution typing result. In this study the sequence of exons 2 and 3 of a panel of 49 cell lines was determined. In addition, the exon 4 region of 35 cell lines was also sequenced to evaluate the exon 4 polymorphism. The HLA-A type of most of the cells could be identified by sequencing only exons 2 and 3. However, the sequence of exon 4 was required to discriminate A*0201 from A*0209 and A*0207 from A*0215N. In this panel, an identical new "HLA-A*0103" was identified in two Caucasian samples.

Parainfluenza virus 3 infection pre- and post-haematopoietic stem cell transplantation: re-infection or persistence?

BACKGROUND: A 5-year-old boy with acute lymphoblastic leukaemia (ALL) received a haematopoietic stem cell transplant (HSCT) from his father and was monitored for the presence of respiratory viruses. STUDY DESIGN: Nasal washes were taken during the transplant period and tested by culture and real-time PCR. RESULTS: Fifteen days prior to transplant parainfluenza virus 3 (PIV3) was isolated by culture and confirmed by immunofluoresence (IF) from a nasal wash specimen. Subsequent samples were negative by both IF and culture so the pre-transplant conditioning was started. One week after the HSCT the patient developed respiratory symptoms which progressively deteriorated. The IF and culture for PIV3 became positive again only after the symptoms deteriorated. However, retrospectively, a real-time PCR assay showed that the PIV3 was detectable throughout the conditioning phase and the amount of virus increased at the time of reappearance of respiratory symptoms post-HSCT. CONCLUSIONS: Management of PIV3 infection is important in HSCT. The use of this real-time PCR assay could improve diagnosis and management of PIV3 infection.

Genetic analysis of short children with apparent growth hormone insensitivity.

BACKGROUND/AIMS: In short children, a low IGF-I and normal GH secretion may be associated with various monogenic causes, but their prevalence is unknown. We aimed at testing GH1, GHR, STAT5B, IGF1, and IGFALS in children with GH insensitivity. SUBJECTS AND METHODS: Patients were divided into three groups: group 1 (height SDS <-2.5, IGF-I <-2 SDS, n = 9), group 2 (height SDS -2.5 to -1.9, IGF-I <-2 SDS, n = 6) and group 3 (height SDS <-1.9, IGF-I -2 to 0 SDS, n = 21). An IGF-I generation test was performed in 11 patients. Genomic DNA was used for direct sequencing, multiplex ligation-dependent probe amplification and whole-genome SNP array analysis. RESULTS: Three patients in group 1 had two novel heterozygous STAT5B mutations, in two combined with novel IGFALS variants. In groups 2 and 3 the association between genetic variants and short stature was uncertain. The IGF-I generation test was not predictive for the growth response to GH treatment. CONCLUSION: In severely short children with IGF-I deficiency, genetic assessment is advised. Heterozygous STAT5B mutations, with or without heterozygous IGFALS defects, may be associated with GH insensitivity. In children with less severe short stature or IGF-I deficiency, functional variants are rare.

HLA-A towards a high-resolution DNA typing.

Sequencing-based typing (SBT) and sequence-specific oligonucleotide probing (PCR-SSOP) are DNA-based typing approaches to identify HLA-A alleles. In this study PCR-SSOP SBT have been evaluated and considered to reach a high-resolution typing. Based upon serological typing, 32 genomic samples were typed by SBT and PCR-SSOP Three main clusters of resolution could be defined. The advantage of the PCR-SSOP approach is the possibility to type numerous samples in a short time. SBT minimizes the number of ambiguous heterozygous combinations and often allows direct detection and identification of new alleles.

Characterization of a novel HLA-A2 variant, A*02012, in the Southern Thai-Muslim population.

Southern Thai Muslims (STM)--from Nakon Si Thammarat, whose ancestors come mainly from Malaysia--constitute more than half of all Thai Muslims which, in total, represent approximately 10% of the country's population. The most common A2 subtypes in STM were A*0203 (n=15), A*0201 (n=8) and A*0207 (n=7). In this study, samples with unexpected amplification patterns were sequenced. Three individuals were indicative of a novel A2 allele, now known as A*02012.

A novel HLA-A24 (A*2420) allele identified in the Atayal tribe of Taiwan.

The Taiwan indigenous population groups are classified into different tribes according their linguistic classification and cultural anthropology. One of the tribes, the Atayal, showed a high frequency of A24 alleles by SSOP analysis. High-resolution sequencing based typing identified a A*2402 variant "A*2420" which was found in 6 unrelated individuals. High-resolution typing is required to identify HLA polymorphism in the Taiwanese minority groups.