Outbreak of a beta-lactam resistant non-typeable Haemophilus influenzae sequence type 14 associated with severe clinical outcomes.

BACKGROUND: During October 2011 several residents and staff members at a long-term care facility (LTCF) for elderly fell ill with respiratory symptoms. Several of the residents required hospitalization and one died. Non-typeable Haemophilus influenzae (NTHi) was identified as the causative pathogen. METHODS: A descriptive analysis of the outbreak and countermeasures was performed. For each identified bacterial isolate implied in the outbreak, standard laboratory resistance testing was performed, as well as molecular typing and phylogenetic analysis. RESULTS: The identified H. influenzae was beta-lactamase negative but had strikingly high MIC-values of ampicillin, cefuroxime and cefotaxime. All isolates displayed the same mutation in the ftsI gene encoding penicillin-binding protein (PBP) 3, and all but one were identified as sequence type 14 by Multilocus Sequence Typing (MLST). In total 15 individuals in connection to the LTCF; 8 residents, 6 staff members and one partner to a staff member were colonized with the strain. CONCLUSION: This report illustrates the existence of non-typeable H. influenzae with high virulence, and furthermore emphasizes the importance of continuous surveillance of possible outbreaks in health care facilities and prompt measures when outbreaks occur.

Detection of Puumala and Rift Valley Fever virus by quantitative RT-PCR and virus viability tests in samples of blood dried and stored on filter paper.

Haemorrhagic fever viruses cause emerging infections worldwide, and blood or serum is the main sample used for diagnosis. However, storage and transportation of such samples from remote areas to regional laboratories may be complicated and expensive. In this study, a novel approach was evaluated for the detection of Puumala hantavirus (PUUV) RNA and Rift Valley fever virus (RVFV) RNA. Whole-blood samples spiked with viable virus particles were tested in parallel with clinical samples from patients with acute haemorrhagic fever with renal syndrome (nephropathia epidemica). Individual blood samples were spotted on filter paper, dried, and used for RNA extraction at later time points. PUUV RNA was detected by RT-PCR after storage at room temperature for up to six weeks. In contrast, only low copy numbers of RVFV RNA were detected after 1-2 days even though viable RVFV was eluted from the dried filter papers after the same time. The use of filter paper to collect and store blood samples for PUUV RNA detection is therefore a simple and reliable procedure. This approach might facilitate sampling and analysis of other RNA viruses from human or animal sources and could be used for field studies in remote areas or in developing countries.

The anti-papillomavirus activity of human and bovine lactoferricin.

Human papillomavirus (HPV) cause common warts, laryngeal papilloma and genital condylomata and is necessary for the development of cervical cancer. We have previously found that lactoferrin has antiviral activity against HPV-16 and others have demonstrated that lactoferricin, an N-terminal fragment of lactoferrin, has inhibitory activities against several viruses. Two cell lines and two virus types, HPV-5 and HPV-16, were used to study if lactoferrin and lactoferricin could inhibit HPV pseudovirus (PsV) infection. We demonstrated that bovine lactoferrin (bLf) and human lactoferrin (hLf) were both potent inhibitors of HPV-5 and -16 PsV infections. Among the four lactoferricin derivatives we analyzed, a 15 amino acid peptide from bovine lactoferricin (bLfcin) 17-31 was the most potent inhibitor of both HPV-5 and HPV-16 PsV infection. Among the other derivatives, the human lactoferricin (hLfcin) 1-49 showed some antiviral activity against HPV PsV infection while bLfcin 17-42 inhibited only HPV-5 PsV infection in one of the cell lines. When we studied initial attachment of HPV-16, only bLfcin 17-42 and hLfcin 1-49 had an antiviral effect. This is the first time that lactoferricin was demonstrated to have an inhibitory effect on HPV infection and the antiviral activity differed depending on size, charge and structures of the lactoferricin.

Lactoferrin inhibits human papillomavirus binding and uptake in vitro.

Lactoferrin (LF), a member of the transferrin family, is a bi-globular protein secreted in milk, saliva, tears, seminal fluid, endocervix and vaginal secretions. LF is an important player in the defence against pathogenic microorganisms and has also been shown to have activity against several viruses including herpesvirus, adenovirus, rotavirus and poliovirus. The antiviral activity of LF is directed against the early steps of viral infection and the LF antiviral effect against herpesvirus is mediated through LF binding to the herpesvirus receptor heparan sulfate. Human papillomavirus (HPV) causes genital warts and is a prerequisite for cervical cancer. HPV can also use heparan sulfate on the cell surface as a receptor. We studied the inhibition by LF on HPV entry by incubating HaCaT cells and HPV-16 virus-like particles (VLPs) with either human (HLF) or bovine lactoferrin (BLF). LF inhibited internalization of HPV-16 particles using CFDA-SE-labelled VLPs that only fluoresce after internalisation. By using a western blot assay we also found dose-dependent LF inhibition of HPV-16 VLP binding to the HaCaT cell surface. BLF was a more potent inhibitor of HPV entry than human LF. It was also clear that LF acted early in the HPV uptake process.

Carboxy-fluorescein diacetate, succinimidyl ester labeled papillomavirus virus-like particles fluoresce after internalization and interact with heparan sulfate for binding and entry.

Human papillomaviruses (HPVs) infect epithelial cells and are associated with genital carcinoma. Most epithelial cell lines express cell-surface glycosaminoglycans (GAGs) usually found attached to the protein core of proteoglycans. Our aim was to study how GAGs influenced HPV entry. Using a human keratinocyte cell line (HaCaT), preincubation of HPV virus-like particles (VLPs) with GAGs showed a dose-dependent inhibition of binding. The IC(50) (50% inhibition) was only 0.5 microg/ml for heparin, 1 microg/ml for dextran sulfate, and 5-10 microg/ml for heparan sulfate from mucosal origin. Mutated chinese hamster ovary (CHO) cell lines lacking heparan sulfate or all GAGs were unable to bind HPV VLPs. Here we also report a method to study internalization by using VLPs labeled with carboxy-fluorescein diacetate, succinimidyl ester, a fluorochrome that is only activated after cell entry. Pretreatment of labeled HPV VLPs with heparin inhibited uptake, suggesting a primary interaction between HPV and cell-surface heparan sulfate.