The role of genotype specific anti-G antibodies in protection against severe human respiratory syncytial virus infection

Human respiratory syncytial virus is a leading cause of lower respiratory tract infection in infants. Current prophylaxis with Palivizumab, a humanised anti-fusion glycoprotein monoclonal antibody, has only achieved moderate success. In animal models, immunization with G glycoprotein has led to largely genotype specific immunity. This suggests the hypothesis that anti-G antibodies to the infecting virus genotype, either acquired transplacentally or induced by immunisation, may contribute to protection of the infant in the early months of life. The aim of this study has been to test this hypothesis by measuring levels of maternally acquired antibody to the G glycoprotein of the infecting virus genotype in an index group of infants with severe HRSV infection and, to the same genotype, in a comparison group of infants with no history of HRSV infection. As only 2% of infants suffer severe disease on primary HRSV infection, the comparison group are assumed to be destined to be resistant to severe infection. A deficiency of anti-G antibodies in the index group will thus indicate a protective role for these antibodies. A phylogenetic study was carried out in Newcastle upon Tyne from autumn 2007 to spring 2010 revealing three circulating genotypes of HRSV, namely GA2, GA5 and BA4 although GA5 was not detected in the third epidemic. The G genes of GA2 and BA4 and the F gene of GA2 were cloned and expressed individually in modified vaccinia Ankara virus. The recombinant proteins were used in the development of a concanavalinA capture ELISA sufficiently sensitive to detect maternal antibodies in the acute sera of infants under 6 months of age. An attempt was made to refine the assay in order to separately detect antibodies to the conserved and variable epitopes of the G glycoprotein. However, mapping of conserved and variable epitopes revealed overlap epitopes precluding the development of a differentiation in a simple ELISA assay. An index group of infants with severe HRSV infection and a comparison group of infants of similar age with no history of HRSV infection were recruited with consent by their legal carer during the epidemic of 2009/2010. The infecting virus lineage of each infant in the index group, either GA2 or BA4, was identified by reverse transciptasepolymerase chain reaction of virus recovered from nasal secretions. Sera were collected from both groups, at the acute stage of infection for the index group, and screened for ii HRSV specific IgA by ELISA. Only those without detectable IgA were included in the study. IgG maternal antibodies to the recombinant G glycoprotein of the infecting virus genotype and the F glycoprotein of the GA2 genotype were measured in the sera of index and comparison group infants whilst maternal antibody levels to both F and G glycoproteins in the sera of both index and control sera decayed at similar rates with age, the index group possessed significantly more anti-G and anti-F antibody at all ages suggesting that infants hospitalized with severe HRSV infection were not deficient in antibodies to either glycoprotein contrary to the hypothesis under test. However, mean maternal antibody levels at birth have been shown to fall across the winter epidemic and the above analysis may be susceptible to bias introduced by uneven sampling of infants across the epidemic. To avoid this potential error, anti-F/anti-G antibody ratios, which give a measure of anti-G immunity independent of age and time of collection, were also compared in the sera of index and comparison group infants. Mean ratios were not significantly different between the two groups also rejecting the hypothesis that severely affected infants have a deficiency in maternal anti-G antibodies. These studies, therefore, fail to establish a role for anti-G glycoprotein antibodies in the protection of infants against severe lower respiratory tract disease.EThOS - Electronic Theses Online ServiceMinistry of Higher Education of Malaysia (MOHE)GBUnited Kingdo

The Extended Stability of Cervical Swabs in careHPV™ Collection Medium

Introduction: The careHPV™ Test is a US FDA approved, CE mark, and WHO prequalified in vitro diagnostic test designed to screen for 14 high-risk human papillomavirus (HRHPV) genotypes. The careHPV™ Test is one of the commercial HPV test validated to be used in low resource settings, boasting the economy of processing a maximum of 90 samples per batch and a near point-of-care turnaround time of 3 hours. According to the manufacturer, cervical swabs stored in careHPV™ Collection Medium are stable for 30 days when stored between 2-8°C. However, we often had difficulty consolidating enough samples for a full batch-test within 30 days, especially when screening women living in the low-density villages in rural Sarawak, Malaysian Borneo. This study aimed to evaluate the stability and repeatability of cervical swabs preserved in careHPV™ Collection Medium stored at 4°C exceeding the recommended 30 days using the careHPV™ Test. Methods: Two groups of confirmed HRHPV-positive and HRHPV-negative cervical swab samples in careHPV™ Collection Medium consisting of 4 samples each were maintained at 4°C and tested using the careHPV™ Test at Day -38, -123, -131, -223, and -395. Results: All cervical swabs in the careHPV™ Collection Medium stored at 4°C remained stable for testing and demonstrated 100% repeatability for at least 395 days from the day of collection. Conclusion: The careHPV™ Test can be successfully performed on cervical swabs preserved in careHPV™ Collection Medium, which were stored at 4°C for at least 395 days

Carriage of upper respiratory tract pathogens in rural communities of Sarawak, Malaysian Borneo

Introduction: Pneumonia is a leading cause of death in Malaysia. Whilst many studies have reported the aetiology of pneumonia in Western countries, the epidemiology of pneumonia in Malaysia remains poorly understood. As carriage is a prerequisite for disease, we sought to improve our understanding of the carriage and antimicrobial resistance (AMR) of respiratory tract pathogens in Malaysia. The rural communities of Sarawak are an understudied part of the Malaysian population and were the focus of this study, allowing us to gain a better understanding of bacterial epidemiology in this population. Methods: A population-based survey of bacterial carriage was undertaken in participants of all ages from rural communities in Sarawak, Malaysia. Nasopharyngeal, nasal, mouth and oropharyngeal swabs were taken. Bacteria were isolated from each swab and identified by culture-based methods and antimicrobial susceptibility testing conducted by disk diffusion or E test. Results: 140 participants were recruited from five rural communities. Klebsiella pneumoniae was most commonly isolated from participants (30.0%), followed by Staphylococcus aureus (20.7%), Streptococcus pneumoniae (10.7%), Haemophilus influenzae (9.3%), Moraxella catarrhalis (6.4%), Pseudomonas aeruginosa (6.4%) and Neisseria meningitidis (5.0%). Of the 21 S. pneumoniae isolated, 33.3 and 14.3% were serotypes included in the 13 valent PCV (PCV13) and 10 valent PCV (PCV10) respectively. 33.8% of all species were resistant to at least one antibiotic, however all bacterial species except S. pneumoniae were susceptible to at least one type of antibiotic. Conclusion: To our knowledge, this is the first bacterial carriage study undertaken in East Malaysia. We provide valuable and timely data regarding the epidemiology and AMR of respiratory pathogens commonly associated with pneumonia. Further surveillance in Malaysia is necessary to monitor changes in the carriage prevalence of upper respiratory tract pathogens and the emergence of AMR, particularly as PCV is added to the National Immunisation Programme (NIP)

The knowledge and awareness of the importance of respirator fit testing

Background: The emergence of the SARS-CoV-2 has caught the world off guard. In the nick of time, respirators and other personal protective equipment turned into highly priced commodities. Many healthcare workers donned respirators for the first time, some donned surgical masks and interchanged them with respirators, depending on their availability. Respirators work by achieving a tight seal with the operators’ face in which respirator fit can be determined via respirator fit testing. Infectious aerosol can be inhaled via the leak, endangering the health of the users. Therefore, the objective of this study is to determine the knowledge and awareness of respirator fit testing among the healthcare workers at the time of the SARS-CoV-2 pandemic in Sarawak, Malaysia. Methods: We conducted an online workshop called “Proper use of facemask & respirator at the time of COVID-19 pandemic” with the objective of providing an authoritative guidance on the selection and use of various face coverings and respirators. The workshop was hosted live via Facebook and YouTube. At the end of it, we provided self-administered questionnaires to gauge the knowledge and awareness of the participants. Results: The workshop was attended by 280 participants, of which 188 responded to the questionnaire. Slightly more than half (52.7%, n=99/188) of the respondents have never heard about respirator fit testing. Most of the participants were healthcare workers (76.6%, n=144/188) and 70.8% (n=102/144) of them mentioned the requirement of respirator use at work. However, only 48% (n=49/102) have been respirator fit-tested. Of the healthcare workers who needed to use respirator for work but were yet to be fit-tested, 22.6% (n=12/53) were still willing to don non-fit tested respirators for work due to the current pandemic circumstances. Conclusions: The awareness of fit testing is inadequate, and each healthcare institution should kindle a robust and active respirator fit testing program to prepare for the next pandemic

Incorrect identification of recent Asian strains of Coxsackievirus A16 as human enterovirus 71: Improved primers for the specific detection of human enterovirus 71 by RT PCR

BACKGROUND: Human enterovirus 71 has emerged as an important pathogen in the Asia Pacific region and it is important to be able to make a rapid and specific diagnosis for outbreak control. Recent Asian strains of Coxsackievirus A16 have changes in the VP1 gene which causes mispriming of widely used primers for human enterovirus 71 specific identification. METHODS: Local strains of Coxsackievirus A16 were sequenced in the VP4 and VP1 genes and using sequence alignment tools, an improved set of primers were designed for specific identification of human enterovirus 71. These primers were evaluated against virus isolates as well as primary clinical specimens. RESULTS: A total of 218 virus strains were tested. All 39 human enterovirus 71 isolates were positive and none of the 38 Coxsackievirus A16, 127 other enteroviruses and 14 prototype flaviviruses and adenoviruses were positive when tested with the new primers. When aliquots of primary specimens known to have yielded human enterovirus 71 were retrospectively tested, we found that within 2 months of collection of the specimens, greater than 90% were positive but that the success rate diminished rapidly to 18% after 2 years storage. CONCLUSIONS: Our new primers will be useful in rapid diagnosis of human enterovirus 71 infection, and can also be used as a screening tool in surveillance programmes for early warning of human enterovirus 71 transmission

Evaluation of The Efficacy of a Phage Cocktail against Gentamicin-Resistant Klebsiella pneumoniae

The bacteria Klebsiella pneumoniae is one of top aetiological agents associated with nosocomial infection, and it has gained its notoriety with the emergence of multidrug resistance strains. In this study, we evaluated the effect of lytic bacteriophage cocktail isolated from our local sewage as potential antimicrobial candidate against Gentamicinresistant Klebsiella pneumoniae. A total of five clinical-acquired K. pneumoniae isolates including a carbapenem-resistant K. pneumoniae (CRKP) strain showed resistance towards gentamicin (GN). Phages were isolated using double-layer agar method against clinicaland community-acquired K. pneumoniae as host strains. Phage characterization using PCR partial sequencing of different viral genes; Lysin, Major Capsid Protein (g23) and Tail Fiber Protein has suggested that these phages possibly belonged to Myoviridae (ɸKPaV04, ɸKPaV08, ɸKPaV12) and Podoviridae (ɸKPaV03, ɸKPaV10). The characterized phages was selected for cocktail have exhibited high titer and broad host range with 22-44% lysis towards a panel of 18 K. pneumoniae strains. The antimicrobial efficacy of a single phage cocktail administration showed 80% growth suppression of GN-resistant K. pneumoniae after 18 h of incubation. Suggesting the possibility of phage cocktail to be used against nosocomial infections by multidrug resistant bacteria including being an alternative to antibiotic GN in the treatment of CRKP infections