Fusobacterium necrophorum an Underrecognized Cause of Petrous Apicitis Presenting with Gradenigo Syndrome: A Case Report.

BACKGROUND With the advent of antibiotics, petrous apicitis (PA), inflammation of the petrous temporal bone, has become a rare complication of otitis media. Even more uncommon is Gradenigo syndrome (GS), a result of PA, characterized by a triad of otitis media or purulent otorrhea, pain within the regions innervated by the first and second division of the trigeminal nerve, and ipsilateral abducens nerve palsy. Recent literature has demonstrated increasing reports of Fusobacterium necrophorum isolated in cases of GS. CASE REPORT A 21-year-old man presented with otalgia, reduced hearing, and severe headache. Examination revealed right-sided purulent otorrhea, anesthesia within the trigeminal nerve distribution, and an ipsilateral abducens nerve palsy. F. necrophorum was isolated from an ear swab and a blood culture. Computed tomography and magnetic resonance imaging (MRI) demonstrated otomastoiditis, PA, cavernous sinus thrombosis, and severe stenosis of the petrous internal carotid artery. He was treated with intravenous benzylpenicillin, underwent a mastoidectomy and insertion of a ventilation tube, and was started on a 3-month course of dabigatran. Interval MRI showed improved internal carotid artery caliber, persistent petrous apex inflammation, and normal appearance of both cavernous sinuses. Follow-up clinical review noted persistent abducens and trigeminal nerve dysfunction. CONCLUSIONS We identified 190 cases of PA; of these, 80 presented with the classic Gradenigo triad. Fusobacterium sp. were cultured in 10% of GS cases, making them the most frequent isolates. Due to the fastidious nature of F. necrophorum, it may be underrepresented in the historical literature, and we recommend that empiric antibiotics cover anaerobic organisms.

A comparison of the performance of saliva and nasopharyngeal nucleic acid amplification testing for the detection of SARS-CoV-2 in New Zealand.

AIM: To compare detection of SARS-CoV-2 from paired nasopharyngeal swabs (NPS) and saliva using molecular methods in common use for testing swabs in New Zealand. METHOD: Samples from individuals testing positive for SARS-CoV-2 in Auckland, Wellington and Dunedin were tested at the local laboratories using methods previously established for these sample types. RESULTS: One hundred and ninety-six paired samples from unique individuals were tested, with 46 (23%) positive from either sample type, of which 43/46 (93%) tested positive from NPS, and 42/46 (91%) from saliva, indicating no significant difference in performance between sample types (p=0.69). The average Δ Ct between saliva and nasopharyngeal swabs overall across the sample set was 0.22 cycles, indicating excellent concordance; however, the difference between NPS and saliva collected from the same individual was quite variable with up to 19 cycles difference between the sample types. CONCLUSION: We found that saliva is an equivalent sample type to nasopharyngeal swab for the detection of SARS-CoV-2 in our laboratories using multiple assay combinations and is suitable for use as a diagnostic and surveillance test for selected groups of individuals.

Lack of N2-gene amplification on the Cepheid Xpert Xpress SARS-CoV-2 assay and potential novel causative mutations: A case series from Auckland, New Zealand.

We describe three cases with viral strains that demonstrate impaired N2-gene detection on the Cepheid Xpert Xpress SARS-CoV-2 assay, with two previously undescribed single nucleotide polymorphisms (SNPs): C29197T and G29227T. We propose that these SNPs are likely responsible since they are in close proximity to the previously described C29200T/C29200A SNPs, already shown to abolish N2-gene detection by the Xpert assay. Whether these SNPs abolish N2-gene detection by the Xpert assay individually or only in combination requires more work to elucidate.

SARS-CoV-2 viral load dynamics and real-time RT-PCR cycle threshold interpretation in symptomatic non-hospitalised individuals in New Zealand: a multicentre cross sectional observational study.

We conducted a multicentre cross sectional observational study of laboratory, public health and hospitalisation data for PCR-confirmed COVID-19 cases within the New Zealand Northern Region, between 12 February and 8 June 2020. The aim of this study was to describe population level SARS-CoV-2 upper respiratory tract (URT) viral load dynamics by stratifying positivity rates and polymerase chain reaction (PCR) cycle threshold (Ct) values of URT samples from COVID-19 cases by days since symptom onset, and to explore utility of Ct values in determining length of time post-infection and thus potential infectivity. Of 123,124 samples tested for SARS-CoV-2 by PCR, 579 samples (407 positive and 172 negative) from 368 symptomatic non-hospitalised individuals with PCR-confirmed infection were included. Sample positivity rate was 61.5% (8/13) for pre-symptomatic samples, rising to 93.2% (317/340) for samples collected during the purported symptomatic infectious period (days 0-10 post-symptom onset), and dropping to 36.3% (82/226) for post-infectious period samples (day 11 onwards). URT viral load peaked shortly after symptom onset, with median Ct values ranging 20.00-29.99 until 15 days post-symptom onset, and >30.00 after this time. Of samples with a Ct value of <20.00, 96.1% were collected during the symptomatic infectious period. However, of samples with a Ct value ≥30.00 and ≥35.00, 46.9% and 18.5%, respectively, were also collected during the symptomatic infectious period. The findings of this study indicate that at or soon after symptom onset represents the optimum time to test for SARS-CoV-2 in the URT, with median Ct values suggesting the useful testing window extends until around 15 days post-symptom onset. In asymptomatic individuals or those with unknown dates of symptom onset, Ct values <20.00 imply recent onset/potential infectivity, but Ct values ≥30.00 or ≥35.00 do not exclude recent onset/potential infectivity. Individual sample Ct values should not be used as an absolute marker of length of time post-infection or to exclude infectivity where date of symptom onset is unavailable.

Comprehensive analysis of SARS-CoV-2 antibody dynamics in New Zealand.

OBJECTIVES: Circulating antibodies are important markers of previous infection and immunity. Questions remain with respect to the durability and functionality of SARS-CoV-2 antibodies. This study explored antibody responses in recovered COVID-19 patients in a setting where the probability of re-exposure is effectively nil, owing to New Zealand's successful elimination strategy. METHODS: A triplex bead-based assay that detects antibody isotype (IgG, IgM and IgA) and subclass (IgG1, IgG2, IgG3 and IgG4) responses against Nucleocapsid (N) protein, the receptor binding domain (RBD) and Spike (S) protein of SARS-CoV-2 was developed. After establishing baseline levels with pre-pandemic control sera (n = 113), samples from PCR-confirmed COVID-19 patients with mild-moderate disease (n = 189) collected up to 8 months post-infection were examined. The relationship between antigen-specific antibodies and neutralising antibodies (NAbs) was explored with a surrogate neutralisation assay that quantifies inhibition of the RBD/hACE-2 interaction. RESULTS: While most individuals had broad isotype and subclass responses to each antigen shortly after infection, only RBD and S protein IgG, as well as NAbs, were relatively stable over the study period, with 99%, 96% and 90% of samples, respectively, having responses over baseline 4-8 months post-infection. Anti-RBD antibodies were strongly correlated with NAbs at all time points (Pearson's r ≥ 0.87), and feasibility of using finger prick sampling to accurately measure anti-RBD IgG was demonstrated. CONCLUSION: Antibodies to SARS-CoV-2 persist for up to 8 months following mild-to-moderate infection. This robust response can be attributed to the initial exposure without immune boosting given the lack of community transmission in our setting.

Evaluation of extraction and amplification assays for the detection of SARS-CoV-2 at Auckland Hospital laboratory during the COVID-19 outbreak in New Zealand.

Utilising diverse molecular platforms has formed a solid foundation in New Zealand's COVID-19 response. We evaluated multiple extraction and PCR assays for the detection of SARS-CoV-2. We included 65 positive samples which were run on the Panther Fusion using a laboratory developed test (LDT, E gene target). Where viral RNA was extracted by MagNA Pure (MP) 96 extraction platform or EpMotion 5075/Geneaid extraction kit, SARS-CoV-2 detection was performed on Light Cycler (LC) 480 using a LDT (E gene) or 3 commercial assays; Certest Viasure (Orf1ab, N genes) GenePro (E, RdRp genes) and A* Star Fortitude (proprietary target). Median Cts on LC 480 LDT for specimens (n = 9) extracted on MP 96 (26.6) were lower than on EpMotion (31.6) whereas median Cts for specimens (n = 10) extracted on the Panther Fusion LDT (23.1) were comparable with MP 96 /LC480 LDT (23.6). Specimens tested on Panther Fusion LDT (n = 28), extracted by MP 96, and amplified using commercial assays showed good concordance with a few exceptions; lower median Ct values were seen for 2 targets on GenePro (16.9, 21.5) and Viasure (19.5, 21.1) than for the Panther Fusion LDT (24.2) and A* Star Fortitude (25.6). Specimens tested on MP 96 (n = 18) had comparable results using commercial assays, with lower median Cts for Viasure (22.2, 23.7) compared with the LC 480 LDT (24.7), GenePro (24.7,25.7) and A*Fortitude (25.1) assays. The study provides an early assessment of the performance characteristics of 3 extraction methods for viral RNA and 5 PCR assays for the detection of SARS-CoV-2.

Collaborative networks enable the rapid establishment of serological assays for SARS-CoV-2 during nationwide lockdown in New Zealand.

BACKGROUND: Serological assays that detect antibodies to SARS-CoV-2 are critical for determining past infection and investigating immune responses in the COVID-19 pandemic. We established ELISA-based immunoassays using locally produced antigens when New Zealand went into a nationwide lockdown and the supply chain of diagnostic reagents was a widely held domestic concern. The relationship between serum antibody binding measured by ELISA and neutralising capacity was investigated using a surrogate viral neutralisation test (sVNT). METHODS: A pre-pandemic sera panel (n = 113), including respiratory infections with symptom overlap with COVID-19, was used to establish assay specificity. Sera from PCR­confirmed SARS-CoV-2 patients (n = 21), and PCR-negative patients with respiratory symptoms suggestive of COVID-19 (n = 82) that presented to the two largest hospitals in Auckland during the lockdown period were included. A two-step IgG ELISA based on the receptor binding domain (RBD) and spike protein was adapted to determine seropositivity, and neutralising antibodies that block the RBD/hACE­2 interaction were quantified by sVNT. RESULTS: The calculated cut-off (>0.2) in the two-step ELISA maximised specificity by classifying all pre-pandemic samples as negative. Sera from all PCR-confirmed COVID-19 patients were classified as seropositive by ELISA ≥7 days after symptom onset. There was 100% concordance between the two-step ELISA and the sVNT with all 7+ day sera from PCR­confirmed COVID-19 patients also classified as positive with respect to neutralising antibodies. Of the symptomatic PCR-negative cohort, one individual with notable travel history was classified as positive by two-step ELISA and sVNT, demonstrating the value of serology in detecting prior infection. CONCLUSIONS: These serological assays were established and assessed at a time when human activity was severely restricted in New Zealand. This was achieved by generous sharing of reagents and technical expertise by the international scientific community, and highly collaborative efforts of scientists and clinicians across the country. The assays have immediate utility in supporting clinical diagnostics, understanding transmission in high-risk cohorts and underpinning longer­term 'exit' strategies based on effective vaccines and therapeutics.

An understanding of discordant SARS-CoV-2 test results: an examination of the data from a central Auckland laboratory.

AIM: The diagnostic sensitivity of the SARS-CoV-2 real time reverse transcription polymerase chain reaction (RT-PCR) test has not been determined. This has led to a degree of uncertainty in the interpretation of results, particularly in patients tested repeatedly. The aim of this study was to explore the characteristics of patients who initially tested negative, and subsequently tested positive for SARS-CoV-2. METHODS: This retrospective observational study utilised data from the LabPlus Virology laboratory, Auckland City Hospital, to identify cases (hospital and community) with initial negative and subsequent positive SARS-CoV-2 RT-PCR results. Their clinical and laboratory characteristics were summarised. RESULTS: From 1 February to 13 April a total of 20,089 samples were received for SARS-CoV-2 testing. Of 2,011 samples from patients with multiple tests, 25 samples were positive. Nine samples were from patients who initially tested negative then tested positive. Reasons for the initial negative test results, which were all from upper respiratory tract samples, included pre-symptomatic presentation or late presentation. All patients had significant risk factors and ongoing or evolving symptoms, which warranted repeat testing. CONCLUSION: Few patients had discordant test results for SARS-CoV-2 RT-PCR. For patients who have a significant risk factor and a negative test result, repeat testing should be performed.

Age-related differences in SARS-CoV-2 testing in the Northern Region of New Zealand.

AIM: There is concern the low incidence of coronavirus disease 2019 (COVID-19) in children reflects under-testing in this population. This study sought to describe the age-distribution of SARS-CoV-2 testing in the Northern Region of New Zealand. METHODS: A retrospective single-centre review of all SARS-CoV-2 tests performed at LabPLUS, Auckland City Hospital, between 12 February and 18 April 2020. RESULTS: A total of 22,333 tests were performed, with 313 (1.40%) positive results. The age-adjusted SARS-CoV-2 testing rate was three times higher in adults than in children. The overall proportion of positive tests was lower in children (0.86%) than adults (1.45%). However, within the paediatric population the proportion of tests positive differed significantly between those <10 years old (0.08%) and those 10-14 years old (2.6%). CONCLUSION: The lower proportion of tests positive in children <10 years of age suggests they are appropriately tested relative to their rates of disease. A large high school-associated cluster makes the higher proportion of tests positive in children 10-14 years old difficult to interpret. Older children may have a higher risk of infection and increasing testing in intermediate and high school aged children may be indicated.

Get the Basics Right: A Description of the Key Priorities for Establishing a Neonatal Service in a Resource-Limited Setting in Cambodia.

BACKGROUND: Worldwide, reduction in under-five mortality has not sufficiently included neonates, who represent 45% of deaths in children of age under five years. The least progress has been observed in resource-limited settings. METHODS: This mixed methods study conducted at a Cambodian non-governmental paediatric hospital described the key priorities of the ongoing neonatal service. Routinely collected data from the hospital and microbiology databases included the number of admissions, discharges and deaths and the number of cases of bacteraemias (2011-2016). Semi-structured interviews with the management staff explored the essential features of the service. RESULTS: There were 2127 neonatal admissions and 247 deaths. The incidence of facility-based neonatal mortality decreased by 81%. Bacteraemic healthcare-associated infections decreased by 68%. A dedicated area for neonatal care was perceived as crucial, allowing better infection control and delivery of staff training. CONCLUSIONS: In this hospital, the neonatal service prioritized basic measures, particularly, having a dedicated neonatal area. Facility-based mortality and bacteraemic healthcare-associated infections decreased.

Healthcare workers' perceptions of an organizational quality assurance program implemented in a resource-limited setting: a qualitative study.

Background:There is increasing awareness of the need to implement quality assurance programs in developing countries. Healthcare staff are the primary drivers of improving the quality of care,but little is known about how they perceive quality assurance programs in resource-limited settings. This study aims to evaluate healthcare workers' perceptions of the organizational quality assurance program (OQA) at Angkor Hospital for Children (AHC), Cambodia. The OQA involves regular data collection and monitoring of quality indicators, to assess whether agreed quality standards are being met. Methods: This qualitative study consisted of four focus group discussions (FGDs) with 29 hospital staff (convenience sampling) from medical, nursing and non-medical departments. Staff members' understanding of quality assurance and perceptions of the strengths and weaknesses of the OQA were explored. Thematic content analysis was used to identify key themes. Results: Participants emphasized that quality indicators must include physical and psychological well-being. Strengths of the OQA included shared understanding amongst all groups of participants of its goals, committed leadership, that it was locally-relevant and that target indicators were developed from a "ground-up" approach. On-going challenges included that there was a gap in understanding of the OQA processes and overall running of the OQA across the organization between managers and staff. Conclusion: The introduction of the OQA at AHC has been well-received by staff members.Overall, the program is perceived to be valuable. Healthcare provision in resource-limited settings increasingly needs to demonstrate quality assurance. The model of OQA developed at AHC is one way to achieve this.

If you come from a well-known organisation, I will trust you: Exploring and understanding the community's attitudes towards healthcare research in Cambodia.

OBJECTIVE: To explore Cambodian community members' understanding of and attitudes towards healthcare research. DESIGN: This qualitative study generated data from semi-structured interviews and focus group discussions. This study was conducted at a non-governmental paediatric hospital and in nearby villages in Siem Reap province, Cambodia. A total of ten semi-structured interviews and four focus group discussions were conducted, involving 27 participants. Iterative data collection and analysis were performed concurrently. Data were analysed by thematic content analysis and the coding structure was developed using relevant literature. RESULTS: Participants did not have a clear understanding of what activities related to research compared with those for routine healthcare. Key attitudes towards research were responsibility and trust: personal (trust of the researcher directly) and institutional (trust of the institution as a whole). Villagers believe the village headman holds responsibility for community activities, while the village headman believes that this responsibility should be shared across all levels of the government system. CONCLUSIONS: It is essential for researchers to understand the structure and relationship within the community they wish to work with in order to develop trust among community participants. This aids effective communication and understanding among all parties, enabling high quality ethical research to be conducted.

Utilization of a clinical microbiology service at a Cambodian paediatric hospital and its impact on appropriate antimicrobial prescribing.

Background: Antimicrobial resistance threatens human health worldwide. Antimicrobial misuse is a major driver of resistance. Promoting appropriate antimicrobial use requires an understanding of how clinical microbiology services are utilized, particularly in resource-limited settings. Objectives: To assess the appropriateness of antimicrobial prescribing and the factors affecting utilization of the established clinical microbiology service (CMS). The CMS comprises the microbiology laboratory, clinical microbiologists (infection doctors) and antimicrobial treatment guidelines. Methods: This mixed-methods study was conducted at a non-governmental Cambodian paediatric hospital. Empirical and post-culture antimicrobial prescriptions were reviewed from medical records. The random sample included 10 outpatients per week in 2016 (retrospective) and 20 inpatients per week for 4 weeks in the medical, neonatal and intensive care wards (prospective). Post-culture prescriptions were assessed in patients with positive blood and cerebrospinal fluid cultures from 1 January 2014 to 31 December 2016. Focus group discussions and semi-structured interviews with clinicians explored barriers and facilitators to use of the CMS. Results: Only 31% of outpatients were prescribed empirical antimicrobials. Post-culture prescriptions (394/443, 89%) were more likely to be appropriate than empirical prescriptions (447/535, 84%), based on treatment guidelines, microbiology advice and antimicrobial susceptibility test results (P = 0.015). Being comprehensive, accessible and trusted enabled CMS utilization. Clinical microbiologists provided a crucial human interface between the CMS and physicians. The main barriers were a strong clinical hierarchy and occasional communication difficulties. Conclusions: Antimicrobial prescribing in this hospital was largely appropriate. A culturally appropriate human interface linking the laboratory and physicians is essential in providing effective microbiology services and ensuring appropriate antimicrobial prescribing in resource-limited settings.

"Know your audience": A hospital community engagement programme in a non-profit paediatric hospital in Cambodia.

OBJECTIVE: The purpose of this evaluation is to explore the impact of the new hospital community engagement programme (comprised of a Young Persons Advisory Group and a Science Café) on community members and other stakeholders, with regard to their attitudes, skills and degree of engagement in a paediatric hospital in Cambodia. DESIGN: Data collection included feedback questionnaires and reflections produced after each YPAG and Science Café event. Further questionnaires and reflective interviews were conducted to gather the views of key stakeholders. Data were analysed by thematic content analysis and numerical data were expressed using descriptive statistics. RESULTS: The vast majority of participants expressed their enjoyment and satisfaction of the hospital community engagement programme. Delivering the programme in the right manner for the target audiences, by prioritising their needs was key to this. Participants valued the programmes in terms of the knowledge delivered around good health practices, the skills developed such as confidence and responsibility for their health, and the provision of opportunities to voice their opinions. All stakeholders recognised the importance of the programme in improving the quality of the healthcare service provided at the hospital. CONCLUSIONS: In order to have a successful hospital community engagement programme, understanding the target audience is essential. The engagement programme must be delivered in the right way to meet the needs of community members, including right communication, right setting, right people and right timing. This will ultimately result in a meaningful programme that is able to empower community members, potentially resulting in lasting change in healthcare practices. In conclusion, the gap between hospitals and the community could narrow, allowing everyone to interact and learn from each other.