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The coronavirus disease (COVID-19) has been established as a major occupational health and safety issue that compounds pre-existing socioeconomic inequalities such as access to basic health services. This is exacerbated in migrant farmworkers who are an essential workforce in maintaining food supply across the country. An outbreak occurred in a remote part of Victoria with limited access to healthcare resources. Existing relationships allowed the Loddon Mallee Public Health Unit to quickly engage farm management and local pathology services and provide cultural and language support. After contact-tracing and comprehensive clinical review, rather than isolate positive cases, those who were asymptomatic and willing to work continued to do so whilst negative workers were in quarantine. Outbreak management and public health actions were quickly implemented even when the nationwide state-testing and contact-tracing systems were experiencing significant strain due to the rapid escalation in case numbers. Despite a large outbreak (68/74 workers), the management of the outbreak allowed asymptomatic cases to perform their work so farm productivity remained uninterrupted. Cases’ health status was closely monitored, with no adverse outcomes in a high-risk population. COVID-19 negative workers safely quarantined away from positive cases until the closure of the outbreak.

Bacterial sepsis characterised by an immunosuppressive and cytokine storm state is a challenge to treat clinically. While conventional antibiotics have been associated with exacerbating the cytokine storm, the role that bacteriophages may play in immune modulation of sepsis remains unclear. Bacteriophages are bacterial viruses that have the capacity to lyse specific bacteria and hence provide a natural alternative to antibiotics. K. pneumoniae is known to cause sepsis in humans, and in this study we isolated two lytic bacteriophages against this pathogen, one of which was a novel jumbo bacteriophage. We employed THP-1 monocyte cell lines, with different functional phenotypes for the interleukin-1 receptor associated kinase 3 (IRAK3- a cytoplasmic homeostatic mediator and prognostic marker of inflammation), to evaluate the role of the K. pneumoniae bacteriophages in modulating the immune response in-vitro. We showed for the first time that bacteriophages did not stimulate excessive production of tumour necrosis factor alpha, or interleukin-6, in THP-1 monocyte cell lines which displayed varying levels of IRAK3 expression.

Bacterial sepsis characterised by an immunosuppressive and cytokine storm state is a challenge to treat clinically. While conventional antibiotics have been associated with exacerbating the cytokine storm, the role that bacteriophages may play in immune modulation of sepsis remains unclear. Bacteriophages are bacterial viruses that have the capacity to lyse specific bacteria and hence provide a natural alternative to antibiotics.

Some cancer treatment failures have been attributed to the tumour microbiota, with implications that microbiota manipulation may improve treatment efficacy. While antibiotics have been used to control bacterial growth, their dysbiotic effects on the microbiome, failure to penetrate biofilms and decreased efficacy due to increasing antimicrobial resistance by bacteria, suggest alternatives are needed. Bacteriophages may provide a precise means for targeting oncobacteria whose relative abundance is increased in tumour tissue microbiomes. Fusobacterium, Streptococcus, Peptostreptococcus, Prevotella, Parvimonas, and Treponema species are prevalent in tumour tissue microbiomes of some cancers. They may promote cancer growth by dampening immunity, stimulating release of proinflammatory cytokines, and directly interacting with cancer cells to stimulate proliferation. Lytic bacteriophages against some of these oncobacteria have been isolated and characterised. The search continues for others. The possibility exists for their testing as adjuncts to complement existing therapies. In this review, we highlight the role of oncobacteria, specifically those whose relative abundance in the intra-tumour microbiome is increased, and discuss the potential for bacteriophages against these micro-organisms to augment existing cancer therapies. The capacity for bacteriophages to modulate immunity and kill specific bacteria makes them suitable candidates to manipulate the tumour microbiome and negate the effects of these oncobacteria.

The bacterial genus Klebsiella includes the closely related species K. michiganensis, K. oxytoca and K. pneumoniae, which are capable of causing severe disease in humans. In this report we describe the isolation, genomic and functional characterisation of the lytic bacteriophage KMI8 specific for K. michiganensis. KMI8 belongs to the family Drexlerviridae, and has a novel genome which shares very little homology (71.89% identity over a query cover of only 8%) with that of its closest related bacteriophages (Klebsiella bacteriophage LF20 (MW417503.1); Klebsiella bacteriophage 066039 (MW042802.1). KMI8, which possess a putative endosialidase (depolymerase) enzyme, was shown to be capable of degrading mono-biofilms of a strain of K. michiganensis that carried the polysaccharide capsule KL70 locus. This is the first report of a lytic bacteriophage for K. michiganensis, which is capable of breaking down a biofilm of this species.

Background Timeliness of cancer care is vital for improved survival and quality of life of patients. Service and care centralisation at larger-volume centres has been associated with improved outcomes. However, there is a lack of systematic data on the impact of tumour stream volume on timeliness of care. Aims To investigate and compare timeliness of care for lung cancer, a high-volume (more commonly diagnosed) tumour stream, and oesophagogastric (OG) cancer, a low-volume (less commonly diagnosed) tumour stream, at a regional health service in Victoria, Australia. Methods A retrospective cohort study comprising random samples of 75 people newly diagnosed with lung cancer (International Classification of Diseases and Related Health Problems-10 [ICD-10] diagnosis codes C34 in the Victorian Cancer Registry [VCR]) and 50 people newly diagnosed with OG cancer (ICD-10 diagnosis codes C15 or C16 in VCR) at one regional Victorian health service between 2016 and 2017. Binary logistic regression was used to calculate odds ratios (ORs) and 95% confidence intervals (CIs) for associations between patient factors and suboptimal timeliness of care. Results In comparison to OG cancer patients, lung cancer patients had reduced odds of suboptimal timeliness of care in reference to times outside OCP for referral to diagnosis (OR [95% CI] = 0.34 [0.14 to 0.83]) but increased odds of suboptimal timeliness for diagnosis to treatment (OR [95% CI] = 2.48 [1.01 to 6.09]). Conclusion In the low-volume OG cancer stream, patients had longer wait times from referral to an MDM, where treatment decisions occur, but shorter time to commencement of first treatment. Conversely in the high-volume lung cancer group, there was delayed initiation of first treatment following presentation at MDM. There is need to explore ways to fast-track MDM presentation and commencement of therapy among people diagnosed with low-volume and high-volume cancers, respectively.

The increase in global warming has favored growth of a range of opportunistic environmental bacteria and allowed some of these to become more pathogenic to humans. Aeromonas hydrophila is one such organism. Surviving in moist conditions in temperate climates, these bacteria have been associated with a range of diseases in humans, and in systemic infections can cause mortality in up to 46% of cases. Their capacity to form biofilms, carry antibiotic resistance mechanisms, and survive disinfection, has meant that they are not easily treated with traditional methods. Bacteriophage offer a possible alternative approach for controlling their growth. This study is the first to report the isolation and characterization of bacteriophages lytic against clinical strains of A. hydrophila which carry intrinsic antibiotic resistance genes. Functionally, these novel bacteriophages were shown to be capable of disrupting biofilms caused by clinical isolates of A. hydrophila. The potential exists for these to be tested in clinical and environmental settings.

Bacteriophages may be formulated into semi-solid bases for therapeutic delivery. This work investigated the effects of a range of preservatives on the viability of Myoviridae and Siphoviridae bacteriophages when these were formulated into a standard semi-solid cream base. The six preservatives tested included: benzoic acid (0·1%), chlorocresol (0·1%), combination hydroxybenzoates (propyl 4-hydroxybenzoates with methyl 4-hydroxybenzoates) (0·1%), methyl 4-hydroxybenzoate (0·08%), 2-phenoxyethanol (1%) and propyl 4-hydroxybenzoate (0·02%). These were each formulated into cetomacrogol cream aqueous to generate six individual semi-solid bases into which Myoviridae and Siphoviridae bacteriophages were added and tested for stability. Optimal bacteriophage stability was seen when the preservative chlorocresol was used. Bacteriophage in the acidic benzoic acid were the least stable, resulting in complete loss of viability after 4-5 weeks. Of the bacteriophages tested, the Myoviridae KOX1 was significantly more stable than the Siphoviridae PAC1 after 91 days in formulations with each of the preservatives. Our results suggest the need for individual testing of specific bacteriophages in pharmaceutical formulations, as their efficacy when exposed to preservatives and excipients in these delivery forms may vary. SIGNIFICANCE AND IMPACT OF THE STUDY: Bacteriophages are being increasingly investigated as alternatives to antibiotics. While bacteriophages can be formulated in diverse ways for therapeutic delivery, there has been scant work on how excipients and preservatives in these formulations affect stability of different bacteriophages. We demonstrate that the nature of preservatives in formulations will affect bacteriophage stability, and that in these formulations, viability of bacteriophage differs according to their morphology. Our work highlights the need for individual testing of specific bacteriophages in pharmaceutical formulations, as efficacy when exposed to preservatives and excipients in these delivery forms may vary.

BACKGROUND Human herpesvirus 6B (HHV-6B) is the causative agent of Roseola infantum, and has also been suggested to play a role in the pathogenesis of febrile seizures in young children, a percentage of whom go on to develop febrile status epilepticus (FSE), but the existing data is conflicting and inconclusive. HHV-6A is a distinct species, rarely detected in most parts of the world, but prior studies suggest a higher prevalence in febrile African children. We describe a case-control study comparing the frequency of HHV-6A and/or HHV-6B infections in children with febrile seizures (including FSE) and a control group of febrile children without seizures. METHODS We recruited children aged 6 to 60 months admitted with a febrile illness with (cases) or without (controls) seizures presenting within 48 hours of commencement of fever. Three milliliters of whole blood was centrifuged and plasma stored at -80°C for pooled screening for HHV-6B and HHV-6A by Taqman real-time polymerase chain reaction. RESULTS 102 cases and 95 controls were recruited. The prevalence of HHV-6B DNA detection did not differ significantly between cases (5.8% (6/102)) and controls (10.5% (10/95)) but HHV-6B infection was associated with FSE (OR, 15; 95% CI, [1.99-120]; P= 0.009). HHV-6A was not detected. CONCLUSION Prevalence of HHV-6B was similar among cases and controls. Within the FS group, HHV-6B infection was associated with FSE, suggesting HHV-6B infections could play a role in the pathogenesis of FSE.

The increase in global warming has favored growth of a range of opportunistic environmental bacteria and allowed some of these to become more pathogenic to humans. Aeromonas hydrophila is one such organism. Surviving in moist conditions in temperate climates, these bacteria have been associated with a range of diseases in humans, and in systemic infections can cause mortality in up to 46% of cases. Their capacity to form biofilms, carry antibiotic resistance mechanisms, and survive disinfection, has meant that they are not easily treated with traditional methods. Bacteriophage offer a possible alternative approach for controlling their growth. This study is the first to report the isolation and characterization of bacteriophages lytic against clinical strains of A. hydrophila which carry intrinsic antibiotic resistance genes. Functionally, these novel bacteriophages were shown to be capable of disrupting biofilms caused by clinical isolates of A. hydrophila. The potential exists for these to be tested in clinical and environmental settings.

Fusobacterium nucleatum is an important oral bacterium that has been linked to the development of chronic diseases such as periodontitis and colorectal cancer. In periodontal disease, F. nucleatum forms the backbone of the polymicrobial biofilm and in colorectal cancer is implicated in aetiology, metastasis and chemotherapy resistance. The control of this bacteria may be important in assisting treatment of these diseases. With increased rates of antibiotic resistance globally, there is need for development of alternatives such as bacteriophages, which may complement existing therapies. Here we describe the morphology, genomics and functional characteristics of FNU1, a novel bacteriophage lytic against F. nucleatum. Transmission electron microscopy revealed FNU1 to be a large Siphoviridae virus with capsid diameter of 88 nm and tail of approximately 310 nm in length. Its genome was 130914 bp, with six tRNAs, and 8% of its ORFs encoding putative defence genes. FNU1 was able to kill cells within and significantly reduce F. nucleatum biofilm mass. The identification and characterisation of this bacteriophage will enable new possibilities for the treatment and prevention of F. nucleatum associated diseases to be explored.

Achromobacter spp. are becoming increasingly associated with lung infections in patients suffering from cystic fibrosis (CF). A. marplatensis, which is closely related to A. xylosoxidans, has been isolated from the lungs of CF patients and other human infections. This article describes the isolation, morphology and characterization of two lytic bacteriophages specific for an A. marplatensis strain isolated from a pneumonia patient. This host strain was the causal agent of hospital acquired pneumonia&ndash, the first clinical report of such an occurrence. Full genome sequencing revealed bacteriophage genomes ranging in size from 45901 to 46,328 bp. Transmission electron microscopy revealed that the two bacteriophages AMA1 and AMA2 belonged to the Siphoviridae family. Host range analysis showed that their host range did not extend to A. xylosoxidans. The possibility exists for future testing of such bacteriophages in the control of Achromobacter infections such as those seen in CF and other infections of the lungs. The incidence of antibiotic resistance in this genus highlights the importance of seeking adjuncts and alternatives in CF and other lung infections.

Background. Betaherpesviruses are established causes of morbidity and mortality in immunosuppressed patient groups but have been little studied in sub-Saharan Africa, the epicenter of the human immunodeficiency virus (HIV) pandemic. In this region, primary infections with human cytomegalovirus (HCMV) and human herpesvirus type 6 (HHV-6) type 6 are endemic in infancy, but the clinical impact of these infections among pediatric inpatient groups is poorly characterized and assumptive, based largely on data from Western populations. Methods. We used TaqMan polymerase chain reaction to screen sera from a group of 303 pediatric inpatients aged between 3 weeks and 2 years, at the University Teaching Hospital in Lusaka, Zambia. We report the prevalence of DNAemia and viral loads within this patient group, and evaluate possible clinical associations/risk factors for betaherpesvirus infections in these hospitalized children. Results. We detected betaherpesvirus DNAemia in 59.1% (179/303) of children. HCMV was the most prevalent (41.3%), followed by HHV-6B (20.5%), HHV-7 (20.1%), and HHV-6A (0.3%). HIV infection (odds ratio OR], 2.31; 95% confidence interval CI], 1.37-3.90; P = .002), being underweight (OR, 1.82; 95% CI, 1.06-3.12; P = .03), and an admission diagnosis of suspected meningitis (OR, 5.72; 95% CI, 1.07-30.5; P = .041) were independently associated with an increased odds of HCMV DNAemia. Conversely, HHV-6B and HHV-7 DNAemia were not associated with HIV, underweight, or admission diagnosis. Median HCMV viral load was moderately but significantly higher in HIV-infected children. Conclusions. Highly prevalent HCMV DNAemia was independently associated with HIV infection and being underweight across all age groups, and was also associated with meningitis, with previously underappreciated implications for the health and development of African children.

Background. Congenital cytomegalovirus (CMV) infection is the major infectious cause of birth defects and hearing loss globally. There is a growing recognition of the potential clinical impact of congenital CMV infections in high-seroprevalence settings. Methods. A cross-sectional study of neonatal admissions at a large referral center in sub-Saharan Africa to determine the prevalence of both symptomatic and asymptomatic congenital CMV infection was performed. Real-time polymerase chain reaction was used to screen DNA-extracted sera, urine, and saliva, and an enzyme-linked immunosorbent assay was used to screen serum samples for anti-CMV immunoglobulin M. Multivariate binary logistic regression was used to identify risk factors associated with increased odds of congenital CMV infection. Results. Congenital CMV was detected in 3.8% (15/395) of neonates. Among these infants, 6 of 15 (40%) presented with jaundice, 1 of whom also had petechiae. Congenital CMV infection was detected in 9 of 79 (11.4%; 95% confidence interval CI], 6.1%-20.3%) neonates born to human immunodeficiency virus (HIV)-infected mothers, and both maternal HIV (odds ratio OR], 6.661 95% CI, 2.126-20.876], P = .001) and jaundice (OR, 5.701 95% CI, 1.776-18.306], P = .003) were independently linked with significantly increased odds of congenital CMV infection. Conclusions. Congenital and early infant CMV infections may have important consequences for child health in sub-Saharan Africa and other high HIV and CMV seroprevalence populations globally.

Background: Autopsy studies are the gold standard for determining cause-of-death and can inform on improved diagnostic strategies and algorithms to improve patient care. We conducted a cross-sectional observational autopsy study to describe the burden of respiratory tract infections in inpatient children who died at the University Teaching Hospital in Lusaka, Zambia. Methods: Gross pathology was recorded and lung tissue was analysed by histopathology and molecular diagnostics. Recruitment bias was estimated by comparing recruited and non-recruited cases. Results: Of 121 children autopsied, 64 % were male, median age was 19 months (IQR, 12-45 months). HIV status was available for 97 children, of whom 34 % were HIV infected. Lung pathology was observed in 92 % of cases. Bacterial bronchopneumonia was the most common pathology (50 %) undiagnosed ante-mortem in 69 % of cases. Other pathologies included interstitial pneumonitis (17 %), tuberculosis (TB; 8 %), cytomegalovirus pneumonia (7 %) and pneumocystis Jirovecii pneumonia (5 %). Comorbidity between lung pathology and other communicable and non-communicable diseases was observed in 80 % of cases. Lung tissue from 70 % of TB cases was positive for Mycobacterium tuberculosis by molecular diagnostic tests. A total of 80 % of TB cases were comorbid with malnutrition and only 10 % of TB cases were on anti-TB therapy when they died. Conclusions: More proactive testing for bacterial pneumonia and TB in paediatric inpatient settings is needed.

Background In sub-Saharan Africa there is scanty data on the causes of neonatal sepsis and antimicrobial resistance among common invasive pathogens, which might guide policy and practice. Methods This was a cross-sectional observational prevalence and aetiology study of neonates with suspected sepsis admitted to the neonatal intensive care unit, University Teaching Hospital, Lusaka, Zambia, between October 2013 and May 2014. Data from blood cultures and phenotypic antibiotic susceptibility testing were compared with multivariate analysis of risk factors for neonatal sepsis. Results Of 313 neonates with suspected sepsis, 54% (170/313) were male; 20% (62/313) were born to HIV-positive mothers; 33% (103/313) had positive blood cultures, of which 85% (88/103) were early onset sepsis (EOS). Klebsiella species was the most prevalent isolate, accounting for 75% (77/103) of cases, followed by coagulase-negative staphylococci (6% (7/103)), Staphylococcus aureus (6% (6/103)), Escherichia coli (5% (5/103) and Candida species (5% (5/103). For Klebsiella species, antibiotic resistance ranged from 96–99% for WHO-recommended first-line therapy (gentamicin and ampicillin/penicillin) to 94–97% for third generation cephalosporins. The prevalence of culture-confirmed sepsis increased from 0–39% from December 2013 to March 2014, during which time mortality increased 29–47%. 93% (14/15) neonates with late onset sepsis and 82% (37/45) with early-onset sepsis aged 4–7 days were admitted >2 days prior to onset of symptoms. Culture results for only 25% (26/103) of cases were available before discharge or death. Maternal HIV infection was associated with a reduced risk of neonatal sepsis (OR 0.46 [0.23–0.93], p=0.029). Conclusions Outbreaks of nosocomial multi-antibiotic-resistant infections are an important cause of neonatal sepsis and associated mortality. Reduced risk of neonatal sepsis associated with maternal HIV infection is counterintuitive and requires further investigation.

Objective/Background: Extrapulmonary tuberculosis (EPTB), which accounts for 10%–40% of the global burden of TB, with the highest incidence in Sub-Saharan Africa, is strongly associated with human immunodeficiency virus infection. Diagnosing EPTB is challenging, and recently, there has been a concerted effort to evaluate the latest molecular diagnostics for diagnosing TB in a range of specimen types. The Xpert MTB/RIF assay (Cepheid, Sunnyvale, CA, USA) is one such technology, which simultaneously detects Mycobacterium tuberculosis and rifampicin resistance. Our objective was to evaluate the accuracy of the Xpert MTB/RIF assay for the diagnosis of EPTB and detection of rifampicin resistance in routinely processed formalin-fixed, paraffin-embedded (FFPE) tissues, compared with histological detection of TB as the gold standard. Methods: A convenience set of 100 biobanked FFPE tissues, including lymph nodes (n = 64), male genital tract tissue (n = 10), abdominal tissue (n = 8), female genital tissue (n = 5), breast tissue (n = 5), synovial tissue (n = 4), skin (n = 2), tongue tissue (n = 1), and thyroid (n = 1), from routine cases of clinically suspected EPTB admitted to the University Teaching Hospital, Lusaka, Zambia, were analyzed using the Xpert MTB/RIF assay and in-house polymerase chain reaction (PCR) assay targeting IS6110, in parallel with Ziehl–Neelsen (ZN) staining, against histology as the gold standard. Results: Some 66% of specimens had histological evidence of TB infection. ZN staining was positive for TB in 8% of cases, and Xpert MTB/RIF was positive for TB in 25% of cases. Taking histology as the gold standard, the sensitivity and specificity were as follows: In lymph tissue the accuracy of the Xpert MTB/RIF assay was 41% (95%CI 27-57), not significantly better than ZN or the in-house PCR assay. In non-lymph tissue the sensitivity of the in-house PCR assay was 82% (95%CI: 56%-95%), significantly higher than the Xpert MTB/RIF assay (P = 0.004). The Xpert MTB/RIF assay indicated rifampicin resistance in just three cases. Conclusion: The Xpert MTB/RIF assay is potentially a useful tool for the diagnosis of TB in routine FFPE tissues.

Background: The World Health Organisation (WHO) estimates that 3 million cases of tuberculosis (TB) are missed every year. Identification and treatment of these are critical to achieving global TB control. Patients with sub-clinical TB, extra-pulmonary TB, and drug-resistant TB are difficult to diagnose and may be missed at all points of healthcare. An autopsy study was conducted to ascertain the burden of TB at post-mortem in adults who died in the inpatient general medical wards at a tertiary care referral center in Lusaka, Zambia. Methods: Complete whole body autopsies were performed on 125 adult inpatients. Pathological examination involved two stages: (1) Gross pathology was recorded, and samples were taken from all organs for histopathology and cryopreservation; and (2) Histopathological examination of tissue after appropriate staining. Specific pathology and diseases identified on examination were recorded. Lung tissues were processed using the GeneXpert MTB/RIF Assay. Primary outcome measures were specific diseases stratified by HIV status. Secondary outcomes were missed TB and drug-resistant TB cases. Findings: Of 125 adults, median age 35. years (IQR: 29-43), 80 (64%) were male and 101 (80.8%) were HIV-positive. Tuberculosis was the most common finding at autopsy with 78/125 cases (62.4%), of which 66/78 (84.6%) were HIV-infected. There were 35/78 cases (44.9%) with extra-pulmonary TB, the odds of which were higher among HIV-infected cases (aOR 5.14 (95% CI: 1.04-25.4), p=. 0.045); 25.6% (20/78) of the TB cases were not diagnosed ante-mortem; and 13/78 (16.7%) of the TB cases had undiagnosed MDR-TB. Other autopsy findings included: pyogenic pneumonia 36.8% (46/125); bacterial meningitis 7.2% (9/125); cardiac failure 7.2% (9/125); and malignancies 8.8% (11/125). Prevalence of HIV did not differ between TB and non-TB cases (84.6% vs. 74.5%: p=. 0.163). Interpretation: TB remains an important cause of death in adult inpatients. A substantial number of inpatients with TB and MDR-TB are not diagnosed by the current cascade of healthcare. Inpatient settings in high TB endemic countries should be included in WHO 'high risk' groups, and heightened clinical awareness and more proactive screening for TB and MDR-TB in all inpatients should be required.