Imported malaria into Australia: surveillance insights and opportunities.

BACKGROUND: Malaria continues to pose a significant burden in endemic countries, many of which lack access to molecular surveillance. Insights from malaria cases in travellers returning to non-endemic areas can provide valuable data to inform endemic country programmes. To evaluate the potential for novel global insights into malaria, we examined epidemiological and molecular data from imported malaria cases to Australia. METHODS: We analysed malaria cases reported in Australia from 2012 to 2022 using National Notifiable Disease Surveillance System data. Molecular data on imported malaria cases were obtained from literature searches. RESULTS: Between 2012 and 2022, 3204 malaria cases were reported in Australia. Most cases (69%) were male and 44% occurred in young adults aged 20-39 years. Incidence rates initially declined between 2012 and 2015, then increased until 2019. During 2012-2019, the incidence in travellers ranged from 1.34 to 7.71 per 100 000 trips. Cases were primarily acquired in Sub-Saharan Africa (n = 1433; 45%), Oceania (n = 569; 18%) and Southern and Central Asia (n = 367; 12%). The most common countries of acquisition were Papua New Guinea (n = 474) and India (n = 277). Plasmodium falciparum accounted for 58% (1871/3204) of cases and was predominantly acquired in Sub-Saharan Africa, and Plasmodium vivax accounted for 32% (1016/3204), predominantly from Oceania and Asia. Molecular studies of imported malaria cases to Australia identified genetic mutations and deletions associated with drug resistance and false-negative rapid diagnostic test results, and led to the establishment of reference genomes for P. vivax and Plasmodium malariae. CONCLUSIONS: Our analysis highlights the continuing burden of imported malaria into Australia. Molecular studies have offered valuable insights into drug resistance and diagnostic limitations, and established reference genomes. Integrating molecular data into national surveillance systems could provide important infectious disease intelligence to optimize treatment guidelines for returning travellers and support endemic country surveillance programmes.

Fatal neural angiostrongyliasis in the Bolivian squirrel monkey (Saimiri boliviensis boliviensis) leading to defining Angiostrongylus cantonensis risk map at a zoo in Australia.

Neural angiostrongyliasis (NA) is a parasitic disease caused by Angiostrongylus cantonensis (rat lungworm). This study presents a case of NA in a captive Bolivian squirrel monkey from a zoo in western Sydney, Australia. The objective was to identify the A. cantonensis cox1 haplotype responsible for the infection and compare its mitochondrial DNA (mtDNA) to known Australian mtDNA. An epidemiological investigation was conducted to assess the risk of infection, focusing on the resident rat population in the zoo. Methods involved trapping rats and collecting rat faeces for Angiostrongylus detection, speciation, and cox1 haplotype confirmation. Various techniques were employed, including necropsy, morphological examination, and molecular methods such as ITS-2 qPCR, cox1 sequencing, and ITS-2 metabarcoding. Cluster analysis of rat faeces distribution and Angiostrongylus detection utilised an equal sampling effort (ESE) approach. Gastropods were collected throughout the study for Angiostrongylus surveillance using a hypersensitive qPCR assay. Results revealed significant clustering of rat faeces near exhibits with fresh food provision and absence of predators. Angiostrongylus-positive faeces were uniformly distributed across the zoo property. Mitochondrial DNA analysis confirmed the presence of the Ac13 haplotype of A. cantonensis in the monkey. Morphology, ITS-2 metabarcoding and partial cox1 sequencing detected only A. cantonensis, with the Ac13 cox1 haplotype predominating. A high prevalence of infection (64%, 9/14) was found in brown rats, with quantification of larvae indicating high shedding rates. Co-infections with both Ac13 and local SYD.1 A. cantonensis cox1 haplotypes were observed. Only three gastropods (all of which were Angiostrongylus-negative) were found in the survey. To minimise the risk of exposure for susceptible species, targeted rodent control was implemented in areas with higher exposure risk. A potential strategy (which requires further exploration) to consider for future zoo design was suggested. This study provides insights into the epidemiology and genetic diversity of A. cantonensis in Australia, emphasising the importance of control measures to prevent future outbreaks.

Magnitude and patterns of severe Plasmodium vivax monoinfection in Vietnam: a 4-year single-center retrospective study.

Introduction: Infection with Plasmodium vivax is a recognized cause of severe malaria including deaths. The exact burden and patterns of severe P. vivax monoinfections is however still not well quantified, especially in P. vivax endemic regions. We examined the magnitude and patterns of severe malaria caused by monoinfections of P. vivax and associated predictors among patients admitted to a tertiary care center for malaria in Vietnam. Methods: A retrospective cohort study was conducted based on the patients' medical records at the Hospital for Tropical Diseases from January 2015 to December 2018. Extracted information included demographic, epidemiologic, clinical, laboratory and treatment characteristics. Results: Monoinfections with P. vivax were found in 153 (34.5, 95% CI 30.3-39.1%) patients of whom, uncomplicated and severe malaria were documented in 89.5% (137/153, 95% CI 83.7-93.5%) and 10.5% (16/153, 95% CI 6.5-16.3%), respectively. Patterns of severe malaria included jaundice (8 cases), hypoglycemia (3 cases), shock (2 cases), anemia (2 cases), and cerebral malaria (1 case). Among 153 patients, 73 (47.7%) had classic malaria paroxysm, 57 (37.3%) had >7 days of illness at the time of admission, and 40 (26.1%) were referred from other hospitals. A misdiagnosis as having other diseases from malaria cases coming from other hospitals was up to 32.5% (13/40). Being admitted to hospital after day 7th of illness (AOR = 6.33, 95% CI 1.14-35.30, p = 0.035) was a predictor of severe malaria. Severe malaria was statistically associated with longer hospital length of stay (p = 0.035). Early and late treatment failures and recrudescence were not recorded. All patients recovered completely. Discussion: This study confirms the emergence of severe vivax malaria in Vietnam which is associated with delayed hospital admission and increased hospital length of stay. Clinical manifestations of P. vivax infection can be misdiagnosed which results in delayed treatment. To meet the goal of malaria elimination by 2030, it is crucial that the non-tertiary hospitals have the capacity to quickly and correctly diagnose malaria and then provide treatment for malaria including P. vivax infections. More robust studies need to be conducted to fully elucidate the magnitude of severe P. vivax in Vietnam.

Rat lungworm (Angiostrongylus cantonensis) active larval emergence from deceased bubble pond snails (Bullastra lessoni) into water.

Angiostrongylus cantonensis (the rat lungworm) is a zoonotic parasite of non-permissive accidental (dogs, humans, horses, marsupials, birds) hosts. The 3rd stage larvae (L3s) in the intermediate host (molluscs) act as the source of infection for accidental hosts through ingestion. Larvae can spontaneously emerge from dead gastropods (slugs and snails) in water, which are experimentally infective to rats. We sought to identify the time when infective A. cantonensis larvae can autonomously leave dead experimentally infected Bullastra lessoni snails. The proportion of A. cantonensis larvae that emerge from crushed and submerged B. lessoni is higher in snails 62 days post-infection (DPI) (30.3%). The total larval burden of snails increases at 91 DPI, indicating that emerged larvae subsequently get recycled by the population. There appears to be a window of opportunity between 1 and 3 months for infective larvae to autonomously escape dead snails. From a human and veterinary medicine viewpoint, the mode of infection needs to be considered; whether that be through ingestion of an infected gastropod, or via drinking water contaminated with escaped larvae.

Angie-LAMP for diagnosis of human eosinophilic meningitis using dog as proxy: A LAMP assay for Angiostrongylus cantonensis DNA in cerebrospinal fluid.

BACKGROUND: Angiostrongylus cantonensis (rat lungworm) is recognised as the leading cause of human eosinophilic meningitis, a serious condition observed when nematode larvae migrate through the CNS. Canine Neural Angiostrongyliasis (CNA) is the analogous disease in dogs. Both humans and dogs are accidental hosts, and a rapid diagnosis is warranted. A highly sensitive PCR based assay is available but often not readily accessible in many jurisdictions. An alternative DNA amplification assay that would further improve accessibility is needed. This study aimed to assess the diagnostic utility of a newly designed LAMP assay to detect DNA of globally distributed and invasive A. cantonensis and Angiostrongylus mackerrasae, the other neurotropic Angiostrongylus species, which is native to Australia. METHODOLOGY/PRINCIPAL FINDINGS: Cerebrospinal fluid (CSF) from dogs with a presumptive diagnosis of A. cantonensis infection (2020-2022) were received for confirmatory laboratory testing and processed for DNA isolation and ultrasensitive Angiostrongylus qPCR targeting AcanR3390. A newly designed LAMP assay targeting the same gene target was directly compared to the reference ultrasensitive qPCR in a diagnostic laboratory setting to determine the presence of A. cantonensis DNA to diagnose CNA. The LAMP assay (Angie-LAMP) allowed the sensitive detection of A. cantonensis DNA from archived DNA specimens (Kappa = 0.81, 95%CI 0.69-0.92; n = 93) and rapid single-step lysis of archived CSF samples (Kappa = 0.77, 95%CI 0.59-0.94; n = 52). Only A. cantonensis DNA was detected in canine CSF samples, and co-infection with A. mackerrasae using amplicon deep sequencing (ITS-2 rDNA) was not demonstrated. Both SYD.1 and AC13 haplotypes were detected using sequencing of partial cox1. CONCLUSIONS/SIGNIFICANCE: The Angie-LAMP assay is a useful molecular tool for detecting Angiostrongylus DNA in canine CSF and performs comparably to a laboratory Angiostrongylus qPCR. Adaptation of single-step sample lysis improved potential applicability for diagnosis of angiostrongyliasis in a clinical setting for dogs and by extension, to humans.

Successful Removal of Angiostrongylus cantonensis Larvae from the Central Nervous System of Rats 7- and 14-Days Post-Infection Using a Product Containing Moxidectin, Sarolaner and Pyrantel Embonate (Simparica Trio™) in Experimental Infections.

Angiostrongylus cantonensis is a nematode with an indirect lifecycle, using molluscs as intermediate hosts. Rats are the definitive host. By administering a suitable anthelmintic, at an appropriate interval, the risk of clinical neuroangiostrongyliasis occurring in paratenic hosts (e.g., dogs, man) can be eliminated. We wanted to determine if infective larvae (L3) of A. cantonensis can be safely killed during their migration through the central nervous system (CNS) by oral administration of an anthelmintic combination containing moxidectin (480 µg/kg, Simparica Trio™; M-S-P), thereby preventing patent infections in rats. Eighteen rats were used: ten received oral M-S-P every four weeks; eight rats were used as controls. Rats were initially given M-S-P as a chew to eat, but an acquired food aversion meant that subsequent doses were given by orogastric lavage. All 18 rats were challenged once or twice with approximately 30 L3 A. cantonensis larvae via orogastric lavage. Infection status was determined by faecal analysis using the Baermann technique and necropsy examination of the heart, pulmonary arteries and lungs. Eight out of ten rats dosed with M-S-P had zero lungworms at necropsy; a single female worm was detected in each of the remaining two rats. No treated rats had L1 larvae in faeces. In contrast, all eight controls were infected with patent infections, with a median of 14.5 worms per rat detected at necropsy. The difference in infection rates was significant (two tailed Fishers Exact; p = 0.0011). Moxidectin given orally once every month killed migrating larvae before they reached the pulmonary arteries in 80% of treated rats, while in 20%, only a single female worm was present. Considering the short half-life of moxidectin in the rat, it is likely that the effectiveness of moxidectin is due to larvicidal action on migrating L3, L4 and L5 larvae in the brain parenchyma or subarachnoid space, either 7 days (L3/L4 in cerebrum and spinal cord) or 14 days (L4/L5 in cerebrum and subarachnoid space) after inoculation. This study is a prelude for future research to determine if monthly moxidectin administration orally as M-S-P could prevent symptomatic neuroangiostrongyliasis in dogs.

Internal transcribed spacer region 1 as a promising target for detection of intra-specific polymorphisms for Strongyloides stercoralis.

Background: Strongyloides stercoralis, the causative agent of strongyloidiasis, is a parasitic worm that has larvae capable of reinfecting the same host. This nematode infection is therefore difficult to treat and to achieve total cure. Information about genetic variation and differences in drug susceptibility between strains is needed to improve treatment outcomes. Aim: To develop a polymerase chain reaction (PCR) to identify the intra-species variation among 13 S. stercoralis isolates collected from Bangladesh, USA and Australia. Material & Methods: PCR assays were designed by using primers targeting S. stercoralis internal transcribed spacer (ITS) regions 1 and 2. Sequence data generated by these PCR products were compared to the existing ITS1/2, 18S and 28S rRNA gene sequences in GenBank for phylogenetic analysis. Results: Intra-species single nucleotide polymorphisms (SNPs) were identified in ITS1 and in the 5.8S rRNA gene. The generated phylogram grouped the 13 isolates into dog, Orangutan and human clusters. Conclusion: This method could be used as an epidemiological tool to study strain differences in larger collections of S. stercoralis isolates. The study forms the basis for further development of an ITS-based assay for S. stercoralis molecular epidemiological studies.

Host transmission dynamics of first- and third-stage Angiostrongylus cantonensis larvae in Bullastra lessoni.

Given the importance of angiostrongyliasis as an emerging infectious disease of humans, companion animals, and wildlife, the current study focused on the transmission dynamics of first- and third-stage larvae of the parasitic nematode, Angiostrongylus cantonensis. The migration of infective larvae and their subsequent distribution within the Lymnaeidae snail, Bullastra lessoni, were investigated over time using microscopic examination of histological sections and fresh tissue. Snails were divided into four anatomical regions: (i) anterior and (ii) posterior cephalopedal masses, (iii) mantle skirt and (iv) visceral mass. The viability of free-swimming third-stage larvae, after their release from snail tissues, was evaluated in vitro by propidium iodide staining and infectivity by in vivo infection of Wistar rats. Snails were sequentially dissected over time to assess the number and anatomical distribution of larvae within each snail and hence infer their migration pathway. Herein, ongoing larval migratory activity was detected over 28 days post-infection. A comparison of infection rates and the larval distribution within the four designated snail regions demonstrated a significant relationship between anatomical region and density of infective larvae, with larvae mostly distributed in the anterior cephalopedal mass (43.6 ± 10.8%) and the mantle skirt (33.0 ± 8.8%). Propidium iodide staining showed that free-swimming third-stage larvae retained viability for between 4 and 8 weeks when stored under laboratory conditions. In contrast to viability, larval infectivity in rats remained for up to 2 weeks only. Knowledge gained from the current work could provide information on the development of new approaches to controlling the transmission of this parasite.

Intensive surveillance, rapid response and border collaboration for malaria elimination: China Yunnan's ''3 + 1''strategy.

BACKGROUND: Eliminating malaria and preventing re-establishment of malaria transmission in border areas requires universal coverage of malaria surveillance and a rapid response to any threats (i.e. malaria cues) of re-establishing transmission. MAIN TEXT: Strategy 1: Intensive interventions within 2.5 km-wide perimeter along the border to prevent border-spill malaria. The area within 2.5 km along the international border is the travel radius of anopheline mosquitoes. Comprehensive interventions should include: (1) proactive and passive case detection, (2) intensive vector surveillance, (3) evidence-based vector control, and (4) evidence-based preventative treatment with anti-malarial drugs. Strategy 2: Community-based malaria detection and screening of migrants and travellers in frontier townships. Un-permitted travellers cross borders frequently and present in frontier townships. Maintenance of intensified malaria surveillance should include: (1) passive malaria detection in the township hospitals, (2) seek assistance from villager leaders and health workers to monitor cross border travellers, and refer febrile patients to the township hospitals and (3) the county's Centre for Disease Control and Prevention maintain regular proactive case detection. Strategy 3: Universal coverage of malaria surveillance to detect malaria cues. Passive detection should be consolidated into the normal health service. Health services personnel should remain vigilant to ensure universal coverage of malaria detection and react promptly to any malaria cues. Strategy + 1: Strong collaborative support with neighbouring countries. Based on the agreement between the two countries, integrated control strategies should be carried out to reduce malaria burden for both countries. There should be a clear focus on the border areas between neighbouring countries. CONCLUSION: The 3 + 1 strategy is an experience summary of border malaria control and elimination, and then contributed to malaria elimination in Yunnan's border areas, China. Nevertheless, Yunnan still has remaining challenges of re-establishment of malaria transmission in the border areas, and the 3 + 1 strategy should still be carried out.

A new subspecies of Trypanosoma cyclops found in the Australian terrestrial leech Chtonobdella bilineata.

Previously, it was suggested that haemadipsid leeches represent an important vector of trypanosomes amongst native animals in Australia. Consequently, Chtonobdella bilineata leeches were investigated for the presence of trypanosome species by polymerase chain reaction (PCR), DNA sequencing and in vitro isolation. Phylogenetic analysis ensued to further define the populations present. PCR targeting the 28S rDNA demonstrated that over 95% of C. bilineata contained trypanosomes; diversity profiling by deep amplicon sequencing of 18S rDNA indicated the presence of four different clusters related to the Trypanosoma (Megatrypanum) theileri. Novy­MacNeal­Nicolle slopes with liquid overlay were used to isolate trypanosomes into culture that proved similar in morphology to Trypanosoma cyclops in that they contained a large numbers of acidocalcisomes. Phylogeny of 18S rDNA/GAPDH/ND5 DNA sequences from primary cultures and subclones showed the trypanosomes were monophyletic, with T. cyclops as a sister group. Blood-meal analysis of leeches showed that leeches primarily contained blood from swamp wallaby (Wallabia bicolour), human (Homo sapiens) or horse (Equus sp.). The leech C. bilineata is a host for at least five lineages of Trypanosoma sp. and these are monophyletic with T. cyclops; we propose Trypanosoma cyclops australiensis as a subspecies of T. cyclops based on genetic similarity and biogeography considerations.

Seropositivity and geographical distribution of Strongyloides stercoralis in Australia: A study of pathology laboratory data from 2012-2016.

BACKGROUND: There are no national prevalence studies of Strongyloides stercoralis infection in Australia, although it is known to be endemic in northern Australia and is reported in high risk groups such as immigrants and returned travellers. We aimed to determine the seropositivity (number positive per 100,000 of population and percent positive of those tested) and geographical distribution of S. stercoralis by using data from pathology laboratories. METHODOLOGY: We contacted all seven Australian laboratories that undertake Strongyloides serological (ELISA antibody) testing to request de-identified data from 2012-2016 inclusive. Six responded. One provided positive data only. The number of people positive, number negative and number tested per 100,000 of population (Australian Bureau of Statistics data) were calculated including for each state/territory, each Australian Bureau of Statistics Statistical Area Level 3 (region), and each suburb/town/community/locality. The data was summarized and expressed as maps of Australia and Greater Capital Cities. PRINCIPAL FINDINGS: We obtained data for 81,777 people who underwent serological testing for Strongyloides infection, 631 of whom were from a laboratory that provided positive data only. Overall, 32 (95% CI: 31, 33) people per 100,000 of population were seropositive, ranging between 23/100,000 (95% CI: 19, 29) (Tasmania) and 489/100,000 population (95%CI: 462, 517) (Northern Territory). Positive cases were detected across all states and territories, with the highest (260-996/100,000 and 17-40% of those tested) in regions across northern Australia, north-east New South Wales and north-west South Australia. Some regions in Greater Capital Cities also had a high seropositivity (112-188/100,000 and 17-20% of those tested). Relatively more males than females tested positive. Relatively more adults than children tested positive. Children were under-represented in the data. CONCLUSIONS/SIGNIFICANCE: The study confirms that substantial numbers of S. stercoralis infections occur in Australia and provides data to inform public health planning.

Plasmodium falciparum Histidine-Rich Protein 2 and 3 Gene Deletions in Strains from Nigeria, Sudan, and South Sudan.

Deletion of histidine-rich protein genes pfhrp2/3 in Plasmodium falciparum causes infections to go undetected by HRP2-based malaria rapid diagnostic tests. We analyzed P. falciparum malaria cases imported to Australia (n = 210, collected 2010-2018) for their pfhrp2/3 status. We detected gene deletions in patients from 12 of 25 countries. We found >10% pfhrp2-deletion levels in those from Nigeria (13.3%, n = 30), Sudan (11.2%, n = 39), and South Sudan (17.7%, n = 17) and low levels of pfhrp3 deletion from Sudan (3.6%) and South Sudan (5.9%). No parasites with pfhrp2/3 double deletions were detected. Microsatellite typing of parasites from Nigeria, Sudan, and South Sudan revealed low relatedness among gene-deleted parasites, indicating independent emergences. The gene deletion proportions signify a risk of false-negative HRP2-RDT results. This study's findings warrant surveillance to determine whether the prevalence of gene-deleted parasites justifies switching malaria rapid diagnostic tests in Nigeria, Sudan, and South Sudan.

HTLV-I and Strongyloides in Australia: The worm lurking beneath.

Strongyloidiasis and HTLV-I (human T-lymphotropic virus-1) are important infections that are endemic in many countries around the world with an estimated 370 million infected with Strongyloides stercoralis alone, and 5-10 million with HTVL-I. Co-infections with these pathogens are associated with significant morbidity and can be fatal. HTLV-I infects T-cells thus causing dysregulation of the immune system which has been linked to dissemination and hyperinfection of S. stercoralis leading to bacterial sepsis which can result in death. Both of these pathogens are endemic in Australia primarily in remote communities in Queensland, the Northern Territory, and Western Australia. Other cases in Australia have occurred in immigrants and refugees, returned travellers, and Australian Defence Force personnel. HTLV-I infection is lifelong with no known cure. Strongyloidiasis is a long-term chronic disease that can remain latent for decades, as shown by infections diagnosed in prisoners of war from World War II and the Vietnam War testing positive decades after they returned from these conflicts. This review aims to shed light on concomitant infections of HTLV-I with S. stercoralis primarily in Australia but in the global context as well.

Transition of radical, preventive and presumptive treatment regimens for malaria in China: a systematic review.

BACKGROUND: Globally, malaria is still a major public health challenge. Drug-based treatment is the primary intervention in malaria control and elimination. However, optimal use of mass or targeted treatments remains unclear. A variety of radical, preventive and presumptive treatment regimens have been administrated in China and a systematic review was conducted to evaluate effectiveness, and discuss experiences, limitations, and lessons learnt in relation to the use of these regimens. METHODS: The search for information includes both paper documents, such as books, malaria control annals and guidelines for malaria prevention and treatment, as well as three computer-based databases in Chinese (CNKI, WanFangdata and Xueshu.baidu) and two databases in English (PubMed and Google Scholar), to identify original articles and reports associated with drug administration for malaria in China. RESULTS: Starting from hyperendemicity to elimination of malaria in China, a large number of radical, preventive and presumptive treatment regimens had been tried. Those effective regimens were scaled up for malaria control and elimination programmes in China. Between 1949 and 1959, presumptive treatment with available anti-malarial drugs was given to people with enlarged spleens and those who had symptoms suggestive of malaria within the last 6 months. Between 1960 and 1999, mass drug administration (MDA) was given for preventive and radical treatment. Between 2000 and 2009, the approach was more targeted, and drugs were administed only to prevent malaria infection in those at high risk of exposure and those who needed radical treatment for suspected malaria. Presumptive therapy was only given to febrile patients. From 2010, the malaria programme changed into elimination phase, radical treatment changed to target individuals with confirmed either Plasmodium vivax or Plasmodium ovale within the last year. Preventive treatment was given to those who will travel to other endemic countries. Presumptive treatment was normally not given during this elimination phase. All cases of suspected were confirmed by either microscopy or rapid diagnosis tests for malaria antigens before drugs were administered. The engagement of the broader community ensured high coverage of these drug-based interventions, and the directly-observed therapy improved patient safety during drug administration. CONCLUSION: A large number of radical, preventive and presumptive treatment regimens for malaria had been tried in China with reported success, but the impact of drug-based interventions has been difficult to quantify because they are just a part of an integrated malaria control strategy. The historical experiences of China suggest that intervention trials should be done by the local health facilities with community involvement, and a local decision is made according to their own trial results.

The controversies surrounding Giardia intestinalis assemblages A and B.

Giardia intestinalis continues to be one of the most encountered parasitic diseases around the world. Although more frequently detected in developing countries, Giardia infections nonetheless pose significant public health problems in developed countries as well. Molecular characterisation of Giardia isolates from humans and animals reveals that there are two genetically different assemblages (known as assemblage A and B) that cause human infections. However, the current molecular assays used to genotype G. intestinalis isolates are quite controversial. This is in part due to a complex phenomenon where assemblages are incorrectly typed and underreported depending on which targeted locus is sequenced. In this review, we outline current knowledge based on molecular epidemiological studies and raise questions as to the reliability of current genotyping assays and a lack of a globally accepted method. Additionally, we discuss the clinical symptoms caused by G. intestinalis infection and how these symptoms vary depending on the assemblage infecting an individual. We also introduce the host-parasite factors that play a role in the subsequent clinical presentation of an infected person, and explore which assemblages are most seen globally.

Using cerebrospinal fluid to confirm Angiostrongylus cantonensis as the cause of canine neuroangiostrongyliasis in Australia where A. cantonensis and Angiostrongylus mackerrasae co-exist.

Both Angiostrongylus cantonensis and Angiostrongylus mackerrasae have been identified along the east coast of Australia. A lack of A. mackerrasae genomic data until 2019, however, has precluded the unequivocal identification of the Angiostrongylus species responsible for neuroangiostrongyliasis in accidental hosts such as dog and man. The availability of a whole-genome data for A. mackerrasae, including mtDNA and ITS2 rDNA, enables discrimination of A. cantonensis from A. mackerrasae. The aim of this study was to develop diagnostic PCR assays to determine the species of Angiostrongylus based on the detection of Angiostrongylus DNA sequences in the cerebrospinal fluid (CSF) of canine patients with eosinophilic meningitis. An in silico workflow utilising available cytochrome c oxidase 1 (cox1) primers streamlined the laboratory work into empirical steps, allowing optimisation and selection of a PCR assay that met the required criteria for discrimination of A. cantonensis and A. mackerrasae DNA in low-template CSF samples. The adopted cox1 qPCR assay specifically amplified and enabled the differentiation of A. cantonensis from A. mackerrasae DNA and confirmed the presence of A. cantonensis DNA in 11/50 archived CSF samples. The DNA sequences demonstrated the presence of two distinct A. cantonensis cox1 haplotypes in dogs from eastern Australia. Species identification was further confirmed via the adoption of an ITS2 rDNA assay, providing confirmation of only A. cantonensis ITS2 rDNA in the CSF samples. To our knowledge, this is the first study to unequivocally demonstrate the antemortem presence of A. cantonensis DNA in CSF from clinically affected dogs. The study confirmed the long-held assumption that A. cantonensis is the causal agent of neuroangiostrongyliasis but refutes the dogma that there was a single introduction of A. cantonensis into Australia by the demonstration of two distinct A. cantonensis cox1 haplotypes.

Magnetic resonance imaging in dogs with neuroangiostrongyliasis (rat lungworm disease).

The magnetic resonance imaging (MRI) appearance of the brain and spinal cord in humans with neuroangiostrongyliasis (NA) due to Angiostrongylus cantonensis infection has been well reported. Equivalent studies in animals are lacking. This case series describes clinical and MRI findings in 11 dogs with presumptively or definitively diagnosed NA. MRI of the brain and/or spinal cord was performed using high-field (1.5 T) or low-field (0.25 T) scanners using various combinations of transverse, sagittal, dorsal and three-dimensional (3D) T1-weighted (T1W), transverse, sagittal and dorsal T2-weighted (T2W), T2W fluid-attenuated inversion recovery (FLAIR) and T2*-weighted (T2*W) gradient echo (GRE), dorsal T2W short tau inversion recovery (STIR) and post-gadolinium transverse, sagittal, dorsal and 3D T1W and transverse T2W FLAIR sequences. In 4/6 cases where the brain was imaged, changes consistent with diffuse meningoencephalitis were observed. Evidence of meningeal involvement was evident even when not clinically apparent. The spinal cord was imaged in 9 dogs, with evidence of meningitis and myelitis detected in regions consistent with the observed neuroanatomical localization. Pathognomonic changes of neural larva migrans, as described in some human patients with NA, were not detected. NA should be considered in the differential diagnosis of dogs with MRI evidence of focal or diffuse meningitis, myelitis and/or encephalitis, especially in areas where A. cantonensis is endemic. If not precluded by imaging findings suggestive of brain herniation, cerebrospinal fluid (CSF) collection for cytology, fluid analysis, real-time polymerase chain reaction (qPCR) and enzyme-linked immunosorbent assay (ELISA) testing should be considered mandatory in such cases after the MRI studies.

Gross, microscopic, radiologic, echocardiographic and haematological findings in rats experimentally infected with Angiostrongylus cantonensis.

Although the gross and microscopic pathology in rats infected with Angiostrongylus cantonensis has been well described, corresponding changes detected using diagnostic imaging modalities have not been reported. This work describes the cardiopulmonary changes in mature Wistar rats chronically infected with moderate burdens of A. cantonensis using radiology, computed tomography (CT), CT angiography, echocardiography, necropsy and histological examinations. Haematology and coagulation studies were also performed. Thoracic radiography, CT and CT angiography showed moderately severe alveolar pulmonary patterns mainly affecting caudal portions of the caudal lung lobes and associated dilatation of the caudal lobar pulmonary arteries. Presumptive worm profiles could be detected using echocardiography, with worms seen in the right ventricular outflow tract or straddling either the pulmonary and/or the tricuspid valves. Extensive, multifocal, coalescing dark areas and multiple pale foci affecting the caudal lung lobes were observed at necropsy. Histologically, these were composed of numerous large, confluent granulomas and fibrotic nodules. Adult worms were found predominantly in the mid- to distal pulmonary arteries. An inflammatory leukogram, hyperproteinaemia and hyperfibrinogenaemia were found in most rats. These findings provide a comparative model for A. cantonensis in its accidental hosts, such as humans and dogs. In addition, the pathological and imaging changes are comparable to those seen in dogs infected with Angiostrongylus vasorum, suggesting rats infected with A. cantonensis could be a model for dogs with A. vasorum infection.

Further studies of neuroangiostrongyliasis (rat lungworm disease) in Australian dogs: 92 new cases (2010-2020) and results for a novel, highly sensitive qPCR assay.

The principal aim of this study was to optimize the diagnosis of canine neuroangiostrongyliasis (NA). In total, 92 cases were seen between 2010 and 2020. Dogs were aged from 7 weeks to 14 years (median 5 months), with 73/90 (81%) less than 6 months and 1.7 times as many males as females. The disease became more common over the study period. Most cases (86%) were seen between March and July. Cerebrospinal fluid (CSF) was obtained from the cisterna magna in 77 dogs, the lumbar cistern in f5, and both sites in 3. Nucleated cell counts for 84 specimens ranged from 1 to 146 150 cells µL-1 (median 4500). Percentage eosinophils varied from 0 to 98% (median 83%). When both cisternal and lumbar CSF were collected, inflammation was more severe caudally. Seventy-three CSF specimens were subjected to enzyme-linked immunosorbent assay (ELISA) testing for antibodies against A. cantonensis; 61 (84%) tested positive, titres ranging from <100 to ⩾12 800 (median 1600). Sixty-one CSF specimens were subjected to real-time quantitative polymerase chain reaction (qPCR) testing using a new protocol targeting a bioinformatically-informed repetitive genetic target; 53/61 samples (87%) tested positive, CT values ranging from 23.4 to 39.5 (median 30.0). For 57 dogs, it was possible to compare CSF ELISA serology and qPCR. ELISA and qPCR were both positive in 40 dogs, in 5 dogs the ELISA was positive while the qPCR was negative, in 9 dogs the qPCR was positive but the ELISA was negative, while in 3 dogs both the ELISA and qPCR were negative. NA is an emerging infectious disease of dogs in Sydney, Australia.