Objectives: To compare two molecular assays (rrs quantitative PCR (qPCR) versus a combined 16SrRNA and LipL32 qPCR) on different sample types for diagnosing leptospirosis in febrile patients presenting to Mahosot Hospital, Vientiane, Laos. Methods: Serum, buffy coat and urine samples were collected on admission, and follow-up serum similar to 10 days later. Leptospira spp. culture and microscopic agglutination tests (MAT) were performed as reference standards. Bayesian latent class modelling was performed to estimate sensitivity and specificity of each diagnostic test. Results: In all, 787 patients were included in the analysis: 4/787 (0.5%) were Leptospira culture positive, 30/787 (3.8%) were MAT positive, 76/787 (9.7%) were rrs qPCR positive and 20/787 (2.5%) were 16SrRNA/LipL32 qPCR positive for pathogenic Leptospira spp. in at least one sample. Estimated sensitivity and specificity (with 95% CI) of 16SrRNA/LipL32 qPCR on serum (53.9% (33.3%-81.8%); 99.6% (99.2%-100%)), buffy coat (58.8% (34.4%-90.9%); 99.9% (99.6%-100%)) and urine samples (45.0% (27.0%-66.7%); 99.6% (99.3%-100%)) were comparable with those of rrs qPCR, except specificity of 16SrRNA/LipL32 qPCR on urine samples was significantly higher (99.6% (99.3%-100%) vs. 92.5% (92.3%-92.8%), p < 0.001). Sensitivities of MAT (16% (95% CI 6.3%-29.4%)) and culture (25% (95% CI 13.3%-44.4%)) were low. Mean positive Cq values showed that buffy coat samples were more frequently inhibitory to qPCR than either serum or urine (p < 0.001). Conclusions: Serum and urine are better samples for qPCR than buffy coat, and 16SrRNA/LipL32 qPCR performs better than rrs qPCR on urine. Quantitative PCR on admission is a reliable rapid diagnostic tool, performing better than MAT or culture, with significant implications for clinical and epidemiological investigations of this global neglected disease. (C) 2017 The Authors. Published by Elsevier Ltd on behalf of European Society of Clinical Microbiology and Infectious Diseases.

The etiology of fever in rural Lao People's Democratic Republic (Laos) has remained obscure until recently owing to the lack of laboratory facilities. We conducted a study to determine the causes of fever among 229 patients without malaria in Savannakhet Province, southern Laos; 52% had evidence of at least one diagnosis (45% with single and 7% with apparent multiple infections). Among patients with only one diagnosis, dengue (30.1%) was the most common, followed by leptospirosis (7.0%), Japanese encephalitis virus infection (3.5%), scrub typhus (2.6%), spotted fever group infection (0.9%), unspecified flavivirus infection (0.9%), and murine typhus (0.4%). We discuss the empirical treatment of fever in relation to these findings.

Although antileptospiral antibodies and leptospiral DNA have been detected in Australian fruit bats, the role of such bats as infectious hosts for the leptospires found in rodents and humans remains unconfirmed. A cohort-design, replicated survey was recently conducted in Far North Queensland, Australia, to determine if the abundance and leptospiral status of rodents were affected by association with colonies of fruit bats (Pteropus conspicillatus spp.) via rodent contact with potentially infectious fruit-bat urine. In each of four study areas, a 'colony site' that included a fruit-bat colony and the land within 1500 m of the colony was compared with a 'control site' that held no fruit-bat colonies and was >2000 m from the nearest edge of the colony site. Rodents were surveyed, for a total of 2400 trap-nights, over six sampling sessions between September 2007 and September 2008. A low abundance of rodents but a high carriage of leptospires in the rodents present were found to be associated with proximity to a fruit-bat colony. For example, means of 0.4 and 2.3 fawn-footed melomys (Melomys cervinipes) were collected/100 trap-nights at sites with and without fruit-bat colonies, respectively (P<0.001), but the corresponding prevalences of leptospiral carriage were 100% and 3.6% (P<0.001). Such trends were consistent across all of the sampling sessions but not across all of the sampling sites.Leptospires were not isolated from fruit bats by culture, and the role of such bats in the transmission of leptospires to rodents cannot be confirmed. The data collected do, however, indicate the existence of a potential pathway for transmission of leptospires from fruit bats to rodents, via rodent contact with infectious fruit-bat urine. Fruit bats may possibly be involved in the ecology of leptospires (including emergent serovars), as disseminators of pathogens to rodent populations. Stringent quantitative risk analysis of the present and similar data, to explore their implications in terms of disease prevalence and wildlife population dynamics, is recommended.

Ticks are obligate haematophagous ectoparasites of various animals, including humans, and are abundant in temperate and tropical zones around the world. They are the most important vectors for the pathogens causing disease in livestock and second only to mosquitoes as vectors of pathogens causing human disease. Ticks are formidable arachnids, capable of not only transmitting the pathogens involved in some infectious diseases but also of inducing allergies and causing toxicoses and paralysis, with possible fatal outcomes for the host. This review focuses on tick paralysis, the role of the Australian paralysis tick Ixodes holocyclus, and the role of toxin molecules from this species in causing paralysis in the host.

Identification of wild animals that harbour the causative leptospires, and the identification of the most important of these 'wild reservoirs' (in terms of threat to human health), are key factors in the epidemiology of human leptospirosis. In an epidemiological investigation in the Australian state of Queensland, in 2007-2008, samples were collected from fruit bats (Pteropus conspicillatus) and rodents (to investigate the potential role of fruit bats in the maintenance and transmission of leptospires to ground-dwelling rodents) and checked for pathogenic leptospires. The results of these studies have now been carefully analysed in attempts to see which method of detection and type of test sample were best. The effects of pentobarbitone sodium used to euthanize wild mammals before collection of necropsy samples, on the survival and detection of leptospires in vitro, were also explored. In the earlier field investigation, serum, renal tissue and urine were collected from wild mammals, for the detection of pathogenic leptospires by culture, the microscopic agglutination test (MAT), real-time PCR and silver impregnation of smears. Although 27.6% of the rodents investigated were found leptospire-positive, culture only yielded four isolates, probably because many cultures were contaminated. The main aims of the present study were to quantify the performance of the individual diagnostic tests and examine the reasons behind the high incidence of culture contamination. The results of sensitivity and specificity analyses for the different diagnostic tests indicated that isolation by culture (the definitive diagnostic test for leptospiral shedding) had perfect (100%) sensitivity when compared with the results of the PCR but a low specificity (40%). The MAT performed poorly, with a sensitivity of 50% when compared against the results of culture. The prevalence of leptospiral carriage revealed by the PCR-based investigation of kidney and urine samples (59.2%) was higher than that revealed using any other method and far higher than the 2.0% revealed by culture. The results of the culture of renal tissue agreed fairly well with those of the PCR-based investigation of such tissue, with a Cohen's unweighted kappa coefficient (κ) of 0.5 (P = 0.04). The levels of agreement between other pairs of tests were generally poor. The presence of pentobarbitone sodium, at final concentrations of 27.8 or 167 mg/ml, did not affect the viability or the detection of leptospires in culture, and is therefore unlikely to reduce the chances of isolating leptospires from an animal that has been euthanized with the compound. It appears that collecting multiple samples from each mammal being checked will improve the chances of detecting leptospires (and reduce the chances of reporting an inconclusive result for any of the mammals). For the identification of a leptospiral carrier, however, the use of just two detection methods (culture and PCR) and one type of sample (renal tissue) may give adequate sensitivity and specificity. Given the robustness of PCR to contamination and its high sensitivity (it can give a positive result when DNA from just two leptospiral cells is present in the sample), a PCR-based serotyping method, to allow the combined detection and characterisation of leptospires from field isolates, would be extremely useful.

Ticks are obligate haematophagous ectoparasites of various animals, including humans, and are abundant in temperate and tropical zones around the world. They are the most important vectors for the pathogens causing disease in livestock and second only to mosquitoes as vectors of pathogens causing human disease. Ticks are formidable arachnids, capable of not only transmitting the pathogens involved in some infectious diseases but also of inducing allergies and causing toxicoses and paralysis, with possible fatal outcomes for the host. This review focuses on tick paralysis, the role of the Australian paralysis tick Ixodes holocyclus, and the role of toxin molecules from this species in causing paralysis in the host.

Hendra virus (HeV) was first isolated in 1994, from a disease outbreak involving at least 21 horses and two humans in the Brisbane suburb of Hendra, Australia. The affected horses and humans all developed a severe but unidentified respiratory disease that resulted in the deaths of one of the human cases and the deaths or putting down of 14 of the horses. The virus, isolated by culture from a horse and the kidney of the fatal human case, was initially characterised as a new member of the genus Morbillivirus in the family Paramyxoviridae. Comparative sequence analysis of part of the matrix protein gene of the virus and the discovery that the virus had an exceptionally large genome subsequently led to HeV being assigned to a new genus, Henipavirus, along with Nipah virus (a newly emergent virus in pigs).The regular outbreaks of HeV-related disease that have occurred in Australia since 1994 have all been characterised by acute respiratory and neurological manifestations, with high levels of morbidity and mortality in the affected horses and humans. The modes of transmission of HeV remain largely unknown. Although fruit bats have been identified as natural hosts of the virus, direct bat-horse, bat-human or human-human transmission has not been reported. Human infection can occur via exposure to infectious urine, saliva or nasopharyngeal fluid from horses. The treatment options and efficacy are very limited and no vaccine exists.Reports on the outbreaks of HeV in Australia are collated in this review and the available data on the biology, transmission and detection of the pathogen are summarized and discussed.

Leptospirosis is an important, re-emerging, infectious disease of animals and humans. Leptospira, a spirochaete and the causative agent of leptospirosis, colonises the renal tissue of infected organisms where it can become endemic or result in clinical infection. Endemicity of a serovar is species specific and defines a reservoir or maintenance host, capable of renal shedding throughout life. Of the several mammalian maintenance hosts that have been described for the transmission of leptospires to humans, rodent reservoirs are the most significant. However, the sources of infection in rodents remain to be elucidated. The detection of antibodies and leptospiral DNA from fruit bats (Pteropus spp.) in Australia has alluded to a previously unrecognised role of fruit bats in the maintenance and transmission of leptospires to other species, in this case to associated ground-dwelling rodents. The primary aim of this thesis was to investigate the effect of the presence of Australian fruit bat (Pteropus conspicillatus) colonies on leptospiral prevalence and population dynamics of associated rodents in a leptospirosis endemic region of Australia. A subsequent aim was to advance the detection and description of leptospires from clinical and field samples. A lower rodent abundance but significantly higher prevalence of leptospiral infection was reported for rodents associated with fruit bat colonies when compared with control rodents not associated with fruit bat colonies. Isolation by culture, which by definition is the most confirmatory test for the detection of leptospires, detected four isolates, which could not be exhaustively compared across sites. On comprehensive analysis of sampling techniques employed for the study, it was evident that a high percentage of samples were contaminated, specifically those collected for examination by culture, which impacted on success of the surveillance study. Detection of leptospiral DNA by real-time Polymerase Chain Reaction (PCR) provided the highest sensitivity when compared to detection by culture, microscopic agglutination test and histopathology. This alluded to the robustness of molecular-based methods to contaminated samples. An additional benefit of collecting multiple samples from each specimen for detection of leptospires by multiple serological and molecular tests was highlighted. Random Amplified Polymorphic DNA (RAPD) markers have been previously used for the characterisation of leptospiral isolates, however, inconsistencies with reproducibility and subjective gel-scoring, have led to its disuse. PCR followed by high-resolution melt (HRM) curve analysis of amplicons from a number of bacterial and viral pathogens, including Campylobacter jejuni, have been successful at characterisation of pathogens. The two molecular methodologies were successfully combined for the serological characterisation of ten reference leptospiral isolates in a pilot trial. For the validation of this technique (RAPD-HRM), 23 strains of leptospires representing eight reference serovars, including the four strains isolated from rodents in this study, were tested. Intra-serovar divergence and inter-serovar convergence were reported during the course of this validation which may be indicative of discrepancies with serological classification. Further, evidence for genetic attenuation was noted in the strains representing the reference collection by the RAPD-HRM curve analysis. This trend was confirmed by analysis of hyper-immune antisera titre comparison between wild-type strains and corresponding reference strains. The attenuation may have occurred due to the high number of passages involved in the maintenance of the reference collections in agar-based, nutrient-rich medium. The data from the field surveillance of this study suggest that transmission of leptospires from fruit bats to rodents in Australia may not be influenced by the density of rodents at a site. Multiple samples collected for multiple tests from a single specimen increased chances of detecting leptospires. However, in the absence of isolation by culture of leptospires from renal samples from fruit bats, renal shedding, and therefore their role as reservoir hosts, cannot be confirmed. A high prevalence and low abundance in rodents associated with a fruit bat colony is suggestive of the potential for transmission which needs to be further evaluated. This surveillance constitutes the only study on the effect of the presence of a fruit bat colony on abundance and/ or leptospiral status in ground-dwelling, small mammals to date. To enhance detection and characterisation of leptospires from clinical and field isolates, the RAPD-HRM is presented here as a modern, closed-tube, reproducible, time and cost efficient method that is novel in bringing the knowledge of serovars to a molecular methodology. However, the diagnostic test could not be accurately validated due to evidence of attenuation in the reference strain collection. This thesis reports potential transmission dynamics from fruit bats to rodents in Australia, provides the foundation for future research into enhanced field-sampling techniques, presents a novel laboratory technique for characterisation of leptospiral isolates and provides a forum to highlight a gap in the quality control of reference strain collection, the effects of which can be mitigated by routine replacement with wild-type strains or in vitro challenge of collection.

Human leptospirosis is a zoonotic disease of global importance that causes significant morbidity and mortality, particularly in developing nations. In this review, the history, epidemiology, transmission, clinical presentation and treatment of this disease, and its impact in Australia, are discussed. Central to this review is the delineation of diagnostic methods for the disease and the challenges that this disease presents for both the clinician and diagnostic laboratory. This information should furnish clinicians with an updated tool to help overcome a number of problems associated with the diagnosis of leptospirosis.

High-resolution melt-curve analysis of random amplified polymorphic DNA (RAPD-HRM) is a novel technology that has emerged as a possible method to characterise leptospires to serovar level. RAPD-HRM has recently been used to measure intra-serovar convergence between strains of the same serovar as well as inter-serovar divergence between strains of different serovars. The results indicate that intra-serovar heterogeneity and inter-serovar homogeneity may limit the application of RAPD-HRM in routine diagnostics. They also indicate that genetic attenuation of aged, high-passage-number isolates could undermine the use of RAPD-HRM or any other molecular technology. Such genetic attenuation may account for a general decrease seen in titres of rabbit hyperimmune antibodies over time. Before RAPD-HRM can be further advanced as a routine diagnostic tool, strains more representative of the wild-type serovars of a given region need to be identified. Further, RAPD-HRM analysis of reference strains indicates that the routine renewal of reference collections, with new isolates, may be needed to maintain the genetic integrity of the collections.

Mosquito-borne diseases continue to be a serious public-health concern in Australia. Endemic alphaviruses (including Ross River and Barmah Forest viruses) account for the majority of the arboviral notifications, while some flaviviruses (Murray Valley encephalitis, Japanese encephalitis and Kunjin viruses) cause occasional outbreaks of encephalitis. Dengue epidemics are increasing in frequency in northern Queensland, with the largest outbreak in 50 years occurring during the 2008-2009 wet season. Of great concern are the threats posed by the importation of exotic arboviruses, such as West Nile, chikungunya and Rift Valley fever viruses, the introduction of exotic vectors, and the potential range expansion of key Australian vectors. Environmental and anthropogenic influences provide additional uncertainty regarding the future impact of mosquito-borne pathogens in Australia. This review discusses the trends, threats and challenges that face the management of mosquito-borne disease in Australia. Topical mosquito-borne pathogens of biosecurity and public-health concern, and the potential impacts of environmental and global trends, are discussed. Finally, a short overview of the public-health response capability in Australia is provided.