Estimating Prevalence and Infection Intensity of Soil-Transmitted Helminths Using Quantitative Polymerase Chain Reaction and Kato-Katz in School-Age Children in Angola.

Quantitative polymerase chain reaction (qPCR) is gaining recognition in soil-transmitted helminth (STH) diagnostics, especially for Strongyloides stercoralis and differentiating hookworm species. However, sample preservation and DNA extraction may influence qPCR performance. We estimated STH prevalence and infection intensity by using qPCR in schoolchildren from Huambo, Uige, and Zaire, Angola, and compared its performance with that of the Kato-Katz technique (here termed Kato-Katz). Stool samples from 3,063 children (219 schools) were preserved in 96% ethanol and analyzed by qPCR, of which 2,974 children (215 schools) had corresponding Kato-Katz results. Cluster-adjusted prevalence and infection intensity estimates were calculated by qPCR and Kato-Katz, with cycle threshold values converted to eggs per gram for qPCR. Cohen's kappa statistic evaluated agreement between qPCR and Kato-Katz. DNA extraction and qPCR were repeated on 191 (of 278) samples that were initially qPCR negative but Kato-Katz positive, of which 112 (58.6%) became positive. Similar prevalence for Ascaris lumbricoides (37.5% versus 34.6%) and Trichuris trichiura (6.5% versus 6.1%) were found by qPCR and Kato-Katz, respectively, while qPCR detected a higher hookworm prevalence (11.9% versus 2.9%). The prevalence of moderate- or high-intensity infections was higher by Kato-Katz than by qPCR. Agreement between qPCR and Kato-Katz was very good for A. lumbricoides, moderate for T. trichiura, and fair for hookworm. Strongyloides stercoralis prevalence was 4.7% (municipality range, 0-14.3%), and no Ancylostoma ceylanicum was detected by qPCR. Despite suboptimal performance, presumably due to fixative choice, qPCR was fundamental in detecting S. stercoralis and excluding zoonotic A. ceylanicum. Further evaluations on sample fixatives and DNA extraction methods are needed to optimize and standardize the performance of qPCR.

Effectiveness of one and two doses of ivermectin mass drug administration in reducing the prevalence and intensity of soil-transmitted helminth (STH) infections in Western Province, Solomon Islands: a cluster-randomised, before-after analysis.

Background: Ivermectin mass drug administration (MDA) is effective for controlling onchocerciasis and scabies, with evidence supporting its role in some species of soil-transmitted helminth (STH) infections. In the context of RISE, a cluster-randomised trial for scabies, this study evaluated the effectiveness of ivermectin MDA in reducing STH burden in the Western Province of Solomon Islands. Methods: Twenty villages were randomised 1:1 to receive ivermectin MDA as one dose (IVM-1) or two doses (IVM-2) for scabies. The effectiveness of one and two doses in reducing STH prevalence and intensity was evaluated before (May 2019) and 21 months after (February 2021) MDA in May 2019. All residents aged 12 months or older in the study villages were eligible to participate and provide stool specimens. Species-specific STH infection and intensity were assessed using quantitative PCR. We compared prevalence and intensity of infection between baseline and 21 months in each intervention arm individually using cluster-level analysis (adjusted for clustering) and individual-level analysis (adjusted for sex, age, and clustering). The primary outcomes were the prevalence risk difference (RD) from the cluster-level analysis, and the change in adjusted odds of infection from the individual-level analysis. Secondary outcomes included change in incident rates of mean eggs per gram (epg) of stool from baseline to 21 months, relative risk difference in prevalence and relative change in odds of infection between arms at 21 months. Sex data (male/female) were self-reported. Findings: Overall, STH infection was assessed in 830 participants from 18 villages at baseline and 1172 from 20 villages at follow-up. Females represented 58% (n = 478) of the sample at baseline and 59% (n = 690) at follow-up. We observed a reduction in Strongyloides spp. prevalence following two doses of ivermectin MDA in the cluster-level analysis from 7.0% (32/458 participants) to 1.2% (8/674 participants), corresponding to a RD of -0.07 (95% CI -0.14 to -0.01, p = 0.036), and in the individual-level analysis (OR 0.11, 95% CI 0.04-0.33, p < 0.001). T. trichiura prevalence decreased following one dose from 19.4% (74/372 participants) to 11.7% (56/505 participants) (OR 0.44, 95% CI 0.26-0.73, p = 0.0040), while egg count reduced in both arms (IVM-1: IRR 0.28, 95% CI 0.11-0.70, p = 0.0070; IVM-2: IRR 0.18, 95% CI 0.08-0.40, p < 0.001), in the individual-level analysis. We did not detect a significant difference in effect measures between the one- and two-dose arms for any species after 21 months. Interpretation: Our study highlights the long-term benefits of ivermectin MDA in reducing the burden of Strongyloides spp. and T. trichiura. STH control programs should leverage the geographical overlap of NTDs, existing drug distribution channels, and broad-spectrum agents. Funding: The National Health and Medical Research Council of Australia.

Community-wide versus school-based targeted deworming for soil-transmitted helminth control in school-aged children in Vietnam: the CoDe-STH cluster-randomised controlled trial.

Background: Soil-transmitted helminth (STH) infection control programs typically consist of school-based preventive chemotherapy (PC) targeted to school-aged children. STH reservoirs in untreated community members contribute to ongoing transmission in children. The CoDe-STH (Community Deworming against STH) trial, conducted in Dak Lak province, Vietnam, between October 2019 and November 2020, aimed to determine whether community-wide mass drug administration (MDA) is more effective than school-based targeted PC in reducing STH prevalence and intensity in children. Methods: In this two-arm cluster randomised controlled trial, 64 primary schools were randomly assigned 1:1 to receive either school-based targeted PC ("school arm") or community-wide MDA ("community arm"). A single dose of albendazole 400 mg was used for deworming. The primary outcome was hookworm prevalence in schoolchildren, measured using quantitative real-time PCR. We also measured infection intensity for Necator americanus only, using qPCR cycle threshold (Ct) values converted into eggs per gram of faeces (EPG). Analysis was by intention to treat. The trial was registered with the Australian New Zealand Clinical Trials Registry (ACTRN12619000309189). Findings: The analysis included 4955 children in the school arm and 5093 children in the community arm. N. americanus was the dominant STH species. The relative reduction in hookworm prevalence was not significantly different between the school arm (30.1%, 95% confidence interval [CI] 20.5-36.9) and the community arm (34.6%, 95% CI 19.9-49.4). Due to lower baseline prevalence than expected, the study was underpowered to detect a difference in prevalence reduction between the study arms. The community arm showed significantly greater relative reduction in N. americanus infection intensity (56.0%, 95% CI 39.9-72.1) compared to the school arm (3.4%, 95% CI -24.7 to 31.4). The community arm also showed greater relative reduction in prevalence of moderate-to-heavy intensity (≥2000 EPG) N. americanus infections (81.1%; 95% CI 69.7-92.6) compared to the school arm (39.0%, 95% CI 13.7-64.2). Interpretation: Although no impact was seen on overall prevalence, community-wide MDA was more effective in lowering N. americanus infection intensity in schoolchildren compared to school-based targeted PC, measured 12 months after one round of albendazole deworming with high coverage. Funding: National Health and Medical Research Council, Australia (APP1139561).

Effect of experimental hookworm infection on insulin resistance in people at risk of type 2 diabetes.

The reduced prevalence of insulin resistance and type 2 diabetes in countries with endemic parasitic worm infections suggests a protective role for worms against metabolic disorders, however clinical evidence has been non-existent. This 2-year randomised, double-blinded clinical trial in Australia of hookworm infection in 40 male and female adults at risk of type 2 diabetes assessed the safety and potential metabolic benefits of treatment with either 20 (n = 14) or 40 (n = 13) Necator americanus larvae (L3) or Placebo (n = 13) (Registration ACTRN12617000818336). Primary outcome was safety defined by adverse events and completion rate. Homoeostatic model assessment of insulin resistance, fasting blood glucose and body mass were key secondary outcomes. Adverse events were more frequent in hookworm-treated participants, where 44% experienced expected gastrointestinal symptoms, but completion rates were comparable to Placebo. Fasting glucose and insulin resistance were lowered in both hookworm-treated groups at 1 year, and body mass was reduced after L3-20 treatment at 2 years. This study suggests hookworm infection is safe in people at risk of type 2 diabetes and associated with improved insulin resistance, warranting further exploration of the benefits of hookworms on metabolic health.

Epidemiology of soil-transmitted helminths using quantitative PCR and risk factors for hookworm and Necator americanus infection in school children in Dak Lak province, Vietnam.

BACKGROUND: Soil-transmitted helminth (STH) infection is driven by a complex interaction of demographic, socioeconomic and behavioural factors, including those related to water, sanitation and hygiene (WASH). Epidemiological studies that measure both infection and potential risk factors associated with infection help to understand the drivers of transmission in a population and therefore can provide information to optimise STH control programmes. METHODS: During October and November 2019, we conducted a cross-sectional survey of the prevalence and intensity of STH infection and associated risk factors among 7710 primary-school-age children from 64 primary schools across 13 districts in Dak Lak province, Vietnam. Quantitative PCR (qPCR) was used to detect and quantify STH infections. RESULTS: The predominant STH species was the hookworm Necator americanus (overall cluster-adjusted prevalence of 13.7%), and its prevalence was heterogeneously distributed across surveyed schools (0% to 56.3%). All other STH species had a prevalence of less than 1%. Using mixed-effects logistic regression, we found that the adjusted odds ratio (aOR) was significantly higher for both infection and moderate-to-heavy-intensity infection with N. americanus among children from multiple ethnic minority groups, compared to children from the majority group (Kinh). Adjusted odds of infection with N. americanus were also higher in children who reported practising open defecation at school (aOR 1.42, 95% CI 1.05, 1.93, P = 0.02) and in those who had an unimproved household water supply (aOR 1.28, 95% CI 1.04, 1.57, P = 0.02). Conversely, children with a flushing household toilet had a reduced risk of infection (aOR 0.58, 95% CI 0.47, 0.70, P < 0.01), as did those whose primary female carer attended secondary (aOR 0.65, 95% CI 0.51, 0.84, P < 0.01) or tertiary education (aOR 0.39, 95% CI 0.24, 0.63, P < 0.01). CONCLUSIONS: This study is the largest reported prevalence survey of STH infections conducted using qPCR as a diagnostic technique. The findings of higher adjusted odds of infection amongst ethnic minority children highlight that STH control programmes may not be reaching certain population groups and that additional culturally appropriate approaches may be required. Additionally, the associations between specific WASH factors and infection indicate potential programmatic targets to complement preventive chemotherapy programmes.

The impact of ivermectin, diethylcarbamazine citrate, and albendazole mass drug administration on the prevalence of scabies and soil-transmitted helminths in school-aged children in three municipalities in Timor-Leste: a before-after assessment.

BACKGROUND: Integrated programmes that use combination mass drug administration (MDA) might improve control of multiple neglected tropical diseases simultaneously. We investigated the impact of Timor-Leste's national ivermectin, diethylcarbamazine citrate, and albendazole MDA, for lymphatic filariasis elimination and soil-transmitted helminth (STH) control, on scabies, impetigo, and STH infections. METHODS: We did a before-after study in six primary schools across three municipalities in Timor-Leste (urban [Dili], semi-urban [Ermera], and rural [Manufahi]) before (April 23 to May 11, 2019) and 18 months after (Nov 9 to Nov 27, 2020) MDA delivery between May 17 and June 1, 2019. Study participants included schoolchildren, as well as infants, children, and adolescents who were incidentally present at school on study days. All schoolchildren whose parents provided consent were eligible to participate in the study. Infants, children, and adolescents younger than 19 years who were not enrolled in the school but were incidentally present at schools on study days were also eligible to participate if their parents consented. Ivermectin, diethylcarbamazine citrate, and albendazole MDA was implemented nationally, with single doses of oral ivermectin (200 µg/kg), diethylcarbamazine citrate (6 mg/kg), and albendazole (400 mg) administered by the Ministry of Health. Scabies and impetigo were assessed by clinical skin examinations, and STHs using quantitative PCR. The primary (cluster-level) analysis adjusted for clustering while the secondary (individual-level) analysis adjusted for sex, age, and clustering. The primary outcomes of the study were prevalence ratios for scabies, impetigo, and STHs (Trichuris trichiura, Ascaris lumbricoides, Necator americanus, and moderate-to-heavy A lumbricoides infections) between baseline and 18 months from the cluster-level analysis. FINDINGS: At baseline, 1043 (87·7%) of 1190 children registered for the study underwent clinical assessment for scabies and impetigo. The mean age of those who completed skin examinations was 9·4 years (SD 2·4) and 514 (53·8%) of 956 were female (87 participants with missing sex data were excluded from this percentage calculation). Stool samples were received for 541 (45·5%) of 1190 children. The mean age of those for whom stool samples were received was 9·8 years (SD 2·2) and 300 (55·5%) were female. At baseline, 348 (33·4%) of 1043 participants had scabies, and 18 months after MDA, 133 (11·1%) of 1196 participants had scabies (prevalence ratio 0·38, 95% CI 0·18-0·88; p=0·020) in the cluster-level analysis. At baseline, 130 (12·5%) of 1043 participants had impetigo, compared with 27 (2·3%) of 1196 participants at follow-up (prevalence ratio 0·14, 95% CI 0·07-0·27; p<0·0001). There was a significant reduction in T trichiura prevalence from baseline (26 [4·8%] of 541 participants) to 18-month follow-up (four [0·6%] of 623 participants; prevalence ratio 0·16, 95% CI 0·04-0·66; p<0·0001). In the individual-level analysis, moderate-to-heavy A lumbricoides infections reduced from 54 (10·0%; 95% CI 0·7-19·6) of 541 participants to 28 (4·5%, 1·2-8·4) of 623 participants (relative reduction 53·6%; 95% CI 9·1-98·1; p=0·018). INTERPRETATION: Ivermectin, diethylcarbamazine citrate, and albendazole MDA was associated with substantial reductions in prevalence of scabies, impetigo, and T trichiura, and of moderate-to-heavy intensity A lumbricoides infections. Combination MDA could be used to support integrated control programmes to target multiple NTDs. FUNDING: National Health and Medical Research Council of Australia and the Department of Foreign Affairs and Trade Indo-Pacific Centre for Health Security. TRANSLATION: For the Tetum translation of the abstract see Supplementary Materials section.

Assessing the efficacy of albendazole against hookworm in Vietnam using quantitative PCR and sodium nitrate flotation.

Preventive chemotherapy (PC), consisting of the regular distribution of anthelmintics to populations or groups of populations at risk, is the primary tool used to control soil-transmitted helminth (STH) infections. This strategy, whilst cost-effective, raises the concern of potential emergence of drug resistance. The efficacy of anthelmintics against STH infections is measured using cure rate (CR) and egg reduction rate (ERR), using microscopy-based techniques such as the Kato-Katz thick smear. However, Kato-Katz has low sensitivity, especially for low-intensity infections, and requires fresh samples that need to be processed quickly. Realtime quantitative PCR (qPCR), which is more sensitive, is emerging as a "gold standard" for STH diagnostics given its higher sensitivity (important in low prevalence settings) and ability to differentiate hookworm species, while sodium nitrate flotation (SNF) may provide a low-cost more sensitive and practical alternative to Kato-Katz in the field. In this study, we examined the efficacy of a locally manufactured brand of albendazole 400 mg ("Alzental") against hookworm in Dak Lak province, Vietnam, using both qPCR and SNF. For qPCR, formulae to convert qPCR cycle threshold (Ct) values into eggs per gram of faeces (EPG) were utilised to determine efficacy calculations, and these values directly compared with efficacy values generated using SNF. Factors associated with CR and ERR were examined, and Alzental tablet quality was assessed by comparing with an Australian TGA-approved equivalent "Eskazole" tablet. We observed a CR and ERR of 64.9% and 87.5% respectively using qPCR, and 68.4% and 67.6% respectively using SNF. The tablet composition of Alzental was comparable to Eskazole in terms of active albendazole drug concentration with no evidence of impurities. This study demonstrates that the efficacy of Alzental against hookworm is within the range of previously reported studies for albendazole 400 mg. The study also demonstrates the value of qPCR and SNF as alternatives to standard Kato-Katz methodology for assessment of anthelmintic efficacy.

Using quantitative PCR to identify opportunities to strengthen soil-transmitted helminth control in Solomon Islands: A cross-sectional epidemiological survey.

BACKGROUND: The Kato-Katz microscopy technique is the global standard for assessment of soil-transmitted helminth (STH) burden. However, major limitations include its poor sensitivity, requirement for rapid sample processing, and inability to differentiate hookworm species nor detect Strongyloides spp. infections. We assessed the prevalence and intensity of STH species in Solomon Islands by conducting a province-wide survey using quantitative PCR (qPCR) for diagnosis, which can provide much better characterisation of STH burden than microscopy. METHODOLOGY/PRINCIPAL FINDINGS: We conducted a cross-sectional survey in 18 villages in Western Province to detect infections with six STH species and quantify intensity with three. We used linear mixed model regression to identify potential water, sanitation, and hygiene (WASH) and environmental risk factors for infection. We collected stool specimens from 830 village residents. Overall STH prevalence was 63.3% (range 27.5 to 91.5% across villages), led by Necator americanus (54.5% [range 17.5-89.4%]), followed by Ancylostoma ceylanicum (15.5% [range 2.8-45.8%]), Trichuris trichiura (9.1% [range 0-79.2%]), and Strongyloides spp. (3.2% [range 0-29.2%]). Most infections were of light intensity for N. americanus (85.7%) and T. trichiura (90.7%). Owning a household latrine was associated with a lower risk of N. americanus infection (AOR 0.41, 95% CI 0.24-0.68) while greater precipitation was linked to more common T. trichiura infection (AOR 1.14, 95% CI 1.04-1.25). CONCLUSION/SIGNIFICANCE: In this first large-scale population survey of STH in the Pacific using qPCR, we found evidence that ivermectin should be incorporated into STH control programmes because of the presence of T. trichiura and Strongyloides spp., both of which are poorly responsive to albendazole. Furthermore, One Health strategies are needed for improved A. ceylanicum and Strongyloides spp. control, WASH access and use should be improved to complement deworming programmes, and control efforts should ideally be expanded to entire communities. TRIAL REGISTRATION: ClinicalTrials.gov Australian and New Zealand Clinical Trials Registry ACTRN12618001086257.

Comparison of the egg recovery rates and limit of detection for soil-transmitted helminths using the Kato-Katz thick smear, faecal flotation and quantitative real-time PCR in human stool.

BACKGROUND: Monitoring the success of soil-transmitted helminth (STH) control programs relies on accurate diagnosis and quantitative assessment of infection prevalence and intensity. As preventative chemotherapeutic program coverage for STH expands, the necessity of gaining insights into the relative or comparative sensitivities, in terms of limits of detection (LOD) and egg-recovery-rates (ERR) for microscopy and quantitative polymerase chain reaction qPCR-based diagnostic techniques becomes imperative to inform suitability for their intended use for large scale STH monitoring and treatment efficacy studies. METHODOLOGY/PRINCIPAL FINDINGS: The diagnostic performance in terms of ERR and LOD of the Kato-Katz (KK) thick smear technique, sodium nitrate (NaNO3) faecal floatation (FF) and qPCR for the accurate detection and enumeration of STH eggs were calculated and expressed in eggs per gram (EPG), by experimentally seeding parasite-free human faeces with Ascaris spp., Trichuris spp. and Necator americanus eggs representing low, medium and high intensity infections. The efficiency of NaNO3 flotation was also calculated over a range of specific gravities (SpGr) for the optimum recovery of STH eggs. FF of SpGr 1.30 recovered 62.7%, 11% and 8.7% more Trichuris spp., Necator americanus and Ascaris spp. eggs respectively, than the recommended SpGr of 1.20. All diagnostic methods demonstrated strong direct correlation to the intensity of seeded EPG. KK and FF (SpGr 1.30) resulted in significant lower ERRs compared to qPCR (p <0.05). qPCR demonstrated significantly (p <0.05) greater sensitivity with an ability to detect as little as 5 EPG for all three STH, compared to 50 EPG by KK and FF (SpGr 1.30). CONCLUSIONS/SIGNIFICANCE: This study compares the diagnostic parameters in terms of LOD and ERRs of STHs for the KK, FF and qPCR. These results indicate that the diagnostic performance of qPCR assays should be considered by control programs in the phase that aims to seek confirmation of transmission break and cessation of preventive chemotherapy in low-transmission settings, in line with the control targets of the WHO neglected tropical diseases 2030 Roadmap.

Molecular Evidence of Novel Spotted Fever Group Rickettsia Species in Amblyomma albolimbatum Ticks from the Shingleback Skink (Tiliqua rugosa) in Southern Western Australia.

Tick-borne infectious diseases caused by obligate intracellular bacteria of the genus Rickettsia are a growing global problem to human and animal health. Surveillance of these pathogens at the wildlife interface is critical to informing public health strategies to limit their impact. In Australia, reptile-associated ticks such as Bothriocroton hydrosauri are the reservoirs for Rickettsia honei, the causative agent of Flinders Island spotted fever. In an effort to gain further insight into the potential for reptile-associated ticks to act as reservoirs for rickettsial infection, Rickettsia-specific PCR screening was performed on 64 Ambylomma albolimbatum ticks taken from shingleback skinks (Tiliqua rugosa) located in southern Western Australia. PCR screening revealed 92% positivity for rickettsial DNA. PCR amplification and sequencing of phylogenetically informative rickettsial genes (ompA, ompB, gltA, sca4, and 17kda) suggested that the single rickettsial genotype detected represented a novel rickettsial species, genetically distinct from but closely related to Rickettsia gravesii and within the rickettsia spotted fever group (SFG). On the basis of this study and previous investigations, it would appear that Rickettsia spp. are endemic to reptile-associated tick species in Australia, with geographically distinct populations of the same tick species harboring genetically distinct SFG Rickettsia species. Further molecular epidemiology studies are required to understand the relationship between these diverse Rickettsiae and their tick hosts and the risk that they may pose to human and animal health.

Screening for Rickettsia, Coxiella and Borrelia Species in Ticks from Queensland, Australia.

Tick bites in Australia are linked to the transmission of a variety of infectious diseases in humans, livestock and wildlife. Despite this recognition, little is currently known about the variety of potential pathogens that are carried and transmitted by Australian ticks. In this study, we attempted to expand knowledge of Australian tick-borne bacterial pathogens by analyzing various tick species from the state of Queensland for potential human pathogens belonging to the Rickettsia, Coxiella and Borrelia genera. A total of 203 ticks, comprising of four genera and nine different tick species, were screened by specific qPCR assays. An overall Rickettsia qPCR positivity of 6.4% (13/203) was detected with rickettsial DNA found in four tick species (Ixodes holocyclus, I. tasmani, Amblyommatriguttatum, and Haemaphysalis longicornis). Amplification and analysis of several rickettsial genes from rickettsial qPCR positive samples identified sequences closely related to but genetically distinct from several previously described cultured and uncultured rickettsial species in the Rickettsia spotted fever subgroup. No ticks were positive for either Coxiella or Borrelia DNA. This work suggests that a further diversity of rickettsiae remain to be described in Australian ticks with the full importance of these bacteria to human and animal health yet to be elucidated.

Domestic dogs are mammalian reservoirs for the emerging zoonosis flea-borne spotted fever, caused by Rickettsia felis.

Rickettsia felis is an obligate intracellular bacterium that is being increasingly recognized as an etiological agent of human rickettsial disease globally. The agent is transmitted through the bite of an infected vector, the cat flea, Ctenocephalides felis, however there is to date, no consensus on the pathogen's vertebrate reservoir, required for the maintenance of this agent in nature. This study for the first time, demonstrates the role of the domestic dog (Canis familiaris) as a vertebrate reservoir of R. felis. The ability of dogs to sustain prolonged periods of rickettsemia, ability to remain asymptomatically infected with normal haematological parameters and ability to act as biological vehicles for the horizontal transmission of R. felis between infected and uninfected fleas provides indication of their status as a mammalian reservoir of this emerging zoonosis.

Development and Evaluation of a Multiplex Quantitative Real-Time Polymerase Chain Reaction for Hookworm Species in Human Stool.

Hookworm disease caused by Necator americanus, Ancylostoma duodenale, and Ancylostoma ceylanicum affects half a billion people worldwide. The prevalence and intensity of infection of individual hookworm species are vital for assessing morbidity and generating targeted intervention programs for their control. The present study aims to evaluate a multiplex real-time quantitative PCR (qPCR) assay to determine the prevalence and egg intensity of all three hookworm species and compare this with standard microscopy and published genus-based conventional and real-time multiplex qPCRs. Performance of the diagnostic assays was evaluated using DNA extracted from 192 fecal samples collected as part of a soil-transmitted helminth (STH) survey in northern Cambodia. The prevalence of hookworms as detected by the multiplex hookworm qPCR of 84/192 (43.8%) was significantly higher than that using microscopy of 49/192 (25.5%). The hookworm multiplex qPCR showed very good agreement for the detection of both N. americanus (Kappa 0.943) and Ancylostoma spp. (Kappa 0.936) with a multiplex STH qPCR. A strong and moderate quantitative correlation between cycle threshold and eggs per gram (EPG) feces was obtained for the hookworm qPCR for seeded DNA egg extracts (R 2 ≥ 0.9004) and naturally egg-infected individuals (R 2 = 0.6848), respectively. The newly developed hookworm quantitative multiplex qPCR has the potential for application in anthelmintic efficacy trials and for monitoring the success of mass deworming programs targeting individual species of anthroponotic and zoonotic hookworms.

The epidemiology of Rickettsia felis infecting fleas of companion animals in eastern Australia.

BACKGROUND: Flea-borne spotted fever (FBSF) caused by Rickettsia felis is an arthropod-borne zoonosis. This study aimed to determine the prevalence, primary species and genotype(s) of R. felis infecting fleas from dogs and cats. RESULTS: All fleas were identified as Ctenocephalides felis felis. All rickettsial DNA detected in fleas was identified as being 100% homologous to R. felis URRWXCal2, with positivity within tropical, subtropical and temperate regions noted at 6.7%, 13.2% and 15.5%, respectively. Toy/small breed dogs were found to be at a lower odds of harboring R. felis-positive fleas compared with large breed dogs on univariate analysis, while DMH and pedigree breed cats were at a lower odds compared to DSH cats. Cooler minimum temperature ranges of between 15 to 20 °C and between 8 to 15 °C increased the odds of R. felis positivity in fleas, as did a constrained maximum temperature range of between 27 to 30 °C on multivariable analysis. CONCLUSIONS: Environmental temperature may play a part in influencing R. felis prevalence and infectivity within its flea host. Regional climatic differences need to be considered when approaching public health risk mitigation strategies for FBSF.

Cats as potential mammalian reservoirs for Rickettsia sp. genotype RF2125 in Bangkok, Thailand.

Rickettsia felis is an obligate intracellular alpha-proteobacteria and the cause of flea-borne spotted fever (FBSF), an emerging zoonosis of global public health importance, for which dogs and cats have been implicated as potential mammalian reservoirs hosts. The purpose of this study was to determine the prevalence and associated risk factors for R. felis-like species in semi-domesticated cats and their fleas in aim of understanding public health risks posed by cats and their fleas in Bangkok, Thailand. Single whole blood samples (n = 432) and where observed, fleas (n = 234), were collected from cats from 53 temple communities in Bangkok. Fleas were morphologically and genetically identified to a species level. Cat blood and fleas were subjected to a spotted fever group (SFG)-specific PCR targeting the partial outer membrane protein B (ompB). Those that were positive, were further characterised using an R. felis-specific nested PCR targeting the partial citrate synthase A (gltA) gene. All fleas were identified as Ctenocephalides felis felis. In total SFG Rickettsiae were detected in the blood of 82/482 (17.01%) cats and 3/234 fleas (1.28%). DNA sequencing of the partial ompB characterised all positive amplicons from cat blood and their fleas as 100% identical to Rickettsia endosymbiont of Ctenocephalides felis orientis isolate (Rickettsia sp. genotype RF2125) and Rickettsia asemboensis (GenBank accession no. KP256362 and KY650699, respectively). The gltA gene targeting R. felis was successfully amplified from 12/82 PCR-positive cat blood samples and these clustered with 99% bootstrap support with isolates within the Rickettsia sp. genotype RF2125 clade. Cats that were permitted to roam freely inside monasteries were more likely to be infected with R. felis compared with cats confined indoors. The results suggest that cats in Thailand are potential mammalian reservoir hosts for Rickettsia sp. genotype RF2125.

Comparison of a new multiplex real-time PCR with the Kato Katz thick smear and copro-antigen ELISA for the detection and differentiation of Taenia spp. in human stools.

BACKGROUND: Taenia solium, the cause of neurocysticercosis (NCC), has significant socioeconomic impacts on communities in developing countries. This disease, along with taeniasis is estimated to infect 2.5 to 5 million people globally. Control of T. solium NCC necessitates accurate diagnosis and treatment of T. solium taeniasis carriers. In areas where all three species of Taenia tapeworms (T. solium, Taenia saginata and Taenia asiatica) occur sympatrically, conventional microscope- and copro-antigen based diagnostic methods are unable to distinguish between these three Taenia species. Molecular diagnostic tools have been developed to overcome this limitation; however, conventional PCR-based techniques remain unsuitable for large-scale deployment in community-based surveys. Moreover, a real-time PCR (qPCR) for the discrimination of all three species of Taenia in human stool does not exist. This study describes the development and validation of a new triplex Taq-Man probe-based qPCR for the detection and discrimination of all three Taenia human tapeworms in human stools collected from communities in the Central Highlands of Vietnam. The diagnostic characteristics of the test are compared with conventional Kato Katz (KK) thick smear and copro-antigen ELISA (cAgELISA) method utilizing fecal samples from a community based cross-sectional study. Using this new multiplex real-time PCR we provide an estimate of the true prevalence of taeniasis in the source population for the community based cross-sectional study. METHODOLOGY/PRINCIPAL FINDINGS: Primers and TaqMan probes for the specific amplification of T. solium, T. saginata and T. asiatica were designed and successfully optimized to target the internal transcribed spacer I (ITS-1) gene of T. solium and the cytochrome oxidase subunit I (COX-1) gene of T. saginata and T. asiatica. The newly designed triplex qPCR (T3qPCR) was compared to KK and cAgELISA for the detection of Taenia eggs in stool samples collected from 342 individuals in Dak Lak province, Central Highlands of Vietnam. The overall apparent prevalence of taeniasis in Dak Lak province was 6.72% (95% confidence interval (CI) [3.94-9.50]) in which T. solium accounted for 1.17% (95% CI [0.37-3.17]), according to the T3qPCR. There was sympatric presence of T. solium, T. saginata and T. asiatica. The T3qPCR proved superior to KK and cAgELISA for the detection and differentiation of Taenia species in human feces. Diagnostic sensitivities of 0.94 (95% credible interval (CrI) [0.88-0.98]), 0.82 (95% CrI [0.58-0.95]) and 0.52 (95% CrI [0.07-0.94]), and diagnostic specificities of 0.98 (95% CrI [0.94-1.00]), 0.91 (95% CrI [0.85-0.96]) and 0.99 (95% CrI [0.96-1.00]) were estimated for the diagnosis of taeniasis for the T3qPCR, cAgELISA and KK thick smear in this study, respectively. CONCLUSIONS: T3qPCR is not only superior to the KK thick smear and cAgELISA in terms of diagnostic sensitivity and specificity, but it also has the advantage of discriminating between species of Taenia eggs in stools. Application of this newly developed T3qPCR has identified the existence of all three human Taenia tapeworms in Dak Lak province and proves for the first time, the existence of T. asiatica in the Central Highlands and the south of Vietnam.

Serological evidence of exposure to Rickettsia felis and Rickettsia typhi in Australian veterinarians.

BACKGROUND: Rickettsia felis and Rickettsia typhi are emerging arthropod-borne zoonoses causing fever and flu-like symptoms. Seroprevalence and risk factors associated with exposure to these organisms was explored in Australian veterinarians. METHODS: One hundred and thirty-one veterinarians from across Australia were recruited to participate in a cross-sectional survey. Veterinarians provided a single blood sample and answered a questionnaire on potential risk factors influencing their exposure to R. felis and R. typhi. Indirect microimmunofluorescence antibody testing (IFAT) was used to identify evidence of serological exposure of the participants to R. felis and R. typhi. Results were analyzed and a logistical regression model performed to predict risk factors associated with seropositivity. RESULTS: In total, 16.0% of participants were seropositive to R. felis, 4.6% to R. typhi and 35.1% seropositive to both, where cross-reactivity of the IFAT between R. felis and R. typhi precluded a definitive diagnosis. Veterinarians residing within the south-eastern states of Victoria and Tasmania were at a higher risk of exposure to R. felis or generalised R. felis or R. typhi exposure. Older veterinarians and those that recommended flea treatment to their clients were found to be significantly protected from exposure. CONCLUSIONS: The high exposure to R. felis amongst veterinary professionals suggests that flea-borne spotted fever is an important cause of undifferentiated fever conditions that may not be adequately recognized in Australia.

Ancylostoma ceylanicum Hookworm in the Solomon Islands.

Although hookworm is highly prevalent in the Solomon Islands, the species involved are unknown. We initiated this study in response to finding Ancylostoma ceylanicum hookworm in a peacekeeper in Australia who had returned from the Solomon Islands. Kato-Katz fecal surveys performed in 2013 and 2014 in 2 village groups in East Malaita, Solomon Islands, identified hookworm-positive samples. These specimens were tested by cytochrome oxidase 1 (cox-1) gene multiplex PCR and sequenced. Of 66 positive specimens, 54 (81.8%) contained only Necator americanus, 11 (16.7%) contained only A. ceylanicum, and 1 (1.5%) contained both species. A. duodenale was not found. Haplotype analysis of cox-1 sequences placed all human isolates (99% bootstrap support) of A. ceylanicum within the zoonotic clade rather than the human-specific clade. This study confirms that A. ceylanicum is endemic in the East Malaita region of this Pacific Island nation. The strain of the A. ceylanicum in this region can be shared among humans, dogs, and cats.

Canine vector-borne pathogens in semi-domesticated dogs residing in northern Cambodia.

BACKGROUND: In Southeast Asia, the canine vector-borne pathogens Babesia spp., Ehrlichia canis, Anaplasma platys, Hepatozoon canis, haemotropic mycoplasmas and Dirofilaria immitis cause significant morbidity and mortality in dogs. Moreover, dogs have also been implicated as natural reservoirs for Rickettsia felis, the agent of flea-borne spotted fever, increasingly implicated as a cause of undifferentiated fever in humans in Southeast Asia. The objective of this study was to determine the prevalence and diversity of canine vector-borne pathogens in 101 semi-domesticated dogs from rural Cambodia using molecular diagnostic techniques. RESULTS: The most common canine vector-borne pathogens found infecting dogs in this study were Babesia vogeli (32.7 %) followed by Ehrlichia canis (21.8 %), Dirofilaria immitis (15.8 %), Hepatozoon canis (10.9 %), Mycoplasma haemocanis (9.9 %) and "Candidatus Mycoplasma haematoparvum" (2.9 %). A high level of co-infection with CVBD agents (23.8 %) was present, most commonly B. vogeli and E. canis. Naturally occurring R. felis infection was also detected in 10.9 % of dogs in support of their role as a natural mammalian reservoir for flea-borne spotted fever in humans. CONCLUSIONS: This study reports for the first time, the prevalence and diversity of CVBD pathogens in dogs in Cambodia. In total, five species of CVBD pathogens were found infecting semi-domesticated dogs and many were co-infected with two or more pathogens. This study supports the role of dogs as natural mammalian reservoirs for R. felis, the agent of flea-borne spotted fever in humans.

Evidence of exposure to Rickettsia felis in Australian patients.

Rickettsia felis is an emerging zoonosis, causing flea-borne spotted fever (FBSF). Serological diagnosis is typically confounded by cross-reactivity with typhus group rickettsiae and prior to the development of specific serological methods, cases of FBSF in Australia were misdiagnosed. Patient sera tested between August 2010 and December 2013 and known to be seropositive to R. typhi by immunofluorescence antibody testing (IFAT) were subsequently retested against R. felis using an R. felis-specific IFAT. Sera of 49 patients were of a sufficient quality to be included in re-analysis. A classification of FBSF and murine typhus (MT) was attributed to fourteen and seven patients respectively, based on a minimum four-fold higher antibody titre to R. felis than to R. typhi and vice versa. Twenty-eight patients were classified as indeterminate for either R. felis or R. typhi (antibody titres within two-fold of one another). Historically, it is likely that Australian patients clinically ill with FBSF were misdiagnosed. It is important that medical practitioners consider FBSF as part of their differential diagnoses, and obtain relevant history with regard to patient's exposure to domestic pets and their fleas. Australian microbiology diagnostic laboratories should include serological testing for R. felis as part of the diagnostic panel for febrile diseases. Veterinarians are encouraged to increase their awareness of this emerging zoonosis and advocate flea control in pets.