Epidemiology and associated risk factors of giardiasis in a peri-urban setting in New South Wales Australia.

Giardiasis is one of the most important non-viral causes of human diarrhoea. Yet, little is known about the epidemiology of giardiasis in the context of developed countries such as Australia and there is a limited information about local sources of exposure to inform prevention strategies in New South Wales. This study aimed to (1) describe the epidemiology of giardiasis and (2) identify potential modifiable risk factors associated with giardiasis that are unique to south-western Sydney, Australia. A 1:2 matched case-control study of 190 confirmed giardiasis cases notified to the South-Western Local Health District Public Health Unit from January to December 2016 was employed to investigate the risk factors for giardiasis. Two groups of controls were selected to increase response rate; Pertussis cases and neighbourhood (NBH) controls. A matched analysis was carried out for both control groups separately. Variables with a significant odds ratio (OR) in the univariate analysis were placed into a multivariable regression for each matched group, respectively. In the regression model with the NBH controls, age and sex were controlled as potential confounders. Identified risk factors included being under 5 years of age (aOR = 7.08; 95% confidence intervals (CI) 1.02-49.36), having a household member diagnosed with a gastrointestinal illness (aOR = 15.89; 95% CI 1.53-164.60) and having contact with farm animals, domestic animals or wildlife (aOR = 3.03; 95% CI 1.08-8.54). Cases that travelled overseas were at increased risk of infection (aOR = 19.89; 95% CI 2.00-197.37) when compared with Pertussis cases. This study provides an update on the epidemiology and associated risk factors of a neglected tropical disease, which can inform enhanced surveillance and prevention strategies in the developed metropolitan areas.

Description of Dientamoeba fragilis cyst and precystic forms from human samples.

Dientamoeba fragilis is a common enteropathogen of humans. Recently a cyst stage of the parasite was described in an animal model; however, no cyst stage has been described in detail from clinical samples. We describe both cyst and precystic forms from human clinical samples.

Bibliometric analysis of parasite vaccine research from 1990 to 2019.

Bibliometric and bibliographic analyses are popular tools for investigating publication metrics and thematic transitions in an expanding codex of biomedical literature. Bibliometric techniques have been employed in parasitology and vaccinology, with only a few malaria-specific literature analyses being reported specifically on parasite vaccines. The pursuit of parasite prophylactics is an important, global endeavour both medically and economically. As such, a comprehensive understanding of the research topics would be a valuable tool in assessing the current status and future directions of parasite vaccine development. Consequently, this study investigated parasite vaccinology from 1990 to 2019 by analysing literature exported from the Web of Science and Dimensions databases using two, commonly used, bibliometric programs: SciMAT and VOSviewer. The results of this study show the common, emerging, and transient themes within the discipline, and where the future lies as vaccine development moves further into the age of omics and informatics.

In vitro susceptibility testing of Dientamoeba fragilis.

Dientamoeba fragilis is a commonly encountered trichomonad which has been implicated as a cause of gastrointestinal disease in humans. Despite the frequency of reports recording infections with this parasite, little research has been undertaken in terms of antimicrobial susceptibility. The aim of this study was to evaluate the susceptibility of D. fragilis to several commonly used antiparasitic agents: diloxanide furoate, furazolidone, iodoquinol, metronidazole, nitazoxanide, ornidazole, paromomycin, secnidazole, ronidazole, tetracycline, and tinidazole. Antibiotic susceptibility testing was performed on four clinical strains of D. fragilis, designated A, E, M, and V, respectively. Molecular testing followed, and all strains were determined to be genotype 1. The activities of antiprotozoal compounds at concentrations ranging from 2 µg/ml to 500 µg/ml were determined via cell counts of D. fragilis trophozoites grown in dixenic culture. Minimum lethal concentrations (MLCs) were as follows: ornidazole, 8 to 16 µg/ml; ronidazole, 8 to 16 µg/ml; tinidazole, 31 µg/ml; metronidazole, 31 µg/ml; secnidazole, 31 to 63 µg/ml; nitazoxanide, 63 µg/ml; tetracycline, 250 µg/ml; furazolidone, 250 to 500 µg/ml; iodoquinol, 500 µg/ml; paromomycin, 500 µg/ml; and diloxanide furoate, >500 µg/ml. This is the first study to report the profiles of susceptibility to a wide range of commonly used treatments for clinical isolates of D. fragilis. Our study indicated 5-nitroimidazole derivatives to be the most active compounds in vitro against D. fragilis.

New advances in the in-vitro culture of Dientamoeba fragilis.

SUMMARYDientamoeba fragilis is an intestinal protozoan in humans that is commonly associated with diarrhoea and other gastrointestinal complaints. Studies conducted to investigate the biology of this parasite are limited by methods for in vitro cultivation. The objective of this study was to improve a biphasic culture medium, based on the Loeffler's slope, by further supplementation in order to increase the yield of trophozoites in culture. The current in vitro culture of D. fragilis is a xenic culture with a mix of bacteria. Three different liquid overlays were evaluated including Earle's balanced salt solution (EBSS), PBS and Dulbecco's modified PBS (DPBS), for their ability to support the in vitro growth of D. fragilis trophozoites. Out of these 3 overlays EBSS gave the highest increase in the trophozoite numbers. The effect of supplementation was analysed by supplementing EBSS with ascorbic acid, ferric ammonium citrate, L-cysteine, cholesterol and alpha-lipoic acid and quantification of in vitro growth by cell counts. A new liquid overlay is here described based upon EBSS supplemented with cholesterol and ferric ammonium citrate that, in conjunction with the Loeffler's slope, supports the growth of D. fragilis trophozoites in vitro. This modified overlay supported a 2-fold increase in the numbers of trophozoite in culture from all 4 D. fragilis isolates tested, when compared to a PBS overlay. These advances enable the harvest of a larger number of trophozoites needed for further studies on this parasite.

Importance of nonenteric protozoan infections in immunocompromised people.

There are many neglected nonenteric protozoa able to cause serious morbidity and mortality in humans, particularly in the developing world. Diseases caused by certain protozoa are often more severe in the presence of HIV. While information regarding neglected tropical diseases caused by trypanosomatids and Plasmodium is abundant, these protozoa are often not a first consideration in Western countries where they are not endemic. As such, diagnostics may not be available in these regions. Due to global travel and immigration, this has become an increasing problem. Inversely, in certain parts of the world (particularly sub-Saharan Africa), the HIV problem is so severe that diseases like microsporidiosis and toxoplasmosis are common. In Western countries, due to the availability of highly active antiretroviral therapy (HAART), these diseases are infrequently encountered. While free-living amoebae are rarely encountered in a clinical setting, when infections do occur, they are often fatal. Rapid diagnosis and treatment are essential to the survival of patients infected with these organisms. This paper reviews information on the diagnosis and treatment of nonenteric protozoal diseases in immunocompromised people, with a focus on patients infected with HIV. The nonenteric microsporidia, some trypanosomatids, Toxoplasma spp., Neospora spp., some free-living amoebae, Plasmodium spp., and Babesia spp. are discussed.

Evaluation of multiplex tandem real-time PCR for detection of Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis in clinical stool samples.

The aim of this study was to describe the first development and evaluation of a multiplex tandem PCR (MT-PCR) assay for the detection and identification of 4 common pathogenic protozoan parasites, Cryptosporidium spp., Dientamoeba fragilis, Entamoeba histolytica, and Giardia intestinalis, from human clinical samples. A total of 472 fecal samples submitted to the Department of Microbiology at St. Vincent's Hospital were included in the study. The MT-PCR assay was compared to four real-time PCR (RT-PCR) assays and microscopy by a traditional modified iron hematoxylin stain. The MT-PCR detected 28 G. intestinalis, 26 D. fragilis, 11 E. histolytica, and 9 Cryptosporidium sp. isolates. Detection and identification of the fecal protozoa by MT-PCR demonstrated 100% correlation with the RT-PCR results, and compared to RT-PCR, MT-PCR exhibited 100% sensitivity and specificity, while traditional microscopy of stained fixed fecal smears exhibited sensitivities and specificities of 56% and 100% for Cryptosporidium spp., 38% and 99% for D. fragilis, 47% and 97% for E. histolytica, and 50% and 100% for G. intestinalis. No cross-reactivity was detected in 100 stool samples containing various other bacterial, viral, and protozoan species. The MT-PCR assay was able to provide rapid, sensitive, and specific simultaneous detection and identification of the four most important diarrhea-causing protozoan parasites that infect humans. This study also highlights the lack of sensitivity demonstrated by microscopy, and thus, molecular methods such as MT-PCR must be considered the diagnostic methods of choice for enteric protozoan parasites.

A case-controlled study of Dientamoeba fragilis infections in children.

Dientamoeba fragilis is a pathogenic protozoan parasite that is implicated as a cause of human diarrhoea. A case-controlled study was conducted to determine the clinical signs associated with D. fragilis infection in children presenting to a Sydney Hospital. Treatment options are also discussed. Stool specimens were collected from children aged 15 years or younger and analysed for the presence of D. fragilis. In total, 41 children were included in the study along with a control group. Laboratory diagnosis was performed by microscopy of permanently stained, fixed faecal smears and by real-time PCR. Gastrointestinal symptoms were present in 40/41 (98%) of these children with dientamoebiasis, with diarrhoea (71%) and abdominal pain (29%) the most common clinical signs. Chronic gastrointestinal symptoms were present in 2% of cases. The most common anti-microbial used for treatment was metronidazole (n=41), with complete resolution of symptoms and clearance of parasite occurring in 85% of cases. A treatment failure rate occurred in 15% of those treated with metronidazole. Follow-up treatment comprised of an additional course of metronidazole or iodoquinol was needed in order to achieve complete resolution of infection and symptoms in this group. This study demonstrates the pathogenic potential of D. fragilis in children and as such it is recommended that all laboratories must routinely test for this organism and treat if detected.

Clinical significance of enteric protozoa in the immunosuppressed human population.

Globally, the number of immunosuppressed people increases each year, with the human immunodeficiency virus (HIV) pandemic continuing to spread unabated in many parts of the world. Immunosuppression may also occur in malnourished persons, patients undergoing chemotherapy for malignancy, and those receiving immunosuppressive therapy. Components of the immune system can be functionally or genetically abnormal as a result of acquired (e.g., caused by HIV infection, lymphoma, or high-dose steroids or other immunosuppressive medications) or congenital illnesses, with more than 120 congenital immunodeficiencies described to date that either affect humoral immunity or compromise T-cell function. All individuals affected by immunosuppression are at risk of infection by opportunistic parasites (such as the microsporidia) as well as those more commonly associated with gastrointestinal disease (such as Giardia). The outcome of infection by enteric protozoan parasites is dependent on absolute CD4(+) cell counts, with lower counts being associated with more severe disease, more atypical disease, and a greater risk of disseminated disease. This review summarizes our current state of knowledge on the significance of enteric parasitic protozoa as a cause of disease in immunosuppressed persons and also provides guidance on recent advances in diagnosis and therapy for the control of these important parasites.

Newly defined conditions for the in vitro cultivation and cryopreservation of Dientamoeba fragilis: new techniques set to fast track molecular studies on this organism.

Dientamoeba fragilis is a pathogen of the human gastrointestinal tract that is a common cause of diarrhoea. A paucity of knowledge on the in vitro cultivation and cryopreservation of Dientamoeba has meant that few studies have been conducted to investigate its biology. The objective of this study was to define, for the first time, in vitro culture conditions able to support the long-term in vitro growth of Dientamoeba. Also, we aimed to define a suitable method for cryopreserving viable Dientamoeba trophozoites. A modified BD medium, TYGM-9, Loeffler's slope medium, Robinson's medium, Medium 199, Trichosel and a Tritrichomonas fetus medium were compared, using cell counts, for their ability to support the growth of D. fragilis at various temperatures and atmospheric conditions. Loeffler's slope medium supported significantly better growth compared to other media. A temperature of 42°C and a microaerophilic atmosphere were also optimum for Dientamoeba growth. To our knowledge, this is the first study to describe and compare different culture media and conditions for the growth of clinical isolates of D. fragilis. This new technology will aid the development of diagnostics for dientamoebiasis as well as facilitate large-scale sequencing projects that will fast track molecular studies on D. fragilis.

Global carbon sinks and their variability inferred from atmospheric O2 and delta13C.

Recent time-series measurements of atmospheric O2 show that the land biosphere and world oceans annually sequestered 1.4 +/- 0.8 and 2.0 +/- 0.6 gigatons of carbon, respectively, between mid-1991 and mid-1997. The rapid storage of carbon by the land biosphere from 1991 to 1997 contrasts with the 1980s, when the land biosphere was approximately neutral. Comparison with measurements of delta13CO2 implies an isotopic flux of 89 +/- 21 gigatons of carbon per mil per year, in agreement with model- and inventory-based estimates of this flux. Both the delta13C and the O2 data show significant interannual variability in carbon storage over the period of record. The general agreement of the independent estimates from O2 and delta13C is a robust signal of variable carbon uptake by both the land biosphere and the oceans.

Genetic diversity amongst isolates of Neospora caninum, and the development of a multiplex assay for the detection of distinct strains.

Infection with Neospora caninum is regarded as a significant cause of abortion in cattle. Despite the economic impact of this infection, relatively little is known about the biology of this parasite. In this study, mini and microsatellite DNAs were detected in the genome of N. caninum and eight loci were identified that each contained repetitive DNA which was polymorphic among different isolates of this parasite. A multiplex PCR assay was developed for the detection of genetic variation within N. caninum based on length polymorphism associated with three different repetitive markers. The utility of the multiplex PCR was demonstrated in that it was able to distinguish amongst strains of N. caninum used as either vaccine or challenge strains in animal vaccination experiments and that it could genotype N. caninum associated with naturally acquired infections of animals. The multiplex PCR is simple, rapid, informative and sensitive and should provide a valuable tool for further studies on the epidemiology of N. caninum in different host species.

Neosporosis and hammondiosis in dogs.

The dog is a definitive host of the protozoan parasite Neospora caninum, and in many parts of the world, infection is relatively common as determined by serology. Reported seroprevalences usually range from 0 to 20 per cent, however, reports of clinically affected dogs are infrequent. Affected dogs are generally less than six months old and predominantly have signs of an ascending hindleg paralysis, with the associated lesions of polyradiculoneuritis and granulomatous polymyositis. Although any organ may be affected, infections are more common in the central nervous system, muscles, lungs and skin. Ante-mortem diagnosis is difficult but serology and cytology can aid diagnosis. The diagnosis can be confirmed by histology, immunohistochemistry, the use of molecular techniques on biopsy material, or on post-mortem examination. Neospora caninum oocysts are rarely found in faeces and must be differentiated from oocysts of related coccidians such as Hammondia heydorni and Toxoplasma gondii. Hammondia heydorni can cause diarrrhoea in immunosuppressed dogs. Neosporosis should be suspected in young pups with an ascending paralysis of the hindlegs. Treatment with clindamycin and potentiated sulphonamides may be useful in cases where muscular atrophy and fibrosis are absent. Feeding of raw meat is a potential risk factor for infection of dogs and should be discouraged.

Prevalence of Neospora caninum infection in Australian (NSW) dairy cattle estimated by a newly validated ELISA for milk.

AIM: To determine the performance characteristics of an Institut Pourquier (IP) enzyme-linked immunosorbent assay (ELISA) for the detection of antibodies against Neospora caninum in bovine milk and subsequent determination of the prevalence of N. caninum infection in New South Wales (NSW) dairy cattle. METHODS: Matching serum and milk samples from 93 cattle were assayed in two commercially available ELISAs for the detection of anti-N. caninum antibodies. Serum test results of one ELISA (IDEXX) were used to determine the N. caninum infection status of the cattle. Optimised cut-off values for the IP ELISA using milk samples were determined by two-graph receiver operating characteristic (TG-ROC) analysis and then applied to a representative sample of 398 milk samples from dairy herds around NSW. RESULTS: When this ELISA was applied to a representative collection of 398 milk samples from dairy cattle across NSW it demonstrated a 21.1% prevalence of N. caninum infection in those cattle. From the TG-ROC analysis an IP ELISA protocol was derived which suggested a cut-off threshold that would allow milk testing with 97% sensitivity and specificity, respectively, relative to serum testing. CONCLUSIONS: The prevalence of N. caninum in NSW dairy cattle was higher than previously believed. When used on individual milk samples this ELISA demonstrated high sensitivity and specificity and so could be used to accurately identify N. caninum infection. TG-ROC analysis of the IP ELISA optimised the protocol and prescribed cut-off values enabling the ELISA to be used for the screening of N. caninum antibodies in the milk of dairy cattle.

Performance characteristics and optimisation of cut-off values of two enzyme-linked immunosorbent assays for the detection of antibodies to Neospora caninum in the serum of cattle.

AIM: To determine the performance characteristics of two enzyme-linked immunosorbent assays (ELISAs) manufactured by Institut Pourquier (IP) for the detection of antibodies against Neospora caninum in bovine sera. METHODS: Sera from 526 cattle were assayed in two ELISAs (IP) for the detection of anti-N. caninum antibodies. Results from a further ELISA (IDEXX) were used to provide the "gold standard"N. caninum infection status of the cattle and the ELISA results assessed by two-graph receiver operating characteristic (TG-ROC) analysis. RESULTS: TG-ROC analysis suggested changes to one of the IP ELISA protocols, arriving at a cut-off threshold that was different to the one recommended by the manufacturer. With that change, both of the ELISAs performed with high sensitivity and specificity (in excess of 98%) for bovine sera. CONCLUSIONS: The analysis of the two IP ELISAs when used on individual bovine sera demonstrated high sensitivity and specificity. TG-ROC analyses optimised the cut-off point suggested by the manufacturer for one of these commercial diagnostic assays and found agreement with the manufacturer's cut-off regarding the other assay. This will help with the accurate identification of infected animals and thereby contributing to the control of neosporosis.

Neospora abortions in dairy cattle: diagnosis, mode of transmission and control.

AIM: To determine the contribution of Neospora caninum to abortions on a dairy farm in NSW (Australia), determine the mode of transmission and develop and trial a control option for infection. METHODS: Two whole herd bleeds were conducted 12 months apart and the association between serological status and abortion events were calculated for a number of bovine abortifacients. Family trees were constructed for N. caninum seropositive cattle in the herd. Some N. caninum seropositive cows were culled from the herd and no female offspring was retained from seropositive cows. RESULTS: At the first whole-herd bleed in December 2002 a seroprevalence of 10.2% for N. caninum infection was detected. Cows with N. caninum infection were 13 times more likely to abort than uninfected ones. Seventy-five percent of seropositive animals in the herd were related, suggesting a high degree of congenital infection/transmission. Only 15% of infections were likely to be postnatally acquired. Selective culling of seropositive cows and not breeding from them reduced the number of seropositive animals. Only one newly sero-converted cow was detected at the second whole-herd bleed 12 months later. CONCLUSIONS: Seroepidemiological approaches were able to establish a high degree of association between N. caninum infection and low-level abortion in the dairy herd. Vertical transmission of infection was the predominant mode of infection and hence control efforts aimed at selectively culling seropositive animals from the herd were highly successful in reducing the level of infection.

The receptor for advanced glycation end products (RAGE) enhances autophagy and neutrophil extracellular traps in pancreatic cancer.

Neutrophil extracellular traps (NETs) are formed when neutrophils expel their DNA, histones and intracellular proteins into the extracellular space or circulation. NET formation is dependent on autophagy and is mediated by citrullination of histones to allow for the unwinding and subsequent expulsion of DNA. NETs have an important role in the pathogenesis of several sterile inflammatory diseases, including malignancy, therefore we investigated the role of NETs in the setting of pancreatic ductal adenocarcinoma (PDA). Neutrophils isolated from two distinct animal models of PDA had an increased propensity to form NETs following stimulation with platelet activating factor (PAF). Serum DNA, a marker of circulating NET formation, was elevated in tumor bearing animals as well as in patients with PDA. Citrullinated histone H3 expression, a marker of NET formation, was observed in pancreatic tumors obtained from murine models and patients with PDA. Inhibition of autophagy with chloroquine or genetic ablation of receptor for advanced glycation end products (RAGE) resulted in decreased propensity for NET formation, decreased serum DNA and decreased citrullinated histone H3 expression in the pancreatic tumor microenvironment. We conclude that NETs are upregulated in pancreatic cancer through RAGE-dependent/autophagy mediated pathways.

Studies of the bone marrow in polycythemia vera and the evolution of myelofibrosis and second hematologic malignancies.

The PVSG study is unique in that it is prospective and composed of 432 patients randomized to three treatment arms. This study also provides the opportunity for serial studies of numerous sequential biopsies. Large numbers of cases with sequential biopsies covering the entire long course are essential to appreciate the full spectrum of tissue changes in this disease. The PVSG was initiated in 1967 and in mid-1985 approximately one third of the patients are alive and on protocol. For these reasons, the results must still be considered preliminary. Pretreatment biopsies from patients randomized in the PVSG have been analyzed for total cellularity, megakaryocyte concentration, and reticulin content. Considerable variation in these elements was found in these biopsies. Sequential posttreatment biopsies from these patients have also been studied and correlated with the clinical course of the disease. None of the morphologic parameters analyzed was shown to be of prognostic significance. Early in the course of PV the marrow reticulin content is almost always normal. The length of the developmental stage is unknown and the precise timing of the clinical onset may be difficult. Therefore, the 11% of patients that showed a significant increase in reticulin on initial evaluation may have had PV longer than was indicated clinically. If large numbers of sequential biopsies are studied, an increase in reticulin content can frequently be demonstrated during the active phase of the disease and before the onset of the spent phase. Currently 39 patients (9%) have developed the spent phase, or PPMM. PPMM occurred in about the same incidence in the patients treated with myelosuppressive therapy as by phlebotomy alone, the spent phase occurring in 16 patients treated by phlebotomy alone, 11 with chlorambucil, and 12 with 32P. The course of the reticulin fibrosis is slowly progressive. There is some evidence for regression in a few patients in the erythrocytotic phase, but sampling variation cannot be completely ruled out. At this time in the study, AL has developed in 37 patients (8.6%). The incidence of AL is quite low in the phlebotomy group (three cases). Presumably this represents the natural incidence in PV unmodified by therapeutic agents. The frequency is approximately equal and quite high in the chlorambucil and 32P groups. There are 19 cases in the chlorambucil-treated group and 15 in the 32P-treated group. The leukemias that developed in the PV patients occurred either de novo or following PPMM.(ABSTRACT TRUNCATED AT 400 WORDS)