COMPARISON OF KNOWLEDGE AMONG THE MEDICAL AND ODONTOLOGY STUDENTS OF DIFFERENT YEARS AT LUHS ABOUT THE MOST LIKELY PATHWAYS OF TOXOPLASMA GONDII INFECTION

(1)

1

COMPARISON OF KNOWLEDGE AMONG THE MEDICAL

AND ODONTOLOGY STUDENTS OF DIFFERENT YEARS AT

LUHS ABOUT THE MOST LIKELY PATHWAYS OF

TOXOPLASMA GONDII INFECTION

A thesis submitted in partial fulfilment for the medical study programme

MASTER'S THESIS OF INTEGRATED MEDICAL STUDIES

Author: Joshua Steve Jacob Supervisor: Dr. Lina Mickienė

KAUNAS 2020

LITHUANIAN UNIVERSITY OF HEALTH SCIENCES

VETERINARY ACADEMY

FACULTY OF ANIMAL SCIENCES

(2)

2

SUMMARY

Author: Joshua Steve Jacob

Scientific supervisor: Dr. Lina Mickienė

Research title: Comparison of knowledge among the Medical and Odontology students of different years of LUHS about the most likely pathways of Toxoplasma gondii infection. Aim: This study seeks to investigate, evaluate and draw comparisons between odontology and medical faculty students (OF and MF) of the Lithuanian University of Health Sciences (LUHS), in terms of their knowledge, attitudes and exposure to the risk factors of Toxoplasma Gondii (T. gondii) infection.

Objectives: To this end, the main objectives of the study have been enumerated as follows; 1. To compare MF and OF student’s knowledge about zoonotic risk as well as the

infectious pathways of T. gondii parasite.

2. To test if self-estimation about the knowledge of possible T. gondii infection pathways relates with the actual knowledge of the MF and OF students.

3. To determine the total number of cat-owners among the students and evaluate attitudes towards relevant aspects of cat ownership.

4. To compare the student’s attitudes regarding personal hygiene measures. Participants: Students of LUHS from both MF and OF.

Material and methods: the investigation was by design, a cross-sectional survey within which students were required to honestly, individually and anonymously answer a variety of topic-specific questions. In order to achieve this, a suitable questionnaire was formulated and disseminated to the target population.

Results: 414 viable responses were analysed meticulously and indicated that 59.7% of the sample population were cat-owners, of which the majority were MF students (77.3%). Cat-handling behaviour was moderately risky across both groups, in comparison to attitudes towards hand hygiene and general T. gondii prophylaxis. The results also indicate that OF students were marginally more pro-active than MF students with regards to these prophylactic measures. In terms of knowledge, OF students outperformed MF students scoring an average

(4)

4 of 8.7 compared to MF’s 8.0. However, OF students predominantly underestimated their performance while MF students, on average, accurately estimated their performance.

Conclusion: it can be inferred from this investigation, that even a well-educated demographic, instructed with T. gondii specific knowledge, can overlook the important details of infection and consequently expose themselves to risk factors of disease. Though there is indeed room for improvement, quite generally attitudes towards prophylaxis are prudent, knowledge is above average and self-estimation is almost accurate.

Recommendations: knowledge of toxoplasmosis and other zoonotic infections should be reinforced through workshops and/or conferences; public institutions should develop and increase funding for improving pathogen control; further research of T. gondii is necessary to bridge current gaps in knowledge and it should be effectively conveyed to all members of the public sphere.

(5)

5

ACKNOWELEDGEMENTS

First and foremost, I would like to express my deepest appreciation to Dr. Lina Mickienė for her patience, guidance, support and encouragement throughout this endeavour. Her assistance enabled me to not only efficiently collect data, but also develop a keen understanding and interest of the subject.

(6)

6

CONFLICT OF INTEREST

(7)

7

ETHICS COMMITTEE CLEARANCE

Ethics approval for this research was granted by Bioethics centre in the Lithuanian University of Health Sciences (LUHS), on the 5th_{of March 2019. The ethics application number is}

(8)

(9)

9

ABBREVIATIONS LIST

2D 2-dimensional

3D 3-dimensional

ABA Abscisic acid

AIDS Acquired immunodeficiency syndrome AMA1 Apical membrane antigen-1

ART Antiretroviral treatment CD Cluster of differentiation CNS Central nervous system COVID-19 Coronavirus disease 2019 CRP C-reactive protein

DC Dendritic cells

DS Double dose

ER Endoplasmic reticulum GPI Glycosylphosphatidylinositol GRA Dense granule proteins

HIV Human immunodeficiency virus IFN-γ Interferon-γ

IL-12 Interleukin-12

IRG Immunity regulated GTPase LC3 Light-chain 3

LD Lethal dose

LUHS Lithuanian University of Health Sciences

MF Medical faculty

MIC Microneme proteins

MJ Moving junction

NO Nitric oxide

NK Natural killer NTZ Nitazoxanide OF Odontology faculty

PCR Polymerase chain reaction PV Parasitophorous vacuole PVM Parasitophorous vacuole

membrane

RON Rhoptry neck proteins ROP Rhoptry bulb proteins ROS Reactive oxygen species SAG Surface antigens

SARS-CoV-2 Severe acute respiratory syndrome coronavirus 2

STAT-1 Signal transducer and activator of transcription 1 TE Toxoplasmic encephalitis TLR Toll-like receptors TMP-SMX

(10)

10

KEY TERMS

T. gondii; obligate intracellular parasite; T. gondii infection pathways; coccidia; cat owner and non-cat owner; life cycle; definitive host; intermediate host; oocyst; sporulated oocyst; unsporulated oocyst; sporozoite; tissue cyst; tachyzoite; bradyzoite; toxoplasmosis; ocular toxoplasmosis; toxoplasmic encephalitis; autophagy; cell-mediated immunity; humoral immunity; CD 4; CD 8; IL-12; seroprevalence.

(11)

11

1. INTRODUCTION

In the era of the coronavirus disease 2019 (COVID-19) pandemic, it has become apparent that the vast majority of emerging infectious diseases afflicting humanity are zoonoses. A firm understanding of the factors that influence pathways of infection is fundamental in their prevention and control. Globally, T. gondii is one of the most prevalent zoonotic pathogens recognised today. Toxoplasmosis is the consequence of T. gondii infection and it affects over one-third of the world’s population [1]. Infection by this coccidian protozoan parasite can lead to congenital and acquired disease, with both forms having a significant effect on patient mortality and their quality of life. Thus, this parasite and its disease process are important public health issues.

T. gondii is an obligate intracellular parasite and it is virulent to all homeothermic animals, of which some birds and most mammals, including humans, are the intermediate host. T. gondii can only complete its life cycle (i.e. sexually reproduce) in members of the family Felidae, which are therefore the definitive hosts. Felids are capable of excreting copious amounts of oocysts that contain the infectious sporozoites into the environment, through their faeces. If so much as a single oocyst or tissue cyst is ingested by an intermediate host, T. gondii may then asexually reproduce and systemically disseminate to all tissues [2]. The life cycle achieves completion when the tissues of the intermediate host are ingested by a feline and sexual reproduction can once again begin [1–5].

In humans, infection generally occurs through 3 routes; undercooked meat consumption, faecal-oral transmission and congenital infections. There are also rare cases of T. gondii transmission through blood transfusions with tachyzoites or organ transplantation with encysted bradyzoites. The toxoplasmic infection is usually asymptomatic due to effective immune system control, however, severe complications, and even death might follow as a result of a congenital infection or in immunocompromised patients (e.g., AIDS, transplantation). This emphasises the significance of immunocompetence in controlling T. gondii infection [2]. Cases of congenital infection and human immunodeficiency virus (HIV) coinfection cause the most serious morbidity, resulting in severe neurologic and ocular diseases.

For any nation, T. gondii infection has substantial socioeconomic implications; estimates suggest that around 2-3 billion people worldwide have been infected by T. gondii and it is

(12)

12 approximated that this ranges from: 8-22% of the people in the USA, a similar prevalence in the UK and 30-90% of the Latin American and continental European populations [2,6]. In the USA alone, it has been estimated that just over 1 million people are infected annually and from foodborne illnesses, T. gondii has been attributed to causing approximately 8% of hospitalisations and 24% of deaths [2]. The infected as well as their families accrue many costs in the lifelong management of the disease and the total burden of illness caused by T. gondii is estimated to be almost $3 billion ($11,000 quality-adjusted life-year) [2,7,8].

There are no simple solutions to mitigate the regional or global problems that toxoplasmosis presents; however, evidence suggests that a greater institutional awareness of the pathways of infection, meticulous peri-natal screening protocols and comprehensive hygiene practices are effective. Lack of knowledge and poor communication of the risks and pathways of exposure have been identified as major factors for infection [2]. It is thus the objective of this study to thoroughly evaluate an educated European demographic’s (i.e. students at LUHS) knowledge of this elusive parasite as well as its treatment and prevention strategies. This investigation also seeks to identify the frequency of cat-owners and evaluate general attitudes towards the various aspects of cat ownership. Students of Odontology and Medical faculties were particularly chosen as they are taught about toxoplasmosis from the first academic year. This investigation will provide invaluable data on determining the effectiveness of the teaching process, as well as the risk factors to which students themselves are most likely exposed.

(13)

13

2. AIMS AND OBJECTIVES

Aim: This study seeks to investigate, evaluate and draw comparisons between OF and MF students of LUHS, in terms of their knowledge, attitudes and exposure to the risk factors of T. gondii infection.

Objectives: To this end, the main objectives of the study have been enumerated as follows; 1. To compare MF and OF student’s knowledge about zoonotic risk as well as the

infectious pathways of T. gondii parasite.

2. To test if self-estimation about the knowledge of possible T. gondii infection pathways relates with the actual knowledge of the MF and OF students.

3. To determine the total number of cat-owners among the students and evaluate attitudes towards relevant aspects of cat ownership.

(14)

14

3. LITERATURE REVIEW

3.1 History of Toxoplasma Gondii

The intracellular protist T. gondii belongs to the phylum Apicomplexa, which is a diverse group of parasitic eukaryotes characterised by their cytoplasmic fluid filled sacs. The parasite was first extracted and isolated in 1908 by Charles Nicolle and Louis Manceaux from the liver of the Tunisian common gundi (Ctenodactylus gundi) [2]. In the same year it was also discovered in Brazil by Alfonso Splendore, from the tissues of a South American species of rabbit. Nicolle and Manceaux named the parasite in reference to its morphology and the original host species: Toxo, from Greek (toxon, “arc, bow”) and plásma (plasma, “shape, form”) and gundi (gondii) [2].

It has been hypothesised that T. gondii originated and evolved in the Amazon rainforest. Currently it is the region with the highest known infection rates with a seroprevalence greater than 90%. The main postulation regarding its current worldwide distribution is that shipping traffic, due to trade, promoted the migration of domestic cats and other infected intermediate hosts, particularly rats and mice [9]. Migratory birds may also have had a significant role in transmission by acting as intermediate hosts for the parasite [2,9,10].

Prior to agriculture, T. gondii transmission is presumed to have occurred through a wide variety of wild cats and intermediate hosts and this parasitic cycle of development has been termed the sylvatic life cycle [9]. During this period, pre-agrarian intermediate hosts were comprised of very low numbers of the rodent house mouse (Mus musculus). Zoo-archaeological studies indicate that 12,000 years ago human societies were largely hunter-gatherers and mice were generally less prevalent [9]. However, in the advent of agriculture around 11,000 years ago, murine species begun to thrive through their commensalism with humans [9]. This association with humans likely developed as a consequence of increased rodent access to highly nutritious grain stores in human settlements. Genetic assessment of domestic cats and their wild progenitors have suggested that their domestication coincided with the rise of agrarian communities in the Middle East where they likely began feeding on the rodent pests that infested grain stores of the first farmers [9,11]. This pattern is conjectured to continue throughout the agricultural era, since food abundance inevitably led to rodent infestation and pest control most probably incentivised cat domestication. Thus, the sylvatic life cycle of T. gondii progressed into the domestic life cycle and the parasite adapted to the new

(15)

predator-15 prey relationship between cats and mice within human settlements. Both cat and mouse population densities within anthropic environments surged globally and are likely to have led to high levels of oocyst contamination and consequently increased infection rates [11]. Although, T. gondii was first recognised in the 1920s in infants presenting with encephalitis, infection in humans was only conclusively described in 1938 when an autopsy in New York revealed lesions in the brain and eyes of a paediatric patient [12]. The child had been delivered by Caesarean section but had begun seizing at the 3 days of age and eventually died at 1 month of age [12]. The lesions found in the autopsy were found to have both free and intracellular T. gondii. Congenital transmission was later observed in a number of other species, especially in sheep and rodents. It was then only in 1954 that transmission through undercooked meat consumption was considered as a possible route of infection [3]. It remained as a hypothetical route of T. gondii transmission until it was empirically tested and verified in Paris by the year 1965. Though postulated in 1959, faecal-oral transmission was only confirmed in humans by 1970. Toxoplasmosis emerged as a major opportunistic infection in AIDS patients in the 1980s, clinically manifesting as severe encephalitis [12]. This highlighted the significance of latent infection reactivation in the setting of HIV-related immunosuppression as well as in generally immunocompromised individuals [12].

3.2 Epidemiology and population structure

Around 2-3 billion people globally are afflicted by latent toxoplasmosis. The incidence of toxoplasmosis differs, however, with less economically developed countries having a higher incidence than developed countries. Regions of high prevalence exist in Latin America, parts of Eastern/Central Europe, the Middle East, and parts of south-east Asia and Africa [2]. As stated before, in the US seroprevalence is 8- 22% which is a slightly less than the previous decade and consistent with the same trend in Europe [2,13]. This variation in prevalence can be due to several factors including the presence and quantity of cats, climate, ethnic and cultural practices. Prevalence tends to be increased in moderately warm and humid countries because oocysts lose their virulence when frozen or dried. The seroprevalence for T. gondii in cats is 30-40% with regional prevalence varying with climate; prevalence is lowest in dry southwestern areas (16.1 % in New Mexico, Utah and Arizona) and highest in wet regions (59.2% for Hawaii) [1,2,14,15]. Cooking methods are also a crucial factor i.e. whether meats are prepared well-done or rare, as well as the washing of fruits and vegetables. In the last decade

(16)

16 investigations in the US have revealed that eating raw oysters, clams or mussels are also a risk factor for T. gondii infection [16].

T. gondii is among the world’s most successful zoonotic pathogens and although it has effectively achieved worldwide spread, the parasite’s distribution remains unique in that it maintains genotypic diversity and virulence through its South American clades whereas all other regions have essentially clonal populations which are also characteristically less virulent [3,25]. Historically, T. gondii isolates were considered to be highly similar, however modern molecular analysis have displayed marked clonality, particularly in North America and Europe where 4 archetypal lineages predominate: types 1 ,2, 3 and 12 [17]. Recent investigations indicate comparable population structures in Africa [17]. Conversely, South America is populated by a variety of lineages which show divergence between groups and diversity within groups [3]. As such, no dominating genotype has been found in South America. This biphasic pattern of global distribution therefore suggests that T. gondii propagates mainly clonally in the northern hemisphere and largely through sexual recombination in South America [17]. The type 1 strain is considered the most virulent with a lethal dose of one single parasite in mice (LD100 = 1), whereas type 2 and type 3 are considered intermediate or low virulence strains

[17]. Type 12 strains are distinct from the other 3 in that it has a range of pathogenesis including those with low virulence as well as those with intermediate to high levels of cumulative mortality. Type 2 strains are responsible for the vast majority of human infections, which probably reflects the abundance of these strains in livestock [17].

3.3 Host-parasite interactions

3.3.1 Domestic life cycle

As aforementioned, T. gondii is an obligate intracellular protozoan parasite capable of infecting all warm-blooded vertebrates [2]. The parasite has a unique and complex life cycle which is characterised by both sexual and asexual reproduction. Sexual reproduction can only take place within wild or domestic felids, while its asexual replication can take place in virtually all warm-blooded animals [18]. Thus, the former are definitive hosts and the latter are intermediate hosts. It is largely because of its remarkably broad spectrum of intermediate hosts, that the parasite was able to become so prominent across the world.

In its primordial form T. gondii strains originally followed the sylvatic life cycle, however at present in most of the world, and particularly the northern hemisphere, the domestic life cycle

(17)

17 predominates. The domestic life cycle allows the parasite to thrive in anthropic environments which are plentiful in domesticated pets -notably felids, house mice, livestock and humans. The parasite has a number of forms, each with morphological peculiarities that enable it to survive in various environments and multiply within hosts. These forms include tachyzoites, merozoites, bradyzoites and sporozoites [19]. There are also the bradyzoite containing tissue cysts and the oocysts within which the sporozoites reside. The asexual phase usually occurs in the intermediate hosts when cyst-contaminated food or water is consumed and T. gondii disseminates throughout the host, stage-converting into tissue cysts [20]. Although this form can manifest in most tissues, they seem to have a predilection for the brain or muscle cells [21]. In contrast, the sexual cycle takes place within feline enteroepithelial cells, terminating when environmentally resistant oocysts are excreted with the feces.

Recent studies indicate that linoleic acid is critical for the gametogony of T. gondii and all Felidae have an excess of this fatty acid due to the absence of delta-6-desaturase in their bowels [22]. In fact, cats are the only mammals deficient of this enzyme and it is for this reason they serve as the parasite’s definitive host. Domestic cats are typically infected when ingesting tissue cysts; the bradyzoites are released from ingested tissue cysts by pepsin and acid digestion within the stomach. Bradyzoites subsequently infiltrate the feline intestinal epithelial cells and undergo sexual reproduction. In 2 days merozoites form which proliferate and then differentiate into micro- and macrogametes that eventually fuse to produce millions of zygote-containing unsporulated oocysts. Eventually infected epithelial cells burst open, releasing these oocysts into the intestinal lumen, whereupon they migrate to the rectum and are passed with the stools [2,20,22,23]. Cats excrete these oocysts 5-10 days post-infection and can expel from 2-20 million oocysts per day through their defecation [23]. Under ambient conditions, the oocysts go through a sporulation process to mature and become infectious. Within each oocyst both meiotic and mitotic division of the zygotes takes place, producing 8 sporozoites encased within the oocyst-walls. These durable thick-walled sporulated oocysts can persist in the environment, remaining infectious for up to 18 months [2]. It is important to note that cats can be infected when ingesting not only tissue cysts but also oocysts and tachyzoites. However, fewer than 30% of cats shed oocysts after ingesting tachyzoites or oocysts, whereas, nearly all cats shed oocysts after ingesting tissue cysts [22,24]. Additionally, the prepatent period (i.e. the time taken until oocysts are produced and excreted) varies depending on which form ingested; tissue cysts being the shortest followed by tachyzoites at 13 days and oocysts taking up to 18 days [22].

(18)

18 Oocysts are efficiently adapted to infect nearly all major orders of placental mammals, excluding fruit eating bats, insectivores and baleen whales [20]. Initial infection of the intermediate host commonly occurs when oocysts cysts are ingested. The cyst wall is broken down by proteolytic enzymes in the bowels releasing sporozoites which readily invade intestinal epithelium [24]. The parasites then differentiate into tachyzoites, the rapidly replicating form of the organism. These tachyzoites then disseminate through systemic circulation and preferentially form bradyzoite tissue cysts in brain and muscle tissue. Intermediate host infection by tissue cysts occurs when raw meat containing the organism is consumed. Post-digestion, bradyzoites will then be released into the gut, penetrate enteroepithelial cells and then in the absence of linoleic acid, will stage-convert into tachyzoites [22]. The tachyzoites will then enact the aforementioned disseminative pattern within the intermediate host. Despite T. gondii’s flexibility in transmission, the oocysts are more infectious for intermediate hosts than the tissue cysts. T. gondii can thus be passed asexually between intermediate hosts indeterminately via a cycle of tissue cyst consumption; however, the lifecycle can only begin and complete when the parasite reaches a feline host within whom it can once again sexually reproduce [19].

3.3.2 Cellular stages

Sporozoite (found in oocysts), bradyzoite (found in tissue cysts), merozoite and tachyzoite all refer to the various cellular morphological forms undertaken by the parasite at different periods of its life cycle. Although, all 4 forms are crescent-shaped, each one has its own unique structural, biochemical and behavioural traits.

Frenkel JK coined the term tachyzoite and it refers to the rapidly multiplying form of the parasite [24]. It quickly replicates in any host cell within intermediate hosts and in non-intestinal epithelial cells of the definitive host [25]. Collections of several tachyzoites are described as terminal colonies, clones or groups. The tachyzoite has a somewhat crescent shape and is around 2x6μm in size. Its anterior portion is conoidal while its posterior end is rounded in shape. The tachyzoite contains a number of distinct organelles including a pellicle, apical rings, rhoptries, micropores, conoid, micropore nucleus, micronemes, dense granules, polar rings, mitochondrion, subpellicular microtubules, endoplasmic reticulum (ER), Golgi complex, ribosomes, rough and smooth ER, amylopectin granules and an apicoplast [26]. The parasite’s nucleus is at the core of the cell and contains chromatin clumps around its central nucleolus. During the acute phase of infection tachyzoites distribute throughout the body via systemic

(19)

19 circulation, whereas in the latent chronic phase they stage-convert into bradyzoites lying within tissue cysts.

Bradyzoite, also coined by Frenkel JK, describes the quiescent slow replicating form of the organism within the tissue cysts [24]. The bradyzoite is morphologically very similar to the tachyzoite with only slight differences. For example, its nucleus is situated closer to the posterior portion of the cell and the rhoptries in bradyzoites are usually electron dense, whereas those in tachyzoites are usually convoluted. These rhoptries also vary depending on the age of the bradyzoite; younger bradyzoites have rhoptries that are more labyrinthine and those in older bradyzoites are electron dense [24]. Bradyzoites are also thinner and smaller than the tachyzoite [25,26]. Tissue cysts are characterised by their thick wall surrounding the slowly replicating bradyzoites [27]. Tissue cysts are intracellular formations that can vary in size from 5 μm containing only 2 bradyzoites to 100 μm containing hundreds of organisms [26]. Intact tissue cysts tend not to cause harm and can persist for the life of the host without causing an inflammatory response i.e. latent toxoplasmosis. The cyst wall forms within the host cell cytoplasm and it is thin, elastic and composed of both host cell and parasite material [26]. Merozoites are morphologically very similar to tachyzoites though they have a less rounded posterior part. It has in common the centrality of the nucleus and low amount of microneme but it is totally lacking polysaccharide granules Merozoites are formed prior to sexual reproduction from ingested bradyzoites inside feline intestinal epithelial cells in the presence of linoleic acid. They form when the definitive host, the cat, consumes a tissue cyst containing bradyzoites. They convert into merozoites inside the intestinal epithelium and undergo rapid population growth within these cells and subsequently convert into the non-infectious sexual stages of the parasite eventually producing oocysts [27].

Sporozoites are similar ultrastructurally to the other parasitic forms but is unique in that it contains both labyrinthine and electron-dense rhoptries. It also has numerous amylopectin granules and lipid bodies. Oocysts can be either unsporulated or sporulated and they are morphologically different. Unsporulated oocysts are subspherical to spherical and can range from 10-12 μm in size, on the other hand, sporulated oocysts are sub-spherical to ellipsoidal and are approximately 11-13 μm [27]. Each sporulated oocysts consists of 2 sporocysts measuring 6-8 μm and within each sporocyst there are 4 sporozoites. Sporulation takes place outside the cat’s body within 1-5 days depending on environmental conditions. The cytoplasm of the unsporulated oocyst has a large nucleus with amorphous nucleoplasm and a distinct

(20)

20 nucleolus. The nucleus divides 2 times, giving rise to 4 nuclei situated peripherally within the zygote [25,26]. At this stage a second limiting membrane is formed and after the cytoplasm divides 2 spherical sporoblasts are formed each with 2 nuclei. As sporulation progresses the sporoblasts lengthen and divide and 2 sporocysts are formed. The 2 outer layers of the sporoblast becomes the outer layer of the sporocyst wall and the plasmalemma becomes the inner layer. Sporozoite formation occurs as each sporocyst continues to develop and the nuclei divide finally producing 4 sporozoites per sporocyst (a total 8 haploid sporozoites) [22].

3.3.3 Pathways of infection

T. gondii infection occurs predominantly via ingestion of oocysts from contaminated food or water, consumption of tissue cysts or congenital transmission. The most important from an epidemiological viewpoint is unclear because there is regional variance, which, depends upon a range of factors including culture, poverty, eating habits and access to healthcare. Infection from tachyzoites through tissue transplants, blood products and unpasteurised milk, though rare, have been recorded [17]. Additionally, recent studies have implicated ticks in their role as a reservoir for T. gondii and their potential as vector in disseminating disease, though more studies are needed to confirm its function in transmission [29].

Infected cats shed oocysts in large numbers through their defecation which occurs 5-10 days following infection. Once shed, the oocyst sporulates in 1 to 5 days, becoming infective and may remain infective for 12-18 months [23]. In fact researchers have found that sporulated oocysts can remain viable for at least 54 months at 4 o_{C in water and have been found to survive}

in seawater for up to 6 months [30]. It appears that oocysts are also more vulnerable to higher temperatures than to freezing, for example, sporulated oocysts can remain infectious in -21 o_C

for 28 days whereas they can be killed when exposed to 55 o_{C for 2 minutes [30]. Low humidity}

is also fatal for oocysts since they perish at 19% humidity for 11 days [30,31]. Due to their impermeable outer shell sporulated oocysts are considered resistant to many chemical disinfectants and physical forces. Worldwide seroprevalence among domestic cats is estimated to range from 30-40% [17]. It was also estimated that in Morro Bay, California domestic cats deposited 77.6 tonnes of faeces and that feral cats in the same area unloaded 30 tonnes, resulting in an annual oocyst load of over 4500 oocysts/m2_{[2]. Thus, the greater the number of cats, the}

greater the quantity of oocysts, and consequently, the greater the possibility of transmission to humans and other domestic animals. Oocyst ingestion by humans is only viable from the consumption of unwashed fruits and vegetables or water contaminated by oocysts. Water-borne

(21)

21 infections were previously considered to be rare until the largescale infection of marine mammals in the USA, as well as, outbreaks linked to contamination of a municipal water reservoir in Canada, led to the recognition of toxoplasmosis as a water-borne disease [1]. Because of the short duration of oocyst shedding, the passing of non-infective oocysts, and the fastidious nature of cats, direct contact with cats is not thought to be a primary risk for human infection [2]. Recently scientists have demonstrated that oocysts can survive in filter feeders such as anchovies and sardines, remaining infectious from within their alimentary canals [32]. As implied in sub-section 3.3.1 tissue cysts form in the tissues of the intermediate hosts as a consequence of oocyst ingestion and successful T. gondii infection. Tissue cysts have a tendency to manifest in the central nervous system (CNS), skeletal muscle and heart of the intermediate hosts. When a tissue cyst is ingested the wall is disrupted by proteolytic enzymes in the stomach and the released bradyzoites are resistant to proteolytic digestion allowing them to survive digestion initiating infection in the small intestine. Cats acquire toxoplasmosis by ingesting infected prey, wherein the enteroepithelial sexual cycle and production of oocysts occurs [1]. Wild carnivorous animals, including bears, wolves, foxes and badgers, also can acquire toxoplasmosis via ingestion of tissue cysts [10]. As omnivores, humans can therefore also acquire the infection via consumption of undercooked contaminated meat products. The tissue cysts are most frequently observed in the meat of infected pigs, sheep and goats. Several epidemiological studies have investigated the seroprevalence of T. gondii infections in livestock; in Slovakia 25%, from 0.1%-92% in Italy, 17.68% of goat and sheep in Ethiopia and 21% of pigs, 16% of sheep and 23% of goats in the West Indies [17,18]. In Swiss meat-producing animals, prevalence was quite low in sheep and pigs ranging from0.1-4.8%, but the highest prevalence was found in cattle at 2.8-7.2% [29]. The role of chicken in T. gondii epidemiology was quite significant; seroprevalence in commercial free-range chickens was found to be 30-50% while in backyard systems this was up to 90% [17].

Congenital toxoplasmosis occurs as a consequence of transplacental transmission of T. gondii from mother to fetus during pregnancy. Tachyzoites present in maternal blood can potentially cross the placenta and infect the fetus. Acute, primary infections usually always result in congenital toxoplasmosis, whereas latent-infections only occasionally transmit the disease [5]. Studies have demonstrated that the trimester of pregnancy in which maternal transmission occurs is a critical factor in the incidence of transmission as well as the severity of congenital infection. During the first trimester, transmission is comparatively low at less than 20% but increases to almost 80% by the end of the pregnancy [33,34]. In contrast, the severity of

(22)

22 congenital infection is inversely proportional to trimester of maternal infection; early in gestation cases are severe with infection resulting in hydrocephaly, mental retardation and spontaneous abortion, while during the last trimester, transmission rates are highest but the majority of these cases are subclinical resulting in asymptomatic infections or recurrent chorioretinitis throughout early adulthood that can lead to vision problems and potentially blindness [33–35]. In studied populations the general occurrence of congenital toxoplasmosis is from 1 in 1000 to 1 in 10,000 live births [33,34]. Contemporary research measuring vertical transmission rates in humans using polymerase chain reaction (PCR) detection of the umbilical cord tissue, obtained at birth, found transmission rates of 19.8% [33,36]. However, in investigations on other species indicate higher rates, for example, in sheep this rate was 65% and in experimental mouse models 75%. This is perhaps indicative of underreporting of human vertical transmission when they are based upon serology [35–37].

Therefore, it can be surmised that both cats and livestock have a significant role in the horizontal transmission of toxoplasmosis. Oocysts excreted by cats in their faeces act as a source of transmission in both humans and farm animals. Infected livestock are also a source of infection for humans through their meat, milk or eggs which contain tissue cysts. And such acute infections in a pregnant woman can lead to vertical transmission. Thus, keeping the environment free from oocysts is imperative, as well as maintaining the health of pet cats as well as keeping them indoors. Farms and sheds must be free from oocysts, cats and prey animals. Animal products must also be processed to inactivate viable toxoplasma.

3.3.4 Lytic cycle

The lytic describes the T. gondii’s process of cell invasion, intracellular replication and subsequent egression from the host-cell [28]. Upon host-cell rupture the parasite tends to repeat the process by infiltrating neighbouring cells until host-immunity intervenes. Generally, host invasion by T. gondii zoites (tachyzoites, bradyzoites, sporozoites and merozoites) appears similar to those described for other coccidian parasites [22]. The mechanical events consist of 5 major steps; gliding motility, attachment, penetration, the formation of the parasitophorous vacuole (PV) and replication/egression.

Gliding motility is a form of movement driven by the actin-myosin system in the absence of cilia or flagella. It enables the zoite to glide across biological barriers thus expanding the range of host cells. T. gondii zoites exhibit 3 basic behavioural patterns on 2-dimensional (2D) surfaces; helical gliding, circular gliding and twirling. Helical gliding is a clockwise spiral

(23)

23 movement where the parasite rotates along its longitudinal axis, whereas circular gliding is a counter-clockwise movement in a circular pattern while remaining in the same place. Twirling is characterised by the parasite spinning its apex while standing on its base. A combination of all 3 results in host-cell invasion. More recent investigations of the parasite in a Matrigel-based environment allowed researchers to also determine its 3-dimensional (3D) irregular cork-screw like movement [28].

The attachment phase of the lytic cycle includes parasitic probing of the host-cell with its conoidal tip. Once the parasite locates a suitable point of entry, it positions apical secretory protein structures in contact with the host-cell membrane [28]. The parasite is covered by glycosylphosphatidylinositol (GPI)-anchored surface antigens (SAGs). Sulphated proteoglycans on the host-cell is recognised by SAG1 and microneme proteins (MIC) are secreted. Following this, the MIC apical membrane antigen (AMA1) and the rhoptry neck proteins (RON2, RON4, RON5 and RON8) bind forming a moving junction (MJ), whereupon host and parasite cell membranes are closely aligned [28]. The microneme proteins MIC4-1-6, MIC2AP and MIC3-8 also have a fundamental function in attachment as well as supporting gliding kinetics. The MJ subsequently moves posteriorly along the zoite as it begins to penetrate into the host-cell [11,28].

Penetration occurs after the MJ repositioning, the actin-based contractile system allows the parasite to lunges frontwards into the host-cell and releases rhoptry bulb proteins (ROPs) into the host-cytosol. It then invaginates the host-cell plasmalemma which is then destined to form the PV membrane (PVM).

The PV is a structure that offers protection from host-cell membrane trafficking. The MJ functions as a sieve in its formation, in effect filtering proteins in the host-cell plasma membrane and excluding transmembrane proteins. This effectively results in a vacuolar compartment with a non-fusogenic membrane resistant to endosomal and lysosomal fusion. The host-cell mitochondria and ER are typically recruited to the PV possibly aiding the parasite in acquiring nutrients [23]. Dense granule proteins (GRAs) are also among those released into the PV, specifically GRA2, GRA4 and GRA6, and they form an intravacuolar tubular network. The parasite finally then employs a unique mode of replication known as endodyogeny. Endodyogeny unfolds through the formation of 2 daughter cells within the boundaries of the mature mother parasite, which is consumed at the end of the process. Egress is triggered when host-immunity responds to T. gondii. This can be following a perforin mediated damage to the

(24)

24 host cell by cytotoxic cluster of differentiation 8 (CD8+) T cells; damage to the host-cell leads to a drop in intracellular K+_{which triggers egression [23]. In the absence of host-immunity,}

egression is triggered by abscisic acid (ABA) production by the parasite. In this situation the parasite will proceed to replicate to maturity and continuously produces ABA [38,39]. When the ABA concentration rises above a threshold (quorum sensing), the parasite initiates egression. Additionally, replication acidifies the PV which also triggers the parasite to egress.

3.3.5 Human immunity

T. gondii infection in immunocompetent individual typically becomes asymptomatic because host innate and adaptive immunity resists its initial proliferation and eradicates most of the parasites. Infection of monocytes by tachyzoites strongly stimulates primary (innate) immune responses such as the production of cytokines, which then activates the secondary (adaptive) humoral response and the cell-mediated response ultimately disrupting the parasite’s intracellular growth. These actions induce T. gondii to stage-convert into the bradyzoite and form a tissue cyst eventually leading to latent toxoplasmosis. Thus, a delicate immunological balance exists between the host and its parasitic occupant and it is significant to developing screening, prevention and treatment strategies.

Innate or primary immunity is the first line of host-defence and it involves the detection of the T. gondii and restricting parasitic multiplication [40]. Monocytes and neutrophils are among the first cell types to arrive at the site of infection. Monocytes restrict the growth of T. gondii by producing nitric oxide (NO), while neutrophils kill the parasite via oxygen-dependent and independent mechanisms [38]. Both initiate interleukin-12 (IL-12) production which is key to interferon-γ (IFN-γ) action; IFN-γ is a key mediator of adaptive immunity in humans [40]. Dendritic cells (DC) also produce IL-12 and, in fact, play an even more central role in bridging innate and adaptive immunity [40]. DCs are the initial antigen presenting cells and are either infected by the parasite or ingest apoptotic enterocytes, wherein they process both for antigen presentation. DCs secrete significantly more IL-12 and thus recruits IFN-γ activated natural killer (NK) cells as well as other inflammatory cells [40]. DCs are also involved in preparing CD8+ T cells to react to T. gondii, thus shaping the early stages of the T cell response. Toll-like receptors (TLR) are pattern recognition receptors and consist of 11 members in humans. Each TLR recognises various ligands which subsequently stimulates different cytokines and chemokines to be produced [40]. In mice, TLR11 and TLR 12 recognises T. gondii profilin proteins and initiates IL-12 production [38]. TLR11 is however, non-functional in humans and

(25)

25 TLR12 does not exist in the human genome, indicating alternative currently unidentified parasite sensing mechanisms in humans [38]. Nevertheless, it is known that within human immune cells nucleic acid recognition by endosomal TLRs such as TLR8 warns of parasite infection and stimulates 12 production [38]. Therefore, innate immune components like IL-12 producing dendritic cells and monocytes, neutrophils and NK cells all have a critical part in modulating the adaptive immunity, which is essential for ultimately controlling parasitic infection.

Once TLRs respond to host infection the eventual IFN-γ production leads to a cascade of events. For example, activated CD4 + cells produce the T-cell mitogen IL-12, which in conjunction with IFN-γ, results in a large-scale recruitment of parasite specific CD4+ and CD8+ T cells [40]. These cells produce even more IFN-γ at the site of infection, which through a STAT-1 dependent pathway, leads to the generation of anti-parasitic NO, reactive oxygen species (ROS) and immunity regulated GTPases (IRGs) [41]. Generally, these are all anti-parasitic effector mechanisms which not only directly kill the parasite but also continue to activate more anti-microbial mechanisms by increasing IFN-γ. IRGs have been recognised to disrupt the T. gondii vacuole through IRG protein loading, which leads to degradation via autophagomal delivery to lysosomes [41]. Parasite specific CD8+ T cells also lyse infected cells through perforin mediated cytotoxic activity [40]. Chronic infection in the brain however, is regulated by CD8+ T cells and resident glial cells, microglia and astrocytes that act as immune effectors [40].

3.3.6 Host-immunity evasion

In order to avoid destruction by the host, T. gondii has evolved several strategies to preserve its existence. Firstly, through the lytic cycle detailed in sub-section 3.3.4, the parasite efficiently and successfully infiltrates host cells which is of utmost importance for its survival. Secondly it avoids cell autophagy by secreting many different effectors that disrupt the canonical CD40-CD40L mediated and the non-canonical IFN-γ dependent autophagy pathways [42]. It does this through its MIC3 and MIC6 proteins, which activates the PI3k/Akt signalling pathway. This event prevents the expression of light-chain 3 (LC3) and thereby inhibits vacuole-lysosomal fusion. Akt phosphorylation also negatively regulates autophagosome formation [42]. T. gondii goes even further to activate a signalling cascade that also inhibits key stimulators of autophagy such as PKR and eIF2α. As a consequence of these events, T. gondii successfully avoids being targeted by host-cell autophagy machinery [42,43].

(26)

26 T. gondii has a variety of other survival techniques that allows it to evade IRGs, resist oxidative stress, regulate host-gene expression and counter host-defence mechanisms. Though the extent to which it can utilise these strategies varies depending on the strain. For example, virulent type I strains can evade IRG-dependent antimicrobial actions through polymorphisms of ROP5, whereas type II and III cannot. Type III strains are also less able to resist oxidative stress, which the other T. gondii strains usually subvert by using an antioxidant network. They utilise cytosolic peroxiredoxins and superoxide mutases to detoxify hydrogen peroxides and ROS. The described approaches relate to acute stages of T. gondii infection; however, the parasite also stays dormant in the chronic stage of its infectious process. After its exponential replication as tachyzoites, increasing host immune responses induce a stage-conversion into the latent bradyzoite stage that resides in tissue cysts [43]. This stage allows the parasite to evade immune responses and common antimicrobial therapies. In addition, when host immunity becomes impaired, the opportunistic pathogen transforms again into the rapidly multiplying tachyzoites and disseminates to immune-privileged organs to form more bradyzoite tissue cysts [42]. This “trojan-horse” strategy is regulated by multiple genes and is one of the keys to this parasite’s success.

3.4 Clinical manifestations

The clinical manifestations of toxoplasmosis vary greatly, producing a range of nonspecific symptoms. For the sake of simplicity, the disease can be classified into several categories: toxoplasmosis acquired in the immunocompetent patient; toxoplasmosis acquired/reactivated in the immunocompromised patient; ocular toxoplasmosis; toxoplasmosis acquired in pregnancy; and congenital toxoplasmosis [36]. Clinical manifestations can vary from asymptomatic or mild cervical lymphadenopathy in an immunocompetent patient to a broad spectrum in immunodeficient patients. There are a few commonalities; encephalitis is prevalent in AIDS patients; posterior uveitis in ocular toxoplasmosis; and chorioretinitis or delayed development in infants in congenital toxoplasmosis [1].

3.4.1 Toxoplasmosis acquired in the immunocompetent patient

The innate and adaptive immune response in immunocompetent individuals usually resists infection well enough such that they are asymptomatic. Despite this, in some immunocompetent hosts, T. gondii infection can present as an acute systemic infection developing 5-23 days post-exposure [36]. This is expressed largely through constitutional

(27)

27 symptoms including fevers, chills, myalgia and headaches. Bilateral cervical lymphadenopathy is also a common clinical manifestation, capable of persisting for several weeks [1]. Laboratory tests are similarly non-specific in acute toxoplasmosis; slight lymphocytosis and moderate increases in C-reactive protein (CRP). In these immunocompetent patients the course of the disease is usually uncomplicated and self-limited, lasting from a few weeks to months [1].

3.4.2 Toxoplasmosis acquired/reactivated in the immunocompromised patient The clinical presentation of reactivated toxoplasmosis in the immunodeficient patient depends on the dissemination of the parasite, though due to the parasite’s affinity for neural tissue it most frequently presents with signs and symptoms of CNS disease [44]. Toxoplasmic encephalitis (TE) is one of the most common manifestations in immunosuppressed patients with a CD4+ count <100 cells/μL [44]. Patients with TE usually present with headaches and other neurological symptoms. Pulmonary and ocular findings are the most common extracerebral manifestations of reactivated toxoplasmosis [44]. Pneumonitis is a pulmonary finding which presents with fever, dyspnoea and a non-productive cough. Toxoplasmic chorioretinitis is a typical ocular finding which presents with eye pain, decreased visual acuity and yellow-white lesions in a non-vascular distribution [44]. Reactivated toxoplasmosis can present in almost every visceral organ, though it rarely does so.

3.4.3 Ocular toxoplasmosis

Ocular disease due to toxoplasmosis is often asymptomatic when patients have what is described as inactive ocular toxoplasmosis [45]. On routine examination inactive ocular toxoplasmosis presents as scarring without significant history of active retinochoroiditis. Posterior uveitis is the typical presentation in patients with active ocular disease. The major

sites of inflammationare the retina and choroid, though secondary vitreal inflammation is the

finding that leads to consultation [45]. Immunocompetent adults with ocular toxoplasmosis typically present with floaters and visual loss, which may be permanent if the lesion affects the macula [44]. Floaters alone are often the presenting sign if the retinochoroidal lesion is not located within the central retina. In immunocompetent hosts, reactivation of a latent infection almost always manifests as retinochoroiditis [44]. Isolated cases of anterior uveitis have been recorded however; they occur rarely.

(28)

28

3.4.4 Toxoplasmosis acquired in pregnancy

Acute maternal infection is generally asymptomatic in greater than 80% of cases [33]. Symptoms of infection are usually non-specific and constitutional i.e. fevers, chills, headaches, myalgia etc. With febrile episodes lasting from 2-3 days. Bilateral lymphadenopathy is among the most common symptoms which can last for a few weeks [36]. Ocular disease can occur in acute cases but are more common with reactivation of a latent infection. If ocular disease manifests, as aforementioned it will present with visual loss and floaters. Most immunocompetent patients are able to limit the spread of the parasite and the associated tissue damage [33].

The main concern in maternal infection are the risk factors for maternal-foetal transmission since congenital toxoplasmosis has poor outcomes. Women infected prior to conception rarely transmit disease to the foetus, whereas the frequency of foetal infection increases steeply with advancing gestational age [33]. However, as mentioned in sub-section 3.3.3, the odds of developing clinical sequelae decreases with gestational age. The main features determining risk by European guidelines include: maternal infection at an advanced gestation age; high parasite load; parasite source, since sporozoites have more foetal infection risk than bradyzoites; virulence of T. gondii strain; and the state of host-immunity [33].

3.4.5 Congenital toxoplasmosis

Congenital infection by T. gondii has a wide spectrum of clinical features, however involvement of the eyes and brain are the clinical hallmarks of congenital T. gondii infection. Mental retardation is a common development in new-borns that survive until child hood and TE is a common neurological consequence of the parasite’s CNS involvement, typically manifesting in seizure activity prior to more serious repercussions. There is a popularised triad of clinical sequelae which includes chorioretinitis (85-92%), hydrocephalus (30-68%) and intracranial calcifications (50-85%) [35]. Recognition of the triad requires detailed ophthalmoscopic examination and formal CNS imaging, thus these abnormalities are often missed. In fact, almost 70-90% of new-borns with congenital toxoplasmosis are asymptomatic upon routine physical examination [33]. Because of T. gondii’s ability to infect virtually any visceral organ, a range of other symptoms of congenital toxoplasmosis may include: jaundice, anaemia, thrombocytopenia, hepatosplenomegaly, microphthalmia and microcephaly.

(29)

29

3.5 Treatment and prevention strategies

Sub-sections 3.3-3.4 describes the pathological pathways and manifestations of the disease and should emphasise the significance of T. gondii in modern society. Due to its prevalence, it stands to reason that prevention of toxoplasmosis is of utmost importance, and if an individual is infected treatment should certainly be sought. Currently treatment protocols predominantly require systemic antimicrobial therapy with or without adjuncts. The main drugs utilised at present include: pyrimethamine and sulfadiazine; trimethoprim-sulfamethoxazole (TMP-SMX); spiramycin; clindamycin and atovaquone. Drugs with activity against T. gondii cysts are highly desirable and as such are still being developed [46]. Treatment strategies vary depending on the clinical presentation and this sub-section will review both prevention strategies, as well as currently approved treatment regimens.

3.5.1 Prevention

Several precautionary measures can be undertaken in order to minimise risk of human toxoplasmosis. Cooking meat to adequate temperatures before consumption is an example of this; most guidelines suggest heating red meat to at least 60 o_{C, ground red meat to 71}o_{C and}

poultry to a minimum of 74 o_{C [46]. If meat is supposed to be served rare it should be frozen}

at sub-zero temperatures for several days prior to preparation [46]. Additionally, all kitchen utensils should be washed and cleaned thoroughly with hot water and soap after contact with raw meat or shellfish. Individuals should avoid unpasteurised non-bovine milk and only consume fruits and vegetables after thoroughly washing them. Gloves should be used and hands should be washed once coming into contact with soil or sand that has been contaminated with cat faeces. Similar hand protection and sanitization efforts should be made when coming into contact with cats that forage outdoors or handling cat litter [46].

Education about risk factors and taking the detailed precautions can significantly reduce the probability of exposure to T. gondii. Often exposure can occur without recognition and data from multiple countries estimates that screening pregnant women may be beneficial for health [36]. Additionally, it is thought to be significantly cost-saving within a broad range of prevalence and cost. A study in Austria suggests that such screening protocols and the prompt initiation of treatment, demonstrated elimination of severe congenital infection as well as a reduction in the numbers infected [36]. Thus, there should be a notable benefit in the practice of prenatal screening.

(30)

30 For patients infected by HIV or suffering from other immunodeficiencies, the risk of developing toxoplasmosis can be reduced by taking the previously described preventative measures to minimise exposure to the pathogen and, in the event of prior infection, by using prophylactic antibiotics to decrease the risk of reactivation. All such individuals ought to be tested for T. gondii exposure by measuring anti-toxoplasma IgG. This primary prophylaxis is indicated when CD4+ counts are less than 100 cells/μL and patients are T. gondii IgG positive. For pregnant women who are immunodeficient the benefits of prophylaxis must be weighed against the risk to the foetus [46,47].

The primary prophylactic medication against reactivation is TMP-SMX. A single double dose (DS) tablet 160/800mg is given to the patient daily [46]. If patients suffer from allergies or are unable to tolerate the primary prophylaxis, they may follow alternative regimens: dapsone 50mg once per day in combination with pyrimethamine 50mg and leucovorin 25mg per week; or atovaquone 1500mg once daily with or without 25mg of pyrimethamine and 10mg of leucovorin daily [46]. Monotherapy is not recommended in prophylaxis. Patients may safely discontinue prophylaxis if their CD4+ count is greater than 200 cells/μL for at least 3 months [46–48].

3.5.2 Toxoplasmosis acquired in the immunocompetent patient

There is limited evidence to support treatment of immunocompetent patients with systemic infection. In these cases, acute toxoplasmosis is often self-limited and treatment is only recommended in those with severe indications or if symptoms are prolonged lasting more than a few weeks. In the situation of severe or prolonged symptoms the antimicrobial regimens are similar to those recommended for immunosuppressed patients described in sub-section 3.5.3, except that the duration is much shorter. The typical medical prescription should be adhered to for 2-4 weeks and includes: pyrimethamine with a loading dose of 100mg and a daily 25-50mg dosage thereafter; followed by sulfadiazine 500-1000mg given 4 times per day; and leucovorin with a daily dosage of 10-25mg [47]. If pyrimethamine is unavailable TMP-SMX may alternatively be used for the same duration at a daily double dosage of 5/25mg per kg, respectively. In case of sulfur allergies, patients are administered atovaquone monotherapy at 750mg 4 times per day [47].

(31)

31 3.5.3 Toxoplasmosis acquired/reactivated in the immunocompromised patient The treatment of toxoplasmosis in patients that are immunosuppressed includes antimicrobial therapy directed against T. gondii. Antimicrobial therapy targeted against the parasite involves an initial phase to treat the acute symptoms, followed by maintenance therapy to reduce the risk of recurrence. Extracerebral toxoplasmosis is treated with the same regimens as TE, although the response may not be as favourable. For certain patients for example, those on antiretroviral treatment (ART), maintenance therapy can be discontinued [46].

An initial regimen containing sulfadiazine and pyrimethamine is usually the most effective; however, it is associated with a higher incidence of cutaneous hypersensitivity reactions. Patients weighing <60kg or >60kg are given sulfadiazine 1000mg or 1500mg, respectively, four times per day [46] [47]. Pyrimethamine is administered with a 200mg loading dose followed by 50/75mg daily, depending on weight [47]. Leucovorin 10-25mg is given daily to prevent pyrimethamine-induced toxicity. Parenteral TMP-SMX can be used to treat TE in severely ill patients for whom oral therapy is inappropriate [46]. Alternatively, for patients intolerant to sulfadiazine, atovaquone can be prescribed; dosed at 1500mg per os twice per day, followed by the previous dosage of pyrimethamine and leucovorin [46].

It is common to co-administer other medications to help manage the patient in case of TE complications. These adjuncts include: corticosteroid dexamethasone 4mg/6h for patients with brain lesions or oedema; and anticonvulsants when presenting with seizures [46]. Both adjuncts should not be given routinely but only administered when such complications arise due to potential adverse drug interactions.

After 6 weeks of the initial phase of therapy treatment is transitioned to the maintenance regimen. The guidelines suggest that a combination of sulfadiazine and pyrimethamine, if taken, should be continued but at a lower dose than for initial treatment. The dosage of sulfadiazine should be 2000-4000mg divided into 2 daily doses with the same previous dosages of pyrimethamine and leucovorin [46]. If there is risk of sulfadiazine allergy, a double dose of atovaquone 750-1500mg can replace sulfadiazine [46]. Atovaquone monotherapy may be used in case of pyrimethamine intolerance, however it has been associated to a 26% relapse rate [46]. Maintenance therapy maybe discontinued once CD4+ counts maintain at >200 cells cells/μL and in case of TE, once neuroimaging studies confirm improvement in patient.

(32)

32 3.5.4 Ocular toxoplasmosis

Inactive ocular toxoplasmosis is typically untreated except in situations where children suffer congenital toxoplasmosis, such cases are reviewed in sub-section 3.5.6. The indications for treatment in patients with active ocular disease include: lesions threatening the optic nerve or fovea; decreased visual acuity; lesions associated with vitreous inflammation or retinal vasculitis; atypical lesions; multiple active lesions; and immunodeficiencies. Treatment is also recommended for pregnant patients, however outcomes in the foetus must be evaluated prior to therapy. Typically, ocular toxoplasmosis is treated with oral antimicrobial therapy with adjunctive glucocorticoids.

Similar to previously described treatment protocols, the preferred regimen involves the administration of TMP-SMX, at a dosage of 160mg TMP and 800mg SMX twice daily for at least 6 weeks [46]. Another rational combination is daily pyrimethamine, sulfadiazine and leucovorin dosed at 100mg, 3000mg and 10mg, respectively [47]. In case patients are intolerant to these first-line regimens atovaquone may be used in combination with pyrimethamine and leucovorin (same previous dosage) at 750mg four times per day [47]. For those responding poorly to systemic antimicrobial therapy, intravitreal clindamycin injections at 1mg/0.1ml are used, also over 6 weeks [46].

In cases of significant vitreal inflammation and retinal vasculitis, glucocorticoids are given as an adjunct to antimicrobial therapy; glucocorticoids are usually given 2-3 days after antimicrobial therapy begins. The delay is to allow the antimicrobial therapy to diminish parasite load and thereby minimise the possibility of glucocorticoids exacerbating the infection. An initial dose of 40mg prednisone is given once per day, which is then tapered down over the following weeks depending on the severity of inflammation [46]. It should be less than 10mg per day by the end of the concurrent systemic antibiotic therapy [46]. In the setting of a patient receiving intravitreal injections, intravitreal dexamethasone injections may be added at a dose of 0.4mg/0.1ml [46]. Glucocorticoids are given as an adjunctive to antimicrobial therapy and as such treatment should also end after 6 weeks [46].

3.5.5 Toxoplasmosis acquired in pregnancy

The primary goals of toxoplasmosis treatment in pregnant women is to prevent congenital toxoplasmosis and, if infected, decrease the progress of the disease within the foetus. To this end, there are no direct maternal benefits. The antimicrobial regimen employed is largely

(33)

33 dependent on foetal gestational age and thus can be classified as either treatment <18weeks of gestation and treatment ≥18 weeks of gestation [36].

Treatment <18 weeks of gestation mainly involves the administration of spiramycin. Spiramycin is a macrolide antibiotic which concentrates in the placenta where it treats placental infection and helps prevent transmission of the parasite to the foetus. It is also preferred as it has less evidence of teratogenic effects than the antimicrobial alternatives. The treatment duration is from diagnosis until 18 weeks of gestation and dosage is 1000mg per day administered orally without food [36]. PCR/ sonography is typically recommended for patients to assess state of the foetus. If such examination is suggestive of congenital toxoplasmosis and the patient plans to continues the pregnancy, it is recommended that the patient transitions to a pyrimethamine, sulfadiazine and leucovorin combination [34,40].

The approach to treatment after 18 weeks of gestation is mainly with pyrimethamine, sulfadiazine and leucovorin [36,47]. This combination of antibiotic therapy is the most studied and effective. It is typically followed until delivery with various orally administered dosing regimens, however the most popular is as follows: pyrimethamine 100mg/day for 2 days then 50mg/day thereafter; sulfadiazine 75mg/day for 1 day and subsequently 100mg/day; and leucovorin 10-20mg/day [36]. Alternatively, in case of intolerance, a pyrimethamine, clindamycin and leucovorin combination may be used, however its efficacy in modern practice is not well-studied [46].

3.5.6 Congenital toxoplasmosis

Antimicrobial therapy is indicated for patients with a confirmed or highly probable diagnosis of congenital toxoplasmosis. The preferred regimen is similar to those mentioned in sub-section 3.5.1-3.5.5 in the choice of drugs i.e. pyrimethamine, sulfadiazine and leucovorin; however, the dosages are adjusted for paediatric tolerance. Pyrimethamine is administered at 2mg/kg once per day and then 1mg/kg daily for 6 months with leucovorin 10mg prescribed 3 times per week throughout [36]. After 6 months, 1mg/kg of pyrimethamine is continued 3 times per week to complete 1 year of therapy and leucovorin administration is sustained until 1 week after pyrimethamine therapy. Additionally, sulfadiazine 100mg/kg is co-administered daily for 1 year [36]. Throughout treatment, infants should be weighed weekly and dosages adjusted. In case of allergies to sulfadiazine, it is suggested to substitute with clindamycin 20-30mg/kg per day [36]. In the event ocular manifestations of the disease, it is common to prescribe prednisone 0.5mg 2 times daily [36].

(34)

34 3.5.7 Treatments in development

Toxoplasmosis continues to be a health threat for the majority of human populations and the currently available treatments detailed in sub-section 3.5 are of limited efficacy in acute infections and are generally ineffective in chronic infection. They also require a prolonged course and demonstrate significant toxicity to patients. These adverse reactions and the questionable efficacy have drawn attention to the need for alternative therapeutic agents. Nitazoxanide (NTZ) is a broad spectrum anti-parasitic agent which has demonstrated considerable effectiveness against a range of apicomplexan parasites. Current trials on mice indicate that it is neither teratogenic or mutagenic and has a significant role in reducing mortality as well as controlling both acute and chronic infection [48]. In fact, it has even been shown to reduce the number of brain cysts by 78-87%, highlighting its potential in the future treatment of both acute and chronic infection [48]. Several other groups of drugs are also being researched including the anticoccidial ponazuril and endochin-like quinolones [46]. There are also several reports regarding immunotherapy in the field of toxoplasmosis. For example, utilising checkpoint inhibitors as immune modulators to boost efficacy of current T. gondii drugs has be shown to reduce cysts in the CNS [46]. Similarly, administration of IL-12 24 hours before infection, followed by drug therapy significantly improves the survival of mice [46]. Nevertheless, much of these are still in the early stages of development and the likelihood of their future practical use lies in the hands of the tentative scientific process.

(35)

35

4. RESEARCH METHODOLOGY AND METHODS

In order to fulfil the objectives outlined in section 2, the questionnaire, except for demographic data, was structured into 3 main parts: cat ownership and handling, hygiene associated with pet ownership, and knowledge concerning the infectious pathways of toxoplasmosis. Various question types were incorporated, including: closed questions (Yes/No), open questions, single-select multiple-choice questions (MCQ), multiple-select MCQs and numerical rating scales. To facilitate the formulation of the questionnaire, articles and published research with a similar methodological process were reviewed [50] [51]. A brief description of the contents of each section is illustrated in table 1.

Once completed, the necessary approval was given by both faculties in February 2019. The Regional Biomedical Research Ethics Committee reviewed the questionnaire and deemed it to be in accordance with the ethical standards of the University on the 5th_{March 2019.}

Subsequently, it was pretested with 5 people close to the target population (Veterinary faculty) before being distributed amongst the students. The survey was then successfully conducted from March 2019 to March 2020.

COMPARISON OF KNOWLEDGE AMONG THE MEDICAL AND ODONTOLOGY STUDENTS OF DIFFERENT YEARS AT LUHS ABOUT THE MOST LIKELY PATHWAYS OF TOXOPLASMA GONDII INFECTION