CHAPTER 1

RESEARCH PROBLEM

Background and Rationale

Primary amoebic meningoencephalitis (PAM) is a rare yet rapidly fatal disease occurring

among healthy recreational swimmers throughout the world who have had a history of recent

swimming in lakes, hot springs, thermal pools and stagnant hyperchlorinated swimming pools

during the summer season. Alarmingly, this disease has a short incubation period of two to seven

days. PAM among humans is characterized by severe vomiting, nausea and dizziness which

progress shortly to a coma and later on, death if left untreated within two weeks (Belizario and

de Leon, 2004).

The most common etiologic agent for PAM is the free living ameboflagellate (FLA)

belonging to the Phylum Sarcomastigophora, Subphylum Sarcodina, Superclass Rhizopoda,

Class Lobosea, Family Vahlkampfiidae which is no other than Naegleria fowleri. Unlike the

other closely related organisms belonging to the same subphylum, N.fowleri is capable of

existing in three morphological forms namely: the cyst, the trophozoite and the flagellate form

therefore, enabling them to survive in environments that may prove fatal to other protozoan

parasites (Mahon et al., 2007). This notorious microbe takes an intranasal route of infection.

Research shows that upon the cysts’ entry into the nares, they immediately convert to their

trophozoite form in order to move towards the central nervous system along the olfactory nerve

(Cabral and Cabral, 2007). Thus, infection occurs in the brain and its meninges in a very short

time span causing hemorrhages and edema.

In the Philippines, Naegleria philippinensis is the only locally occurring pathogenic

species identified. Due to the lack of attention given to PAM causing Naegleria spp. here in our

country, only a few studies have been documented with regards to the description of this

Philippine isolate, and that would include the researches done in 1986 by Castro and in 1991 by

Matias (Belizario and de Leon, 2004). This could probably be the reason why only one case of

PAM was reported here in the Philippines amidst the fact that Naegleria spp. are virtually

ubiquitous in freshwaters worldwide (Mahon et al., 2007).

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In Thailand, N.fowleri was easily isolated from different fresh water environments which

could’ve posed as a great risk for recreational swimmers (Ithoi et al., 2011). And yet despite this

fact, only six patients were able to recover from PAM between the period of 1970 and 2001

(Tiewcharoen et al., 2002). Indeed, the disease has a high mortality rate. One the other hand, in

Malaysia, a neighboring country that shares a similar climate with Thailand; the free living

Naegleria amoeboflagellates were still almost unknown to laboratory and health care workers

until 2010. PAM was rarely considered in the diagnostic differentiations of CNS infections (Ithoi

et al., 2011). Therefore, this only tells us that indeed, the body of knowledge concerning PAM is

still very limited particularly here in the tropics.

Since the Philippines is rich with both artificial and natural aquatic environments that

would cater the needs of free living Naegleria spp. for survival, not to mention the fact that these

sites are advertised as tourist destinations during the summer season, there is a probability that

the PAM causing Naegleria spp. are present in these bodies of water; thus posing a potential

harm for young and adult swimmers alike.

Hence, it is vital and inevitable to study these free living amoeboflagellates here in our

country. Thus, this present study generally and hopefully aims to determine the occurrence of

PAM caused by Naegleria spp. isolated from selected artificial and natural bodies of water in

Leyte and Biliran as a stepping stone in truly understanding these elusive yet deadly protozoan

parasites here in the Philippines.

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Statement of the Problem

This study generally aims to determine the occurrence of primary amoebic

meningoencephalitis (PAM) caused by Naegleria spp. isolated from selected waters in Leyte and

Biliran. Specifically, this study intends to undertake the following objectives:

1. Determine the physical properties of the different artificial and natural bodies of

water before the collection of the water sample. These factors are the following:

A.) pH

B.) Temperature

C.) Salinity

2. Find out if there is a significant difference in the isolation of free living

amoeboflagelllates (FLA) of the genus Naegleria if the water sample would be

subjected to milipore filtration or centrifugation before culture.

3. Identify the prevalence of Naegleria spp. isolated from the selected artificial bodies

of water from the provinces of Leyte and Biliran regardless of the separation

techniques applied to the water specimens before culture.

4. Determine the number of Naegleria isolates that causes PAM among 2-6 week old

Albino mice.

5. Find out the average number of hours that takes for the PAM causing Naegleria

isolates to kill a single Albino mouse.

6. Identify the location and the physical characteristics of the artificial and natural

bodies of water wherein the pathogenic Naegleria spp. were isolated.

Null Hypotheses

1. There is no significant difference on the number of viable free living Naegleria

ameboflagellates isolated from water samples filtered with 1.2 µm milipore and from

water samples subjected to 2000 rpm centrifugation.

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SIGNIFICANCE OF THE STUDY

The general purpose of this study is to determine the occurrence of primary amebic

meningoencephalitis (PAM) caused by Naegleria species isolated from selected waters in Leyte

and Biliran. The results will be used to supplement the data that is currently possessed by St.

Scholastica’s College-Tacloban, the World Health Organization (WHO) and some government

agencies in the Philippines namely the Department of Health (DOH) and Department of Science

and Technology (DOST). At the same time it aims to cater a reliable source of information for

the following benefactors:

A.) Private Swimming Pool and Hot Spring Businesses– since recreational swimming

proves to be a major source of income during the summer season, the information

gathered in this study will enable them to take the appropriate measures in preventing

the accumulation and growth of pathogenic amebolagellates if there are any in their

swimming pools and hot springs. Thus, further improving the quality of their

businesses.

B.) Provincial and Regional Government- if natural bodies of water are utilized for

tourism and swimming in order to raise government funds but are proven to be

positive for pathogenic Naegleria spp, it is their responsibility to take precautionary

measures in handling the situation. Therefore, the data gathered in this study will

enable them to protect the general public and prevent the further growth of

pathogenic FLA in these contaminated waters.

C.) General Public- the results gathered in the course of this study will serve as a guide

to tourist spots in Leyte and Biliran that are safe for recreational swimming if and

only if there any of these bodies of water proven to be positive for pathogenic FLA.

D.) Physicians and other Health Workers – since PAM is a rapidly fatal disease

capable of causing death within one week, in one way or another the findings of this

research will help them develop techniques in handling emergency cases.

E.) Future Researchers - due to the rapid growth of the body of microbiological

sciences and the enormous possibilities that these researchers may undertake the same

closely related research problems in the near future, this research study will serve as a

reliable reference material.

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Scope and limitation of the Study

Scope

The general purpose of this study will only be limited to determine the occurrence of

primary amebic meningoencephalitis (PAM) caused by Naegleria species isolated from selected

waters in Leyte and Biliran.

Water samples are going to be collected from nineteen randomly selected areas in Leyte

and Biliran with artificial and natural bodies of water that may be suitable for the growth and

survival of Naegleria spp. and at the same time promoted for tourism purposes. These areas are

the following: Alangalang, Albuera, Babatngon, Baybay, Barugo, Biliran, Burauen, Carigara,

Dolores, Dulag, Jaro, Lapaz, Leyte-Leyte, Naval, Ormoc, Palompon, Sta.Fe, Tacloban and

Tolosa. Water samples maybe collected from swimming pools, lakes and hot springs from these

areas depending on their availability for each locale. Prior to collection, the physical properties

of the water source that may intervene with the viability of the organisms will be measured

quantitatively namely: pH, salinity and temperature.

Specimen processing will be done in the Medical Laboratory Science (MLS) Laboratory

of St.Scholastica’s College-Tacloban. For each water sample, the researchers will utilize two

methods of separating the sediments. First method will be the use of 1.2 micrometer millipore

filter. The second method will be centrifugation under 2000 rpm. The water samples will be

cultured using nutrient agar seeded with Escherichia coli colonies to find out the presence of

viable amoebae. In order to determine if the amoebae isolated can cause PAM, albino mice will

be infected artificially by a suspension of Naegleria isolates. If the mice will die two to seven

days prior to infection, histopathologic analyses will be done to further identify the cause of

death.

Limitations

Since the water samples are going to be collected from areas that are kilometers away

from the research laboratory, the lack of the necessary equipments that will preserve the viability

of the amoebae that may be present in the water samples may intervene with the results of the

study.

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Conceptual Framework

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The Occurrence of Primary Amoebic Meningoencephalitis (PAM) caused by Naegleria spp. Isolated from Selected Waters in Leyte and Biliran

INDEPENDENT VARIABLES

INTERVENING VARIABLES

DEPENDENT VARIABLE

The Selected Waters in the Provinces of Leyte

and Biliran

Physical Properties of

the Water Sample

namely:

pH

temperature

salinity

Occurrence of Primary Amoebic

Meningoencephalitis caused by the Naegleria

spp. isolates

Figure 1.0: The scheme shows the variables that influence the occurrence of PAM caused by Naegleria spp. isolated from selected waters in Leyte and Biliran.

REVIEW OF RELATED LITERATURE

Parasitic organisms are generally categorized into two major groups with medical and

public health importance namely: the unicellular protozoans and the multicellular metazoans

(Mahon et al., 2007). Primarily, metazoan parasites could either be helminthes or arthropods.

Class Trematoda (Flukes), Class Cestoda (Tapeworms) and Class Nematoda (Roundworms)

comprise the wide array of helminthes parasitic to both humans and animals (Belizario and de

Leon, 2004). While arthropods are characterized to be bilaterally segmented organisms protected

by chitinous exoskeletons with jointed appendages. These include centipedes, millipedes,

spiders, scorpions and a few crustaceans.

On the other hand, the unicellular protozoans are characterized to be eukaryotic

organisms that belong to Kingdom Protista. Therefore, they have a true nucleus, a cytoplasm, a

cell membrane, cellular elaborations and locomotory organelles such as the pseudopodia, cilia

and flagella. Most of these organisms require moist environments for survival. Thus, many

protozoan parasites utilize the human body for feeding and reproduction. Apicomplexa,

Microspora, Ciliophora and Sarcomastigophora are the four Phyla of protozoan parasites

reputed and proven to be most pathogenic to humans (Belizario and de Leon, 2004).

In the case of this research study, it is essential to have a general background of Phylum

Sarcomastigophora. This phylum is further classified into two subphyla namely: Sarcodina and

Mastigophora. Most organisms under Subphylum Mastigophora move through their flagella’s

whipping motion, thus they are called flagellates. Flagellates reproduce sexually. Intestinal

flagellates may exist as non-motile environmentally resistant cysts and motile, feeding

trophozoites. On the other hand, the protozoans belonging to Subphylum Sarcodina move by

means of hyaline-footlike structures called pseudopodia. These organisms are generally referred

to as amoebae. Amoebae reproduce asexually and may exist as either trophozoites or cysts

(Mahon et al., 2007).

Amoebae have a virtually simple life cycle, infective cysts are ingested via the fecal-oral

transmission through contaminated food or water. Unlike most parasitic infections, amebic

infections do not require intermediate hosts. After ingestion, the infective cysts excyst in the

intestinal tract, multiply by binary fission and colonize the cecal area. In the case of Entamoeba

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histolytica infections, the organisms may migrate to the liver after eroding the intestinal wall and

cause hepatic abscesses which may be fatal to patients if left untreated (Mahon et al., 2007).

Unlike the other closely related organisms belonging to the same subphylum, the

pathogenic Naegleria spp. take a different route of transmission and they can exist in three

morphological states. They live as free living flagellates in warm, stagnant waters, as feeding

trophozoites as well as resistant cysts. They can be acquired through forceful entry of

contaminated water or soil into the nostrils. These organisms colonize the nasal cavity in their

trophozoite form, penetrate the cribiform plate, move along the olfactory nerve towards the

central nervous system (CNS) and begin invading the brain and its meninges (Belizario and de

Leon, 2004).

Studies show that the pathogenic Naegleria species are capable of tolerating temperatures

of up to 460 C and a pH range of 4.0 to 9.5 (Villa et al., 1993). These conditions are present in

stagnant swimming pools during the summer season and geothermal plant effluents which flows

into lakes, rivers and streams. Studies have also shown that these species were isolated from

hyperchlorinated waters of 0.5 µg/mL (Init et al, 2010). Since these parasites can withstand the

effects of chlorination in drinking waters and swimming pools, amebic gastritis and

meningoencephalitis may easily be established within the host’s CNS.

If forcefully inhaled when swimming in contaminated waters, these protozoan parasites

will immediately multiply as trophozoites in the nasal cavity and consume the olfactory bulb

(Sandoval et al., 2008). Since the pathogenic Naegleria spp. can rapidly infect the mitral cell

axons and the rest of the cerebrum, PAM then is characterized by a sudden onset of frank

encephalitic symptoms, severe cephalgia, vomiting, nausea, and rigidity of the neck muscles

which may eventually progress to delirium, seizures and worst- irreversible coma. If left

untreated, death may result shortly after 7-14 days of incubation due to respiratory failure caused

by the destruction of the autonomic nerve cells of the medulla oblongata.

Diagnosis may be made prior to microscopic examination of cerebrospinal fluid (CSF) or

hematoxylin and eosin stained brain tissues from patients suspected of PAM (Cabral and Cabral,

2007). The presence of motile amebic trophozoites that move by means of very active

pseudopodia could mean a neurological infection caused by a pathogenic Naegleria species. The

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trophozoites can also be identified through their characteristic large karyosome that may be

covered by a halo (Mahon et al., 2007). Identifying PAM causing Naegleria at the species level

requires polymerase chain reaction (PCR) - today’s golden standard of identifying the wide array

of microorganisms present worldwide.

Since PAM is an exceptionally rare disease, many physicians and health workers all over

the world know little about the diagnosis of the disease. Thus, poor diagnosis and prognosis of

infection are established. Researches show that up to 2010, Naegleria was seldom given prior

importance in the differential diagnosis of CNS infections (Ithoi et al., 2011). Therefore,

questions arise if PAM is under-diagnosed or that N.fowleri, the most pathogenic species is rare

in the environment. With these, only 300 confirmed cases were recorded in 2008, with an in-

hospital fatality case of 97%.

From the time when PAM was first described in Australia, until December 1980, a period

of 15 years; only 49 cases were reported in the United States. Historical records have it that in

1974, DeJonckheere and co-workers isolated pathogenic N. fowleri from a canal where a boy

contracted PAM in 1973. This contaminated canal received warm effluent from a lead and zinc

factory in Belgium. During a 2-year period (1980-1981), the severity of PAM contracted from

thermal effluents, receiving waters and cooling towers of eight power plants located in Western

Pennyslvania were investigated (Sykora et al., 1982). And it was always proven that PAM leads

to death after 1 to 2 weeks prior to the time of entry if no medical treatment applied.

Other thermotolerant species, N. lovaniensis and N. australiensis were previously isolated

from thermal waters in Mexico. Thermotolerant Naegleria strains were also identified. Five of

which were from patients from the warm valley of Mexicalli in 1990, from California, from

Mexico and from a water sample of an artificial canal in which a victim was swimming before

contracting the disease (Villa et al., 1992).

In the last four decades, 200 cases of meningoencephalitis caused by N. fowleri were

reported (Cermeño et al., 2006). The first case of Naegleria infection in Asia was found at Si Sa

Ket, Thailand in 1985. The second case was in Bangkok, the third and fourth cases were from

Nakhon Pathom province, with a mortality rate of 100% (Tiewchaloren & Junnu, 1999). During

the period 1970 – 2001, only six patients have been reported to survive. The last unpublished

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case was reported in a Thai patient in 2001 at the Siriraj Hospital Faculty of Medicine, Mahidol

University (Tiewcharoen et al., 2002).

Molecularly, the pathogenesis of PAM is still not fully understood. Both the pathogenic

and non-pathogenic Naegleria species have been easily isolated from the environment and yet

the virulence factors and the pathogenicity of these microorganisms are still unknown (Cabral

and Cabral, 2007). Prolonged growth of N.fowleri in pure cultures results in the alternation of

their virulence factors whereas several passages through mice can restore and maintain their

pathogenicity (Cabral and Cabral, 2007). On the other hand, heat-shock protein 70 (HSP70)

found in N.fowleri also plays an important role in adaptive responses for its survival. At the same

time it regulates the cytolytic effect of the host’s immune system (Cabral and Cabral, 2007).

During entry into the host’s nasal cavity, the adherence of the pathogenic Naegleria

species to the host cell also plays a key role in the establishment of infection. Previous studies

had shown that trophozoites can immediately gain access to the extracellular matrix glycoprotein

(EMC) such as fibronectin, collagen, and lamina that are found in the basal lamina and

surrounding tissues of the host’s CNS (Cabral and Cabral, 2007). The ability of the trophozoites

to attach to the nasal mucosa, increase their rate movement and chemotactic responses to the

nerve cells also play important roles in the disease progression (Cabral and Cabral, 2007).

As of today, in vitro cell culture systems are being used to study the behavior of N.fowleri

in mammalian cell hosts. Findings show that during the course of infection, N.fowleri

trophozoites can potentially destroy the nerve cells and the other cells in the host CNS by

trogocytosis- a primitive feeding mechanism that uses “food-cup” found in the surface of the

amoebae that releases cytolyitc molecules that facilitate easier ingestion of food particles (Cabral

and Cabral, 2007).

In vitro cell culture studies had also shown that there are membrane-associated pore-

forming proteins on the surface of N.fowleri. These two membrane-associated pore forming

proteins are designated as Naegleriapore A and B. Both of these molecules exhibit cytotoxicity

to human erythrocytes. Naegleriapores enable the amoebae to secrete proteases, acid hydrolases,

and phospholipolytic enzymes that can degrade sphingomyelin (Cabral and Cabral, 2007). It has

also been reported that N. fowleri can produce higher levels of phospholipase A and

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lysosphospholipase compared to other non-pathogenic Naegleria species (Cabral and Cabral,

2007). It was also detected that N. fowleri can produce elastase, an enzyme that degrades a wide

range of tissue proteins such as collagen and proteoglycan (Cabral and Cabral, 2007).

Previous studies have also shown that once in the CNS, the amoebae can induce an

inflammatory response that will lyse leukocytes.

In order to fully understand the amoebae’s pathogenicity, it is inevitable that the host’s

immunological responses to N. folweri be studied. Thus researches were conducted on how N.

fowleri induces excessive mucus production in mice. In order to show mucus secretion in mice

infected with N. fowleri, histological analyses of the animals’ nasal cavity were performed. One

hour prior to infection, increased mucus production and secretion were already observed.

Trophozoites were also mixed with the mucus in the nasal cavity. Six hours prior to infection,

neutrophils were observed (inflammatory reaction) with the amoebae in the mucus secretions.

But after twelve hours, the amoebae had already adhered and penetrated to the neuroepithelium.

This only shows that the trophozoites can evade the primary inflammatory response and establish

infection (Sandoval et al., 2008).

Aside from the ability of trophozoites to attach to the nasal mucosa, N. fowleri has also

developed mechanisms to further evade the host’s immune system. It has been presented that the

amoebae are resistant to cytolytic molecules such as tumor necrosis factor (TNF)-α, IL-1, and

membrane attack complex (MAC) C5b-C9 of the complement system.

The components of the innate immune system that have been shown to respond

effectively to N. fowleri infection include complement, neutrophils and macrophages (Cabral and

Cabral, 2007).

On the other hand, only minor protective effects are attributed to antibodies during an

immune response to N. fowleri infection. These molecules can only immobilize the amoebae,

enhance complement-mediated lysis, and neutralize the amoebae’s cytotoxins. Indeed, humoral

immunity is not a major line of defense against Naegleria (Cabral and Cabral, 2007).

Naegleria are known to feed on bacteria, yeasts and algae. The interaction of bacteria and

amoebae can result in the destruction of amebae, lysis of the batceria or development of a

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symbiotic relationship. In this relationship N. fowleri are not only known as agents of human

disease. They can also serve as reservoirs for pathogenic bacteria sequester them from biocides

in the environment (Cabral and Cabral, 2007).

Therefore, the use of non-nutrient agar seeded with Escherichia coli for the recovery and

isolation of Naegleria spp. had become a part of the protocol of most microbiology laboratories

studying these elusive organisms. Their predation for bacteria is reflected by the fact that most of

these organisms were isolated from water samples gathered one meter away from the walls of

swimming pools where bacterial biofilms grow abundantly (Init et al., 2010).

As of today, the medically accepted treatment for PAM is the administration of

amphotericin B. Its amebicidal properties are characterized by the alteration of the amoebae’s

nuclei and mitochondria. The drug’s mode of action highly decreases the amoebae’s food

vacuoles thus depriving them of nutrients (Belizario and de Leon, 2004). Research studies

conducted in Mahidol University, Thailand shows that the combination of 5-fluorouracil and

amphotericin B had also promising amebicidal properties in vitro (Tiewcharoen et al., 2002).

Since Naegleria spp. are virtually ubiquitous in fresh water environments and there are

still no scientifically proven means of prevention and control, it still safe and advisable to avoid

diving and swimming in bodies of water suspected for contamination of these free living

ameboflagellates.

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Definition of Terms

The following terms are conceptually and operationally defined for the better

understanding of the study:

1.) Occurrence – a general extension of an event considered as simply presenting itself

to notice without obvious cause.

2.) Selected Waters- refer to the different artificial and natural bodies of fresh water

such as swimming pools, lakes and hot springs that may cater the growth of

Naegleria spp. present in the locale.

3.) Leyte and Biliran– two provinces in Eastern Visayas utilized by the researchers for

specimen gathering due to its closeness to the microbiology laboratory wherein data

gathering and specimen processing are to be performed.

4.) Primary Amebic Meningoencephalitis –a disease of the central nervous system

caused by infection of Naegleria fowleri and is characterized by inflammation of the

brain and its meninges which may rapidly develop to coma and even death if left

untreated.

5.) Naegleria spp. – a suspected group of amoebae that can cause a very rare, but severe,

infection of the brain. These amoebae are commonly found in warm freshwater (for

example, lakes, rivers, and hot springs) and even in the soil.

6.) Isolates- pure culture of parasites that are going to be identified if they can potentially

cause PAM among 2-6 week old albino mice.

7.) Millipore filter - a thin membrane of modified cellulose that is used as a filter in the

bacteriological examination of water or waste water. In the course of this study, it will

be used to separate bacteria and amoebae that may be present in the water sample.

8.) Centrifugation- a separation technique that uses centrifugal force. In the course of

this study, it will be compared to millipore filtration to find out if there is a significant

difference in the viability of amoebae recovered from the water samples collected.

9.) Escherichia coli – A gram negative bacilli utilized by the researchers as a food

source for free living ameboflagellates present in the water samples.

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