University of Santo Tomas

Faculty of Pharmacy

Department of Medical Technology

A Comparative Study of Acquiring Pneumonitis among Smokers and Passive Smokers

with Ascariasis

A Thesis Submitted to the

Faculty of Pharmacy

Department of Medical Technology

University of Santo Tomas

In Partial Fulfillment of the Requirements

For the Degree Bachelor of Science

in Medical Technology

By

Asenci, Jim Carlos G.

Dela Cruz, Mariella Mhae Q.

Pondevida, Lean Miguel B.

Rosales, Gersa May S.

Zacarias, Yenna Mae D.

OCTOBER 2017
 CHAPTER 1

THE PROBLEM AND ITS BACKGROUND

Introduction

According to the Philippine Statistics Authority, 28% or 17.3 million Filipino adults

of age 15 years or older are currently tobacco smokers. Cigarettes, which include

manufactured cigarettes and hand-made rolled cigarettes, are mainly the type of tobacco

that Filipinos smoke. Among WHO regions, the Western Pacific Region which covers East

Asia and the Pacific has the highest smoking rate, with nearly two-thirds of men smoking

(WHO, 2002). Based on a previous survey conducted by the World Health Organization on

May 27, 2002, about 60% of Filipino men smoke. According to the survey, more than half of

Filipino households are not smoke-free. People who smoke in public areas as well as

communal areas inside their homes cannot prevent the spread of secondhand smoke as well

as third hand smoke, which is residual nicotine stuck to clothing and other surfaces even

after the smoke clears. Those people who are exposed to secondhand smoke are considered

to be passive smokers.

Smoking is a silent killer and is one of the leading causes of death worldwide. Ten

people die every hour because of smoke-related diseases. This translates to 240 deaths every

day or 87,600 death every year (Manila Times, 2015). The Centers of Disease Control and

Prevention (CDC) have stated numerous smoke-related diseases, which include developing

heart disease, stroke, and lung cancer (2017). However, this study focuses primarily on the
development of pneumonitis or inflammation of the lungs, taking into account whether the

persons involved are smokers or passive smokers.

Pneumonitis, in the general context, is the inflammation of lung tissue. It is also

commonly called as pulmonitis, in which the lungs air sacs become inflamed due to the

inhalation of certain substances, thereby causing difficulty in breathing. Statistics from a

previous study conducted by NSVR (2001) have indicated that death from pneumonitis

accounted 15,268 in the United States of America. Pneumonitis, when left untreated, can

cause irreversible lung damage. Pneumonitis is caused by various factorsincluding, but not

limited to, drugs, molds and bacteria, bird exposure, radiation treatments, as well as parasitic

infections. This study focuses on pneumonitis caused by larval migration of Ascaris

lumbricoides to the lungs.

Ascariasis is an infection caused by the soil-transmitted helminth A. lumbricoides, also

known as the giant roundworm. According to Centers for Disease Control and Prevention

(CDC), it is one of the most common intestinal helminths in the world, affecting

approximately 807 to 1,121 million people. Ascaris infections are obtained through ingestion

of embryonated eggs mainly through food and water contaminated by human feces. Poor

sanitation and personal hygiene are one of the main predisposing factors to A. lumbricoides

infections.

Ascaris larvae residing in the small intestine cause further complications through

extraintestinal migration. Commonly, larvae undergo lung migration and result in multiple

pulmonary manifestations, more specifically, Ascaris pneumonitis or ascariasis

pneumonitis. Also, other terms used for this disease are eosinophilic pneumonitis, Loefflers

syndrome, and pulmonary ascariasis.

In a previous study conducted by Heggers, J.P., Muller, M.J., Elwood, E., and Herndon,

D.N. (1995), ascariasis pneumonitis has been proven to be a potentially fatal complication in

smoke inhalation injury patients burn victims with lung injury caused by smoke. In line

with this, this study aims to establish whether this complication spans out to cigarette

smokers as well as passive smokers.

Statement of the Problem

The study aims to determine the risk of having pneumonitis among patients with

ascariasis who are either smokers or passive smokers. It aims to provide answers to the

following queries:

1. In the condition of both having ascariasis, who has the greater probability of

developing pneumonitis?

2. How much smoke inhaled affects the possibility of developing pneumonitis?

3. What is/are the components of inhaled smoke that indicate the development of

pneumonitis?

Objectives of the Study

General Objective:

The primary aim of this research is to determine the prevalence of acquiring

pneumonitis among smokers and passive smokers diagnosed with ascariasis.

Specific Objectives:

This study specifically seeks to:

1. determine whether passive smoking or direct smoking increases the

risk of developing pneumonitis among patients diagnosed with

ascariasis,

2. establish how much smoke intake affects the development of

pneumonitis, and

3. identify whether cigarette smoking is a major risk factor in ascariasis

pneumonitis in order to formulate hypotheses and develop preventive

intervention strategies.

Significance of the Study

Previous studies correlated the fatal complication of pneumonitis in ascariasis

patients with smoke inhalation injuries, wherein two of three ascariasis patients with smoke

inhalation injuries developed severe pneumonitis. This study will assess whether being a

smoker or a passive smoker will increase the burden of the disease, ascariasis pneumonitis.

With this, the pervasiveness of smoking in the Philippines along with its harmful effects were

taken into account as well as A. lumbricoides being one of the most common helminthic

infections in the world.

Scope and Limitations

This comparative study is limited to smokers and passive smokers with A.

lumbricoides. The study will be conducted to a total of 50 randomly selected men

presumptively diagnosed with ascariasis, with the age bracket of 30 to 45. All the randomly

selected men are residents of Tondo, Manila. The study is limited to determining the

prevalence of having pneumonitis only. This will be conducted from April to May 2018.

The study will not be determining other diseases associated with the migration of

A.lumbricoides to the lungs as well as infections caused by other helminthic parasites.

Definition of Terms

The following terms are defined for the readers to have a better understanding of the content

of this research:

Ascariasis pneumonitis- inflammation of the lungs caused by A. lumbricoides, also called

as Ascaris pneumonitis, eosinophilic pneumonitis, Loefflers

syndrome, and pulmonary ascariasis

Embryonated egg- A. lumbricoides egg containing a larva. This is the infective stage of A.

lumbricoides to humans.

Extraintestinal migration- movement of A. lumbricoides from the small intestine into

other organs in the body, more commonly in the lungs

Larvae- a form of A. lumbricoides that causes infection in humans; commonly resides in the

small intestine but can undergo extraintestinal migration

Passive smoking- involuntary inhalation of secondhand cigarette smoke; may also be

thirdhand smoke, which is contact with nicotine residue that remains in

clothes and surfaces long after a cigarette smoke has cleared.

Susceptibility- a lack of immunity or resistance by a person, thus increasing the risk of being

infected or having a disease

 CHAPTER 2

Review of Related Literature

2.1 Ascaris lumbricoides

Figure 1. Left/Right: Fertilized eggs of A. lumbricoides in unstained wet mounts of stool. Center: Adult

female A. lumbricoides. Credit: DPDx, Orange County Public Health Laboratory, Santa Ana, CA.

A. lumbricoides is a common parasite in various parts of the world, especially among

the lower socioeconomic groups (Vedjani & Namvar, 2009). A. lumbricoides parasites cause

ascariasis, one of the most widespread parasitic diseases in the world. Its most

characteristic effects are perforation of the intestinal wall and pulmonary abscesses (Lupo,

Leguto, Bartolato & Korol, 2016).

The human parasitic nematode A. lumbricoides is of major medical importance,

infecting 1.5 billion people worldwide (OLorcain and Holland, 2000; Elliott et al., 2007).

Infection by A. lumbricoides causes acute effects such as intestinal obstruction and chronic

infection impacts upon growth, appetite, physical fitness, work capacity and cognitive

development in populations who are already compromised by poor nutrition, and other
educational and health disadvantages (Bradley and Jackson, 2004; Holland, 2009). Ascaris

infection is characterised by a predominant Th2 immune response and resistance to

infection is associated with high levels of the Th2 cytokines, IL-4 and IL-5, and low levels of

Th1 cytokines (Cooper et al., 2000; Geiger et al., 2002). However, there are few studies to

date that have examined the interaction of A. lumbricoides-derived molecules with DCs.

2.1.1 Life cycle of A. lumbricoides

Figure 2. Ascaris lumbricoides life cycle. Retrieved from

https://www.cdc.gov/parasites/ascariasis/biology.html
 Adult worms live in the lumen of the small intestine. A female may produce

approximately 200,000 eggs per day, which are passed with the feces. Unfertilized

eggs may be ingested but are not infective. Fertile eggs embryonate and become

infective after 18 days to several weeks, depending on the environmental conditions

(optimum: moist, warm, shaded soil). After infective eggs are swallowed, the larvae

hatch, invade the intestinal mucosa, and are carried via the portal, then systemic

circulation to the lungs. The larvae mature further in the lungs (10 to 14 days),

penetrate the alveolar walls, ascend the bronchial tree to the throat, and are

swallowed. Upon reaching the small intestine, they develop into adult worms.

Between two and three months are required from ingestion of the infective eggs to

oviposition by the adult female. Adult worms can live one to two years (Belizario &

De Leon, 2004).

2.2 Ascariasis

Stewart and Hoys study (as cited in Mendes, Rebeiro, Severo & Niza-Ribeiro, 2017)

states that Ascariasis is the most common parasitosis of swine worldwide and is mainly

caused by the helminth Ascaris suum. The striking feature of this nematodiasis is the

migration of the parasite through the liver, which affects animal welfare and productivity

(Stewart & Hoyt, 2013).

Yoshihara et al. (as cited in Mendes, Rebeiro, Severo & Niza-Ribeiro, 2017) describes

that the pathological consequence is a chronic multifocal interstitial hepatitis that is usually

asymptomatic. However, at post-mortem inspection, the liver will show multiple, spherical,
and whitish foci, which are commonly called milk spots, rendering it unfit for human

consumption.

Ascariasis is the term used to describe the spectrum of disease symptoms observed

in infected humans and pigs. Morbidity and mortality increase with worm burden, and

those who harbour light infections tend to be asymptomatic. Aggregation leads to relatively

few individuals harbouring sufficient worms to precipitate life-threatening or severe

morbidity (Dold & Holland, 2010).

Adults with ascariasis are more likely to develop biliary complications due to

migration of adult worm. Intestinal obstruction, usually in the terminal ileum, is the most

commonly attributed fatal complication, resulting in 8000-100,000 deaths per year,

according to World Health Organization.

In previous studies, that in the Philippines, it was also indicated that infection rates

for intestinal parasites were higher in male than female. Tricuris trichiura and A.

lumbricoides remain to be the most prevalent helminths (Baldo, Belizario, De Leon, Chung,

& Chung, 2004).

2.2.1 Major risk factors of acquiring ascariasis

Globally, intestinal parasitic infections (IPIs), including helminths and

protozoa, remain endemic. About one-third of the worlds populations, more

than 2 billion people, are infected with the largest majority being children.1,2

Helminths parasites are mostly A. lumbricoides, Trichuris trichiura and

hookworms, and are commonly referred to as soil-transmitted helminths

(STHs). A recent report implicating STHs in over thirty-nine million disability

adjusted life years (DALYs), and increased mortality rate, highlights the

burden of these diseases (Gyang et al., 2017).

Ascariasis or A. lumbricoides infection is associated with poor

sanitation and hygiene, among other biological and social factors, according

to a study conducted by Quntero et al. (2012) in North Central Venezuela.

3,388 individuals among 4,675,433 residents in the region were interviewed

using a probabilistic, stratified, cross-sectional survey. The study shows that

ascariasis has a significantly higher prevalence among people living in

vulnerable housesthose that are characterized by rural households with

inappropriate building materials, insufficient basic public services such as

water and waste disposal, and surrounding-disease prone environments. In

addition, according to Ugbomoiko (2009), the number of biological parents

living with a child and the number of playmates a child has are socio-

environmental risk factors that play a role in disease transmission.

Moreover, in a study performed by Huat et al. (2012) among children

in Beris Lalang, Malaysia, poor education of mothers (primary education or

less), eating raw salad, and physical inactivity were determined to be

independent risk factors. Also, a higher percentage of those with ascariasis

complained of tiredness and fatigue as compared to those without such

condition.
 2.2.3 Intensity of ascariasis

Ascariasis is preventable, it is on its way of disappearing completely

in developed societies where there are high standards of sanitation.

However, it remain problems in most of still developing countries, where the

methods of disposal of human excreta are inadequate. The intensity of

invasion is regulated by specific or non-specific responses of the host to

migrating A. lumbricoides larvae. The role of ascariasis in the prevalence of

allergic asthma still remains unclear. Complications due to migration of adult

worms into the biliary duct system and intestinal obstruction are the major

cause of mortality and acute morbidity in ascariasis. (Pawlowski, 1982).

According in some studies of > or = 100 patients are admitted due to

ascariasis, intestinal obstruction was the most common complication and

accounted for 38-87.5% of all complications. The proportion of intestinal

obstruction caused by ascariasis was identified in 14 studies from 7 countries

with varying degrees of endemic ascariasis.

2.3 Infection rates of Ascaris lumbricoides in relation with the seasonal variation in

the Philippines

The infection rate of A. lumbricoides is also dependent on the climatic conditions. In

a study conducted by Cabrera (1984) of the 203 Ascaris worms collected 69% were females
and 31% were males with a sex ratio of 2.4 females to one male; 73% were mature and 27%

were immature females; 70% were mature and 30% were immature males; mean number

of worms per child was 2.6. The mean length and weight of female was 22.9 cm and 3.6 gms

respectively while for males it was 16.1 cm and 1.7 gm respectively. The graph showing

amount of rainfall, reinfection and infection rates of ascariasis revealed the presence of two

major peaks both in the reinfection and infection rates noted in December-January and

May-June. Likewise, the rainfall also peaked twice namely in August and October. It appears

that ascariasis reinfection or transmission is highest when rainfall is minimal and lowest

when rainfall is at its highest peak.

2.4 Intestinal migration of Ascaris lumbricoides

Larval migration in the host lung tissue induces pulmonary distress in both porcine

and human hosts. The respiratory distress experienced during pulmonary ascariasis is

referred to Loefflers syndrome, which is a recognised eosinophilic disease. In A.

lumbricoides infection, dyspnoea (difficult or laboured breathing) and bronchospasm may

be severe. Severe dyspnoea has also been documented in porcine infections. Short dry

coughs are also a typical feature of A. suum-induced respiratory distress in pigs and have

been reported in experimental infections of cows (Dold & Holland, 2010).

2.5 Ascaris Pneumonitis

 Ascaris pneumonitis in areas of endemic infestation is considered a benign

condition. Smoke inhalation with any burn injury can be potentially fatal. A heavy

infestation of Ascaris could further exacerbate the smoke-induced lung injury. After

ingested eggs hatch in the small intestine, the larvae penetrate the mucosa and invade the

bloodstream and are then carried to the lungs. The larvae break out into the alveolar spaces

as they too large to cross the capillary bed and are carried up the bronchial tree and

eventually swallowed (Heggers, Muller, Elwood, & Herndon, 1995).

In the study conducted by Heggers, Muller, Elwood, & Herndon (1995), it described

three cases of Ascaris infection in thermally injured children. While the burns were < 30

per cent total body surface area, two patients who were injured in the same fire had a

further complication of smoke inhalation which necessitated sophisticated therapy in order

to promote survival. All patients were treated initially with Vermox. The one patient

without smoke inhalation did not develop ascariasis pneumonitis even with positive stool

samples and was discharged with no complications, whereas the two with smoke inhalation

developed severe pneumonitis. One patient was placed on ECMO and did not receive a full

course of the Vermox treatment. This patient died after several weeks of ECMO treatment.

The third patient received a full course of Vermox, slowly recovered, and went home.

Supportive therapy only is recommended during the lung migration phase of the Ascaris

lifecycle. We feel that continuation of chemotherapy (Vermox) would have been beneficial

in the fatal case based on the survival of the second patient. Ascaris-induced lung injury

superimposed upon severe smoke-induced lung injury may have had an additive effect that

precipitated severe, unrecoverable respiratory failure.

2.6 Pneumonitis

Pneumonitis is a general term for that refers to inflammation of lung tissue. Common

causes of pneumonitis include airborne irritants from your surroundings. If you have

worked with asbestos, handled birds, owned hot tubs or a humidifier, or worked in the

farming industry, you are at a higher risk. In addition, certain chemotherapy drugs can

increase your risk as well as radiation therapy targeting the respiratory system.

The annual incidence of pneumonitis has been estimated as 30:100,000 with

hypersensitivity pneumonitis accounting for fewer than 2% of these cases. Some risk of

developing side-effects is not only from radiation dose but also may be affected by patient-

related risk factors such as older age and the presence of morbidity (Dr. Colin Tidy, 2015).

2.6.1 Effects of Pneumonitis

Radiation-induced pneumonitis and fibrosis are compli- cations that

may arise in the lung after irradiation of any kind of malignant disease in the

thorax, such as radiation treatment after breast cancer surgery and as a

primary treatment for esophagus cancer patients. Based on a study

conducted by Johannsona, Bjermerb, Franzena, & Henrikssona (1998), 6

breast cancer patients had spontaneously reported pneumonitis, which was

radiation- induced, after ongoing smoking. Five of them were non-smokers

(P=0.182) and the other was a former smoker. Eight of the oesophagus cancer
 patients had spontaneously reported radiation-induced clinical pneumonitis

and they were all non-smokers (P=0.022), except one, who was a pipe

smoker. None of the patients who were cigarette smokers were recorded as

developing clinical pneumonitis after irradiation. The inflammatory process

of the lung tissue in broncho- alveolar lavage (BAL) after radiation treatment

of breast cancer patients. Ongoing smoking seemed to suppress the early

inflammatory reactions in BAL after radiation treatment and these data have

been confirmed by experi- mental studies on animals as well. As far as we

know, radiation-induced pneumonitis has not previously been evaluated in

oesophagus cancer patients (Johansson, Bjermer, Franzen, & Henriksson,

1998) .

2.7 Smokers and Passive Smokers

2.7.1 Prevalence of Smoking in Middle-aged Men

Ng et al. (2014) stated that in men, prevalence increased rapidly in the

15- to 19-year and 20- to 24-year age groups in both developed and

developing countries. The highest prevalence rates were seen at age 30 to 34

years in developed countries and age 45 to 49 years in developing countries.

After age 35 to 39 years, prevalence was consistently higher in developing

countries.

2.7.2 Secondhand smoking

 Lee et al. (2010) indicated that exposure to secondhand smoke (SHS)

is a major threat to public health. Asian countries having the highest smoking

prevalence are seriously affected by SHS.

The mechanisms by which secondhand smoke affects others is

debated; they probably resemble the pathway in the primary smoker but may

have other effects. Mainstream smoke, which is inhaled by the primary

smoker, differs from sidestream smoke, which is released into the

environment. It is possible that sidestream smoke may be more toxic, and

physiologic changes among nonsmokers who are regularly exposed to

cigarette smoke could have widespread effects. These include lower levels of

high-density lipoprotein cholesterol, increased atrial fibrillation, and platelet

abnormality. Also observed are endothelial dysfunction and lower exercise

tolerance. All these factors are associated with increased incidence of

cardiovascular disease and, coupled with the positive effects of smoking bans

on non smokers, reinforce the importance of reducing exposure to

secondhand smoke (Luepker, 2016).

2.7.3 Thirdhand smoking

According to Protano and Vittali (2011), thirdhand smoke is a

complex phenomenon caused by residual tobacco smoke which adhere to

clothing, surfaces and dust indoors. Such pollutants persist long after

secondhand smoke has cleared. In addition, they are reemitted into the gas

phase, or even react with other oxidants or compounds in the environment

 to produce secondary contaminants, some of which are carcinogenic or

otherwise toxic for human health (Matt et al., 2011).

Thirdhand smoke exposure consists of unintentional intake of

tobacco smoke and other related chemicals, mainly through inhalation as

well as ingestion and intradermal routes. This exposure can also take place

long after smoking has stopped, through close contact with smokers and in

indoors where tobacco is smoked frequently.

2.7.4 Component of cigarette that causes disease

Cigarette smoking is associated with an increased risk of respiratory

tract infections, chronic airway disease, and cardiovascular diseases, all of

which may be modulated by endogenous nitric oxide (NO). Based on the

study of Kharitonov, Robbins, Yates, Keatings, & Barnes (1995), 41 current

cigarette smokers with normal lung function and 73 age-matched non-

smoking controls were compared. Peak exhaled NO levels were measured by

a modified chemiluminescence analyzer. The effects of inhaling a single

cigarette in smokers were also measured. In control subjects we also

measured the effects of inhalation of NO itself and carbon monoxide, both

constituents of tobacco smoke. Peak exhaled NO concentrations were

significantly reduced in smokers (42 +/- 3.9 compared with 88 +/- 2.7 parts

per billion in nonsmokers, p < 0.01), with a significant relation between the

exhaled NO and cigarette consumption (r = 0.77, p < 0.001). Smoking a single

cigarette also significantly (p < 0.02), but transiently, reduced exhaled NO.
Inhalation of carbon monoxide and NO had no effect on exhaled NO in normal

subjects. Cigarette smoking decreased exhaled NO, suggesting that it may

inhibit the enzyme NO synthase. Since endogenous NO is important in

defending the respiratory tract against infection, in counteracting

bronchoconstriction and vasoconstriction, and in inhibiting platelet

aggregation, this effect may contribute to the increased risks of chronic

respiratory and cardiovascular disease in cigarette smokers.

According to a study conducted by Ezzati and Lopez (2003) estimated

that in 2000, 483 (uncertainty range 394593) million premature deaths

in the world were attributable to smoking; 241(180315) million in

developing countries and 243 (213278) million in industrialised

countries. 384 million of these deaths were in men. The leading causes of

death from smoking were cardiovascular diseases (169 million deaths),

chronic obstructive pulmonary disease (097 million deaths), and lung

cancer (085 million deaths

It is assumed that the effects of smoking on the vasculature are

mediated by smoke constituents that are spilled over from the lung into the

circula- tory system . More than 5300 individual constituents have been

identified in cigarette smoke; however, their individual con- tributions and

potential mechanisms of action are largely un- known. Cigarette smoke has

also been shown to enhance oxidative stress in the endothelium and decrease

the release or activity of nitric oxide (NO) in vascular ECs (Steffen et al., 2011)
2.8 The relevance of the review to the present study

A. lumbricoides is one of the most common soil-transmitted helminths worldwide.

Ascaris infections or ascariasis possess multiple risk factors, primarily sanitation and

hygiene. In its life cycle, A. lumbricoides normally undergoes lung migration, which can

cause a variety of conditions, most commonly pneumonitis, also called Loefflers syndrome.

In a previous study, it has been shown that smoke inhalation injury further increases the

chance of occurrence of pneumonitis in Ascariasis patients. In contrast, this study aims to

establish the relationship between cigarette smoking in ascariasis-infected patients.

CHAPTER 3

RESEARCH METHODS
 This chapter discusses the various procedures that will be conducted in order to

accomplish the objectives of the study. In the study, the research method that will be utilized

is experimental. The tests that will be employed in the methods are direct fecal smear (DFS),

white blood cell count (WBC), differential white blood cell count, and chest X-rays.

3.1 Study Participants

The participants of this study are smokers, passive smokers, and non-smokers who

are initially presumed to be diagnosed with Ascariasis. Furthermore, this study will be

conducted to a total of 50 randomly selected men within the age bracket of 30 to 45 years

old residing in the slums of Tondo, Manila.

3.2 Study Locale

The study will be conducted in the slums of Tondo, Manila. Among the 16 districts in

Manila, it is the largest in terms of land area, which has a total of 5.64 km. Moreover, it is the

most densely populated district with a census-estimated 631,313 people in 2015 and further

consists of two congressional districts. Tondo is known for being one of the poorest and most

underdeveloped areas of the country, rendering it to be prone to helminthic parasites such

as A. lumbricoides; this is according to the study conducted by Baldo, Belizario, Kong, and

Chung (2004).

3.3 Materials

3.3.1 Reagents

3.3.1.1 Direct Fecal Smear (DFS)

 The reagents to be used for the Direct Fecal Smear (DFS) are the

following: 10% formalin, ether (ethyl acetate), and iodine. The sole

function of formalin is to fix the eggs, larvae, oocysts, and spores to

make the organism no longer infectious, as well as, to preserve the

morphology of the organism (CDC, 2016).

3.3.1.2 WBC Count

The reagents to be used for the White Blood Cell Count (WBC) are WBC

diluting fluids; 1% to 3% Acetic acid with Gentian violet, 1% Hydrochloric acid,

and Tuerks solution.

3.4 Methods

Before all of the tests will be done, consent forms and waivers will be given to the 50

randomly selected men who are presumptively suffering from Ascariasis.

3.4.1 Initial test for the presence of A. lumbricoides

Direct fecal smear (DFS) will be used in checking for the presence of

A. lumbricoides. According to CDC (2013), the standard method for diagnosing

ascariasis is by identifying A. lumbricoides eggs in a stool sample using a

compound light microscope. Because eggs may be difficult to find in light

infections, a concentration procedure is recommended. The concentration

procedures are divided into the two kinds, namely sedimentation technique
and floatation technique. In this study method, the sedimentation technique

will be practiced. In this technique, solutions of lower specific gravity relative

to the parasitic organism will be used, thus concentrating the latter in the

sediment. The reagents to be used, as stated in the section above (see Materials

section 3.3.1.1), are 10% formalin, ether, and iodine.

The stool samples will be collected from the 50 randomly selected men

who are presumptively suffering from the disease. After the collection, 5mL of

each stool suspension will be strained through wetted cheesecloth-type gauze

placed over a disposable paper funnel into a 15 mL conical centrifuge tube.

Then, 10% formalin will be added through the debris on the gauze to bring the

volume in the centrifuge tube to 15 mL. This will be centrifuged at 500 x g for

ten minutes and after the process, the supernatant will be decanted. Another

10 mL of 10% formalin will be added to the sediment and will be mixed

thoroughly using a wooden applicator stick. Subsequently, four milliliters of

ethyl acetate will be added, a tube stopper will be placed, and will be shaken

vigorously in an invert position for 30 seconds. After the said time, the stopper

will be removed, then centrifuged at 500 x g for 10 mins. Then, after

centrifugation, the top layers of the supernatant will be decanted, and several

drops of 10% formalin will be added to resuspend the concentrated specimen.

After performing the formalin-ethyl acetate sedimentation technique,

the sediments will be examined on a wet mount. This will allow the

examination of A. lumbricoides ova, larvae, and adult form.

 3.4.2 Division of Study Participants According to Smoke Intake

After conducting the test for the presence of A. lumbricoides, the

following step that will be done is the observation of each individual who

tested positive for A. lumbricoides in their stool. Each individual will be

assessed regarding their smoking habits. A survey form will be sent out to the

individuals containing questions that establish whether they are a smoker,

passive smoker with a succeeding question of whether they are passive or

non-smokers.

When all of the individuals have responded, the next step will be the

division of the groups. These groups are the smoker group, passive smoker

group, and the controlled non-smoker group.

3.4.3 Test for the larva migration of A. lumbricoides to the lungs and checking

for the development for the pneumonitis

Ascariasis infection is acquired by ingestion of embryonated eggs that

penetrate the small intestine and are passed out to the liver, and larval lung

migration begins. Lung migration happens 10-15 days after ingestion. This

may cause several manifestations that includes pneumonitis. A number of

tests can be performed for diagnosis of A. lumbricoides including chest

radiography (x-ray) and laboratory tests.

 Chest x-ray would reveal round or oval infiltrates. Patients physical

examination and blood test will also reveal eosinophilia. The white blood cell

count (WBC) will be performed by extracting blood from the patient and

placing it in an ethylenediaminetetraacetic acid (EDTA) tube. Blood will be

drawn to the 0.5 mark of the WBC pipette and be mixed with diluting fluid up

to the 11 mark. WBC diluting fluid are 1% to 3% Acetic acid with Gentian

violet, 1% Hydrochloric acid, and Tuerks solution. Then, the contents of the

pipette will be mixed for five minutes in a horizontal position. After mixing,

two to three drops of the diluted blood will be discarded, and the Neubauer

counting chamber will be carefully charged. The chamber will be allowed to

stand for five to ten minutes to facilitate settling of white blood cells. Under

the low power objective, the ruled areas for WBC which are the four secondary

squares will be located. Strip differential method will be used in counting WBC.

In order to assure the accuracy of the test, WBC count for each ruled are should

only be 12 or less. For the computation, the formula will be used. As an

alternative for WBC count, differential count of WBC may also be used. In this

test, a peripheral blood smear is needed. A two-slide method can be used in

preparing a peripheral blood. Procedure of staining are as follows: 30 seconds

in methanol, six seconds in eosin, four seconds in methylene blue, and 45

seconds in distilled water or buffered solution. Counting of WBC must be done

on the feathery edge part of the blood smear under the Oil immersion

objective. Strip differential method will also be used in counting 100 WBC, and
 at the same time differentiating them. The normal value of eosinophil is one to

three.

3.5 Data Analysis

Data found from the study will be written accordingly using the following table.

Table 1. Format for the data collected from the study

Presence of Pneumonitis

Smoke intake Positive Negative TOTAL

Smokers

Passive Smokers

Non-Smokers

TOTAL

In case the frequency of patients positive for pneumonitis among smokers and

passive smokers is small (less than five), then the following table will be used instead for the

data, wherein the data for smokers and passive smokers are merged.

Table 1. Alternative format for the data collected from the study
 Presence of Pneumonitis

Smoke intake Positive Negative TOTAL

Smokers & Passive smokers

(With smoke intake)

Non-Smokers
(Without smoke intake)

TOTAL

For data analysis, the chi-square test of association will be used. This test is commonly

used to compare 2 variables one variable having nominal data and the other ordinal data.

The nominal data in the study is the presence of pneumonitis caused by the ascariasis

infection. On the other hand, the ordinal data will be the intake of cigarette smoke, which is

divided into 3 categories, namely, smoker, passive smoker, and non-smoker.

The following hypotheses will be used in the test:

Null Hypothesis: Ascariasis pneumonitis is not associated with cigarette

smoke.

Alternative Hypothesis: Ascariasis pneumonitis is associated with cigarette

smoke.

The following formulae will be used in the data analysis:

Chi-square test formula:

()2
2 = ; whereas O = observed frequencies; E = expected frequencies

Degree of frequency (used for the determination of critical value)

 = ( 1)( 1); whereas i and j are total no. or rows and columns,

respectively

The necessary calculations using the formulae above will be done and compared to

the critical value at 5% significance level. This comparison will determine whether to reject

the null hypothesis or not. Beyond the critical value, it is imperative to reject the null

hypothesis and conclude that there is sufficient evidence that smoking is associated with

ascariasis pneumonitis.

Finally, the odds ratio will be computed using the data collected from the study:
.
.
Odds ratio = .
.
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