MEKELLE UNIVERSITY

COLLEGE OF HEALTH SCIENCES

DEPARTMENT OF PHARMACY

COMPARATIVE IN VITRO QUALITY EVALUATION ON DIFFERENT

BRANDS OF THE COMMONLY PRESCRIBED ANTHELMENTIC DRUGS,
MEBENDAZOLE TABLETS, FROM DRUG RETAIL OUTLETS IN MEKELLE
TOWN, TIGRAY, ETHIOPIA

By: Abraham Gebrezgabiher

Advisor: Mr. zewdu
.
A RESEARCH PROPOSAL SUBMITTED TO MEKELLE UNIVERSITY
COLLEGE OF HEALTH SCIENCES DEPARTMENT OF PHARMACY FOR
THE PARITIAL FULLFILMENT OF DEGREE IN CLINICAL PHARMACY
December, 2014

Mekelle Ethiopia
Title: COMPARATIVE IN VITRO QUALITY EVALUATION ON DIFFERENT BRANDS
OF THE COMMONLY PRESCRIBED ANTHELMENTIC DRUGS, MEBENDAZOLE
TABLETS, FROM DRUG RETAIL OUTLETS IN MEKELLE TOWN, TIGRAY, ETHIOPIA

Name of investigator Abraham

Name of adviser ADVISORS: MR. ZEWDU

Full title of the research COMPARATIVE IN VITRO QUALITY

project EVALUATION ON DIFFERENT BRANDS OF

THE COMMONLY PRESCRIBED
ANTHELMENTIC DRUGS, MEBENDAZOLE
TABLETS, FROM DRUG RETAIL OUTLETS IN
MEKELLE TOWN, TIGRAY, ETHIOPIA
Duration of the study JAN 1ST TO JAN 20TH 2015 G.C
Study area AYDER REFERRAL HOSPITAL
Total cost of the project
Address of investigator Mob +251914485290
Contents
1. INTRODUCTION ......................................................................................................................................... 4
1.1 BACK GROUND .................................................................................................................................... 4
2. Statement of the problem ........................................................................................................................ 5
3. Literature review ....................................................................................................................................... 6
4. Significance of the study ......................................................................................................................... 13
5. Objectives................................................................................................................................................ 14
5.1 General Objectives ............................................................................................................................ 14
5.2 Specific Objectives ............................................................................................................................ 14
Bibliography ................................................................................................... Error! Bookmark not defined.
Bibliography ................................................................................................... Error! Bookmark not defined.
1. INTRODUCTION

1.1 BACK GROUND

Helminthes, roundworm and flatworm parasites are among the most widespread infectious
agents that have affected and still affect human populations, particularly in the marginalized low-
income and resource-constrained regions of the world (hotez P, molyneux DH, & fenwick A,
2007). It is estimated that over two billion people worldwide are infected with helminthes with
about 300 million suffering severe pathological manifestations associated with these diseases
(De silva NR, Brookers S, & Hotez P, 2003).

Helminths affect mostly school-age children from 1-14 years. In Cameroon and India more than
two thirds of children at this age group are affected (WHO, Eliminating soil transmitted
helminthiasis as a public health problem in children ; progress report 2001-2010 and strategic
plan 2011-2020., 2012). It is estimated that more than 610 million school-age children are at risk
of contracting helminths. The prevalence of these diseases in Cameroon and India is between
(WHO, Eliminating soil transmitted helminthiasis as a public health problem in children ;
progress report 2001-2010 and strategic plan 2011-2020., 2012).

Infection with intestinal helminthes is one of the most common health problems of children in
developing countries. Moreover, chronic intestinal helminthic infections have been identified as
one of the possible risk factors that contribute to the pathogenesis as well as the widespread of
other infectious agents such as HIV and mycobacterium in the tropical countries ( (Bentwich,
Kalinkovich, & Weisman, 1995)).

In developing countries, it is difficult to prevent infection with geo-helminthes because

improvements in environmental sanitation are not easily achievable. However, treatment of
infected individuals with effective and broad-spectrum anthelmintic can minimize problems that
arise from intestinal helminthic infections (Thein-Hlaing, 1991).
Several studies have shown that albendazole and mebendazole from different brands are drugs
of choices for the treatment of single or mixed infections with intestinal helminthes (Muttalib,
1981). In contrast, others have reported that these drugs are not equally effective in curing all
intestinal helminthic infections (Ndenecho, 2002).
WHO recommends regular deworming as an effective way to reduce both morbidity due to
these parasites and the occurrence of serious complications using the following drugs:
praziquantel, albendazole, mebendazole and levamisole. The benzimidazole drugs,
albendazole and mebendazole are the most widely used drugs for the control of helminths.

Mebendazole is related chemically to the Benzimidazole antihelmintic which is Tubulin-

disrupting drug and has Antitumour activity in vitro and in vivo by Inhibits growth and induces
apoptosis. Its chemical name is N-(6-Benzoyl-1H-benzimidazol-2-yl) - carbamic acid methyl
ester and its Molecular Weight is 295.30. Mebendazole is a synthetic drug which is soluble in
DMSO to 10Mm, and stored at room temperature (WHO, 2003).
Oral absorption in all species is limited; from 17-22% in healthy adult human. The plasma
concentration of mebendazole is 15-49 times lower than that of albendazole. Clearance, which is
dependent upon metabolism in the liver, occurs predominantly as metabolites in bile and urine
and there is much unchanged drug in the faeces. Co administration with a fatty meal enhances
absorption. Excretion in breast milk has not been reported (WHO, 2003).
The oral LD50 in the rat is 1434mg/kg in males and 714mg/kg in females and for other species,
such as the rabbit, is >1280mg/kg. In repeated dose testing, rats receiving doses up to
127.3 (males) - 151.6 (females) mg/kg/d for 13 weeks demonstrated growth retardation, anaemia
and some deaths. Testicular damage was also observed in male rats receiving the
highest doses (WHO, 2003).

2. Statement of the problem

Soil-transmitted helminths (STHs); roundworms, whipworms, and hookworms) infect millions
of children in sub-tropical and tropical countries, resulting in malnutrition, growth stunting,
intellectual retardation, and cognitive deficits. To fight against STH, large-scale deworming
programs are implemented in which anthelmintic drugs (either albendazole (ALB) or
mebendazole (MEB)) are administered. Currently, these large-scale programs are intensifying,
highlighting the need to closely monitor the efficacy of anthelmintic drugs to detect changes in
drug efficacy that may arise through the evolution of anthelmintic drug resistance in the parasites
(Bruno levecke, 2014).
The availability of numerous brands of mebendazole in our drug market today places clinicians
and pharmacists in a difficult situation of choice of a suitable brand or the possibility of
alternative use. Besides, there are growing concerns that various mebendazole formulations may
have different bioavailability and that development of resistance will accelerate if suboptimal
doses are used. Despite the considerable use in Ethiopia, there are no reports on the
bioavailability and bioequivalence of the various brands of mebendazole tablets marketed in
Ethiopia.

To my knowledge, there are no studies conducted on comparative in vitro quality evaluation on

different brands of mebendazole tablet in Ethiopian in general and in Mekelle in particular.
Therefore it is very important to study the comparative in vitro quality evaluation on different
brands of mebendazole tablets, with respect to their quality, safety and efficacy.

3. Literature review
It is imperative to gain a mebendazole tablet with good bioavailability and bioequivalence
properties. In this context, studies conducted on comparative in vitro quality evaluation on
different brands of mebendazole tablets is a prerequisite to get a good quality of mebendazole
tablet
Therapeutic effectiveness and quality of pharmaceuticals

For a drug to be effective, enough of it needs to reach its site(s) of action and stay there long enough to be
able to exert its pharmacological effects. Many factors have been found to influence the rate and extent of
absorption, and hence the time course of a drug in the plasma and, therefore, at its site(s) of action. These
include the foods eaten by the patient, the effect of the disease state on drug absorption, the age of the
patient, the sites of absorption of the administered drug, the co-administration of other drugs. In addition,
the physical and chemical properties of the drug, the type of dosage form, the composition and method of
manufacture of the dosage form, the size of the dose and frequency of administration are among the
factors, which are almost addressed in quality assessment studies (Ashford, 2002).

The presence of substandard pharmaceutical products in the drug distribution chain may produce a danger
to public health. Drug quality reports by the United States Pharmacopoeia Drug Quality and Information
Program in different countries e.g. Benin, Ghana, Nigeria, Bangladesh, Cambodia, China revealed that a
large number of drugs failed quality testing. Some of these drugs were found to contain active ingredients
outside the appropriate limits and most of them below the limits. Such drug products have therapeutic as
well as social and economic implications (World Health Organization, 1999).

Pharmaceutical quality
Pharmaceuticals play an important role in improving human health and promoting well- being.
However, to produce the desired effect, they have to be safe, efficacious and of acceptable quality, and
have to be used rationally. The use of ineffective and poor quality drugs will endanger therapeutic
treatment and may lead to treatment failures. Thus, the production, storage, and distribution of drugs in
each country need to be regulated by the government drug regulatory authority. Challenges to these drug
regulatory authorities are the flourishing of many pharmaceutical industries and distribution channels
during the past few years in the world, leading to an increased number of products circulating in national
and international drug markets. In the same manner, the presence of counterfeit and substandard drugs in
those markets has increased substantially as a result of ineffective regulation of the manufacturers and
trading of pharmaceutical products by both exporting and importing countries (World Health
Organization, 1999).

Marketing of poor quality drugs is high in developing countries, especially of Africa and Asia because of
weak drug regulatory systems. Thus, in countries like Ethiopia, where drug regulatory control is weak, the
quality of marketed drug products cannot be guaranteed. Quality assessment studies on some of the
marketed drug products could give an insight into the quality of the pharmaceutical products marketed
within the distribution chain and consumed. Such studies could provide basis for corrective measures
taken by drug regulatory authorities.
Quality control parameter and properties and properties of tablet dosage forms

The need for precisely defined and acceptable specifications for production control during
manufacturing processes and for the final products, in order to assure reproducibility in the wide
context of drug safety, is recognized. The assurance of quality of medicines is the primary
responsibility of the manufacturers (internal quality control).
However, it is recognized in most countries that the national health authorities must exercise
comprehensive surveillance by legislative methods over pharmaceutical manufacturers within
their jurisdiction, in order to ensure observance of good manufacturing practices and quality
control of products. During research, development and formulation, physicochemical analysis
and analytical profiles of drug substances provide good quality control data on which good
decisions can be established (deasy & Timoney, 1976).
Assessment of quality, safety and efficacy constitutes an important component of pharmaceutical
product evaluation, which is based on quality control tests. The minimum quality control tests for
pharmaceutical preparations are illustrated in Table1.1 (Ayeres G, 1981)

Table 1.1 Minimum Quality Control Tests for Pharmaceutical Preparations

Types of pharmaceutical preparations Types of tests

1. Tablet, Capsule, Lozenges Identification of active ingredient (s), Uniformity of
weight, Friability, Hardness, Disintegration, Dissolution,
Assay of active ingredient (s).
2. Injectable Identification of active ingredient(s), pH, Extractable
(Liquid) volume, Particle Count, Pyrogen / LAL, Sterility,
Effectiveness of microbial preservatives, Assay of active
ingredient (s)
3.Cream, Ointment Identification of active ingredient (s), Viscosity,
Homogeneity, pH, Release rate, Sterility, Microbial limit
test, Effectiveness of preservatives (if present)
4. Aerosol, Inhalation, Spray Identification of active ingredient (s), Net contents,
Particle size and tests for foreign particles, Delivery rate,
Leak testing, Pressure testing, Limit test for degradation
products/ impurities (where applicable), Moisture
determination, Assay of active ingredient (s)

Tablets have some apparent features, like certain amount of hardness and resistance to friability,
tablet dosage form uniformity (weight variation, content uniformity), disintegration, and drug
dissolution. Each property will influence the other as hardness has influence on both friability and
drug dissolution. All are tablet properties that can be utilized as parameters for drug quality control.
The following physicochemical parameters (hardness and friability, content of uniformity,
weight variation, disintegration, assay of active ingredient, and dissolution test of tablet
have been taken directly from United State Pharmacopeia (USP, 2014)
Hardness and friability
Tablets require certain degree of strength and resistance to friability to withstand mechanical
shocks of handling during manufacturing, packaging, shipping and utilization by the patient.
Hardness is a force required to break a tablet across the diameter. The hardness of a tablet is an
indication of its strength. The hardness was measured using Schleuniger-2E Hardness tester. The
values were expressed in Newton (N).
The friability of tablets is determined by using ERWEKA TA Friabilator. Ten tablets will
weighed and placed in the Friabilator and rotated at 25 rpm for 4 minutes. Then the tablets will
take out, dusted and reweighed. The percentage friability of the tablets will calculated by the
formula, Percentage Friability = [(Initial Weight – Final Weight)/ Initial Weight] × 100. Three
trials per brand will perform.

Uniformity of dosage units: content uniformity and weight variation

This term includes both the mass of the dosage form and the content of the active substance in
the dosage form. These are expressed in terms of content uniformity or weight variation; which
are among the parameters of tablet quality control.

Drug content estimation

Weighed accurately 250 mg of drug then dissolve in 3 ml of anhydrous formic acid and 30 ml of
anhydrous glacial acetic acid and titrate with 0.1 M perchloric acid. The end point will
potentiometrically determined (1 ml of 0.1 M perchloric acid = 29.53 mg of C16H13N3O3).

Weight variation
Twenty tablets of each formulation were selected at random and weighed individually. The
weight of individual tablets was noted. Average weight was calculated and the individual
weights were compared with the average weight.

Disintegration
For the active medicinal agent in a tablet to become fully available for absorption, the tablet must
first disintegrate and discharge the drug to the body fluids for dissolution.
Disintegration test was carried out by using ERWEKA ZT 3 Disintegration test apparatus. One
tablet is placed in each tube, and the basket rack was positioned in a 1-litre beaker of distilled
water, at 37°C ±2°C. A standard motor-driven device is used to move the basket assembly
containing the tablets up and down through a distance of 5 to 6 cm at a frequency of 32 cycles
per minutes. The time taken for the tablet to disintegrate completely was noted.
Assay for the active ingredients
Assay is a critical step in analytical sciences. It is the determination of the strength or content of
the active ingredient within the dosage form. The quantitative determination of a drug dosage
form is preferentially performed by physicochemical methods. Such analytical techniques are
very diverse and successfully applied to the assay of the active medicinal agent in the
pharmaceutical tablets. The technique should provide specificity, accuracy, precision and
sensitivity to the particular ingredient of interest within the dosage form.
Dissolution Test as Quality Control Parameters
Drug absorption from a solid dosage form after oral administration depends on the release of the
drug substance from the drug product, the dissolution or solubilization of the drug under
physiological conditions, and the permeability across the gastrointestinal tract. Thus in vitro
dissolution may be relevant to the prediction of in vivo performance. In vitro dissolution tests for
immediate release dosage forms, such as tablets, are used to assess lot to lot quality of drug
product and ensure continuing drug product quality and performance after certain changes such
as changes in formulation, the manufacturing process and the site of manufacture (FDA, 1997).
In general, in vitro dissolution rate measurements have been used in a variety of ways including:
 To study the effects of physicochemical variables of the pure drug on dissolution rates.
 To study variables such as manufacturing processes, tablet coating, excipients, on
dissolution characteristics of the dosage form.
 To screen potential dosage form candidates for in vivo bioavailability studies.
 As a retrospective study to explain clinical failures of a particular dosage form.
 As a sensitive quality control procedure to detect changes in the release characteristics
due to lot-to-lot variations, formulation changes, or storage conditions, which may or
may not be detected in less sensitive in vivo absorption.
Dissolution, Bioavailability and Bioequivalence
Literatures showed that there might be variations in clinical response among orally administered
drug products that contain chemically equivalent amounts of a drug due to differences in their
dissolution rates. This is a useful indication of the influence of dissolution rate on absorption and
bioavailability of drugs for clinical use (Banakar UV, 1992).
‘’Bioequivalence’’ is a comparison of the bioavailability of two or more drug products. Thus,
two products or formulations containing the same active ingredient are bioequivalent if their
rates and extents of absorption are the same. For a drug product to be considered bioequivalent to
a pioneer product there must be no statistical differences (as specified in the accepted criteria)
between their plasma concentration-time profiles, with some degree of acceptable tolerance
limits.
Bioavailability assessment and in vitro/ in vivo correlations
The term commonly used to describe such relationship is in vitro/in vivo correlation. If such
correlation could be established, it would be possible to use in vitro data to predict a drug’s in
vivo bioavailability. This would drastically reduce, or in some cases, completely eliminate the
need for bioavailability tests. The desirability for this becomes clear when one considers the cost
and time involved in administering drugs to healthy subjects or patients. It would certainly be
preferable to be able to substitute quick, inexpensive in vitro tests for in vivo bioavailability
studies (Banakar UV, 1992).
In vitro methods of assessing bioavailability
Evidences provided us with formulation variables that can greatly affect the bioavailability and
thus the clinical efficacy of many important therapeutic agents. Various in vivo and in vitro
physical methods have been developed to assess these effects. The release of a drug from the
dosage form into solution in the gastrointestinal fluids is often the rate-limiting step in
determining the rate and extent of absorption and thus in vitro measurements on disintegration
and dissolution provide a rapid, sensitive and reproducible means to study and assess the
bioavailability without involvement of human experimentation (Jollow, 1972).
Dissolution test for assessing bioavailability
Dissolution test is currently considered to be the sensitive and reliable in vitro parameter most
likely to correlate with bioavailability. Since dissolution of a dosage form in vivo is often a rate
limiting factor determining the physiologic availability of a drug, measurement of the in vitro
dissolution rate is more likely to offer a meaningful indication of physiologic availability.
Dissolution testing is implemented in the assessment and evaluation of the release rates and
bioavailability of a variety of conventional tablets. It is believed that if a tablet does not dissolve
properly in in-vitro test, it certainly will not do so in in-vivo tests for dissolution (Banakar UV,
1992).
Official dissolution methods
Dissolution tests provide an indication for differences in in-vivo absorption characteristics of the
drug and serve as a secondary standard to detect dosage forms with a potential for poor
bioavailability. It involves official methods. There are two official USP dissolution methods:
Apparatus-1 (Basket method) and Apparatus-2 (Paddle method) (Figure 1.5 and 1.6).

Figure 1.5: Schematic representation of dissolution Apparatus I (Basket method)

Figure 1.6: Schematic representation of dissolution Apparatus II (Paddle method)

In vitro-in vivo correlations
When dissolution tests are utilized to assess possible differences in bioavailability of
pharmaceutical products, the main requirement is a high degree of correlation between in vitro
and in vivo methods and the selectivity of the method to reject poor products but not acceptable
ones. It will be much more difficult to find general methods that will be applicable to all
products as a compendia standard to assure comparable performance of many different brands of
drugs than a method that can be used by a particular manufacturer for his product alone (Banakar
UV, 1992).
There are, thus two important characteristics of a given drug dissolution method: the sensitivity
of the method to detect differences and the correlation of observed differences to those in in-vivo
bioavailability. The sensitivity and extent of correlation between in vitro dissolution methods and
in vivo absorption parameters will depend on the complex inter-relationship between the
numerous variables of the drug and the dosage form, the apparatus and experimental conditions
of the in vitro method and the mechanism of absorption in the species tested
A study conducted in Cameron showed that all brands comply with official requirements for
uniformity of weight and hardness. One brand failed the friability test with more than 1% of
weight loss. The disintegration test revealed that two brands disintegrate in less than 2 min while
other brands in more than 1 hour. Titrimetric assay of mebendazole content showed only two
brands containing not less than 90% (w/w) of labeled chemical content. Brand MBZ3 (Diamap,
india) showed better characteristics of chewable tablet. Consumers need to pay attention to the
manufacturer information and chewable tablets have to be treated as such.

4. Significance of the study

In Ethiopia The availability of numerous brands of mebendazole in our drug market today places
clinicians and pharmacists in a difficult situation of choice of a suitable brand or the possibility
of alternative use. Currently, large-scale deworming programs are intensifying, highlighting the
need to closely monitor the efficacy of anthelmintic drugs to detect changes in drug efficacy that
may arise through the evolution of anthelmintic drug resistance in the parasites (Bruno levecke,
2014) Therefore, the current study is aimed to evaluate the bioequivalence of the different brands
of mebendazole in Mekelle town, Tigray Regional State. To the best of my knowledge, this study
is the first to evaluate the bioequivalence of the different brands of mebendazole. It will also use
as a reference for further studies in the future.

5. Objectives

5.1 General Objectives

To assess the physical properties and the quality control parameters of the imported, marketed
and locally manufactured brands of mebendazole 100mg found in illegitimate pharmacy outlets.

5.2 Specific Objectives

The specific objectives of the study are:
 To identify the active pharmaceutical ingredient of different brands of the commonly
prescribed anthelmintic drugs: mebendazole 100mg tablets.
 To evaluate the physical properties [hardness, friability, uniformity of dosage form (Weight
variation or content uniformity)] of the different brand of the mebendazole tablets.
 To evaluate and compare disintegration time of the different brand of the mebendazole
tablets.
 To assay and quantity of Active Pharmaceutical Ingredient (API) in different brand of the
mebendazole tablets.
 To study the dissolution profiles of the tablets of different manufacturers.

6. EXPERIMENTAL
6.1. Materials and Methods
6.1.1. Materials
6.1.1.1. Tablets and reference standards
6.1.1.2. Solvents / Chemicals/ Reagents
6.1.1.3. Instruments
6.1.2. Methodology
6.1.2.1. Identification
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