IN VITRO SENSITIVITY STUDY OF DIFFERENT BRANDS OF ANTIAMOEBIC SECNIDAZOLE
AND TINIDAZOLE ORAL PREPARATIONS AGAINST ENTAMOEBA HISTOLYTICA INFECTED
BANGLADESHI POPULATION.
Farhana Rizwan1, Affifah Mustafa Mim1, Amna Rasul1, Rashidul Haque2, Abdulllah
Siddique2, Md. Mehedi Aziz Sarker1 and Sufia Islam1*.
1
Department of Pharmacy, East West University, 43 Mohakhali C\A, Dhaka-1212;
2
Parasitology Laboratory, ICDDR, B.
*Corresponding author [sufia@ewubd.edu]
Abstract
The aim of the study was to evaluate in vitro sensitivity of different brands of secnidazole and
tinidazole tablets from different pharmaceutical companies against Entamoeba histolytica.
Secnidazole is an anti-amoebic drug with higher serum half-life effective against intestinal
amoebiasis. Tinidazole, at higher concentration, is an equally efficacious congener of
secnidazole. In Bangladesh, different pharmaceutical companies manufacture secnidazole and
tinidazole in different dosage form. In this study, we tried to find out the efficacy of these
drugs against clinical isolates of Entamoeba histolytica.
Different concentrations of secnidazole and tinidazole were prepared after collection from
different drug stores in Bangladesh. The parasite count was adjusted to 3×106 parasites/ml in
a medium. In vitro drug sensitivity assay of the samples was carried out by using microtiter
plates after treatment with different concentrations of these drugs. The viable parasites were
counted by haemocytometer.
The results show that secnidazole tablet of all brands are highly effective whereas the
tinidazole tablet was effective only in higher concentration against Entamoeba histolytica.
Keyword: Amoebiasis, Secnidazole, Tinidazole, Entamoeba histolytica
IN VITRO SENSITIVITY STUDY OF DIFFERENT BRANDS OF ANTIAMOEBIC SECNIDAZOLE
AND TINIDAZOLE ORAL PREPARATIONS AGAINST ENTAMOEBA HISTOLYTICA INFECTED
BANGLADESHI POPULATION.
Introduction
Entamoeba histolytica, associated with major public health problem throughout the world
(Wkly Epidemiol Rec 1997). Poverty, ignorance, overcrowding, poor sanitation and
malnutrition favor the transmission and increased disease burden (Walsh 1988).
Entamoeba histolytica produces a broad-spectrum illness in human. The majority of those
infected experience have symptoms, less than 10% have a few loose stools, and a much
smaller percentage suffer from bloody, febrile dysentery or hepatic abscess (Walsh 1986).
Parasites strains were originally isolated from patients with invasive intestinal amoebiasis
from an endemic area (Mirpur), Dhaka, Bangladesh. Then the clinical isolates of E.histolytica
were cultured and maintained axenically in (LYI-S-2) medium.
Antiamoebic drugs chiefly treat amoebiasis in symptomatic patients (Martinez-Palomo and
Martínez-Báez 1983). In asymptomatic individuals, inappropriate usage of drugs or
overdosing could lead to drug resistance The substandard and/or spurious drugs is the result
of addition of incorrect amount of active ingredients; date expired sub-potent active
ingredients and excipients; poor stability of active ingredients in the finished product and so
on. The treatment with this type of substandard and/or spurious drug could also lead to drug
resistance and could endanger patient’s life.
The drugs, which are used in the amoebiasis, are metronidazole, secnidazole, tinidazole,
ornidazole, diloxanide furoate, emetine, chloroquine, etc. to destroy amoeba that have
invaded tissue. Despite extreme potency of metronidazole against E. histolytica, it has to be
taken for a relatively long period and has various side effects including headache, metallic
tongue, nausea, diarrhoea and abdominal discomfort. Secnidazole has a much longer
biological half-life compared to metronidazole, tinidazole and ornidazole (Bassily 1987).
Secnidazole is extensively used in the treatment of amoebiasis with 2 g single dose. The halflife of tinidazole is 12 hrs, thus it is used for once daily therapy.Tinidazole is given in the
doses of 50 mg/kg/d for 3 days for mild to moderate and even severe cases of intestinal
amoebiasis (Gupta 2004).
We realize that there is a need to check the in vitro drug sensitivity test of secnidazole and
tinidazole tablets in order to understand the effectiveness of these drugs against the E.
histolytica. Therefore, these studies were carried out to investigate the in-vitro drug
sensitivity study of different brands of secnidazole and tinidazole tablets manufactured by
different pharmaceuticals in Bangladesh against E. histolytica.
Materials and Methods
Collection of sample
Six brands of secnidazole tablets and suspensions are manufactured by four different
pharmaceuticals and three pharmaceutical companies manufacture tinidazole tablets. Three
different brands of secnidazole tablets and one brand of tinidazole tablet were included in this
study.
The samples were collected from different retail medicine shops. The secnidazoles are coded
as S-1, S-2 and S-3. The tinidazole is coded as T-1. At the purchasing time the manufacturing
date, date of expiry, batch number, manufacturing license number, retail price had been
checked properly.
This study was performed at the Parasitology Laboratory, International Centre for Diarrhoeal
Disease Research, Bangladesh (ICDDR, B).
Preparation of antimicrobial agents: Standards and samples
The standard secnidazole was collected as pure salt form, the newest among the
nitroimidazole group, from Sanofi-Aventis Bangladesh Ltd. and the standard tinidazole was
collected from Renata Limited. Standard secnidazole and tinidazole (8 mg) were weighed and
dissolved in two different tubes with distilled water (1 mL). The stock solutions were stored
in a refrigerator. All the samples of secnidazole (S-1 to S-3) and tinidazole (T-1) were
prepared in a similar manner. Clinical isolates of E. histolytica were harvested from 24 hours
old cultures and suspended in an Axenic medium (LYI-S-2) medium. LYI-S-2 consists of
liver digest, yeast extract, iron and serum (Graham and Diamond 2002).
The parasite count was adjusted to 3×106 parasites/ml in medium by haemocytometer
(Mukhopadhyay and Chaudhuri 1996, Bansal et al 2004). Isolation is usually achieved by
growing the species in an environment that was previously sterilized, and was thereby rid of
contaminating organisms.
In vitro drug sensitivity assay
Drug sensitivity assay of the samples were carried out by using microtiter plates. In row “A”
200 µl of the standard and the samples were given. In all other rows the 100 µl medium was
added and dilutions of the drugs were performed down the plate mixed properly.100 µl of the
medium from the last row was discarded to maintain the equality of the concentration of the
drugs. The final concentrations of the secnidazole were 1.08, 2.16, 3.24 and 4.32 µM. The
final concentrations of tinidazole were 0.4, 0.8, 1.6, 3.2 µM. Further 100 µl of parasite
suspension (3×106 parasites/ml) was added to all the rows. Each test included blank control.
Then plastic strip was used to cover the plate, incubated at 37 º C, and examined after 1, 2
and 4 hours under a microscope to check for the presence of amoebae. After 4 hours, the
plate was taken from the incubator. Then the viable parasites were counted by
haemocytometer under microscope in each of the rows.
Statistical analysis
The data were analyzed using SPSS for windows version 12.0 (SPSS, Lead Technologies,
Inc, USA). Descriptive statistics were done by one-way ANOVA & Post Hoc Tests, a
probability level of 0.05 was considered statistically significant.
Results and Discussion
Table I shows the mean viable parasites count from isolates after treatment with different
brands of secnidazole tablets (S-1 to S-3). No statistical significance difference has been
observed in terms of viable parasites with the brands S-1, S-2, and S-3 when compared with
the standard secnidazole at the concentrations of 1.08, 2.16, 3.24 and 4.32 µM.
Table 1. Mean viable parasites after treatment with different brands of
secnidazole at different concentrations.
Concentrations Standard
(Mean±SD)
Sample (S-1)
Sample (S-2)
Sample (S-3)
4.32 µM
7±2.52
6± 1.00(p=0.991)
7± 0.58(p=1.000)
11±2.08(p=0.700)
3.24 µM
8±2.52
8±1.00(p=1.000)
8±0.58(p=1.000)
14±3.27(p=0.061)
2.16µM
9±3.06
11±0.58(p=0.861)
10±1.00(p=0.997)
15±3.46(p=0.113)
1.04 µM
13±3.06
15±2.08(p=0.932)
14±1.15(p=0.994)
19±4.36(p=0.256)
Figure 1 shows the percentage of viable E. histolytica after exposure to different solvents. For
performing the in vitro sensitivity assay of the secnidazole and tinidazole, DMSO was used.
It has been shown that the DMSO has insignificant effect on damaging the parasites, which
means the parasites are mainly attacked by antiamoebic drugs.
Figure 1. Percentage of viable E. histolytica after exposure to different solvents.
% of Viable Parasites
120%
100%
80%
60%
40%
20%
0%
W
S
H
AA M
Solvents
E
DMSO
W=Distilled Water; S=0.1 Mol Sodium Hydroxide; H=0.1 Mol Hydrochloric Acid;AA=10% Acetic Acid;
M=10% Methanol; E= 10% Ethanol; DMSO=10% Dimethyl Sulfoxide).
Figure 2 shows the percentage of inhibition of non-viable E. histolytica by different brands of
secnidazole tablets (S-1, S-2 and S-3). At concentration 4.32, about 95% of E. histolytica is
inhibited by standard, S-1 and S-2. However, 89% of E. histolytica is inhibited by S-3.
Figure 2. Percentage inhibition of E. histolytica after exposure to different brands
of secnidazole at different concentrations.
100
% Inhibition
95
90
Std
S1
85
S2
S3
80
75
70
1.08
2.16
3.24
4.32
Concentration of secnidazole (µM)
Figure 3 shows the percentage inhibition of non-viable E. histolytica by tinidazole tablet. At
concentration 3.2 (µM), 95% of E. histolytica is inhibited by standard tinidazole and 89% of
E. histolytica is inhibited by the drug.
Figure 3. Percentage inhibition of E. histolytica after exposure to tinidazole at
different concentrations.
100
90
% Inhibition
80
70
60
Std
50
Sample
40
30
20
10
0
0.4
0.8
1.6
Concentration of tinidazole (µM)
3.2
Treatment failure among amoebiasis patients often raises the possibility of drug resistance
(Ayala P 1990). In the present study, the E. histolytica isolates maintained by in vitro
cultivation in axenic medium were subjected to drug susceptibility tests against secnidazole
and tinidazole tablets.
Our results showed that all the secnidazole tablets are as good as standard secnidazole at
different concentrations as 4.32, 3.24, 2.16 and 1.04 µM. It is seen that all secnidazole tablets
are having high efficacy on E. histolytica. They can effectively inhibit the trophozoite of E.
histolytica that causes the disease. It has been shown from another study that secnidazole is
more active than metronidazole (Symonds 1979).
The tinidazole tablet is effective against the parasite. However, the lower concentrations of
this drug are not as sensitive as the higher concentration in terms of their in vitro sensitivity
against the E. histolytica.
Conclusion
We conclude that the three brands of secnidazole and one brand of tinidazole of different
pharmaceuticals in Bangladesh are as sensitive (in vitro) as standard secnidazole and
tinidazole against E. histolytica. The solvent in which secnidazole drug was dissolved has no
lethal effect on E. histolytica. Secnidazole and tinidazole are the newer member, of 5nitroimidazole group; they can play a better role to cure the patients suffering from
amoebiasis.
Monitoring the random drug sensitivity of different brands of antiamoebic drugs was helpful
to get an impression about the possible emergence of resistance of antiamoebic drugs in
future in Bangladeshi patients. Further studies are needed to test other brands of different
antiamoebic drugs available in Bangladesh. Increased awareness and continued surveillance
for the possible emergence of resistance are necessary for the ultimate prevention and control
of amoebiasis. The findings of this study may be helpful to make awareness the physicians to
select quality products.
Acknowledgements
The authors acknowledge to the authority of Parasitology Laboratory, ICDDR, B for
providing support to carry out the study. We also acknowledge Sanofi-Aventis Bangladesh
Ltd. and Renata Limited, Dhaka, Bangladesh for providing standard secnidazole and
tinidazole for conducting our study.
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