Abstract: The aim of the study was to evaluate in vitro sensitivity of different metronidazole tablets from Bangladeshi pharmaceuticals against clinical isolates of E. histolytica. Metronidazole tablets of 12 different brands were randomized from some big and small pharmaceuticals according to their business. The parasite count was adjusted to 3x106 parasites mL-1 in a medium. In vitro drug sensitivity assay of the samples was carried out by using microtiter plates after treatment with different concentrations of metronidazoles. The viable parasites were counted by haemocytometer. No statistical significance was observed in terms of viable parasites with the metronidazole tablets from three big pharmaceuticals at the concentration of 2.3, 3.5 and 4.6 μM when compared with the standard metronidazole. We conclude that brands from some big pharmaceuticals showed in vitro sensitivity against E. histolytica.
INTRODUCTION
Entamoeba histolytica, associated with high morbidity and mortality continues to be a major public health problem throughout the world. Asymptomatic individuals account for almost 90% of the infections (WHO, 1997). Poverty, ignorance, overcrowding, poor sanitation and malnutrition favour the transmission and increased disease burden (Walsh, 1988). Prevalence varies from country to country and within a country (Tanaka and Kaneda, 1991; Haque et al., 2000; Blessmann et al., 2002; Kang et al., 1998).
Diarrheal diseases are the major causes of morbidity and mortality in children in developing countries. For example, in Bangladesh 1 in 30 children dies of diarrhea or dysentery by his or her fifth birthday (Petri et al., 2000). Bacillary dysentery is most commonly caused by microorganisms belonging to the genus Shigella, whereas amoebic dysentery is an infection caused by the protozoan parasite Entamoeba histolytica (Kotloff et al., 1999).
It is now generally accepted that what was earlier known as E. histolytica actually comprises two genetically distinct but morphologically indistinguishable species, E. histolytica and E. dispar, previously known as pathogenic and non-pathogenic E. histolytica, respectively. The World Health Organization has reaffirmed the definition of amebiasis as infection with E. histolytica sensu stricto with or without clinical manifestations (WHO, 1997). Only E. histolytica can cause intestinal and extraintestinal disease. Amebiasis is a common problem in the developing world. Based on the Mexican National Serosurvey at least 8.4% of the population of Mexico had evidence of prior invasive amoebiasis with an estimated one million cases of amoebiasis and 1,000 deaths annually (Caballero-Salcedo et al., 1994).
The amoebic infection is primarily treated by instituting antiamoebic therapy. The major drug of choice for treating invasive amoebiasis is metronidazole which is derived from 5-nitroimidazole. This agent kills the trophozoites by alterations in the protoplasmic organelles of the amoeba and by fatal destabilization of the DNA helix (Tocher and Edwards, 1994; Öztas et al., 2003; Bansal et al., 2004).
Amoebiasis in symptomatic patients is chiefly treated by antiamoebic drugs (Martínez-Palomo and Martinez Baez, 1983). The limited number of drugs available to treat amoebiasis needs a new approach to treat infected individuals (Pillai et al., 1999). In asymptomatic individuals, inappropriate usage of drugs or overdosing could lead to drug resistance although drug resistance in E. histolytica is not common yet. The quality of medicines available in some less-developed countries is inadequate in terms of content of active ingredient (Taylor et al., 2001). 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. Poor stability can also result from the excessive decomposition of active ingredient at elevated temperature and humidity in tropical countries like Bangladesh. The treatment with this type of substandard and/or spurious drug could also lead to drug resistance and could endanger patient`s life.
Previous study has shown that preschool children in urban slum of Bangladesh are at great risk of acquiring E. histolytica infection, with almost half infected by five years of age (Haque et al., 1999). There are about 100 pharmaceutical companies manufacture metronidazoles in Bangladesh. To our knowledge, no study is undertaken in Bangladesh till date to evaluate the in vitro drug sensitivity of metronidazole. Since amoebiasis is a major public health problem in Bangladesh, we realize that there is a need to check the in vitro drug sensitivity of metronidazole. Therefore, this pilot study was carried out to investigate the in vitro drug sensitivity of different brands of metronidazole manufactured by different pharmaceutical companies in Bangladesh against clinical isolates of E. histolytica.
MATERIALS AND METHODS
Collection of sample: About 160 products of metronidazole (tablets and suspension) are manufactured by different pharmaceutical companies in Bangladesh. All the pharmaceutical companies are divided into two groups as big and small pharmaceutical according to their business in Bangladesh. Name of the pharmaceuticals from each group was arranged alphabetically. Four big and 8 small companies producing metronidazole tablets were selected randomly for this study. Thus 12 brands of metronidazole tablets were collected from retail medicine shop from different areas of Bangladesh.
Metronidazole tablets of 12 different brands were coded as MT01, MT02, MT03, MT04, MT05, MT06, MT07, MT08, MT09, MT10, MT11 and MT12. The products of big pharmaceutical companies are coded as MT01, MT02, MT03 and MT04. From MT05 to MT12 are the products of small pharmaceutical companies. The samples were properly checked for their physical appearance, name of the manufacturer, batch number, manufacturing date, expiry date, manufacturing license number, D.A.R. number and maximum retail price at the time of purchase. This investigation was performed in the Parasitology Laboratory, Laboratory Sciences Division, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) during March, 2006 to December, 2007.
Preparation of antimicrobial agents (standard and sample): The standard metronidazole drug used in the study was collected as pure salt from Sanofi-Aventis Ltd. Dhaka, Bangladesh. Standard metronidazole (8 mg) was weighed and dissolved in 1 mL of distilled water. The stock solution was stored in a refrigerator. All the samples of metronidazoles (MT01 to MT12) were prepared in a similar manner.
Clinical isolates: Clinical isolates of E. histolytica were harvested from 24 h old cultures and suspended in a LYI-S-2 medium. Axenic medium (LYI-S-2) consists of liver digest, yeast extract, iron, serum (Table 1).
The parasite count was adjusted to 3x106 parasites mL-1 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 was carried out by using microtiter plates. In row A 200 µL of the standard
and the samples were given. In all other rows (B-H) the 100 µL medium was added
and dilutions of the drugs were performed down the plate mixed properly. One
hundred microlitre of the medium from the last row (H) was discarded to maintain
the equality of the concentration of the drugs. The final concentrations of
the drugs were 0.07, 0.14, 0.29, 0.58, 1.1, 2.3 and 4.6 µM. Further 100 µL of
parasite suspension (3x106 parasites mL-1) was added to
all the rows (A-H). Each test included control (without drug) and blank wells
(medium only). Then plastic strip was used to cover the plate. Plates were incubated
at 37 °C and examined after 1,2 and 4 h under a microscope to check for the
presence of amoebae. After 4 h the plate was taken from the incubator. Then
the viable parasites were counted by haemocytometer under microscope in each
of the rows. For another sets of experiments the final concentrations of the
drugs were 1.1, 2.3, 3.5 and 4.6 µM.
| Table 1: | Quantitative composition of LYI-S-2 |
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 and Post Hoc Tests, a probability level of 0.05 was considered statistically significant.
RESULTS AND DISCUSSION
The study was conducted with different brands of metronidazole tablets manufactured by different pharmaceutical companies. Twelve brands are checked for their in vitro drug sensitivity against clinical isolates of E. histolytica.
Table 2 shows the mean viable parasites count from clinical isolates after treatment with different brands of metronidazole tablets (MT01 to MT04). No statistical significance has been observed in terms of viable parasites with the brands MT01, MT02 and MT03 when compared with the standard metronidazole at the concentration of 2.3, 3.5 and 4.6 µM. MT02 at the concentration 1.1 µM and MT04 at the concentrations of 1.1, 2.3 and 3.5 µM are significantly different when compared with the standard metronidazole in terms of viable parasite count.
Table 3 and 4 shows the mean viable parasites count from clinical isolates after treatment with different brands of metronidazole tablets (MT05 to MT12). All the values in terms of viable parasites are significantly different when compared between samples and standard metronidazole at different concentrations.
Figure 1 show percentage inhibition of non-viable E. histolytica by different brands of metronidazole tablets (MT01, MT02, MT03 and MT04). The mean IC50 of standard metronidazole tablet is 0.07 µM. The mean IC50 of MT01, MT03 and MT4 against E. histolytica are less than 0.14 µM. However, IC50 of MT02 is more than 0.14 µM.
Figure 2 shows percentage inhibition of non-viable E. histolytica by different brands of metronidazole tablets (MT05, MT06, MT07 and MT08). The mean IC50 of MT05, MT06, MT07 and MT08 against E. histolytica is 0.29 µM and above but less than 0.30 µM.
Figure 3 shows percentage inhibition of non-viable E.
histolytica by different brands of metronidazole tablets (MT09, MT10, MT11
and MT12). The mean IC50 of MT09 and MT10 are about 0.58 µM. The
mean IC50 of MT11 and MT12 is about 0.14.
| Table 2: | The mean viable parasites count from clinical isolates after treatment with different brands of metronidazole tablets (MT01 to MT04) |
| Results expressed as mean ± SD, *p = 0.047, **p = 0.003, ***p = 0.007, ****p = 0.000 vs. standard metronidazole | |
| Table 3: | The mean viable parasites count from clinical isolates after treatment with different brands of metronidazole tablets (MT05 to MT08) |
| Results expressed as mean ± SD, p = all values are significantly different vs. standard metronidazole | |
| Table 4: | The mean viable parasites count from clinical isolates after treatment with different brands of metronidazole tablets (MT09 to MT12) |
| Results expressed as mean ± SD, p = all values are significantly different vs. standard metronidazol | |
This study with different brands of metronidazole in Bangladesh showed different sensitivity of the drugs against clinical isolates of E. histolytica. Results showed that the metronidazole tablets from three big pharmaceutical companies are as good as standard metronidazole at different concentrations (2.3, 3.5 and 4.6 µM).
Treatment failure among amoebiasis patients often raises the possibility of drug resistance (Ayala et al., 1990). In the present study, the E. histolytica clinical isolates maintained by in vitro cultivation in axenic medium were subjected to drug susceptibility tests against metronidazole tablets.
The IC50 value (µM) against E. histolytica after treatment
with standard metronidazole is about 0.07 µM. The IC50 values of
all the drugs except MT09 and MT10 are comparable with the standard metronidazole.
However, all the products of small pharmaceutical companies are not as sensitive
as the products of some big pharmaceuticals in terms of their in vitro
sensitivity against the clinical isolates. Out of four big pharmaceuticals only
one is a multinational and rests of them are local pharmaceuticals.We conclude
that the three big pharmaceuticals including the two local and one multinational
companies in Bangladesh are manufacturing the metronidazoles which are as sensitive
(in vitro) as standard metronidazole against the clinical isolates of
E. histolytica. Monitoring the random drug sensitivity of different brands
of metronidazole was helpful to get an impression about the possible emergence
of resistance of metronidazole in future in Bangladeshi patients. Further studies
are needed to test other brands of metronidazole available in Bangladesh. Increased
awareness and continued surveillance for the possible emergence of resistance
among clinical isolates is necessary for the ultimate prevention and control
of amoebiasis. The findings of this study are also very helpful to make awareness
of both physicians and consumers to select quality products.
| Fig. 1: | Percentage inhibition of non-viable E. histolytica by different brands of metronidazole tablets (MT01 to MT04) |
| Fig. 2: | Percentage inhibition of non-viable E. histolytica by different brands of metronidazole tablets (MT05 to MT08) |
| Fig. 3: | Percentage inhibition of non-viable E. histolytica by different brands of metronidazole tablets (MT09 to MT12) |
ACKNOWLEDGMENTS
The authors acknowledge to the authority of Parasitology Laboratory, Laboratory Sciences Division, International Centre for Diarrhoeal Disease Research, Bangladesh (icddr,b) for providing support to carry out this study. We also acknowledge Sanofi-Aventis Ltd., Dhaka, Bangladesh for providing standard metronidazole. The authors acknowledge Professor ABM Faroque, Department of Pharmaceutical Technology, University of Dhaka for his guidelines to find out different brands of metronidazole tablets.