Monitoring the Response of Plasmodium falciparum and P. vivax to Antimalarial Drugs in the Malarious Areas in South-East Iran

Gh. H. Edrisian PharmD,* M. Nateghpoor PhD,* A. Afshar,* A. Sayedzadeh MSc,** Gh. Mohsseni,** M.T. Satvat BSc,* A.M. Emadi DVM**

*School of Public Health and Institute of Public Health Research, Tehran University of Medical Sciences and Health Services, **Provincial Health Departments of Sistan-Baluchestan and Hormozgan and the General Office for Prevention and Control of Diseases, Ministry of Health and Medical Education, Iran

Introduction

Malaria is the most lethal of all vector-borne diseases in the world and about 500 million cases occur each year killing at least two million people.¹

The emergence of vector resistance to widely used insecticides and parasite resistance to first-line drugs have resulted in a rise in incidence in many endemic areas, resulting in and also the need to resort to more costly chemotherapeutic agents with greater toxicity.²

In Iran, although the extensive anti-malarial campaign carried out during the past 4 to 5 decades has considerably decreased the endemicity of the disease in most parts of the country, malaria is still the most important parasitic disease in Iran and one of the main public health concerns in the south-east of the country.

The resistance of Plasmodium falciparum to chloroquine (CHL) was first observed in Iran-Shahr of the Sistan-Baluchestan Province in 1983³ and later in Bandar-Abbas, Hormozgan Province⁴ both situated in south-eastern Iran.⁴

In the present combined studies, the susceptibility of P. falciparum to CHL and some other common blood schizontocides and also P. vivax to CHL has been monitored in the malaria problem areas in south-east Iran during 1990-1996 to determine the response of Plasmodia to anti-malarial drugs.

Materials and Methods

The malaria endemic areas of Iran are located in the south-eastern part of the country, bordered in the south by the Persian Gulf and the Gulf of Oman, and to the east by Pakistan. These areas include the provinces of Sistan-Baluchestan, Hormozgan and Kerman (mainly the Kahnoudj Distinct). The weather in these areas is generally warm and humid.

These areas incorporate less than 5% of Iran's total population, but contain more than 85% of the total incidence of malaria in the whole country with P. vivax and P. falciparum being prevalent species.

The annual parasite index (API) and percentage of P. falciparum (%P.f) during 1990-1996 are shown in Fig. 1.

Five species of Anopheles (A. stephensi, A. cutlicifaices, A. fluviatilis, A. superpictus and A. d'thali) are considered as malaria vectors in these areas.⁵

Active and passive case detection and treatment, intradomiciliary spraying with propoxure and other insecticides such as pirimiphos-methyl and larviciding with fuel oil or abate, have been the main control measures applied in these areas. In these studies, in vivo and in vitro susceptibility tests were performed according to World Health Organization guidelines^6,7 in the selected malaria patients referred to the Malaria Laboratories at the Bandar-Abbass and Iran-Shahr Health Research and Training Centers.

The Dill-Glazko urine test⁸ was used to determine prior use of CHL, and patients with positive tests were excluded from the study.

Falciparum and vivax malaria patients were treated with standard doses of CHL ie; 25 mg/kg over 3 days. Primaquine was administered in a single dose of 0.75 mg/kg on day 2 with the third dose of CHL as a gametocytocidal drug in falciparum malaria and as an hypnozoitocidal (anti-relapse) drug in vivax malaria. In vivax malaria patients, administration of primaquine was

continued at the same dose weekly up to and including the 8th week.²

In falciparum malaria patients whose parasites were resistant to CHL, further treatment, either with one dose of 25 mg/kg sulfadoxine plus 1.25 mg/kg pyrimethamine or 10 mg/kg of quinine, 8 hourly for 2 days plus the above dose of sulfadoxine/pyrimethamine (SDR/PYR), was instituted.

Micro in vitro tests were conducted using blood samples collected from falciparum malaria patients on plates pre-dosed with CHL, amodiaquine (AMO), SDX/PYR, mefloquine (MEF) and quinine (QNN), using kits supplied by the World Health Organization.

Results and Conclusions

The results of the in vivo response of P. falciparum to CHL in Sistan-Baluchestan, Hormozgan and Kerman Provinces during 1990-1993 and 1994-1996 are summarized in Table 1.

In total, of 808 selected patients tested during 1990-1993, 593 cases (73.4%) were resistant at RI (45.%%), RII (16.7%) and RIII (11.2%) levels. The parasites were sensitive in 90 patients (11.1%) and were considered as sensitive or RI in 125 individuals (15.5%).

During 1994-1996, of 493 falciparum malaria patients tested, 297 cases (60.2%) were resistant to CHL at RI (46.9%), RII (10.5%) and RIII (2.8%) levels (Table 1).

Generally, the rate and levels of resistance of P. falciparum to CHL during 1994-1996 were lower than 1990-1993. The incidence and levels of resistance in Hormozgan and Kerman Provinces were higher than those in Sistan-Baluchestan.

The in vivo resistance to SDX/PYR was observed in 12 of the 88 falciparum CHL-resistant cases (13.6%) at RI (6.8%) and RII (6.8%) levels. The rate of SDX/PYR-resistant cases was also much higher in Hormozgan and Kerman Provinces.

The results of the in vitro assessment of the response of P. falciparum carried out during 1990-1993 against CHL, AMO, SDX/PYR, MEF and QNN are shown in Table 2 and the number of successful tests and rates of resistance are shown in Fig. 2. Resistance rates of P. falciparum to the above anti-malaria drugs were 33.4%, 15.2%, 17.9%,2.2% and 0.0%, respectively.

The successful growth rate of the parasites in a total of 1,011 tests was 50.2%.

In 118 malaria patients examined by both the extended in vivo and the micro in vitro tests for sensitivity to CHL, correlation of the drug resistance by the two methods was poor; corresponding in only 54%. The resistance rate in the in vivo tests was much higher than that of the in vitro tests. This discrepancy may be due to the selection of falciparum malaria patients who had previously been treated for malaria and already had traces of CHL in their blood by the time the in vitro test was carried out. Although the Dill-Glazko urine test was usually negative in the selected patients, this test probably was not sufficiently sensitive for our purposes. The greatest discrepancy was between cases who showed the RI resistance level in vivo but were sensitive to in vitro at levels below £ 4 pmol CHL/well.

Although the successful growth of P. falciparum in the SDX/PYR-treated plates was low (20.1%), the incidence of in vitro resistance of the parasites to SDX/PYR was rather high in samples from both Sistan-Baluchestan and Hormozgan Provinces (18.2% and 17.6%, respectively). The determinants of resistance to the drug combination was the growth of schizonts with ³ 8 normal divided nuclei in a drug concentration of ³ 1000 pmol of sulfadoxine and ³ 12.5 pmol of pyrimethamine per well.⁹

The in vitro resistance to amodiaquine was observed in samples from both Sistan-Baluchestan and Hormozgan Provinces. In addition to cross-resistance, which is usually expected between chloroquine and amodiaquine, the use of amodiaquine in Hormozgan Province for a short time and in limited areas probably caused the increase of amodiaquine-resistant cases in that province in 1990-1993 (Table 2).

Some treatment failures in falciparum patients given amodiaquine were also observed in Hormozgan.

Mefloquine has not yet been used for malaria control in Iran. In the study of the micro in vitro assessment of the response of P. falciparum to this drug, one exceptionally high in vitro resistant case was found in a 15-year-old Afghan immigrant boy in Bandar-Abbass, in whom the infection was most probably contracted in Afghanistan or Pakistan. Parasites were totally sensitive in vitro to mefloquine in 33 samples taken from patients from the Iran-Shahr area during 1990-1993. In most cases, no growth was observed in any mefloquine treated well. However, sporadic cases of innate resistance to mefloquine in vitro has previously been reported in Iran-Shahr and Bandar-Abbass.^4,10

All isolates of P. falciparum were sensitive to quinine in vitro.

Both in vivo and in vitro monitoring of the susceptibility of P. falciparum to chloroquine during 1990-1993 showed rapid development and rather high resistance of the parasite to this drug in the malarious areas of Iran as compared to previous studies.^3,4 However, the rate and levels of resistance were lower during 1993-1996. Such decreasing trends are apparently in accordance with the decrease of the annual parasite index (API) and percentages of falciparum malaria cases in the studied areas (Fig. 1).

In this combined study, the data were obtained from a highly selective group of falciparum malaria patients referred to provincial health centers. Some of these cases might have been previous drug failures. Therefore, the rather low rate of clinical failures in the treatment of falciparum malaria with chloroquine, both in this group and the population in general, indicates that chloroquine can still be considered as the first-line treatment for malaria in Iran.¹¹

The results of the in vivo assessment of P. vivax to chloroquine in the studied malarious areas during 1995-1996 are summarized in Table 3. These findings indicate that the parasite is relatively sensitive; however, the Mean of Parasite Clearance Time (MPCT) in Hormozgan province was somewhat higher than that in Sistan-Baluchestan (2.81 and 1.95 days, respectively). The MPCT in 64 Afghan immigrants and 228 Iranian vivax malaria patients was 2.79 and 2.45 days, respectively.

Acknowledgments

We would like to express our gratitude to the directors, scientific and administrative staff of all organizations who supported, gave guidance and cooperated in these studies. We would also like to thank our colleagues in the Training and Research Centers; Malaria Units of Health and Medical Networks in Bandar-Abbass and Iran-Shahr Districts. We also thank the Protozoology Unit; Computer Center and other divisions of the School of Public Health and Institute of Public Health Research for their sincere collaboration in this research work.

References

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