• View in gallery

    Map of Assam and the other northeastern states of India. The study site, located south of the Brahmaputra River, is denoted by a dot (•) The inset shows the geographic location of the northeastern region of India.

  • 1

    Kondrashin AV, 1992. Malaria in the WHO Southeast Asia region. Indian J Malariol 29 :129–160.

  • 2

    Sehgal PN, Sharma MID, Sharma SI, Gogal S, 1973. Resistance to chloroquine in falciparum malaria in Assam state, India. J Commun Dis 5 :175–180.

    • Search Google Scholar
    • Export Citation
  • 3

    Lal S, Dhillon GPS, Sonal GS, Sita Rama Rao B, 1998. Country Scenario – Malaria and Its Control in India. National Malaria Eradication Programme (Enhanced Malaria Control Project). Delhi: Ministry of Health and Family Welfare, Government of India publication.

  • 4

    Dev V, Hira CR, Rajkhowa, 2001. Malaria-attributable morbidity in Assam, North-Eastern India. Ann Trop Med Parasitol 95 :789–796.

  • 5

    Alvan G, Ekman L, Lindstrom B, 1982. Determination of chloroquine and its desethyl metabolite in plasma, red blood cells and urine by liquid chromatography. J Chromatogr 229 :241–247.

    • Search Google Scholar
    • Export Citation
  • 6

    Dua VK, Brohult J, Ericsson O, Sharma VP, 1986. High performance liquid chromatographic determination of chloroquine in finger-tip blood dried on filter paper: sample handling problems. Indian J Malariol 23 :151–154.

    • Search Google Scholar
    • Export Citation
  • 7

    Dua VK, Sarin R, Sharma VP, 1994. Sulfadoxine concentrations in plasma, red blood cells and whole blood in healthy and Plasmodium falciparum malaria cases after treatment with Fansidar using high performance liquid chromatography. J Pharm Biomed Anal 12 :1323–1328.

    • Search Google Scholar
    • Export Citation
  • 8

    Dua VK, Sarin R, Prakash A, 1993. Determination of quinine in serum, plasma, red blood cells and whole blood in healthy and Plasmodium falciparum malaria cases by high-performance liquid chromatography. J Chromatogr 614 :87–93.

    • Search Google Scholar
    • Export Citation
  • 9

    Desjardins RE, Doberstyn EB, Wernsdorfer WH, 1988. The treatment and prophylaxis of malaria. Wernsdorfer WH, McGregor SI, eds. Malaria: Principles and Practice of Malariology. Volume I. New York: Churchill Livingstone, 827–864.

  • 10

    Hellgren U, Kihamia CM, Mahikwano LF, Bjorkman A, Eriksson O, Rombo L, 1989. Response of Plasmodium falciparum to chloroquine treatment: relation to whole blood concentrations of chloroquine and desethylchloroquine. Bull World Health Organ 67 :197–202.

    • Search Google Scholar
    • Export Citation
  • 11

    Na-Bangchang K, Karbwang J, 1993. Clinical pharmacology of chloroquine. Karbwang J, Wernsdorfer WH, eds. Clinical Pharmacology of Antimalarials. Bangkok: Mahidol University Publications, 85–125.

  • 12

    Dua VK, Gupta NC, Kar PK, Nand J, Sharma VP, Subbarao SK, 2000. Chloroquine and desethylchloroquine concentrations in blood cells and plasma from Indian patients infected with sensitive or resistant Plasmodium falciparum.Ann Trop Med Parasitol 94 :565–570.

    • Search Google Scholar
    • Export Citation
  • 13

    Ward SA, Bray PG, Hawley SR, 1997. Quinoline resistance mechanisms in Plasmodium falciparum: the debate goes on. Parasitol. 114 (Suppl): 125–136.

    • Search Google Scholar
    • Export Citation
  • 14

    Sharma VP, 2000. Status of drug resistance in malaria in India. Mahajan RC, ed. Multi-Drug Resistance in Emerging and ReEmerging Diseases. Delhi: Narosa Publications, 191–202.

  • 15

    Smithuis FM, Monti F, Grundl M, Oo AZ, Kyaw TT, Phe O, White NJ, 1997. Plasmodium falciparum: sensitivity in vivo to chloroquine, pyrimethamine/sulfadoxine and mefloquine in western Myanmar. Trans R Soc Trop Med Hyg 91 :468–472.

    • Search Google Scholar
    • Export Citation

 

 

 

 

MULTI-DRUG RESISTANT PLASMODIUM FALCIPARUM MALARIA IN ASSAM, INDIA: TIMING OF RECURRENCE AND ANTI-MALARIAL DRUG CONCENTRATIONS IN WHOLE BLOOD

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  • 1 Malaria Research Centre, Field Station, Sector III, Ranipur, Hardwar, India; Malaria Research Centre, Field Station, Sonapur, Kamrup, Assam, India; Malaria Research Centre, Delhi, India

The susceptibility of 23 cases of Plasmodium falciparum malaria from the Sonapur primary health center in the Kamrup district of Assam, India to different antimalarials was investigated using the 28-day World Health Organization in vivo test. Whole blood concentrations of chloroquine, sulfadoxine, and quinine were determined at different intervals and at the time of parasites recrudescence after completion of treatment with the respective drugs to confirm the status of drug sensitivity. A case of multi-drug resistant P. falciparum malaria was found where recrudescence occurred, despite standard oral treatment with chloroquine, sulfadoxine/pyrimethamine, and quinine sequentially. Whole blood concentrations of chloroquine, sulfadoxine, and quinine at the time of recrudescence were 0.35 μg/ml (day 7), 18 μg/ml (day 14), and 0.009 μg/ml (day 14), respectively. Therefore, monitoring of drug-resistant P. falciparum malaria and its proper treatment should be intensified to check the spread of multi-drug resistant strains in other parts of the country.

INTRODUCTION

The problem of multiple drug resistance in Plasmodium falciparum malaria is increasing in the countries in southeast Asia.1 It is highly prevalent in Myanmar and Thailand, and there is a strong possibility of it entering India through the northeastern states of this country. Chloroquine resistance in P. falciparum was first reported from Assam, India in 1973, and it has spread gradually to other parts of India.2 Resistance to other antimalarial drugs such as sulfadoxine is rare, but a few cases have been reported.3 Malaria outbreaks are a yearly phenomena in Assam and are associated with high morbidity and mortality.4 Therefore, there is a need to monitor the presence of multiple drug-resistant stains of P. falciparum in Assam to contain its spread to other parts of India. We have investigated one of 23 cases of P. falciparum malaria in which parasites reappeared despite treatment with chloroquine, sulfadoxine/pyrimethamine, and quinine sequentially. This case demonstrates the emergence of multiple drug-resistant malaria in Assam.

MATERIALS AND METHODS

Study area.

Assam is one of the eight northeastern states of India and borders Bhutan, Bangladesh, and Myanmar. Most districts in this state are endemic for malaria. The study was performed at the Sonapur Primary Health Center in the Kamrup district of Assam, which is located 25 km east of Guwahati (the capital of Assam) on the southern bank of the Brahmaputra River (Figure 1). This area is endemic for malaria and contributes nearly 50% of the total cases in the district. A predominance of P. falciparum (> 70%), along with many deaths due to malaria, has been reported in this area.

Subjects.

Twenty-three cases of P. falciparum malaria (18 males and 5 females) were selected for the study. The Institutional Review Committee of the Malaria Research Center in New Delhi reviewed and approved the study protocol. The protocol was explained to the patients and all gave their consents voluntarily. The mean age of the patients was 18.8 years (range = 8–40 years) and the mean day 0 parasitemia was 28,361 asexual forms/μL of blood (range = 1,680–81,280/ μL). Any patient found to have evidence on presentation of recent use of chloroquine, sulfadoxine/pyrimethamine, or quinine in whole blood samples dried on filter paper5–8 was excluded from the study.

Treatment.

Each enrolled patient was first treated orally with 25 mg of chloroquine base/kg of body weight over a three-day period (10 mg/kg on day 0, 10 mg/kg on day 1 [24 hours], and 5 mg/kg on day 2 [48 hours]) under active supervision while swallowing the tablets and for 30 minutes afterwards. Cases with a reappearance of parasites after chloroquine therapy (chloroquine-resistant cases) were treated with three tablets of Fansidar® (F. Hoffmann-La Roche, Basel, Switzerland) (500 mg of sulfadoxine and 25 mg of pyrimethamine per tablet). Quinine hydrochloride was given at a dose of 10 mg/kg as a slow intravenous infusion in 5% dextrose solution over a four-hour period for three days, followed by an oral dose of 600 mg twice a day on days 3 to 6, to the patients whose parasites were resistant to chloroquine and sulfadoxine/pyrimethamine. A slightly lower loading dose of quinine was given to one patient to avoid toxicity because this patient had taken other antimalarials before receiving quinine therapy.9 Quinine-resistant cases were treated with artesunate (100 mg on day 0 followed by 50 mg on days 1 to 4). All resistant cases were treated by a doctor at the primary health center. Children were given proportionately lower dosages. Each case was followed for 28 days and analyzed with the World Health Organization extended in vivo test to study the sensitivity of P. falciparum to different antimalarials. Blood smears were collected on days 0, 2, 7, 14, 21, and 28 and also whenever the patient complained of fever after completing antimalarial treatments. These smears were stained with Giemsa to determine asexual parasitemia and clinical response.

Drug analysis.

Concentrations of chloroquine, sulfadoxine (one component of the combination drug Fansidar®), and quinine were determined in whole blood samples collected on filter paper at days 0, 2, 7, 14, and 28, or when the parasites reappeared after starting treatment. Finger prick blood (100 μL) was drawn from each patient into a heparinized capillary tube at various intervals as stated earlier, and the concentrations were determined by high-performance liquid chromatography. The methods developed for chloroquine5,6, sulfadoxine,7 and quinine8 were used.

RESULTS

Twenty-three cases of P. falciparum malaria were successfully followed after chloroquine therapy. The results of in vivo study indicated that 16 (69.56%) of the 23 infections were sensitive to CQ and 7 (30.43%) were resistant; 6 (26%) of the resistant cases showed RI resistance and one (4.34%) showed RII resistance. In all six RI resistant cases, parasites reappeared on day 14 with a mean parasite density of 6,773.33/μL (range = 1,120–25,440/μL), while the parasite density of the one RII case on day 7 was 10,080/μL. The mean ± SD chloroquine concentration in the six RI cases at the time of reappearance of parasites on day 14 was 0.56 ± 0.32 μg/mL (range = 0.20–0.95 μg/mL), while the one RII resistant case on day 7 showed a concentration of 0.35 μg/mL (Table 1). Hellgren and others10 showed a mean whole blood chloroquine concentration of 0.47 μg/mL on day 7 in RII resistant cases. The mean ± SD chloroquine concentration of sensitive cases on day 2 was 1.75 ± 0.57 μg/mL (Table 2), while resistant cases showed a concentration of 0.78 ± 0.43 μg/mL (range = 0.25–1.40 μg/mL). Chloroquine concentrations on day 2 were much higher in whole blood from sensitive cases than from resistant cases (t = 3.85, P < 0.01). A chloroquine whole blood concentration of 90 ng/mL was found to be the minimum inhibitory concentration for P. falciparum malaria cases.11 The relatively low concentrations of resistant cases were due to reduced accumulation in resistant parasites and not because of changes in adsorption and inter-individual variability in chloroquine pharmacokinetics, as previously reported.12,13 All seven chloroquine-resistant cases were treated with sulfadoxine/pyrimethamine. In vivo results (Table 1) indicated that six (85%) were sensitive to sulfadoxine/pyrimethamine, and in one case parasites reappeared on day 14 (RI resistance) with a parasite density of 18,240/μL. The mean sulfadoxine concentration on day 2 in sensitive cases was 70.40 μg/mL (range = 45.0–115.0 μg/mL); a resistant case showed a concentration of 55 μg/mL. It is interesting to note that the minimum inhibitory concentration of sulfadoxine/pyrimethamine required to clear P. falciparum parasites from whole blood on day 2 was 20.15 μg/mL (Hellgren U, 1990. Drug concentrations during antimalarial treatment and prophylaxis - relation to in vivo outcome and in vitro susceptibility. Ph.D Thesis, Karolinska Institute, Stockholm, Sweden). The sulfadoxine concentration on the reappearance of parasites on day 14 was 18 μg/mL. A case with P. falciparum resistant to chloroquine and sulfadoxine was treated with quinine. A blood smear from this patient was negative on day 2 with a whole blood quinine concentration of 0.70 μg/mL. However, parasites reappeared on day 14 when the whole blood concentration of quinine was 0.009 μg/mL. The case was confirmed as having a parasite with RI resistance to quinine. The patient was treated with artesunate and was cured.

DISCUSSION

Chloroquine continues to be the drug of choice for the treatment of malaria in India, despite decreased sensitivity reported in P. falciparum.2 Few P. falciparum cases resistant to other antimalarials such as quinine and sulfadoxine-pyrimethamine have been reported.14 Multidrug-resistant strains of P. falciparum are common in the neighboring countries of Myanmar and Thailand, and there is a strong possibility of the development of resistant foci in the northeastern states of India.15 We detected a case of multidrug-resistant P. falciparum malaria who did not respond to standard treatments with chloroquine, sulfadoxine/pyrimethamine, and quinine sequentially. The concentrations of chloroquine, sulfadoxine, and quinine on day 2 in this patient were greater than the minimum inhibitory concentrations prescribed for clearance of P. falciparum parasites. The concentrations of antimalarials at the time of reappearance of parasites were 0.35 μg/mL for chloroquine (day 7, RII), 18 μg/mL for sulfadoxine (day 14, RI), and 0.009 μg/mL for quinine (day 14, RI) after the respective treatments. This confirms the presence of multidrug-resistant strain in this area. Therefore, monitoring of drug-resistant P. falciparum malaria and its proper treatment must be intensified to check the spread of multidrug-resistant strains to other parts of India.

Table 1

Parasite density and concentration of antimalarials in whole blood during in vivo follow-up of Plasmodium falciparum malaria cases*

ChloroquineSulfadoxineQuinine
Case no. (age, years/sex)D0D2D7D14D0D2D7D14D0D2D7D14Sensitivity status
* Values are parasite densities per microliter of blood. Values in parentheses are drug concentrations (μg/mL). Only the concentration of sulfadoxine (one component of the combination drug Fansidar®) was measured. D = day; CQ = chloroquine; R = resistant; SP = sulfadoxine/pyrimethamine; S = sensitive; ND = not determined; Neg = negative.
1 (22/F)70,7201,920 (0.95)25,440 (0.70)25,440240 NDCQ(RI) SP (S)
2 (13/M)28,9601,280 (0.85)1,120 (0.20)800Neg (45.0)CQ(RI) SP (S)
3 (11/M)4,960480 (ND)10,080 (0.35)10,080320 (55.0)18,240 (18.0)18,240Neg (0.70)Positive (0.009)CQ(RII) SP (RI) Quinine (RI)
4 (18/M)10,720160 (1.0)3,840 (0.25)Neg (80.0)CQ(RI) SP(S)
5 (9/M)36,4003,040 (0.90)40,800 (0.70)42,400Neg (115.0)CQ(RI) SP(S)
6 (10/M)5,360Neg (0.80)2,9602,960Neg (52.0)CQ(RI) SP(S)
7 (12/F)12,4002,480 (1.40)4,240 (0.75)4,240Neg (90.0)CQ(RI) SP(S)
Table 2

Parasite density and concentration of chloroquine in whole blood of Plasmodium falciparum–sensitive cases*

Parasite density/μL
Case no. (age, years/sex)D0D2Concentration ± SD (μg/mL) D2
* D = day.
1 (12/F)59,68001.40
2 (19/M)55,2005,2801.00
3 (9/M)36,4001,4401.50
4 (10/M)32,1604001.50
5 (25/M)1,68002.40
6 (25/M)81,28017,4411.61
7 (40/M)10,240802.26
8 (13/M)35,6802,4801.86
9 (12/F)18,72011,5202.20
Mean36,782.224,293.331.75 ± 0.46
Figure 1.
Figure 1.

Map of Assam and the other northeastern states of India. The study site, located south of the Brahmaputra River, is denoted by a dot (•) The inset shows the geographic location of the northeastern region of India.

Citation: The American Journal of Tropical Medicine and Hygiene Am J Trop Med Hyg 69, 5; 10.4269/ajtmh.2003.69.555

Authors’ addresses: Virendra K. Dua and N. C. Gupta, Malaria Research Centre, Field Station, Sector III, BHEL, Ranipur, Hardwar 249 403, India, E-mail: virendradua@hotmail.com. Vas Dev and S. Phookan, Malaria Research Centre, Field Station, Sonapur, Kamrup, Assam 782 402, India. V. P. Sharma and S. K. Subbarao, Malaria Research Centre, 22 Sham Nath Marg, Delhi 110 054, India.

Acknowledgments: We are thankful to Dr. B. Das (Primary Health Center, Sonapur) for overall clinical supervision and appropriate treatment of malaria cases. Thanks are also due to Dr. G. Edwards (Department of Pharmacology University of Liverpool, Liverpool, United Kingdom) for critical comments on the manuscript.

REFERENCES

  • 1

    Kondrashin AV, 1992. Malaria in the WHO Southeast Asia region. Indian J Malariol 29 :129–160.

  • 2

    Sehgal PN, Sharma MID, Sharma SI, Gogal S, 1973. Resistance to chloroquine in falciparum malaria in Assam state, India. J Commun Dis 5 :175–180.

    • Search Google Scholar
    • Export Citation
  • 3

    Lal S, Dhillon GPS, Sonal GS, Sita Rama Rao B, 1998. Country Scenario – Malaria and Its Control in India. National Malaria Eradication Programme (Enhanced Malaria Control Project). Delhi: Ministry of Health and Family Welfare, Government of India publication.

  • 4

    Dev V, Hira CR, Rajkhowa, 2001. Malaria-attributable morbidity in Assam, North-Eastern India. Ann Trop Med Parasitol 95 :789–796.

  • 5

    Alvan G, Ekman L, Lindstrom B, 1982. Determination of chloroquine and its desethyl metabolite in plasma, red blood cells and urine by liquid chromatography. J Chromatogr 229 :241–247.

    • Search Google Scholar
    • Export Citation
  • 6

    Dua VK, Brohult J, Ericsson O, Sharma VP, 1986. High performance liquid chromatographic determination of chloroquine in finger-tip blood dried on filter paper: sample handling problems. Indian J Malariol 23 :151–154.

    • Search Google Scholar
    • Export Citation
  • 7

    Dua VK, Sarin R, Sharma VP, 1994. Sulfadoxine concentrations in plasma, red blood cells and whole blood in healthy and Plasmodium falciparum malaria cases after treatment with Fansidar using high performance liquid chromatography. J Pharm Biomed Anal 12 :1323–1328.

    • Search Google Scholar
    • Export Citation
  • 8

    Dua VK, Sarin R, Prakash A, 1993. Determination of quinine in serum, plasma, red blood cells and whole blood in healthy and Plasmodium falciparum malaria cases by high-performance liquid chromatography. J Chromatogr 614 :87–93.

    • Search Google Scholar
    • Export Citation
  • 9

    Desjardins RE, Doberstyn EB, Wernsdorfer WH, 1988. The treatment and prophylaxis of malaria. Wernsdorfer WH, McGregor SI, eds. Malaria: Principles and Practice of Malariology. Volume I. New York: Churchill Livingstone, 827–864.

  • 10

    Hellgren U, Kihamia CM, Mahikwano LF, Bjorkman A, Eriksson O, Rombo L, 1989. Response of Plasmodium falciparum to chloroquine treatment: relation to whole blood concentrations of chloroquine and desethylchloroquine. Bull World Health Organ 67 :197–202.

    • Search Google Scholar
    • Export Citation
  • 11

    Na-Bangchang K, Karbwang J, 1993. Clinical pharmacology of chloroquine. Karbwang J, Wernsdorfer WH, eds. Clinical Pharmacology of Antimalarials. Bangkok: Mahidol University Publications, 85–125.

  • 12

    Dua VK, Gupta NC, Kar PK, Nand J, Sharma VP, Subbarao SK, 2000. Chloroquine and desethylchloroquine concentrations in blood cells and plasma from Indian patients infected with sensitive or resistant Plasmodium falciparum.Ann Trop Med Parasitol 94 :565–570.

    • Search Google Scholar
    • Export Citation
  • 13

    Ward SA, Bray PG, Hawley SR, 1997. Quinoline resistance mechanisms in Plasmodium falciparum: the debate goes on. Parasitol. 114 (Suppl): 125–136.

    • Search Google Scholar
    • Export Citation
  • 14

    Sharma VP, 2000. Status of drug resistance in malaria in India. Mahajan RC, ed. Multi-Drug Resistance in Emerging and ReEmerging Diseases. Delhi: Narosa Publications, 191–202.

  • 15

    Smithuis FM, Monti F, Grundl M, Oo AZ, Kyaw TT, Phe O, White NJ, 1997. Plasmodium falciparum: sensitivity in vivo to chloroquine, pyrimethamine/sulfadoxine and mefloquine in western Myanmar. Trans R Soc Trop Med Hyg 91 :468–472.

    • Search Google Scholar
    • Export Citation
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