In vitro responses of P. Jalciparum parasites to ...

Reproduced by Sabinet Gateway under licence granted by the Publisher (dated 2009).

CENTRAL AFRICAN

Vol. 39, No.7, 1993

JOURNAL OF MEDICINE

4.

5. 6.

7. 8.

granulocytic leukaemia Shew MT, ed. Esst Sussex: U.K.: Praeger,1982;137-67. Craig O. Case of disease of the spleen in which death took place in consequence of the presence of purulent matter in the blood. Edinbourgh Med Sur J 1945;64:400. Keaney L, Orchard KH, Hibbin JA. Goldman 1M. T. cell cytogenetics in chronic granulocytic leukaemia. Lancet 1981;1:858. Bartran CR, Ragbavachar A, Angar B et al. T. lymphocytic lack rearrangement of the bcP gene i. Philadelphia chromosome positive chronic myelogenous leukaemia. Blood 1985;69:1682. Lawler SO. The cytogenetics of chronic granulocytic leukaemia. Clin HaematoI65:55. Stephen K. Canter, Eli Glastein, Robert B. Livingston. Principles of cancertreatmenl New York: McGraw-Hill BookCompany,1982;760-

3. 9.

10.

Medical Research Council Working Party. Chronic granulocytic leukaemia comparison of radiotherapy and Busulphan therapy. Br MedJ 1968;1:201. Minot lB, Buckman TE, Isaacs R. Chronic myelogenous leukaem~ age incidence, duration and benefit from irradiation. JAm Med

Assoc 1924;82:1489. 11.

12.

Steel RGO, Torrie JH. Principles and procedures of stastitics. Tokyo: McGraw-Hill International Book Company, 633. Williams J Williams, Ernest Beutler, Allan J Erslev, Marshall Lichtman. Haematology. 4th ed.1990;202-3.

In vitro responses of P. Jalciparum parasites to chloroquine, amodiaquine and quinine in two ecological zones in Ghana EA AFARII, BO AKANMORP, T NAKANOI, o OFORI-ADJEP SUMMARY Blood specimens were taken from 318 school children' with at least 1 000 and not more than 80 000 P. falciparum asexual parasites per IJ..l of blood for a 30 hour in vitro microtest of P. falciparum asexual parasites responses to chloroquine, amodiaquine and quinine. The study was conducted in primary schools in four urban and three rural communities in the coastal and forest zones in Ghana between June 1988 and December 1990. Chloroquine resistance was present in 58,7 pc (541 92) and 3,9 pc (41103) of the successful in vitro tests in the coastal and forest wnes respectively. Resistance to amodiaquine was recorded in 28,6 pc (12142) of the successful tests in coastal zone. There was no resistance to quinine in any of the ecological zones. Concentrations of the three drugs in pmol required for 90 pc inhibition of schizont maturation were generally higher in communities in the coastal zone than those in the forest zone. The results suggest an increase in sensitivity or a reduction in resistance of P.falciparum to the drugs from the coast to the forest zone. Correspondence to:

FAA/an l'Epidemiology Unit Noguchi Memorial Institute for Medical Research University of Ghana PO Bo;c25 Legon, Ghana 2. Immunology Unit Noguchi Memeriallnsitute for MMical ResUlrch University of Ghana PO Bo;c25 Legon, Ghana "Centre for Tropical Clinical Pharmacology and Therapeutics University of Ghana Medical School PO Bo;c4236 Accra, Ghana

136


Vol. 39, No.7, 1993

CENTRAL AFRICAN JOURNAL OF MEDICINE INTRODUCTION Malaria is endemic in Ghana, and the commonest parasite species responsible is P. /alciparum. It is the commonest disease treated at all health facilities in the country and accounts for about 40 pc of all out-patient attendances. Chloroquine (CQ) is the drug of choice for the treatment of malaria in Ghana. Other antimalarial drugs like amodiaquine (AQ) sulfadoxinel pyrimethamine (SDZlPYR) and quinine (QNN) are also available in the country and are sometimes used on clinically suspected cases of P. /alciparum resistant to CQ. There has been no evidence of in vivo or in vitro resistance of P~ /alciparum to CQ until late 1987 when reduced susceptibility to CQ was noticed and subsequently resistance to RI and RIT levels was reported in semi-immune in-patient children at the Korle-Bu Teaching Hospila1. 1.2 The presence of chloroquine resistant P. /alciparum has since been confIrmed in community based in vivo studies in southern Ghana.l.4 We are currently studying P. /alciparum sensitivity to antimalarial drugs in vivo and in vitro in Ghana and the objecti ve of this study was to determine P.falciparum asexual parasites responses in vitro to chloroquine, amodiaquine and quinine in two ecological zones in Ghana. MATERIALS AND MEfHODS Ghana is broadly divided into three ecological zones:coastal, forest and northern savannah. A multi-stage sampling method was used to select three primary schools (one urban, one peri-urban and one rural) from the coastal zone and four primary schools (two urban and two rural) from the forest zone. All indigenous children aged six to 15 years who were present on the day of the study in the selected primary schools were screened for the study which was conducted from June 1988 to December 1990. Study in ~ilro: Children with single infection of P. falciparum parasitaemia of at least 1 000 and not more than 80 000 asexual parasites per 8 000 white blood cells and negative urine test for 4-aminoquinolines were included in the study. The modifIed Haskins test' was used for the urine test. The standard WHO microtest (Mark IT) was used for the in vitro test. 6 Preculture thick and thin blood films were made for each study child. Simultaneously 100 microIitres of blood was drawn into a herparinized capillary tube and immediately transferred into 900

microlitres of RPM I 1640 medium supplemented with L-glutamine HEPES buffer, sodium hydrogen carbonate and gentamicin sulphate. Specimens were transported to the laboratory within three hours of coUection and 50microlitres each added to weUs of microlitre plates prcdossed with varying concentration of CQ, AQ and QNN. All plates were placed in candle jars and incubated at 37,5°C for 30 hrs. The culture supemant was then carefully siphoned off with pasteur pipettes and separate smears made of the contents of each well. Slides were stained with Giemsa stain. Counts of mature schizont (schizont with three or more nuclei) per 200 asexual parasites (schizonts and trophozoites) were made. The cut off points for the antimalarial drugs sbldies were as follows:- CQ - schizont growth at eight pmol or more, AQ and QNN-schizont growth at four pmol or more and 256 pmol or more respectively were taken as indication of P./alciparum resistance to the drugs. RESULTS

co: CQ resistance was present in 58,7 pc (54192) and 3,9 pc (41103) of the successful tests in the coastal and forest zones respectively. A concentration of four pmol CQ was required to effect 90 pc inhibition of schizont maturation in communities in the forest zone while higher concentrations (16 and 32 pmoI) were required to achieve the same results in the coastal zone (Table I). AQ: Resistance to AQ was recorded in 28,9 pc (12142) of the successful tests in the coastal zone. There was no resistance to AQ in the forest zone. One pmol concentration of amodiaquine was required to effect 90 pc inhibition of schizont maturation in CODlmu nities in the forest zone while higher concentrations (four and eight pmol) were required to achieve the same results in the coastal zone (Table 11). QNN: All the successful tests were sensitive to QNN. However, concentrations of QNN in pmol required for 90 pc inhibition of schizont maturation were generally higher in communities in the coastal than in the forest zone (Table 111). DISCUSSION The results of the study confumed the existence of resistant P.falciparum in Ghana. The emergence of P. /alciparum resistance to AQ has also been demonstrated. However, there was no resistance to QNN in any of the communities studied. The results of this study suggest an increase in the sensitivity or reduction

137


Vol. 39, No. 7, 1993

CF.NfRAL AFRICAN JOURNAL OF MEDICINE

Table I: III .ilro responses ojP. jalcipllI'um asexual parasites to chloroquine in children in two ecological wnes in Ghana 1988-1990. Ecological No. of No. of ZatWIIII lelia tasIs Communi.. performed successflJ COlaW Nina (Urban) Maclna (P.....urban) GomoaFet\eh (RIAl) Fo,.at Sunyani (Urban) Chiraa (RuraQ Ho(Urban) NdoIBagbie (RIAl)

TOTAL

No. Sensitive

No. Resistant

Percentage of inhiblion of schizont maturation in concer-.ration of chIoroqUN in prnoI 4 K(cont) 8 1 2

16

32

64

48

24 (50,0)

9

15 (62,5)

0

10,60

20,59 45,66

62,50

87,40

99,26 100

59

36 (61,0)

13

23(63,9)

0

36,20

52,30 65,20

71,70

82,40

95,70 100

49

32 (65,3)

16

16 (50,0)

0

28,44

59,14 79,80

86,15

92,498 96,95 100

36 42 48

22(61,1) 24 (57,1) 34 (70,8)

19 23 34

3 (13,6) 1 (4,2) 0(0,0)

0 0 0

54,2 55,05 28,57

82,90 96,78 74,90 96,91 82,87 97,67

99,15 99,70 99,70 100 99,93 100 100 100 100 100 100 100

36

23(63,9)

23

0(0,0)

0

41,87

70,93 93,93

100

318

195(61,3)

137

58 (29,7)"

100

100

100

Percentage. In bracket.. • Pen:entage .uccellful te.t ,..,I.tant.

Table II: III .itro responses of P. jalcipllI'um aseXJJ.al parasites to amodiaquine in children in two ecological wnes in Ghana 1988--1990. ~

Ecological

ZonesJ Communities

Coaatal Nina (Urban) Maclna (P.....urban) Gomoa Felleh (Rural) Sunyani (Urban) Chiraa (RuraQ Ho(Urban) NdoIBagbie (Rural)

TOTAL

No. of No. 01 tests tests performed successflJ

No. Sensitive

No. Resistant

Percentage 01 inhiblion of schizont maturation in c:oncentration of amodiaquine in prnoI 0,50 K(cont) 0,25 2 1

4

8

48

20 (41,7)

14

6(30,0)

0

13,16

42,27 70,87

83,59

95,94 100

59

14 (23,7)

9

5 (35,7)

0

20,50

43,54 52,29

69,48

84,26

49

8 (16,3)

7

1 (12,5)

0

18,08

49,71

75,15

79,67

88,12 100

36 42 48

23(63,9) 23(54,8) 39 (81,3)

23 23 39

0(0,0) 0(0,0) 0(0,0)

0 0 0

40,75 51,73 39,00

70,90 95,15 79,93 92,60 75,30 94,06

98,73 98,53 99,79

36

23(63,9)

23

0(0,0)

0

30,14

68,00 85,60

95,11

318

150 (47,2)

138

12 (8,0)"

Pen:entage. in bracketl. • Pen:entage .uccellful ta.t ,..,I.tant. 138

100 100 100

16

100

99,84 100 100

100 100 100

100 100 100

98,88 100

100


Vol. 39, No.7, 1993

CENTRAL AFRICAN JOURNAL OF MEDIC1NE

Table Ill: In vitro responses of p, falciparum asexual parasites to quinine in children in two ecological

zones in Ghana Ecological Zooesl Communities

19~I990.

No. of

No. of

tests

tests

performed successflJ

No. Sensitive

Percentage of inhibition of schizont mahntion in concentration of amociaquine in pmoI

No. Resistant

K (conI)

4

8

16

32

64

128

256

100

100

100

Coa.tal Nima (Urban) Madina (Peri-urban) Gomoa Fettah (Rural) Sunyani (Urban) Chiraa (Rura~ Ho (Urban) Ndo&gble (Rural)

TOTAl..

48

21 (43,8)

21

0

0

16,95

38,97 63,10

92,29

59

17 (28,8)

17

0

0

28,49

44,70 62,15

75,28

99,44 100

100

49

10 (20,4)

10

0

0

18,08

36,30 67,04

73,33

85,19 100

100

36

48

19 (52,8) 20 (47,6) 39 (81,3)

19 20 39

0 0 0

0 0 0

37,89 39,75 29,96

48,65 63,53 63,95 88,21 71,44 92,81

90,08 93,86 98,95

95,53 99,35 100 98,58 99,76 100 100 100 100

36

26 (72,2)

26

0

0

36,32

76,93 86,32

97,63

100

318

152 (47,8)

152

42

100

100

0(0,0)"

Percentage. in bracket•• • Percentage .ucce..ful te.t re.i.tsnt

in resistance of P. /alciparum to antimalarial drugs from the coast to the forest zones and from the urban to the rural communities especially in the coastal zone. Drug pressure through misuse of antimalarial drugs may be the most important factor favouring the selection of P. /alciparum resistant asexual parasites to antimalarial drugs. The higher levels of resistance to CQ and AQ and the higher concentrations of the three drugs in pmol required for 90 pc inhibition of schizont maturation in communities in the coastal zone compared to those in the forest zone are a reflection of accessibility to and use of antimalarial drugs in the two zones. The 100 pc sensitivity of P./alciparum toQNN in all the communities studied is very important since quinine remains the drug of choice for the treabnent of severe and complicated malaria in Ghana Although treatment policies are usually based on results of in vivo tests. in vitro tests could be used to test P. /alciparum sensiti vity to new antimalarial drugs and monitor any changes in parasite sensitivity to antimalarial drugs in use in the country-. Such results could also be used to guide the ~se of alternati ve antimalarial drugs to avoid the rapid development and

spread of drug resistant parasites in Ghana ACKNOWLEDGEMENTS The work was supported in part by the Japan International Cooperative Agency (JICA). and the Government of Ghana. We are grateful to Professor FK Nkrumah for useful suggestions during the preparation of the manuscript. We also thank Messrs Andrew Adjei. Ben Gyan and Augustine Badasu of the Noguchi Memorial Institute for Medical Research for technical assistance. REFERENCES

1.

2.

3.

139

Neequaye JE. Ofori-Adjei O. Odame I. Coker L. Mensah-Annan. Falciparum malaria not sensitive to chloroquine emerges in Accra in 1987. Ghana Med J 1988;22:6-10. Ofori-Adjei O. Adjepon-YamoahKK. Commey JOO. Ofori-Adjei E. In vivo sensitivitiy of P. /alciparum to chloroquine in Accra, Ghana. Ghana Med J 1988;22: 114. Mari EA. Akanmori BO. Nakano T. OforiAdjei E. Owusu-Adjei S. Gyan B. Adjei A. In vivo and in vitro sensitivity status of P.


Vol. 39. No.7. 1993

CENTRAL AFRICAN JOURNAL OF MEDICINE

falciparum to chloroquine in three communities in Ghana. Goon Med J 1989;23:233-7. 4.

5.

6.

Afari EA., Akanomri BD, Nakano T, OforiAdjeiD. Plasmodiumfalciparum: sensitivity to chloroquine in vivo in three ecological zones in Ghana. Trans R Soc Trop Med Hyg 1992;86:231-4. MountDL, PatchenLC, Williams SB, Churchill FC. Colorimetric and thin-layer chromatographic methods for field assay of chloroquine and metabolites in urine. Bull WHO 1987;65: 615-23. WHO. MAP/87.2 Revision I, June 1990. In vitro micro-test (MARK II) for the assessment of the response of Plasmodium /alciparum to chloroquine, mefloquine, quinine, sulfadoxine pyrimethamine and amodiaquine.

Antifungal activity of essential oil from Artemisia afra Jacq MGUNDIDZA

SUMMARY

Anemisia afra is indigenous to the eastern highlands of Zimbabwe where it is used in folk medicine. Hydrodistilled volatile oil from the aerial parts of the plant was tested for antifungal activity against 10 fungal species using the dry weight method. The results obtained showed that the essential oil exhibited significant activity against Aspergillus ochraceus, Candida

albicans, Ahernaria alternata, Geotrichum candidum, Aspergillus niger, Penicillium citrium and Aspergillus parasiticus. INTRODUCTION

Artemisia afra Jacq belongs to the compositae family of plants. It is also known as African wormwood, wild wormwood or wild also It is widely used in folk meiliDepaTtmellt of Pha17flllCJ

Facully 0/MediciM Univenity o/Zimbabwe PO BoJC MP 167 Mount Plearanl

Harare, Zimbabwe

cines in southern Africa., mainly for coughs and cold, chills, stomach ache and dry dyspepsia and as a purgative. I The leaves are used in tea, for a bath. or as a leaf poultice, or the vapours from boiling leaves can be inhaled. The plant is also used as an insect repellent. It is distributed in the mountainous region of eastern Zimbabwe and western Mozambique. The plant is bushy and grows up to one metre in height. It is propagated easily from seeds, cuttings and rooted pieces and grows well both in the wild and in cultivation. Previous gas chromatographic studies showed that the main components of the volatile oil are a-thujone (52,5 pc), 6-thujone (13,1 pc). 1.8-cineole (13,0 pc), and camphor (6,6 pc), with other monoterpenes and sesquiterpenes under two pc (camphene, y-terpene, pcymene, a-terpinolene and a-pinene. l .... The essential oil yield was found to be ranging from 0,25 to 1,2 pc. The preservative nature of plants has been realised for centuries as a variety of herbs and spices have traditionally been used to extend the shelf-life of foods. Aromatic plant volatile oils have been known since antiquity to possess biological activities, notably antibacterial and antifungal properties.'·' In this study, the fungal species were chosen to represent several major groups that include filamentous fungi which encompass a number of spoilage and mycotoxigenic strains and the Candida which usually infect the skin. Plant volatile oils with antifungal activity can be incorporated into lotions that can be used tropically to treat fungal skin infections that usually accompany AIDS. These types oflotions are currently being developed in the Department of Pharmacy at the University of Zimbabwe. This forms the basis of this study. MA1ERIALS AND METIlODS Plant material: The fresh aerial parts of Anemisia afra were collected from the Nyanga district ofZimbabwe. The plant was identified by the botanists from the National Herbarium and Botanical Gardens of Zimbabwe. One voucher specimen is kept at the Deparlment of Pharmacy, at the University of Zimbabwe and the other at the National Herbarium and Botanical Gardens of Zimbabwe. Volatile oil distillation: The volatile oil was obtained by hydrodistillation of fresh aerial parts. In this method, one kg ofleaves was placed in a four litre flask containing one litre ofdistilled water. A condenser was fIXed to the flask according to the method outlined in the British Pharmacopoeia.' The oil was distilled for

140