514

TIUNSACT~N~ OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1987) 81, 514-515

1 Short Report (

Absence of crisis form factor in subjects immune to Plasmodium falciparum in The Gambia,

West Africa

KEVIN MARSH*, LIZABETH 0~00, BRIAN M. GREENWOOD

MRC Laboratories, Fajara, Banjul, The Gambia Despite the rapid advance in our understanding of

the antigenic structure and molecular biology of malaria parasites, little is known of the mechanisms underlying the development of natural immunity to the most harmful human malaria parasite, Plasmo- dium falciparum.

The development of techniques for in vitro culture of P. falciparum (TRAGER & JENSEN, 1976) has facilitated the analysis of possible effector mechan- isms. Whole sera obtained from artificially immu- nized non-human primates or from subjects living in endemic areas inhibit parasite growth ii vitro (C&P- BELL et al.. 1979: WILSON & PHILLIPS. 1976). Purified IgG’antibo&es from such sera are inhibitory but there is little or no correlation between the presence of such inhibitory antibodies detected in vitro and functional immunity in viva (FANDEUR et a!., 1984). Crisis form factor (CFF) is a non- dralysable, non-antibody factor originally detected in sera of highly immune subjects living in the Sudan (JENSEN et al., 1983,1984; VANDE WAA et al., 1984). In contrast to antibody it acts as a toxin, leading to the death of parasites within infected host erythrocytes. Its anti-parasite properties and its epidemiological characteristics suggest that CFF plays an important role in acquired immunity to P. falciparum in some endemic areas of Africa. We have? therefore, investi- gated the role of CFF in malarial unmunity in a rural population living in an area of Sudan savanna on the north bank of The Gambia river. The studv oonula- tion, which is the subject of a longitudindsiudy of factors involved in immunity to malaria, will be described elsewhere. The study area is one of high malaria transmission unmodified by chemoprophyla- xis or mosquito control measures. Malaria is a major cause of childhood morbidity and mortality in this community but there is little morbidity due to malaria in adults. The sickle cell trait is carried by over 20% of the population, indicating that P. falciparum has exerted strong selective pressure. The absence of malarial morbidity in adults indicates that they have developed an effective immune response. In these respects our study population seems comparable to those in the Sudan in which CFF has been described.

We examined sera from 130 members of the Gambian village population; 20 sera from non- immune British blood donors acted as controls. The assay for CFF was based closely on that of JENSEN et al. (1983). Sera were stored at -70°C. Before testing they were dialysed thrice against phosphate-buffered saline and once against RPM1 1640 medium, heated to 56°C for 30 &in and passed through fillipore filters. A culture-adawed Gambian narasite, 85/355, was cultured by the-method of TUGGER & JENSEN (1976) and tightly synchronized by sequential sorbitol

treatments and physiogel flotations. Red cells contain- ing young rings at a parasitaemia of 1% and a haematocrit of 1.5% were suspended in test sera diluted 1:4 in RPM1 1640 supplemented with 75% non-immune serum to provide basic nutritional re- quirements. 200$ of suspension were placed in the wells of a flat bottomed 96-well plate in quintuplicate and cultured in candle jars. Test media were reple- nished after 24 h and 1 @i of tritiated hypoxanthine (Amersham) added to each well. The assay was terminated after a further 16 h. Intraerythrocytic retardation was sought by microscopic examination of slides prepared from duplicate wells and by measure- ment of the incorporation of tritiated hypoxanthine by parasites in the other three wells. The use of highly synchronized parasites allowed exposure to test sera to occur throughout one entire cycleof intraerythrocytic develonment whilst excluding the effects of antibodv after s&izont rupture. We dowed for the possibilit$ of seasonal variation in levels of CFF by testing adult sera collected at the beginning of the rainy season (August) and shortly after its end (January).

Table-Incorporation of tritiated hypoxantbine by parasites grown ia test sera. Means for triplicate wells arc shown together with 1 standard deviation

Source of Age Incorporation of sera hears) NUIllblX label (coml

Europeans Gambians Gambians Gambians Wwst) Gambians (Jaw)

Adult 20 l-5 20

6-15 20 Adult 40

Adult 50

45 737 f 9084 47 439 k 4512 44 726 k 4249 44 903 k 5756

44040~9091

Parasites grew normally to mature schizonts in all Gambian sera and no crisis form was seen. There was no significant difference between growth of parasites in Gambian sera and Eurooean sera (Table). To test the possibility that culture adapted p&asites’might be resistant to CFF we tested a further 20 adult sera using a wild parasite isolate; once again there was no evidence of intraerythrocytic retardation. Our results show that CFF is not detectable in the sera of functionally immune subjects in the Gambian popula- tion we studied, nor is it present in the sera of younger subjects who are developing immunity. The differ- ences between this population and those studied in the Sudan are intriguing. In the Sudan the ability of individuals to oroduce CFF auoeared to be related to a particularly kfficient immune response (JENSEN et al., 1984). If such differences are genetically based we would expect the ability to develop such responses to be widespread in the malaria endemic areas of Africa, as is the case with the sickle cell trait. Further investigations in other endemic areas are indicated.

References Campbell, G. H., Mrema, J. E. K., O’Leary, T. R.,O:, Jost,

R. C., & Rieckmann, K. H. (1979). In vizro inhlhtion of

*Present address: Nuffield Dept. of Medicine, John Radcliffe Hospital, Oxford, OX3 9DU, UK.

SHORT REPORTS 515

the growth of Plasmodium fakiparum by Aotus serum. ~;$~2; of the World Health Qrganlzatron, 57 (Suppl.),

Fandeur, T:, Dubois, P., Gysm,, J., Deded, J. P. & Pereira Da Silva, L. (1984). In vttro and in viva Studies on protective and inhibitory antibodies against Plasmodium falciparum in the Saimiri monkey. 3oumal of Immunology, 132, 432-437.

Jensen, J. B., Boland, M. T., Allan, S. J., Carlin, J. M., Vande Waa, J. A., Divo, A. A. & Akood, M. (1983). Association between human serum induced crisis forms in cultured Plasmodium falciparum and clinical immunity to malaria in Sudan. Infection and Immunity, 41, 1302- 1311.

(1984). Comparison of immunity to malaria in Sudan and Indonesia: crisis-form versus merozoite invasion inhibi- tion. Proceedings of the National Academy of Sciences of the USA, 81, 922-925.

Trager, W. & Jensen, J. B. (1976). Human malaria parasites in continuous culture. Science, 193, 673-675.

Vande War+ J. A., Jensen, J. I&, Akood, M. A. S. & Bayourm, R. (1984). Longitudmal study on the in vifro immune response to Plasmodium falciparum in Sudan. Infection and Zmmuniq, 45, 505-510.

Wilson,, R. J. M. & Philhps, R. S. (1976). Method to test inhibitory antibodies in human sera to wild populations of Plusnwdium falcipancm. Nature, 263, 132-134.

Jensen, J. B., Hoffman, S. L. Boland, M. T., Akood, M. A. S., Laughlin, L. W., Kurniawan, L. & Marwato, H. A. Accepted for publication 30 October 1986

TRANSACTIONS OF IHE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE (1987) 81, 515

Short Report. 1 I

Quality control for mal laboratories in south-west Tanzania

B. COOPER-POOLE British Technical Co-operation, Southern Regions

Health Project, Mbeya, Tanzania Last year I reported the first stage of our quality

control programme, a year later the exercise was repeated to monitor continued performance on uri- nalysis in these laboratories (COOPER-POOLE, 1986).

During the year most of the laboratories have been visited by myself or a colleague. All laboratories had been sent a newsletter giving the correct results for comparison with their own as well as comments about common errors. In one region attendants from 10 centres had attended a refresher course.

The samples sent out were prepared in the same way as in the previous year; 3 samples each for albumen, glucose, ketones and blood, 5 for microsco- pic examination of deposits.

Specimens were sent to 70 laboratories; 46 returned results. The chemical analysis was done better this year, 30 (65%) of the laboratories scored 100% compared with 12 (29%) last year. There was a reduction in the number of false negatives, fewer

expired “stix” are being used and most staff now understand about checking “stix” with known con- trols before use.

Again microscopic examination of deposits was not well done though there was a slight improvement, 32 (70%) scored more than 50% compared with 23 (56%) last year. There is still much careless microscopy esoeciallv with casts being missed and WBCs and RIKs confused, but otherwise fewer imaginary ob- jects are being reported.

Our efforts have produced near satisfactory results for chemical analysis, but training must be continued on microscopy. The laboratory staff themselves can also see where their weaknesses lie. After repeating the procedure I am convinced that a continuous monitoring programme, such as this, is the only way to control results and improve standards in rural laboratories where there is little supervision.

I would be interested to hear, from anybody who has tried a similar programme, what their limlings were.

Reference Cooper-Poole, B. (1986). Quality control for rural laborator-

ies in south-west Tanzania. Transactions of the Royal Society of Tropical Medicine and Hygiene, 80, 823.

Accepted for publication 17 February 1987