COMPARISON OF A DIGESTION-SEDIMENTATION TECHNIQUE
WITH THE K A T O - K A T Z TECHNIQUE IN THE DETECTION AND QUANTIFICATION OF S. MANSONI EGGS IN LIGHT TO MODERATE INFECTIONS
BOREL E.*, ETARD J.-F.**, ADDO Α.* & DIAKITE M.***
S u m m a r y :
A comparison between a digestion-sedimentation technique (DST) and the Kato-Katz thick smear technique (KKT) in the detection and quantification of Schistosoma eggs in stool was carried out in 5 5 1 subjects. Specimen were collected one or two years after treatment with praziquantel from subjects living in a schistosomiasis endemic area of Mali. One hundred infections missed by the KKT were detected by the DST. Conversely, 35 infections missed by the DST were detected by the KKT (88 % were light infections).
More subjects were classified as lightly infected by the DST (p < 10-3) and more subjects were classified as moderately infected (101-400 epg) by the KKT (p = 0.02). The KKT produced higher counts than the DST among the youngest age group which was also the most infected. The principal advantage of the DST over the KKT was its better sensitivity to detect light infections resulting from a larger amount of stool processed.
KEY WORDS : Schistosoma mansoni, diagnosis, sedimentation, Kato, Mali.
MOTS CLÉS : Schistosoma mansoni, diagnostic, sédimentation, Kato, Mali.
Résumé : COMPARAISON DE LA TECHNIQUE DE DIGESTION-
SÉDIMENTATION À LA TECHNIQUE DE KATO-KATZ POUR LA DÉTECTION ET LA QUANTIFICATION DES ŒUFS DE S. MANSONI
Une technique de digestion-sédimentation a été comparée à la technique de Kato-Katz pour la détection des œufs de
Schistosoma mansoni chez 551 sujets vivant en zone d'endémie bilharzienne au Mali et traités par du praziquantel un à deux ans auparavant. Cent infections, non identifiées par la technique de Kato-Katz, l'ont été par digestion-sédimentation. Inversement, 35 infections non diagnostiquées par digestion-sédimentation l'ont été par la technique de Kato-Katz (faible intensité dans 88 %). Plus de sujets ont été classés dans la catégorie des faibles intensités par digestion-sédimentation (p < 10-3) et plus de sujets dans la catégorie des intensités modérées par Kato-Katz (p = 0,02). La technique de Kato-Katz a produit des comptes d'œufs plus élevés que la digestion-sédimentation chez les sujets les plus jeunes (6- 9 ans), par ailleurs les plus intensément infectés. Le principal avantage de la digestion-sédimentation sur le Kato-Katz sur un prélèvement unique de selles était sa meilleure sensibilité pour détecter les faibles infections du fait d'une quantité plus importante de selles examinées.
P
r e v a l e n c e rate a n d intensity o f infection with S. mansoni, m e a s u r e d b y direct parasitological t e c h n i q u e s , are w i d e l y u s e d to c o l l e c t b a s e l i n e infection data in a c o m m u n i t y o r to m o n i t o r a c o n t r o l p r o g r a m m e . T h e simplicity a n d l o w c o s t o f the K a t o - Katz t e c h n i q u e justify its r e c o m m e n d a t i o n for field w o r k . H o w e v e r , single-stool surveys s h o w a l o w s e n - sitivity in detecting light s c h i s t o s o m e infections (Knight et al., 1 9 7 6 ) . U n d e r e s t i m a t i o n o f the p r o p o r t i o n o f* Université С. Bernard/Laboratoire de Parasitologie et Mycologie Médicale, 8, avenue Rockefeller, 69373 Lyon Cedex 08, France.
** Institut de Recherche pour le Développement (IRD, anciennement ORSTOM)/ Institut National de Recherche en Santé Publique (INRSP), BP 1771, Bamako, Mali.
*** Faculté de Médecine de Pharmacie et d'Odonto-stomatologie/
Département d'Epidémiologie des Affections Parasitaires, BP 1805, Bamako, Mali.
Correspondence:
Jean-François Etard. IRD, BP 1386, Dakar, Sénégal.
Tél. : +221 832 34 80 - Fax : +221 832 43 07 E-mail : [email protected]
infected individuals, particularly w h e n l o w infections are e x p e c t e d , s u c h after c h e m o t h e r a p y , is t h e n likely.
Part o f the bias is e x p l a i n e d b y the short-term varia
bility in e g g output within individuals ( B a r r e t o et al, 1 9 9 0 ) . Although t h e sensitivity o f the s e d i m e n t a t i o n t e c h n i q u e s in d e t e c t i n g h e a v y e g g s , s u c h as schisto
s o m e eggs, has b e e n r e c o g n i z e d for m a n y years (Faust, 1 9 4 6 ) , the p r o c e s s i n g time and the n u m e r o u s mani
p u l a t i o n s p r e v e n t e d their u s e in large p o p u l a t i o n - b a s e d surveys.
W e report h e r e a c o m p a r i s o n o f a d i g e s t i o n - s e d i m e n tation t e c h n i q u e ( D S T ) with the Kato-Katz t e c h n i q u e ( K K T ) in a c o n t e x t o f l o w intensity o f infection.
MATERIALS AND METHODS
STUDY DESIGN
F
ield w o r k w a s c a r r i e d out in the "Office du Niger", Mali, an irrigated rice-growing area in Central Mali w h e r e b o t h S. mansoni a n d S. hae-Parasite, 1999, 6, 175-178
Note de recherche 175
Article available athttp://www.parasite-journal.orgorhttp://dx.doi.org/10.1051/parasite/1999062175
matobium infections are present ( B r i n k m a n n et al., 1 9 8 8 ) . This work was part o f a larger study o n the s o c i o - e c o n o m i c impact o f schistosomiasis a p p r o v e d by the "Programme National de Lutte Contre les Schis
tosomiases (PNLCS)" (Audibert & Etard, 1998). A subset o f 6 2 3 subjects from the main random sample w e r e included in this study. T h e y had previously received a single d o s e o f 4 0 m g . k g- 1 o f praziquantel under medical supervision either in D e c e m b e r 1989 (mass treatment) and D e c e m b e r 1 9 9 0 (selective treatment) or in D e c e m b e r 9 0 (mass treatment). In D e c e m b e r 1 9 9 1 , egg counts using the D S T and the K K T w e r e per
formed. T h o s e found infected r e c e i v e d a close o f pra
ziquantel.
L A B O R A T O R Y P R O C E D U R E
Individuals w e r e a s k e d to provide a stool sample o n the night before the examination. T h e s p e c i m e n w e r e transported to the laboratory o n the morning and pro
c e s s e d . T h e K K T and the D S T w e r e performed in a blind fashion by two i n d e p e n d e n t teams.
T h e K K T was performed b y the PNLCS team: 4 0 mg o f calibrated sieved faeces, c l e a r a n c e o f the smears for o n e to two hours, reading b y three e x p e r i e n c e d tech
nicians.
T h e D S T followed the procedure d e s c r i b e d b y Shidam ( 1 9 9 1 ) : 3 0 0 mg stool s p e c i m e n using 4 % potassium hydroxide for digestion, o n e or two smears depending o n the v o l u m e o f the sediment, reading b y o n e inves
tigator ( E . B . ) at a magnitude o f x 10 or x 2 5 , addition o f the egg counts in c a s e o f two smears.
STATISTICAL A N A L Y S I S
T h e n u m b e r o f eggs per gram ( e p g ) was c o m p u t e d after multiplication o f the eggs counts b y 3-33 ( D S T ) or 2 5 ( K K T ) . Intensity o f infection was categorized in four levels: n o infection, light infection ( 1 - 1 0 0 e p g ) , m o d e r a t e infection ( 1 0 1 - 4 0 0 e p g ) and severe infection (> 4 0 0 e p g ) . Geometric means after Ln (egg counts + 1) t r a n s f o r m a t i o n , M c N e m a r , p a i r e d t-test, W i l c o x o n signed-ranks test for paired-sample and correlation coefficient w e r e used.
RESULTS
O
n the average, 8 0 samples w e r e p r o c e s s e d per day. D S T data w e r e o b t a i n e d from 6 2 3 subjects and K K T from 5 5 4 . Analysis was per
formed o n 551 c o m p l e t e data set.
More c a s e s w e r e detected by the D S T than b y the KKT ( T a b l e I ) . T h e overall difference in the proportion o f d e t e c t e d infections was 11.8 % ( M c N e m a r X2 = 3 1 . 3 , ρ < 1 0- 3) . O n e hundred infections detected by the D S T
KKT* Total DSTf 0# 1-100 101-400 > 400 No %
0 233 31 4 0 268 48.6
1-100 97 86 40 1 224 40.7
101-400 3 17 19 <4 47 S.5
> 400 0 0 4 8 12 2.2
Total
No 333 134 67 17 551 100
% 60.4 24.3 12.2 3.1 100
* Kato-Katz technique
† Digestion-sedimentation technique Eggs/gram (epg)
Table I. - Cross-classification of the infection grades, Office du Niger, Mali, 1991, 551 subjects.
w e r e missed b y the KKT, 97 % o f these missed infec
tions being classified as light infection by the D S T . O n the other hand, 35 infections detected by the KKT w e r e left undetected by the DST. However, 7 5 % o f the DST- false negatives c o r r e s p o n d e d to o n e or t w o eggs per Kato s m e a r and 8 8 % c o r r e s p o n d e d to light infections in KKT. In terms o f infection grades, significantly m o r e infections w e r e classified as light b y the D S T than b y the K K T ( 4 0 . 6 % vs 2 4 . 3 %, M c N e m a r X2 = 4 3 . 5 , ρ < 1 0- 3) . Conversely, the n u m b e r o f infections clas
sified as moderate was 1.4 time higher by the KKT than by the D S T ( 1 2 . 1 % vs 8.5 %, McNemar X2 = 5.3, ρ = 0 . 0 2 ) . A b o v e 4 0 0 epg, n o difference w a s s h o w n . C o r r e l a t i o n b e t w e e n t h e Ln-transformation o f e g g counts was g o o d (r = 0 . 6 8 ; ρ < 1 0- 3) .
W h e n the results w e r e stratified b y a g e , the propor
tion of positive c a s e s d e t e c t e d b y the D S T was greater in all age groups e x c e p t a m o n g the six to nine years o l d c h i l d r e n w h e r e n o d i f f e r e n c e w a s o b s e r v e d ( T a b l e II). In terms o f intensity o f infection, the a s s o ciation was reversed: the overall intensity o f infection did not differ and the stratification b y a g e revealed a difference a m o n g the six to nine years old children, the K K T intensity being higher than the D S T intensity (p < 1 0- 2) .
DISCUSSION
T
h e assessment o f light infections with S. man- soni within an e n d e m i c c o m m u n i t y or the evaluation o f a p o p u l a t i o n - b a s e d c h e m o t h e r a p y require a sensitive and easily performed parasitological t e c h n i q u e under the field conditions.
Our results s h o w that, e x c e p t a m o n g the six to nine years old children, a single digestion-sedimentation detected m o r e infections than a single Kato-Katz smear and that both t e c h n i q u e s reported the s a m e overall
176 Note de recherche Parasite, 1999, 6, 175-178
« G E S T I O N - S E D I M E N T A T I O N T E C H N I Q U E A N D S C H I S T O S O M A E G G S I N S T O O I
No. Infected Mean Ln (SD)
% +ve СМ ;
Age No. o f patients DST KKT X2 DST KKT t-test/nonpar φ
6-9 юз 77 73 NS 2.74 (2.01) 3.33 (2.32)
...
74.8 70.9 15.5 27.9
10-14 82 66 51 *** 2.73 (1.82) 2.80 (2.34) NS
80.5 6 2 . 2 14.8 17.1
15-19 5 5 3 3 23 ** 1.85 (1.86) 1.94 (2.37) VS
6 0 . 0 Í1.8 6.4 6.9
> 20 311 107 71
....
1.05 (1.69) 0.98 (1.86) NS34.4 22.8 2.8 2.7
Total 283 218 **** 1.69 (1.95) 1.78 (± 2.29) NS
51.4 39.6 5.4 5.9
t Geometric mean including zero egg counts; φ Paired l-test and Wilcoxon signed-ranks for paired sample; ** 0.01 < ρ < 0.05;
*** 10-3 < ρ < 1 0- 2; **** ρ < 10-3.
Table II. - Number of detected infections and density of infection by age group and technique. Office du Niger, Mali, 1991. 551 subjects.
intensity o f infection. Although age was clearly related to p r e v a l e n c e and intensity o f infection, it could not have g e n e r a t e d a differential bias b e t w e e n the tech
niques: the laboratory technicians did not know the age and g e n d e r o f the subjects and the laboratory p r o c e dures w e r e standardized. It has b e e n reported that the KKT tends to overestimate the intensity o f infection and has a better sensitivity in groups o f heavily infected subjects (Sleigh et al., 1982). This is what probably hap
p e n e d a m o n g the youngest children.
T h e overall better sensitivity o f the D S T could b e pri
marily e x p l a i n e d b y the larger amount o f stools pro
c e s s e d ( 3 0 0 mg w?40 m g ) . Indeed, assuming a Poisson distribution o f eggs in stool and n o loss o f eggs due to the t e c h n i q u e , o n e can s h o w that, for a given den
sity o f o n e e g g / 4 0 mg o f faeces, the probability to detect o n e e g g is 9 9 % and 6 4 % for p r o c e s s i n g 3 0 0 mg and 4 0 mg o f faeces respectively.
O f m o r e c o n c e r n w e r e the 3 5 subjects left undetected by the D S T but classified as infected by the KKT. O u r preliminary testing o f the DST, in the experimental set
ting o f a university laboratory, have shown that n o eggs r e m a i n e d in the sieving device after processing a stool sample, so that a loss in eggs during the manipulation w a s ruled out. However, under field conditions, this hypothesis must b e considered. Given the e x p e r i e n c e o f the technicians, Kato-Katz false positives is not a plausible alternative.
T h e implementation o f the DST in a field laboratory presented n o practical difficulty and required n o high- qualified personnel. T h e time required for the diges
tion and sedimentation phases was rewarded by an e a s e o f m i c r o s c o p y reading, e v e n at a magnitude o f χ 10. T h e D S T allowed a perfect distinction b e t w e e n the s p e c i e s o f s c h i s t o s o m e s , w h i c h c o u l d b e worth in
areas w h e r e S. mansoni and S. haematobium are both present, such as the Office du Niger in Mali.
Although the lack o f sensitivity o f a single Kato-Katz smear is widely recognized ( D e Vlas & Gryseels, 1992), this technique remains useful in large screening surveys in highly e n d e m i c areas w h e r e high sensitivity is not crucial and remains the first c h o i c e for community investigation o f soil-transmitted nematodes. T o improve the case detection rate o f the KKT, several stool samples obtained on consecutive days can b e e x a m i n e d (Bar
reto et al., 1990); however, this procedure is time-consu
ming, e x p e n s i v e and often c o m e up against a refusal from the villagers. Assuming that the parasite popula
tion is best described by the negative binomial distri
bution (Bliss & Fisher, 1 9 5 3 ) helps to infer true preva
lences from observed data. A p o c k e t chart, based on this distribution, has b e e n proposed to estimate the true S. mansoni prevalences from single-stool surveys ( D e Vlas et al., 1993). T h e use o f this chart is appealing but requires comparison o f the predicted true prevalence with observed prevalence derived from sensitive tech
nique, such as the D S T described here.
ACKNOWLEDGEMENTS
T
he study was supported by grants from CFD, GTZ, ECC. UNICEF and OSI Laboratory. W e wish to thank Dr M. Traoré (INRSP, Mali) and Dr M. Audibert (CNRS/CERDI, Clermont-Ferrand) w h o permitted this study. Kato-Katz examinations and treatment w e r e carried out by the team o f the "Programme National de Lutte Contre les Schistosomiases" (INRSP, Mali) and the "Département d'Epidémiologie des Affec
tions Parasitaires" (Faculté de Médecine, Mali). T h a n k s are due to Dr D. d e Clercq for reading o f the manus
cript.
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