Shigella flexneri O-antigen-specific enzyme immunoassay: class-specific antibody titres against lipopolysaccharide antigens in healthy Vietnamese and Swedish populations

Serodiagnosis

and Immunotherapy

( 1988) 2, 47-61

#zigella flexneri O-antigen-specific enzyme immunoassay: class-specific antibody titres against lipopolysaccharide antigens in healthy Vietnamese and Swedish populations

Erik Ekwall*, Phung Dac Cam?, Dang Due Tracht, Adam Taubef and Alf A. Lindberg*$

* Karolinska institute, Department of‘ Clinical S-141 86 Huddinge, Sweden; tNationa1 Institute Yersin, Hanoi, Vietnam; $ Universit.v of Uppsala, S-751 20 Uppsala.

Bacteriology*. Huddinge Hospital, of Hygiene and Epidemiology, I Pho Department of‘ Statistics. PO Box 513 Sweden

Sera collected from 397 healthy Vietnamese individuals, living in two districts in the surroundings of Hanoi, and 62 healthy Swedish individuals, living in the greater Stockholm area were analysed in enzyme-immunoassays (EIA) for their class-specific immunoglobulin titres against a panel of IO O-antigen containing lipopolysaccharides (LPS) from Shigella flexneri, Shigella dvsenteriar serotype 1, Shigella sonnei and Salmonella serogroups A and B. The Vietnamese population was divided into age groups: 0.551 year, l-2, 34, S-9, IO-14 years, etc. Young Vietnamese children had low anti-S.,fiexneri LPS IgA. IgG and IgM titres which reached adult levels for IgA in the IO- to I6year-old group, and for IgG and IgM in the 3- to-4-year-old group. The IgA, IgG and IgM titres were significantly higher (P < 0.00 I) in sera from Vietnamese individuals than in sera from Swedish individuals. The EIA titres against S dysenteriue serotype I and S. sonnei LPSs were higher in the Vietnamese than in the Swedish population, but to a lesser extent than seen for S. ,jfe.uneri. The titres against Salmonella serogroups A and B LPS antigens did not differ significantly in the study populations. These results suggest that the incidence of S.jle.xneri infections is high in Vietnam, followed in frequency by S. +senteriae serotype I and S. sonnei infections. On the contrary the incidence of salmonellosis appears to be low. Kelrwords: Shigella Vietnamese.

jkneri.

enzyme

immunoassay,

lipopolysaccharides,

healthy

Introduction In many developing countries diarrhoeal diseasescause considerable morbidity and mortality, especially in infants and children ‘. Much of the diarrhoeal illness is due to shigella infection in children living in crowded areas with limited safe water availability and poor sanitation’,‘. An ongoing community-based study of the aetiological structure of diarrhoea in children younger than 5 years was started in the Tu Liem district west of Hanoi, Vietnam. in 1980. The specific objectives included collection of epidemiological data on t;To whom correspondence should be addressed 41 088880786/88/010047

+ I5 $03.00/O

‘( 1 1988 Academic

Press Limited

48

E. Ekwall et al.

distribution, morbidity and mortality patterns of diarrhoeal diseases and identification of causative pathogens. As expected, shigellae were found to be common aetiological agents with ShigelluJlexneri as the most common species isolated. Isolation and identification of the bacteria in faecal samples is the basis for laboratory diagnosis of shigellosis. Optimal recovery of the shigellae requires short transport periods between sampling and cultivation. These conditions are often difficult, if not impossible, to meet in developing countries. Alternative methods for the diagnosis of shigella infections would be worthwhile. Detection of the humoral antibody response after a shigella infection has so far been little used. One reason for this may be sought in the fact that the traditional use of whole, killed bacteria as antigen in assays has made them both insensitive and unspecific. As a result of increasing knowledge of the chemistry of Shigella 0-antigens4, with methods of their isolation in pure form’ and with the development of sensitive immunoassay#, there is the possibility of a more reliable serodiagnostic test for shigellosis. This has been demonstrated by our development of sensitive and specific enzyme immunoassays (EIA), for class-specific immunoglobulin determinations after S. dysenteriae serotype I and S. sonnei infections7,‘. In both-assays characterized and defined O-antigens containing lipopolysaccharides (LPS) were used as antigens. In this paper we report on the development and use of an EIA for S. jlexneri using LPS as antigens. A total of 321 sera collected from healthy individuals in the Tu Liem district were studied. The control was 76 sera from healthy individuals in the Tranh Tri district, Vietnam, and 62 sera from healthy individuals in Stockholm, Sweden. As well as the class-specific immunoglobulin titre determinations against S.Jexneri LPS antigens, titres were estimated in EIA against LPS antigens from S. dysenteriue serotype 1, S. sonnei and Salmonella serogroups A and B. Materials

and methods

Tu Liem District Tu Liem is a district situated approximately 15 km west of Hanoi, Vietnam, consisting of 25 separate villages. A population of 145,691 individuals (1982) live in an area of 105 km2, a population density of 1388 per km2 (average for Vietnam 164 per km*). Within the population, 18,035 (12.4%) are infants and children less than 5 years old. The Tu Liem district is rural with farming (rice and vegetable crops) as the main occupation. The environmental sanitation profile is assessed periodically by medical students with regard to water supply, waste disposal, feeding habits, etc. [Department of Epidemiology, National Institute of Hygiene and Epidemiology (NIHE), Hanoi]. The climate in the area is tropical with an average low temperature during the winter season of 16.8”C and an average high of 29.3”C during the summer. The average humidity varies between 79 and 85%. Sample surveys were performed by cluster sampling (NIHE, Hanoi): 2 185 households with 13,762 inhabitants were visited. The annual incidence of diarrhoeal disease was 23.1 per thousand in the entire population, with a rate of 108.3 per thousand in infants and children less than 5 years old. The annual diarrhoeal morbidity rate was, in the group less than 5 years old, 2400 per thousand with 2.4 diarrhoeal episodes per child per year. Diarrhoea was defined as > 3 abnormal stools per day. Each latrine is used by an average of two households and approximately 42% of the latrines were considered to be of good quality.

Shigefla

jlexneri

49

enzyme immunoassay

Stud)! population.~ Test populations. Tu Liem district: one single serum sample each was collected from February to November 1982 from 321 healthy individuals (no diarrhoeal episode within the last 3 months) living in 20/25 villages of the Tu Liem district (Table 1). Serum was collected after the blood had been allowed to clot, aliquoted in tubes and immediately frozen and stored at - 20°C. Frozen sampleswere transported to Sweden where the EIA was done. Control populations. Thanh Tri district: the Vietnamese group consisted of 76 persons (48 males and 28 females) living in the Thanh Tri district (a rural district south-east of Hanoi with a population of the samesize and similar socio-economic conditions as in Tu Liem). Their age varied from 8 to 51 years with a median age of 26 years. A Swedishcontrol group consisted of 62 persons(25 malesand 37 females), all healthy individuals, aged 2-56 years with a median of 24 years. All individuals lived in the greater Stockholm area which has a population of 1.5 million. Preparution

of antigens

For lipopolysaccharide (LPS) preparation bacteria were grown in batch culture, LPS extracted by hot phenol-water or phenol-chloroform-light petroleum ether (PCP). Each LPS preparation was chemically and immunochemically characterized before use as described: ShigellaJIexneri LPSs9, Shigella dysenteriae serotype I LPS’, LPS from Plesiomonasshigelloideswith an 0-antigenic polysaccharide chain identical to that of Shigella sonnei’ and Salmonella LPSs”‘. Immunoglohulin-enzyme conjugate,for EIA The following alkaline phosphatase conjugates were used: swine anti-human IgA (I; 800), swine anti-human IgM (l/800), swine anti-human IgG (l/800) made by Orion Oy. Helsinki, Finland.

Table 1. Numberof seraanalysedwithin the different agegroupsof the inhabitantsin the Tu Liem

district Age (years) 0.5--1.o l-2 3-4 559 lo-14 15-19 20--24 25-29 30-34 35-39 4c49 > 50

Total

Male 13 13 8 21 21

Female

12 12 15 12 9

9 IS 18 18 31 23 14 8 5

15 24 20 30 36 29 26 43 35 29 20 14

155

166

321

11 8

2

Total

I1 12

50

E. Ekwall et al,

EIA procedure The EIA was performed according to the method of Engvall & Perlmann’, as previous]y described”. Wells in disposable microtitre plates (Dynatech) were coated with 100pl of LPS antigen (10 ug ml ’ in 0.05 M carbonate buffer, pH 9.6) at 20-2X for 18h. Control wells were reacted with coating buffer only. Plates not used immediately were wrapped in aluminium foil and stored at -4°C for up to 6 months without notable variation. Before use the plates were washed three times with washing buffer, 0.15 M NaCl containing 0.05% (v/v) of Tween 20. The sera to be assayed were diluted in incubation buffer, phosphate-buffered saline, pH 7.4, containing 0.5% (v/v) of Tween 20 (PBS-T). Aliquots (100 ~1) of the diluted sera to be tested, and of one positive control serum, one negative control serum and incubation buffer only, all in duplicates, were added to the microtitre plates. They were incubated at 22°C for 2 h and washed as before. Samples (100 ~1) of alkaline phosphate conjugate antibodies, diluted in incubation buffer, were added to the wells and the microtitre plates incubated at 22°C for 18 h. After washing, the wells were filled with 100 pl enzyme substrate solution (paranitrophenyl phosphate 1 mg mll’) in 1.OM diethanolamineeHC1 buffer, pH 9.8, containing 0.5 mM HgCl, and incubated. The colour was determined in a Titertek Multiscan photometer (Flow Laboratories Ltd, Irvine, Scotland) at 405 nm after 25, 50 and 100 min. The results were expressedas relative titres being the absorbance value (mean of duplicates) of the lo-’ dilution of the serum multiplied by the dilution factor (1000). The amount of unspecific reaction of the sera seenin control wells corresponded to an absorbance of< O-100 at 405 nm per 100min and was not accounted for. The intra-assay variation was lessthan & 5% and the inter-assay variation lessthan f 15%, as estimated with the positive and negative control sera. No adjustment of the titres in individual sera was therefore considered necessary. Statistical meth0d.y Primarily, the aim of the investigation was to describe a pattern which might be related to the individuals’ ages. Therefore, the sample of healthy individuals (n = 321) was selected so that a fairly even distribution over all ageswas obtained (seeTable 1). A number of variables are presented in Figures 1 and 2, and for each the median values of the respective age-groups are plotted as a function of age. In Figure 3, one variable is displayed in each part; the distributions within the various age groups are presented by their “ogives” (distribution functions). Using the Kolmogorov-Smirnov test, each distribution is compared with the distribution in healthy Swedish individuals [Figure 3 (a)-(f)]. For descriptive purposes, the resulting P-values are displayed, but it should be noted that the underlying comparisons are not orthogonal. A number of pairwise correlations of special interest are presented in Table 3. Results Anti Shigella and Salmonella titres in a normal Swedishpopulation Serum samplescollected from 62 healthy Swedish blood donors were analysed for IgA. 1gM and IgG antibody titres against the panel of IO different shigella and salmonella antigens (Figure I). All IgA- and IgM-titres were low (median values< 150). The IgGtitres also were low in most instances (median values < 160). Only against S. JEexneri

Figure I. Median enzyme immunoassay IgG, IgM and IgA antibody titres in sera collected from healthy Vietnamese (divided into age groups) and Swedish individuals. Antigens (hpopolysaccharides) used (a)(c): x , Shigellaflexneri serotype I b; 0. S.jexneri serotype 2a; 0, S. Jlexneri serotype 3a: V, S. flexneri serotype Y: 0. S. ,flexneri serotype X: A, S. .jkxneri serotype 4b (rough mutant). Antigens (lipopolysaccharides) used in Figures I (d)-(f): v, Salmonella serogroup A; A. Salmonella serogroup B; 0, Shigella &senteriae serotype 1; W, Shigella sonnei.

vI 3

52

E. Ekwall et al.

serotypes 1b and Y were the median titres higher, 330 and 260, respectively. These titre values could at least partially be explained by the known cross-reactions between certain E. coli serotypes and Shigellu species12. The mean titre as well as the standard deviations were calculated for all antigens and in general the means were 610% higher than the medians (exemplified in Figure 2). Aetiological structure of diarrhoeal diseasein Tu Liem The Tu Liem district has been the subject of a study to collect data on the prevalence of diarrhoeal diseasein a rural Vietnamese community since 1980(NIHE, Hanoi). Between 1980and 1982, approximately 1500faecal cultures were investigated annually. The most prevalent pathogens isolated were rotavirus (49-66%), Shigellu spp. (l&20%), Escherichia coli ETEC and EPEC (5-15% each) and Salmonella (l-2%). Attempts to isolate Campylobacter jejuni were not done. In 1981 and 1982 Shigella spp. were the second most prevalent pathogen isolated, 44 and 52 isolates, respectively. Shigellaflexneri was the most common species within the genus, accounting for 9&95% of the Shigella isolated. Among S.Jlexneri serotypes, 1b, 2a and 4a accounted for more than 90% of the isolates. Each year lessthan 10 isolates of each of S. sonnei, S. boydii and S. dysenteriae were found. During 198&1983 no epidemic outbreak of shigella infection was observed in the Tu Liem district. Anti Shigella flexneri titres in the Tu Liem population A total of 321 serum sampleswere collected from persons of different agesliving in the Tu Liem district (Table 1). None of them had reported a diarrhoeal episode within the last 3 months. The population was divided into age groups with 5-year intervals. Children under 5 years old were separated into three groups: 0.5-1.0 year, l-2 years and 334 years old. This makes it possible to study whether there is an age dependent appearance of anti-Shigella titres. Individuals older than 50 years were lumped into one group. LPSs from five S.Jexneri serotypes (lb, 2a, 3a, X and Y) and the rough mutant 4bR which has the complete core but no 0-antigenic polysaccharide were used in the panel. The S. JEexneri IgG titres were low in the youngest age group (0.5-1.0 year) with median values 5&l 10 against the six antigens [Figure 1 (a), Table 21. A marked rise in titre with age group was then noted, up to 3-4 years old, when a plateau was reached with median values of 2OG-1200.This level was then maintained in all age groups. The highest titres were seen against the serotypes lb, 3a, Y and X LPS antigens. Titres against the core LPS were uniformly the lowest with median values in the range of 15& 200. The median titres in sera from the Swedish comparison group were two- to threefold lower. The S.JEexneri IgM titres were also low in the youngest age group, median values 3070, and then increasedto reach their highest levels in the 3- to 4-year-old groups, median values 23&600 [Figure I (b)]. Thereafter the titres declined slightly over the years. Titres in the > 50-year-old group were in the range 20&300. Again the highest titres were seen against S.Jexneri serotypes lb, 3a, Y and X LPS antigens, and lowest titres against the serotype 2a and core LPS antigens. The median titres in sera from the Swedish comparison group were two- to four-fold lower. The S.j%xneri IgA titres were lowest in the youngest age group, with median titres in

Shigela jlexneri

-3 r-Ye

-

enzyme immunoassay

lb

110 440 1210 1040 1040 940

1030

1050

330

Tu Liem: 0.5-1.0 (n= 15) l-2 (n = 24) 34 (n = 20) 5-9 (n = 30) 2&24 (n = 26) 30-34 (n = 35)

Total (n = 321)

Thanh Tri (n = 76)

Swedish group (n = 62) 70

450

260

50 130 230 220 240 260

2a

S. jexneri

150

630

600

60 240 630 630 530 530

3a X

180

570

510

50 110 530 480 520 500

serotype

TgG titres in healthy individuals

Age (years)

Table 2. Median

260

880

960

60 300 910 980 890 860

Y

Antigens

50

270

200

50 70 120 120 200 230

S. dvsenteriae serotype 1

60

300

130

60 80 230 110 110 160

Sweden

40

150

180

40 60 120 150 140 180

A

Salmonella

and Stockholm,

S. sonnei

Tri, Vietnam

used in EIA

from Tu Liem. Thanh

20

170

160

20 40 100 120 160 160

B

serogroup

Shigella Jexneri

enzyme

immunoassay

55

the range 2&40 [Figure 1 (c)l. The increase in relative titres was slow and a plateau was reached in the lo- to 14-year-old group, with median titres in the range 12&250. Thereafter the titres remained on the samelevel. The highest titres were seenagainst the S.Jlexneri serotype 1b and X LPS antigens, and the lowest against the serotype 2a and the core LPS antigens. The median titres against the Swedish comparison group were two- to four-fold lower. No differences in antibody titres were seenbetween males and females for any immunoglobulin class(data not shown). Anti Shigella sonnei, S. dysenteriae serot-vpe I and Salmonella serogroups.4 and B titres in the TM Liern population The samesera from the Tu Liem and the Swedish comparison groups were assayed for their class-specificantibody titre against two other Shigella LPS antigens: S. dysentwiac serotype 1 and S. sonnei. Within Tu Liem during 1980-1982. no S. dysenteriae serotype 1, and few S. sonnei, had been isolated. The relative IgG titres against S. dysenteriae serotype I were lowest in the youngest age group and gradually climbed to their highest level in individuals > 3 -4 years old. The median titres were low, never exceeding 300 [Figure 1 (d), Table 21. A few individuals. however, had high titres > 1000(data not shown). The S. dysenteriae serotype 1 IgM and IgA titres [Figure 1 (e) and (f)] were low. The development of titres followed the same pattern as seenfor the S. Jiesneri LPS antigens [Figure 1 (a)-(c)]. The panel of Swedish sera had low S. dysenteriae serotype 1 median titres, two- to five-fold lower than seenin the sera from Tu Liem. Titres against the S. sonnei LPS antigen followed the same pattern as seenfor the S. dysenteriae serotype I LPS antigen [Figure I (d)-(f), Table 21. Titres in all immunoglobulin classeswere generally low but with an age-dependent increase up to levels of 2OC 220 for IgG and IgM, and lo&130 for IgA. Titres in sera from the Swedish group were two- to five-fold lower. Salmonella are infrequently isolated in the Tu Liem district (seeabove). Titres against LPSs from serogroups A (S. paratyphi A) and B (S. parat.vphi B, S. typhimurium and others) were generally low [Figure I (d)-(f), Table 21. The IgG median titres developed gradually but, with one exception, were < 200 [Figure 1 (d)]. The IgM titres from age 34 years were around 200 and the IgA titres gradually climbed to approximately the same level [Figure 1 (e) and (f)]. Again titres seen in the Swedish group were lower in all immunoglobulin classes. Individual S. Bexneri serotype lb and Y titres For all Shigella and Salmonella LPS antigens the mean and standard deviations were calculated as well as the median values. In Figure 2 we illustrate the two S.,fle.xneri LPS antigens (serotypes lb and Y) which gave the highest titres within the Tu Liem population. Also shown are the individual titres in sera from the Swedish group. In the Vietnamese group the mean and median titres were numerically almost equal in all instances.This indicates that the EIA titres found within the different age groups were close to being normally distributed. In turn, this suggeststhat epidemic outbreaks of S. flrxneri serotype 1b or Y infections affecting the study population had not occurred in recent years. The samewas seenwith all the other Shigella and the two Salmorwlla LPS antigens (data not shown). The individual titre values seen in sera from the Swedish comparison group showed

56

E. Ekwall

et al.

that, for IgG, all titres fell below the median values in the Tu Liem population for both the S.$exneri LPS antigens [Figure 2 (a) and (d)]. With a few exceptions the same was also true for the IgM and IgA titres [Figure 2 (b), (c), (e) and (f)]. These results clearly indicate that the Swedish group has been much less exposed to S. Jexneri serotypes I b and Y bacteria than has the Vietnamese groups. The individual serum EIA titres from the Tu Liem and Stockholm populations were then analysed by the KolmogorovSmirnov test. The results are illustrated for two LPS antigens: S.Jiexneri serotype I b, which is common in the Hanoi area, and Salmonella serogroup B, which is uncommon. In the comparisons the Stockholm population (n = 62, median age 24 years) was treated as one group and compared with the 12 different age groups in Tu Liem. The cumulative frequencies for four of the groups (0.5I .O, l-2, 334, 20-24 years) are shown (Figure 3). The anti S. jlexneri serotype lb LPS titres in the Tu Liem population were significantly higher (P
between the titres against the diflerent S. flexneri LPS antigens

The polysaccharide chain of the LPS of S.flexneri serotype Y is basal for the S.JEexneri serotypes l-5 and serotype X. These latter all have their antigenic specificities as a result of additions of D-glucosyl and or 0-acetyl groups to the tetrasaccharide repeating unit of the Y polysaccharide (see Figure 1 in our preceeding paper13). This means that, besides type-specific antigenic determinants, each serotype could share epitopes common to all the S. jlexneri strains. As a consequence, a S. JZexneri serotype-specific immunodiagnosis may be difficult to deve10p’3. We have studied the EIA titres against the various S. jexneri LPS antigens in sera from Tu Liem by a correlation analysis. The results for the IgG titres for five age groups are given in Table 3. There were significant correlations, P
Figure 3. Comparison of enzyme immunoassay Vietnamese individuals of different ages (0,0,05-

IgG. IgM and IgA antibody titres expressed as cumulative percentages in sera from I ,O years: 0. l-2 years; A, 3-4 years; A, 20-24 years). Antigen (lipopolysaccharides) serotype lb; (d)+f) Salmonella serogroup B.

healthy Swedish (x---x) and used; (at(c) Shige//ujle.weri

Y

58 Table

E. Ekwall et al. 3. Correlations between the IgG-titres against five different Shigeflajexneri antigens (LPSs) in some age groups in the Tu Liem district (r-values) S. Jlexneri serotype

Age group and no.

S. flexneri

2a

3a

Y

X

0.5-1.0 year (n = IS)

lb 2a 3a Y

0.68*

O-65 0.86

0.51

o-43 0.62 0.79 0.66

l-2 years (n = 24)

lb 2a 3a Y

0.73

0.95 0.70

0.86 0.83 0.86

0.53 0.19 0.61 0.48

34 years (n = 20)

lb 2a 3a Y

0.58

O-69 0.19

0.89 0.58 0.75

0.71 o-31 0.78 0.61

2624 years (n = 26)

lb 2a 3a Y

0.51

0.69 0.30

0.66 044 0.60

0.73 0.59 0.50 0.23

25-29 years (n = 43)

lb 2a 3a Y

0.59t

0.79 0.57

0.83 0.59 0.82

0.48 0.43 0.63 0.51

* n=15 and r=0.43 P>O.OS;r=0.50 0.025
0.80 O-76

rz0.86 P
S. dysenteriae serotype 1, S. sonnei, and Salmonella serogroup A and B LPS antigens. In all instances with the two Salmonella LPS antigens no correlation was found, i.e. P> O-05 (data not shown). For S. dysenteriae serotype I LPS in 14/20 comparisons no correlation was found, i.e. P> O-05. Five of the six exceptions were in the 25 to 29-yearold group. For S. sonneino correlation was found for lo/20 instances, again exceptions were found in the higher age groups (data not shown).

Discussion The aims of this study were several. (i) Could a species-specificS. flexneri EIA be developed based on the 0-antigenic specificity of the polysaccharide chain of the cell envelope LPS? (ii) Could a serotype-specific S. Jlexneri based on LPS antigens be developed? (iii) Are the titres seenin a Vietnamese population, where the prevalence of shigellosisis high, significantly higher than in a comparable Swedish population, where the prevalence is low? (iv) Can high anti-S.Jexneri LPS titres be used for estimation of the prevalence of S. JEexneri infection? The studies of a total of 397 sera from healthy individuals in two districts outside Hanoi, Vietnam, and 62 sera from healthy individuals in Stockholm. Sweden, using 10 different S. jkxneri, S. dysenteriae serotype 1, S. sonnei

Shigelia jlexneri

enzyme

immunoassay

59

and Salmonella serogroup A and B LPSs. have given at least partial answers using immunoglobulin class-specificEIA. In 1982 approximately 18,000 of the inhabitants of Tu Liem were O-5 years of age. With the reported annual incidence of diarrhoeal disease (two or three diarrhoeal episodes)approximately 50,000 faecal cultures should have been taken in this age group for isolation/identification of a causative micro-organism. However, no more than about 1500 faecal specifimens had been subject to isolation of pathogens annually: rotavirus. Shigella sp., Salmonella sp. and enteropathogenic E. coli (EPEC). From approximately one-third of the faecal specimens either a viral or bacterial pathogen was isolated. Although S.fEexneri was the second most prevalent micro-organism isolated, only 44-51 strains were found annually between 1980 and 1982 in a district with approximately 145,000inhabitants. Low figures for both faecal sampling and isolation of pathogens are commonly seenin developing countries. The EIA with S.JIexneri LPS antigens showed that Vietnamese children less than I year old had low titres in all immunoglobulin classes[Figures 1 (a)-(c), 21.The titre levels increased in older children. The IgG titres reached a level in the 3- to 4-year-old group which was maintained in all older age groups. These IgG titres were significantly higher (P
60

E. Ekwall et al.

An interesting question is whether the children in the Tu Liem district have S.Jtexneri infections in their early childhood. In an forthcoming paper we present data from a prospective study at St. Paul’s hospital in Hanoi. Of children followed for up to 9 months after the shigella infection, diagnosed by faecal isolation of S.Jlexneri, S. sonnei or S. dysenteriae serotype I, no lessthan 85% were younger than 3 years. The youngest age-group. < 1-year-old, appears to be protected by breast-feeding which is practised for 6-10 months by most Vietnamese mothers (NIHE, Hanoi). The correlation between the agesat which S.fEexneri infections are most prevalent and the appearance of high anti S. flexneri LPS titres is striking. Correlation analyses showed a highly significant similarity between the titres determined with the serotype lb, 3a, 2a, X and Y LPSs. This must be interpreted to mean that antibodies are formed against more epitopes which are common on the basic serotype Y conformation than against epitopes which are the result of the additons of D-glucosyland- */or 0-acetyl-creating serotypes 1b, 2a, etc. (seeFigure 1 in our preceeding paper13). Such common epitopes have been next to impossible to identify and define using classical immunoabsorption techniques. Studies of monoclonal antibodies directed against the S. flexneri 0-antigenic polysaccharide chain have, however, permitted the identification of such common epitopes”. Acknowledgements This work was supported by the Swedish Medical Research Council (grant no 16x-656) and the Swedish Agency for Research Cooperation with Developing countries. The skilled technical assistanceof Maj Ringman and Kerstin Forsell is gratefully acknowledged. We are grateful to Cecilia and Jakob Lindberg for transfering all the EIA data to computer discs. References I. Appendix D- 15.The prospectsfor immunizingagainstShigella spp.In: Institute of Medicine.

New Vaccine Development: EstablishingPriorities. Vol. II. Diseasesof Importance in DevelopingCountries.WashingtonDC: National Academy Press,1986:329-36. 2. Stoll BJ, GlassRI, Huq MI, Khan MU, Banu H, Holt J. Epidemiologicalandclinical features of patientsinfected with Shigelfa who attendeda diarrhea1diseasehospitalin Bangladesh.J Infect Dis 1982;146: 177-83. 3. Black RE, Merson MH, Rahman ASMM, Yunus M, Alim ARMA, Huq 1, Yolken RH, Curlin GT. A two-year study of bacterial,viral, and parasiticagentsassociatedwith diarrhea in rural Bangladesh.J Infect Dis 1980;142:66&4. 4. Carlin NIA, Lindberg AA, Bock K, Bundle DR. The Shigeflajexneri 0-antigenic polysaccharidechain. Nature of the biologicalrepeatingunit. Eur J Biochem1984;139: 189-94. 5. Lindberg AA, Holme T. Evaluation of someextraction methods for the preparation of bacterial lipopolysaccharidesfor structural analysis.Acta Path Microbial Stand (1972)(B); 80: 751-9. 6. Engvall E, PerlmannP. Enzyme-linkedimmunosorbentassay.Quantitative assayof immunoglobulin G. Immunochemistry1971;8: 8714. 7. Lindberg AA, HaeggmannS, KarlssonK, Dac CamP, Due Trach D. The humoral antibody responseto Shigella dysenteriae type I infections,asdeterminedby ELISA. Bull WHO 1984; 62 (4): 597606. 8. Ekwall, E, HaeggmannS, Kalin M, SvenungssonB, Lindbcrg AA. Antibody responseto Shigeila sonnei infection determinedby an enzyme-linkedimmunosorbentassay.Eur J Clin Microbial 1983;2: 20&5.

Shigella jfexneri

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