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Shigella flexneri O-antigen specific enzyme immunoassay: a prospective study of class-specific antibody titres against lipopolysaccharide antigens in Vietnamese children and adults with serotype 1b or 2a dysentery
Serodiagnosis and Immunotherapy in Infectious Disease (1988) 2, 17l-l 82
O-antigen specific enzyme immunoassay: a prospective study of class-specific antibody titres against lipopolysaccharide antigens in Vietnamese children and adults with serotype lb or 2a dysentery Shigelfa flexneri
Erik Ekwall*, Phung Dac Cam?, Nguyen Chani, Le Kim PhuJ, Dang Due Tracht and Alf A. Lindberg* *Karolinska Institute, Department of Clinical Bacteriology, HuddingeHospital, S-141 86 Huddinge.Sweden;tNationa1Institute of Hygieneand Epidemiology,I Pho Yersin, Hanoi, Vietnam: $St. Paul’sHospital, Hanoi, Vietnam
A total of 278 serawere collectedfrom 97 patientswith a bacteriologicallyverified Shigellaflexneri serotype lb or 2a infection. Of the patients,65 werechildren below the ageof 5 yearsand admittedto the Departmentof Infectious Diseases at St. Paul’s Hospital, Hanoi, Vietnam; 32 were adults, aged2@-24years, and infected during a dysentery outbreak at Son Tay technicalschool,Hanoi. The serawereanalysedfor their specificimmunoglobulinA (IgA), M (IgM) and G (IgG) titres againstphenolwater-extracted and chemically defined lipopolysaccharide(LPS) antigens of S. fiexneri and other shigellaeby an enzymeimmunoassay(EIA). The titres estimatedin serafrom patientswere comparedwith titres seenin serafrom age-matchedhealthy peopleliving in the Tu Liem district, Hanoi. Positive titres were definedas greater than the meantitre + 2 SD in serafrom healthy persons.Children l-2 yearsold and infected with S.Jlexneri serotype 1b respondedwith significantly elevatedIgA titres up to 60 days after falling ill (P-values0.06 to
* To whom
correspondence
should
be addressed. 171
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Introduction Shigeflu Jlexneri is still one of the most common causes of diarrhoea, particularly in developing countries’. ShigelluJlexneri infections occur predominantly in children with a peak incidence in the 2- to 4-year-old group’. Spread is commonly via person to person contact’, with the faecal-oral route, using food and water as vehicles, as another mode of transmission’. The diagnosis of S.jexneri infections is usually based on the isolation of the pathogen from faecal specimens. The poor recovery of the shigellae from faecal specimens means that the direct plating of a sample, or at least within 24 h, is necessary. Such conditions are often difficult to meet, particularly in developing countries. Since S. Jiexneri is an invasive organism, multiplying intracellularly in the intestinal mucosa, measurement of an elicited immune response should be a feasible alternative. In two proceeding communications we have reported on the development of an enzyme immunoassay (EIA) based on the use of the antigenic specificity of the cell envelope lipopolysaccharide (LPS)3.4. In these studies it was apparent that a species-specific, but not serotype-specific, EJA could be used3, and that in a S.Jlexneri endemic region near Hanoi, Vietnam, the normal, healthy population has significantly higher IgA, IgM and IgG titres than a corresponding population in a non-endemic country like Sweden (PC 0.001)4. This communication reports on a prospective study of 65 children, less than 5 years old, admitted to St. Paul’s Hospital, Hanoi, for bacillary dysentery caused by S.Jlexneri serotype 1b or 2a. Another group consisted of 32 students, 20-24 years old, from a technical school in Son Tay outside Hanoi who became ill in a S. jexneri serotype 1b epidemic. Serum samples collected for up to 9 months after the infection were analysed for the class-specific immunoglobulin response against S.Jlexneri, S. sonnei, S. dysenteriae serotype 1 and Salmonella serogroup A and B LPS antigens. The sensitivity and specificity of the EIAs were analysed. Materials
and methods
Patients Department of Infectious Diseases, St. Paul’s Hospital. St. Paul’s Hospital, located in central Hanoi, is a district hospital serving a population of approximately 200,000 inhabitants. From February 1982 to May 1983 a total of 83 children less than 5 years old were admitted to the hospital with dysentery, and from whom two or more serum samples were collected up to 9 months after falling ill. Fifty-four children had S.Jlexneri serotype lb, eleven serotype 2a, seven serotype 4a, seven S. sonnei and four S. dysenteriae serotype 1. Only patients with S. Jlexneri serotype 1b or 2a infections were included in the study, and the characteristics of these patients are seen in Table 1. The children were kept in the hospital for up to 2 weeks and when discharged they were clinically well and had negative faecal cultures. Technical school, Son Tuy. The school, with approximately 1000 students enrolled, is situated 40 km outside Hanoi. The students live in dormitories with an average of 30 students in each room. In the spring of 1982 there was an outbreak of dysentery caused by S. Jlexneri serotype 1b affecting approximately 200 students. The mode of transmission of the serotype 1b strain was never established, but contaminated drinking water
Shigella flexneri
O-antigen
enzyme immunoassay
Table 1. Characteristicsof children with S. JIexneri
173
1b or 2a bacillary dysentery No. of patientswith:
Rectal Species isolated
Age (years) and
in faeces
no. of children
S. Jtexneri
S. jlexneri
1b
2a
temperature > 10daily >38” stools
Blood
Mucus
in stool
in stool
0.5-l .O year; 13 1-2 years; 33 \ 54 34 years; 8 I
45
32
43
49
year; 0.5-1.0 1-2 years; 5 4 1 II 34 years; 2 J
IO
2
5
8
was suspected. No isolation of S. jlexneri from the water was made. A total of 32 students (25 males,seven females; aged 2&24 years), had a 1b strain isolated from faecal samples. Tu Liem district. A reference material of blood samples,collected from individuals of all ageswho had not had any diarrhoeal episode within the last 3 months, was obtained from the Tu Liem district outside Hanoi4. Bacteriological methods Faecal sampleswere collected and plated on desoxycholate-citrate (DC) agar within 3 h at the bacteriology laboratory at St. Paul’s Hospital. In the Son Tay study rectal swabs were immediately plated on Dc-agar and transported to the National Institute of Hygiene and Epidemiology (NIHE) where they were incubated within 6 h after sampling. The Dc-agar plates were incubated for 48 h at 37°C suspect colonies picked and identified by biochemical and serological tests according to established methodology5. Most strains were preserved as agar stab cultures and subsequently shipped to the Department of Bacteriology at the National Bacteriological Laboratory, Stockholm, Sweden, for verification, antibiotic susceptibility testing and phage-typing. Phage-typing was done according to Slopek6. Of the S. Jexneri serotype 1b isolates from St. Paul’s, 77% belonged to phage-type 8, others were either of phage-types 3,9,28 or non-typable. All isolatesfrom the Son Tay outbreak belonged to phage-type 8. All S. ftexneri serotype 2a isolates from St. Paul’s belonged to phage-type 12. Enzyme immunoassay (EIA ) The EIA and the phenol-water-extracted lipopolysaccharide (LPS) antigens from shigellae and salmonellae were performed as described previously3.4. Statistical analyses For statistical analysesthe Wilcoxon’s signed-rank and the Kolmogorov-Smirnov were used when appropriate.
tests
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et uf.
Results
Children with S. tlexneri serotype lb infection The analysis of sera from the Tu Liem healthy individuals, by an EIA with the LPS antigens from S. JEexneri, S. sonnei and S. dysenteriae serotype 1, has shown an agedependent increase in class-specific antibody titres4. Therefore patients from St. Paul’s Hospital and Son Tay were separated into age groups corresponding to the age groups in the Tu Liem study. A total of 162 sera from the 54 children with S. $exneri serotype 1b infection were analysed. The results for the homologous LPS lb antigen are given in Figure l(aHc), (e)-(g) and (i)-(l) and Table 2. Comparisons between the different groups were done by the Kolmogorov-Smirnov test. The S.Jlexneri serotype 1b IgA titres were generally low as measured by median titres, never exceeding a relative value of 250 (Table 2). The cumulative frequencies indicate that in the youngest age groups significant (P= O-03; 0.5-l year old) or highly significant (P < 0.001; l-2 years old) titre increases compared to the healthy population were seen in the earliest serum samples [Figure l(aXb); Table 31. It is evident that some of the younger children responded much better than others. The IgA titres in the upper quartile were higher than in the healthy Tu Liem population and particularly in the 0*5- to l*Oyear-old group The S. flexneri serotype lb IgM titres were only higher in the youngest patients, although this was not significant [Figure l(e)-(g); Table 21. The S. JEexneri serotype lb IgG titres were generally high, and significantly higher than in the Tu Liem population [Figure 1(+(l); Tables 2 and 31. As expected, the highest titres were seen in samples collected 29-59 and 60-160 days after the start of clinical symptoms. Adults with S. flexneri serotype lb infection A total of 86 sera were analysed from the 32 students aged 20-24 years at the Son Tay technical school who fell ill with dysentery in an epidemic outbreak. The S. Jlexneri serotype lb IgA titres in the Son Tay population remained virtually unchanged throughout the 9 month follow-up period: median values 17&220, range 401050 [Figure l(d); Table 21. The titres were not different from those seen in the agematched control group from the Tu Liem district [Figure l(d); Table 31. The S.JEexneri serotype 1b IgM titres also remained virtually unchanged [Figure 1(h); Table 21. Compared with the titres seen in the Tu Liem population no difference whatsoever could be observed. The S. jexneri serotype 1b IgG titres in the Son Tay population were elevated as a result of infection [Figure l(m); Table 21. The increase was particularly notable in samples collected 4 weeks or more after the students fell ill, where the differences against the Tu Liem population were statistically significant, 0.01
a w
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et al.
Table 2. Median enzyme immunoassay IgA, IgM and IgG antibody titres against S. JEexneriserotype 1b lipopolysaccharide in sera collected from healthy Vietnamese individuals and S. flexneri serotype I b infected patients at various intervals after onset of disease Age (years) 0.5-1.0
I-2
34
2&24
130 250
240 170 220
IgA normal < 12 days 29-59 days 6(r160 days 161-250 days
30 80 90 40 80
40 170 150 90 50
IgM normal <: 12 days 29-59 days 6&160 days 161-250 days
70 140 190 140 II0
300 360 360 440 340
IgG normal -C 12 days 29-59 days 60-160 days 161-250 days
110 500 1000 940 680
440 1070 1840 1740 920
130 250
210
670 310
490 360 380
370 420
430
1210 1740
1040 1380 1450
1940 1840
1520
Entries left blank = too few sera (less than three) were available.
Table 3. Levels of significance” in the differences in immunoglobulin serotype 1b infected patients and healthy Vietnamese Healthy Vietnamese Sera collected from S. Jlexneri serotype lb patients (Days after onset) < 12 29-59 60-160 161-250
titres between S. jlexneri individuals
individuals w
IgA 0.5-1.0 years
l-2 years
34 years
2&24 years
0.5-1.0 years
l-2 years
34 years
* n.s. n.s. ns.
*** ** n.s. ns.
n.s.
n.s. n.s.
** *** *** ***
** *** *** *
n.s.
n.s. n.s.
n.s.
*** *
___. --~ 20-24 years n.s. * **
9l.s. = c-0.05, ‘0.01
number of S. Jlexneri serotype 2a infected patients did not permit statistical analyses. Therefore the individual EIA titres determined by the homologous S. Jlexneri serotype 2a LPS antigen are shown in Figure 2(a)-(c). The S.Jlexneri serotype 2a IgA titres [Figure 2(a)] were higher in the patients than in the healthy Tu Liem groups. The greatest difference between the patients and the healthy groups were seenwithin the first 11 days.
The low
Shigella flexneri
O-antigen
enzyme immunoassay
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The S.JEexneri serotype 2a IgM titres [Figure 2(b)] were of about the samemagnitude as those seenin the healthy populations. The S. jexneri serotype 2a IgG titres [Figure 2(c)] were higher in the patients than in the healthy populations at all time periods. The greatest differences were seenin the 1-2 month interval. SensitivitJl of the S. flexneri serotype lb LPS EIA The comparisons performed by the Kolmogorov-Smirnov test describe differences between groups of 1b infected patients and healthy individuals but give no information of the diagnostic value of a single serum sample. Therefore the number of serum specimens showing positive titres, i.e. greater than the mean + 2 SD in the healthy population, were tabulated for S.JIexneri lb patients (Table 4). In the two youngest age groups, 0.5-1.0 and l-2 years, a high number of patients had positive IgA titres, 9113 (69%) and 17/33 (52%), respectively. The IgM titres were positive in 4/13 (30%) in the youngest age group and in 9/33 (27%) in the l- to 2-year-old group. The IgG titres were positive in a high percentage: 12/l 3 (92%) in the 0.5 to 1.O-year old group and in 29133 (88%) in the l- to 2-year-old group. However, the number of positive titres in the patients older than 3 years was lower. The highest number was seenfor IgG in the 3- to 4-year-old group (2/8; 25%).
I
I
I
50
100
Relative
A*-
Relattve
EIA
200 EIA
500 1000 tltre
161-250
titre
Figure 2. Enzyme immunoassay IgA (a), IgM (b) and IgG (c) antibody titres against S.&meri serotype 2a lipopolysaccharide in sera from healthy Vietnamese individuals (expressed as cumulative frequencies) of different ages (il, 0.5 I.0 year; 0, l-2 years: a, 34 years) and from patients with bacteriologically verified S. /kmeri srrotype 2a infections (W. O.S- I .O year: 0, I-2 years; A. 3 4 years) at various intervals post-infection.
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E. Ekwall et al.
Table 4. Number of enzyme immunoassay IgA, IgM and 1gG antibody titres against S.Jexneri serotype 1b lipopolysaccharide in sera collected from S.JIexneri serotype 1b infected Vietnamese patients of different ages at various intervals after onset of disease exceeding the mean titre+ 2 SD (in parenthesis) in sera from healthy Vietnamese individuals Age (years) 0.551 .o (n= 13)
W
l-2 (n=33)
3-4 (n=8)
2&24 (n = 32)
(70) 6/11 519 6/17” 519
(270) IO/23 6125 6133 2116
(800)
< 12 days 29-59 days 60- 160 days 161-250 days
017
l/26
Number
9113
17133
I/S
3132
(400) l/l 1 219 l/17” 319
(770) 6123 O/25 3133 I,‘16
(1970) O/4
(1340) l/32 0128
4113
913.7
(320) S/II 919 14/17” 8/9
(1380) 8123 20125 23133 5116
(2280) l/4 O/7
1126
12/13
29133
2/8
4/32
of patients with positive titre@
kM
< 12 days 29959 days 6@160 days 161-250 days Number
of patients with positive titresh
kG
< 12 days 29-59 days 60- 160 days 161P2S0 days
Number
of patients with positive titre?
-
l/4
(720) 2132 2128
O/8
O/8 O/7
0126
O/8
Ii32
l/8
(2030) l/32 2128
“From four patients aged 0.5-1.0 year, two blood samples were available in the 60-160 day period hThe individual patient had at least one positive titre at any of the time periods. Entries left blank = too few sera (less than three) were available.
Becauseof the low number of S.Jlexneri serotype 2a infected patients no calculations of this kind could be done for the S.flexneri serotype 2a LPS EIA. SpecSficity of the S. fiexneri serotype lb and 2a LPS EIAs The serotype-specificity of S.flexneri residesin the 0-antigenic polysaccharide chain of the LPS molecule. Since all serotypes from lb to 5b are the result of additions of Dglucoseand/or 0-acetyl groups to each repeating unit of the polysaccharide chain, crossreactions based on epitopes in the common repeating unit are abundant3x7.However, antibodies elicited against the D-glucose and 0-acetyl epitopes, in S.jexneri serotype 1b LPS (Figure 3) may permit a serotype-specific diagnosis. To test the hypothesis the IgA, IgM and IgG titres against the lb LPS antigen were compared with the corresponding titres against the S.Jlexneri serotype 2a, 3a, X and Y LPS antigens. Using Wilcoxon’s signed-rank test we found that the S.jIexneri serotype 1b LPS titres differed significantly
Shigella jlexneri
O-antigen
179
enzyme immunoassay
(PC 0.01 or less) in all immunoglobulin classes at all time periods and against all four LPS antigens. The specificity of the S.j?exneri serotype 1b LPS EIA was further studied by making a comparison of the 1b LPS titres in sera from S. jkxneri serotype 1b and S. Jlexneri serotype 2a infected patients at St. Paul’s Hospital. Because of the low number of 2a infected individuals these patients were combined into one group. Using the Kolmogorov-Smirnov test we found no difference at any time for the IgA and IgM titres (data not shown). For the IgG titres significant differences were found only in the 1- to 2-yearold group in samples collected 6C-160 days after infection (P= 0.01) and in the 3- to 4year-old group in samples collected 60-160 (P~O.001) and 161-250 days (P=O.Ol) after start of the dysentery. Specificity analyses of the S.Jlexneri serotype 2a LPS EIA were done as for the 1b LPS EIA: first a comparison between 2a and 1b, 3a, X and Y LPS titres in the same serum samples and, second, a comparison between the 2a LPS titres in sera from S. JEexneri serotype 1b and from S.jlexneri serotype 2a infected patients. No statistically significant difference could be observed for any comparison in any of the immunoglobulin classes at any time. Spec!jicit.v of the S. flexneri LPS EIA
The S.,flexneri EIAs based on LPSs extracted from the various serotypes apparently do not permit a serotype-specific diagnosis. We next tested the hypothesis that the use of S. flexneri LPS will allow a species-specific EIA. The hypothesis was tested in comparisons between IgA, IgM and IgG titres seen in sera from St. Paul’s patients with S.Jexneri serotype 1b or 2a infections using S. dysenteriae serotype 1, S. sonnei-specific and Salmonella serogroup A and B antigens and titres in sera from the Tu Liem age-matched groups. In none of the instances were titres significantly elevated (P> 0.05) against the S. dj-senteriueserotype 1, S. sonnei or Salmonella LPS antigens (data not shown).
lb
+ 0
(I.61
3Ahc.l
-,
I
cl
3GlcNAcl
+ 44
Q
I 208~
20 (II;
3,4)
+ a
I
GIG
3Rhol
T 4
a
p
+ a
3GlcNACI
+ 13
GiC
y (-;3,4)
Figure
(Rho1
3. 0-Antlgenic
+ a
2Rhol
epitopes
-, a
3Rhol
in serolypes
+ a
3GlcNAcl)
lb. Za and Y
-) P
180
E. Ekwall et al. Discussion
Previous investigations with a S. flexneri EIA and defined LPSs from the different serotypes as antigens showed that in two healthy Vietnamese populations there was an age-dependent increase in IgA, IgM and IgG titres4. For IgM and IgG the titres increased 5- to IO-fold from 6 months up to the 3- to 4-year-old age group where they reached plateaux which remained steady throughout life. For IgA the plateau was not reached until the children were 10-14 years old4. The titres in all the immunoglobulin classes were significantly higher against the S. Jlexneri LPS antigens in the Vietnamese populations than in a Swedish popu1ation4. Since titres against LPS antigens from Salmonella serogroups A and B were not different the results made us conclude that S. flexneri is endemic in the areas studied4. Since the bacteriological isolation of shigellae can be difficult, unless direct plating of or short transportation periods for faecal specimens are possible, we have evaluated the use of the S.Jlexneri LPS-based EIAs for a serological instead of a bacteriological diagnosis. The two populations studied, children below 5 years of age and admitted to the Department of Infectious Diseases at St. Paul’s Hospital, and adults aged 2c-24 years falling ill in an epidemic outbreak, were sampled for up to 9 months of their dysentery and the titres compared to age-matched groups in the healthy Tu Liem population. The youngest children, 0.5-l .O and l-2 years old, who had a bacteriologically verified S. jfexneri serotype 1b infection. responded with significantly elevated IgA titres in samples collected within 2 months after falling ill [Figure l(a) and (b)]. The most pronounced differences in the comparisons with healthy children were seen in the I- to 2year old group. In children below 1 year old, approximately one-quarter were IgA nonresponders, presumably an age-dependent effect, whereas in the uppermost quartile high IgA titres were seen [ > 200, Figure l(a)]. Children in the 3- to 4-year-old group and adults did not respond with elevated IgA titres. The IgM responses against the S. fiexneri serotype lb LPS antigen were less pronounced [Figure l(e)-(h)]. In particular, increases were seen in children below 1 year old, but the titre increases were not statistically significant. At least two factors may account for this. Firstly, it is not unreasonable to assume that B cells in the Peyer’s patches have been committed and switched to IgA production before migration, and hence IgA titre increases are seen. Secondly, in the EIA, low affinity IgM antibodies are at a selective disadvantage compared with IgA and IgG antibodies, and incubation at 22°C instead of at 37°C also discriminates against IgM.8. The IgG responses against the lb LPS antigen were highly significant [P
Shigellafixneri
O-antigenenzymeimmunoassay
181
revealed that the children responded with elevated IgA responses,particularly in samples collected within 2 months of the onset of disease[Figure 2(a)]. For IgM, small titre increases,or no increases,were seen. IgG titre increaseswere also seen[Figure 2(c)]. The antibody patterns seen with significant IgA and IgG (and to some extent IgM) responses in children 0.5-2.0 years old suggest that they experienced a primary S. jkxneri infection. Older children and adults showed only IgG titre increases which suggesteda secondary response. The fact that we saw differences in the antibody responsesin children who had been infected with S. JIexneri serotype 1b compared with children infected with S. Jlexneri serotype 2a (the 1b infected responded better) raised the question of whether S.Jlexneri serotype 1b infections were more virulent. Several observations suggest that this may indeed have been the case: (i) Of the children who initially entered the study at St. Paul’s Hospital, three out of four who died were infected with S. JEexneri serotype lb strains (none of these were included in the study). (ii) Of the patients enrolled in this study 80% with lb infection had bloody stools compared with 45% with 2a infection (Table 1). (iii) Two-thirds of the patients admitted for shigellosiswere infected with S. Jexneri serotype 1b. (iv) Most Shigelfu isolates in the Tu Liem district (population of 160,000inhabitants) between 1980 and 1982 were S.,ftexneri serotype 1b (unpublished data). The results showed that in the youngest age groups S.Jiexneri serotype 1b LPS EIAs estimates of IgA titres were diagnostic in the first 2 months after the onset of disease (Table 3). When measuring specific IgG titres the EIA was of diagnostic value from 4 weeks and onwards after the infection started and in children up to 3 years of age. IgM titre determinations were of little diagnostic value. Lipopolysaccharides cannot be used to produce a S. fiesneri serotype-specific diagnosis. The antibody responsesagainst epitopes present in the repeating unit and common to all S. jlexneri of serotypes l--5 prevent this. The result obtained so far suggest that the LPS-based ElA is suitable for a species-specificS. jlexneri immunoassay.
Acknowledgements This work was supported by the Swedish Agency for Research Co-operation with Developing Countries (SAREC) and the Swedish Medical Research Council (grant no. 16X-656). The skilled technical assistanceof MS Kerstin Forsell, Kerstin Karlsson and Gunilla Lindberg is gratefully acknowledged. We also thank the phage-section at the National Bacteriological Laboratory, Stockholm, Sweden, for the phage-typing of the S. ,fiexneri serotype lb isolates. We are indebted to Assistant Professor Adam Taube for fruitful statistical discussions.
References 1. Black RE, Merson MH, Rahman ASMM, Yunus M, Alim ARMA, Huq I, Yolken RH, Curlin GT. A two-year study of bacterial, viral, and parasiticagentsassociatedwith diarrhea in rural Bangladesh.J Infect Dis 1980;142:660-4.
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2. Appendix D-i 5. The prospectsfor immunizingagainstShigellaspp.In: Institute of Medicine. New Vaccine Department: Establishing Priorities. Vol. II. Diseasesof Importance in DevelopingCountries.WashingtonD.C.: National Academy Press,1986. 3. Lindberg AA, Ekwall E, Carlin NIA. S. jlexneri O-antigen specificenzyme immunoassay: lipopolysaccharidesand synthetic glycoconjugatesas antigens.SerodiagImmunother Infect Dis 1988;2: 63-78. 4. Ekwall E, Dac Cam P, Due Trach D, Taube A, Lindberg AA. S.flexneri O-antigen specific enzyme immunoassay:class-specificantibody titres against lipopolysaccharideantigensin healthy Vietnameseand Swedishpopulations.SerodiagImmunother Infect Dis 1988;2: 477 61. 5. EdwardsPR, Ewing WH. Identification of Enterobacteriaceae.Minneapolis: Burgess,1972. 6. SlopekS. Epidemiologieund Laboratoriumsdiagnostik.In: SedlakJ, RischeH, eds.Enterobacteriaceae-infektionen.Leipzig: VEB Georg Thieme, 1968:3754. 7. Ewing WH, Lindberg AA. Serology of Shigella. In: Methods in microbiology, Vol. 14. London: AcademicPress,1984:11342. 8. Karlsson K, Granstriim M, Lindberg AA. Salmonellasp. antibodies. XI. Antigens and Antibodies 2. In: BergmeyerHW, ed. Methods of enzymaticanalysis.Weinheim,FRG: VCH, Verlagsgesellschaft, 1986:85-98. 9. Formal SB, Levine MM. Shigellosis.In: Germanier R, ed. Bacterial vaccines.New York: Academic Press,1984:167-86. 10. Shaugnessy HJ, OlssonRC, BassK, Friewer F, LevinsonSO. Experimentalhumanbacillary dysentery:polyvalent dysenteryvaccinein its prevention.J Am Med Assoc1946;132:352-68. 11. Higgins AR, Floyd TM, Kader MA. Studiesin shigellosis.III. A controlled evaluation of a monovalentShigella vaccinein a highly endemicenvironment.Am J Trop Med Hyg 1955;4: 281-8. (Manuscript
accepted
22nd February
1988)
O-antigen specific enzyme immunoassay: a prospective study of class-specific antibody titres against lipopolysaccharide antigens in Vietnamese children and adults with serotype lb or 2a dysentery Shigelfa flexneri
Erik Ekwall*, Phung Dac Cam?, Nguyen Chani, Le Kim PhuJ, Dang Due Tracht and Alf A. Lindberg* *Karolinska Institute, Department of Clinical Bacteriology, HuddingeHospital, S-141 86 Huddinge.Sweden;tNationa1Institute of Hygieneand Epidemiology,I Pho Yersin, Hanoi, Vietnam: $St. Paul’sHospital, Hanoi, Vietnam
A total of 278 serawere collectedfrom 97 patientswith a bacteriologicallyverified Shigellaflexneri serotype lb or 2a infection. Of the patients,65 werechildren below the ageof 5 yearsand admittedto the Departmentof Infectious Diseases at St. Paul’s Hospital, Hanoi, Vietnam; 32 were adults, aged2@-24years, and infected during a dysentery outbreak at Son Tay technicalschool,Hanoi. The serawereanalysedfor their specificimmunoglobulinA (IgA), M (IgM) and G (IgG) titres againstphenolwater-extracted and chemically defined lipopolysaccharide(LPS) antigens of S. fiexneri and other shigellaeby an enzymeimmunoassay(EIA). The titres estimatedin serafrom patientswere comparedwith titres seenin serafrom age-matchedhealthy peopleliving in the Tu Liem district, Hanoi. Positive titres were definedas greater than the meantitre + 2 SD in serafrom healthy persons.Children l-2 yearsold and infected with S.Jlexneri serotype 1b respondedwith significantly elevatedIgA titres up to 60 days after falling ill (P-values0.06 to
* To whom
correspondence
should
be addressed. 171
0888--0786/88/030171+
12 $03.00/O
Q 1988Academic PressLimited
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Introduction Shigeflu Jlexneri is still one of the most common causes of diarrhoea, particularly in developing countries’. ShigelluJlexneri infections occur predominantly in children with a peak incidence in the 2- to 4-year-old group’. Spread is commonly via person to person contact’, with the faecal-oral route, using food and water as vehicles, as another mode of transmission’. The diagnosis of S.jexneri infections is usually based on the isolation of the pathogen from faecal specimens. The poor recovery of the shigellae from faecal specimens means that the direct plating of a sample, or at least within 24 h, is necessary. Such conditions are often difficult to meet, particularly in developing countries. Since S. Jiexneri is an invasive organism, multiplying intracellularly in the intestinal mucosa, measurement of an elicited immune response should be a feasible alternative. In two proceeding communications we have reported on the development of an enzyme immunoassay (EIA) based on the use of the antigenic specificity of the cell envelope lipopolysaccharide (LPS)3.4. In these studies it was apparent that a species-specific, but not serotype-specific, EJA could be used3, and that in a S.Jlexneri endemic region near Hanoi, Vietnam, the normal, healthy population has significantly higher IgA, IgM and IgG titres than a corresponding population in a non-endemic country like Sweden (PC 0.001)4. This communication reports on a prospective study of 65 children, less than 5 years old, admitted to St. Paul’s Hospital, Hanoi, for bacillary dysentery caused by S.Jlexneri serotype 1b or 2a. Another group consisted of 32 students, 20-24 years old, from a technical school in Son Tay outside Hanoi who became ill in a S. jexneri serotype 1b epidemic. Serum samples collected for up to 9 months after the infection were analysed for the class-specific immunoglobulin response against S.Jlexneri, S. sonnei, S. dysenteriae serotype 1 and Salmonella serogroup A and B LPS antigens. The sensitivity and specificity of the EIAs were analysed. Materials
and methods
Patients Department of Infectious Diseases, St. Paul’s Hospital. St. Paul’s Hospital, located in central Hanoi, is a district hospital serving a population of approximately 200,000 inhabitants. From February 1982 to May 1983 a total of 83 children less than 5 years old were admitted to the hospital with dysentery, and from whom two or more serum samples were collected up to 9 months after falling ill. Fifty-four children had S.Jlexneri serotype lb, eleven serotype 2a, seven serotype 4a, seven S. sonnei and four S. dysenteriae serotype 1. Only patients with S. Jlexneri serotype 1b or 2a infections were included in the study, and the characteristics of these patients are seen in Table 1. The children were kept in the hospital for up to 2 weeks and when discharged they were clinically well and had negative faecal cultures. Technical school, Son Tuy. The school, with approximately 1000 students enrolled, is situated 40 km outside Hanoi. The students live in dormitories with an average of 30 students in each room. In the spring of 1982 there was an outbreak of dysentery caused by S. Jlexneri serotype 1b affecting approximately 200 students. The mode of transmission of the serotype 1b strain was never established, but contaminated drinking water
Shigella flexneri
O-antigen
enzyme immunoassay
Table 1. Characteristicsof children with S. JIexneri
173
1b or 2a bacillary dysentery No. of patientswith:
Rectal Species isolated
Age (years) and
in faeces
no. of children
S. Jtexneri
S. jlexneri
1b
2a
temperature > 10daily >38” stools
Blood
Mucus
in stool
in stool
0.5-l .O year; 13 1-2 years; 33 \ 54 34 years; 8 I
45
32
43
49
year; 0.5-1.0 1-2 years; 5 4 1 II 34 years; 2 J
IO
2
5
8
was suspected. No isolation of S. jlexneri from the water was made. A total of 32 students (25 males,seven females; aged 2&24 years), had a 1b strain isolated from faecal samples. Tu Liem district. A reference material of blood samples,collected from individuals of all ageswho had not had any diarrhoeal episode within the last 3 months, was obtained from the Tu Liem district outside Hanoi4. Bacteriological methods Faecal sampleswere collected and plated on desoxycholate-citrate (DC) agar within 3 h at the bacteriology laboratory at St. Paul’s Hospital. In the Son Tay study rectal swabs were immediately plated on Dc-agar and transported to the National Institute of Hygiene and Epidemiology (NIHE) where they were incubated within 6 h after sampling. The Dc-agar plates were incubated for 48 h at 37°C suspect colonies picked and identified by biochemical and serological tests according to established methodology5. Most strains were preserved as agar stab cultures and subsequently shipped to the Department of Bacteriology at the National Bacteriological Laboratory, Stockholm, Sweden, for verification, antibiotic susceptibility testing and phage-typing. Phage-typing was done according to Slopek6. Of the S. Jexneri serotype 1b isolates from St. Paul’s, 77% belonged to phage-type 8, others were either of phage-types 3,9,28 or non-typable. All isolatesfrom the Son Tay outbreak belonged to phage-type 8. All S. ftexneri serotype 2a isolates from St. Paul’s belonged to phage-type 12. Enzyme immunoassay (EIA ) The EIA and the phenol-water-extracted lipopolysaccharide (LPS) antigens from shigellae and salmonellae were performed as described previously3.4. Statistical analyses For statistical analysesthe Wilcoxon’s signed-rank and the Kolmogorov-Smirnov were used when appropriate.
tests
174
E. Ekwd
et uf.
Results
Children with S. tlexneri serotype lb infection The analysis of sera from the Tu Liem healthy individuals, by an EIA with the LPS antigens from S. JEexneri, S. sonnei and S. dysenteriae serotype 1, has shown an agedependent increase in class-specific antibody titres4. Therefore patients from St. Paul’s Hospital and Son Tay were separated into age groups corresponding to the age groups in the Tu Liem study. A total of 162 sera from the 54 children with S. $exneri serotype 1b infection were analysed. The results for the homologous LPS lb antigen are given in Figure l(aHc), (e)-(g) and (i)-(l) and Table 2. Comparisons between the different groups were done by the Kolmogorov-Smirnov test. The S.Jlexneri serotype 1b IgA titres were generally low as measured by median titres, never exceeding a relative value of 250 (Table 2). The cumulative frequencies indicate that in the youngest age groups significant (P= O-03; 0.5-l year old) or highly significant (P < 0.001; l-2 years old) titre increases compared to the healthy population were seen in the earliest serum samples [Figure l(aXb); Table 31. It is evident that some of the younger children responded much better than others. The IgA titres in the upper quartile were higher than in the healthy Tu Liem population and particularly in the 0*5- to l*Oyear-old group The S. flexneri serotype lb IgM titres were only higher in the youngest patients, although this was not significant [Figure l(e)-(g); Table 21. The S. JEexneri serotype lb IgG titres were generally high, and significantly higher than in the Tu Liem population [Figure 1(+(l); Tables 2 and 31. As expected, the highest titres were seen in samples collected 29-59 and 60-160 days after the start of clinical symptoms. Adults with S. flexneri serotype lb infection A total of 86 sera were analysed from the 32 students aged 20-24 years at the Son Tay technical school who fell ill with dysentery in an epidemic outbreak. The S. Jlexneri serotype lb IgA titres in the Son Tay population remained virtually unchanged throughout the 9 month follow-up period: median values 17&220, range 401050 [Figure l(d); Table 21. The titres were not different from those seen in the agematched control group from the Tu Liem district [Figure l(d); Table 31. The S.JEexneri serotype 1b IgM titres also remained virtually unchanged [Figure 1(h); Table 21. Compared with the titres seen in the Tu Liem population no difference whatsoever could be observed. The S. jexneri serotype 1b IgG titres in the Son Tay population were elevated as a result of infection [Figure l(m); Table 21. The increase was particularly notable in samples collected 4 weeks or more after the students fell ill, where the differences against the Tu Liem population were statistically significant, 0.01
a w
E. Ekwall
et al.
Table 2. Median enzyme immunoassay IgA, IgM and IgG antibody titres against S. JEexneriserotype 1b lipopolysaccharide in sera collected from healthy Vietnamese individuals and S. flexneri serotype I b infected patients at various intervals after onset of disease Age (years) 0.5-1.0
I-2
34
2&24
130 250
240 170 220
IgA normal < 12 days 29-59 days 6(r160 days 161-250 days
30 80 90 40 80
40 170 150 90 50
IgM normal <: 12 days 29-59 days 6&160 days 161-250 days
70 140 190 140 II0
300 360 360 440 340
IgG normal -C 12 days 29-59 days 60-160 days 161-250 days
110 500 1000 940 680
440 1070 1840 1740 920
130 250
210
670 310
490 360 380
370 420
430
1210 1740
1040 1380 1450
1940 1840
1520
Entries left blank = too few sera (less than three) were available.
Table 3. Levels of significance” in the differences in immunoglobulin serotype 1b infected patients and healthy Vietnamese Healthy Vietnamese Sera collected from S. Jlexneri serotype lb patients (Days after onset) < 12 29-59 60-160 161-250
titres between S. jlexneri individuals
individuals w
IgA 0.5-1.0 years
l-2 years
34 years
2&24 years
0.5-1.0 years
l-2 years
34 years
* n.s. n.s. ns.
*** ** n.s. ns.
n.s.
n.s. n.s.
** *** *** ***
** *** *** *
n.s.
n.s. n.s.
n.s.
*** *
___. --~ 20-24 years n.s. * **
9l.s. = c-0.05, ‘0.01
number of S. Jlexneri serotype 2a infected patients did not permit statistical analyses. Therefore the individual EIA titres determined by the homologous S. Jlexneri serotype 2a LPS antigen are shown in Figure 2(a)-(c). The S.Jlexneri serotype 2a IgA titres [Figure 2(a)] were higher in the patients than in the healthy Tu Liem groups. The greatest difference between the patients and the healthy groups were seenwithin the first 11 days.
The low
Shigella flexneri
O-antigen
enzyme immunoassay
177
The S.JEexneri serotype 2a IgM titres [Figure 2(b)] were of about the samemagnitude as those seenin the healthy populations. The S. jexneri serotype 2a IgG titres [Figure 2(c)] were higher in the patients than in the healthy populations at all time periods. The greatest differences were seenin the 1-2 month interval. SensitivitJl of the S. flexneri serotype lb LPS EIA The comparisons performed by the Kolmogorov-Smirnov test describe differences between groups of 1b infected patients and healthy individuals but give no information of the diagnostic value of a single serum sample. Therefore the number of serum specimens showing positive titres, i.e. greater than the mean + 2 SD in the healthy population, were tabulated for S.JIexneri lb patients (Table 4). In the two youngest age groups, 0.5-1.0 and l-2 years, a high number of patients had positive IgA titres, 9113 (69%) and 17/33 (52%), respectively. The IgM titres were positive in 4/13 (30%) in the youngest age group and in 9/33 (27%) in the l- to 2-year-old group. The IgG titres were positive in a high percentage: 12/l 3 (92%) in the 0.5 to 1.O-year old group and in 29133 (88%) in the l- to 2-year-old group. However, the number of positive titres in the patients older than 3 years was lower. The highest number was seenfor IgG in the 3- to 4-year-old group (2/8; 25%).
I
I
I
50
100
Relative
A*-
Relattve
EIA
200 EIA
500 1000 tltre
161-250
titre
Figure 2. Enzyme immunoassay IgA (a), IgM (b) and IgG (c) antibody titres against S.&meri serotype 2a lipopolysaccharide in sera from healthy Vietnamese individuals (expressed as cumulative frequencies) of different ages (il, 0.5 I.0 year; 0, l-2 years: a, 34 years) and from patients with bacteriologically verified S. /kmeri srrotype 2a infections (W. O.S- I .O year: 0, I-2 years; A. 3 4 years) at various intervals post-infection.
178
E. Ekwall et al.
Table 4. Number of enzyme immunoassay IgA, IgM and 1gG antibody titres against S.Jexneri serotype 1b lipopolysaccharide in sera collected from S.JIexneri serotype 1b infected Vietnamese patients of different ages at various intervals after onset of disease exceeding the mean titre+ 2 SD (in parenthesis) in sera from healthy Vietnamese individuals Age (years) 0.551 .o (n= 13)
W
l-2 (n=33)
3-4 (n=8)
2&24 (n = 32)
(70) 6/11 519 6/17” 519
(270) IO/23 6125 6133 2116
(800)
< 12 days 29-59 days 60- 160 days 161-250 days
017
l/26
Number
9113
17133
I/S
3132
(400) l/l 1 219 l/17” 319
(770) 6123 O/25 3133 I,‘16
(1970) O/4
(1340) l/32 0128
4113
913.7
(320) S/II 919 14/17” 8/9
(1380) 8123 20125 23133 5116
(2280) l/4 O/7
1126
12/13
29133
2/8
4/32
of patients with positive titre@
kM
< 12 days 29959 days 6@160 days 161-250 days Number
of patients with positive titresh
kG
< 12 days 29-59 days 60- 160 days 161P2S0 days
Number
of patients with positive titre?
-
l/4
(720) 2132 2128
O/8
O/8 O/7
0126
O/8
Ii32
l/8
(2030) l/32 2128
“From four patients aged 0.5-1.0 year, two blood samples were available in the 60-160 day period hThe individual patient had at least one positive titre at any of the time periods. Entries left blank = too few sera (less than three) were available.
Becauseof the low number of S.Jlexneri serotype 2a infected patients no calculations of this kind could be done for the S.flexneri serotype 2a LPS EIA. SpecSficity of the S. fiexneri serotype lb and 2a LPS EIAs The serotype-specificity of S.flexneri residesin the 0-antigenic polysaccharide chain of the LPS molecule. Since all serotypes from lb to 5b are the result of additions of Dglucoseand/or 0-acetyl groups to each repeating unit of the polysaccharide chain, crossreactions based on epitopes in the common repeating unit are abundant3x7.However, antibodies elicited against the D-glucose and 0-acetyl epitopes, in S.jexneri serotype 1b LPS (Figure 3) may permit a serotype-specific diagnosis. To test the hypothesis the IgA, IgM and IgG titres against the lb LPS antigen were compared with the corresponding titres against the S.Jlexneri serotype 2a, 3a, X and Y LPS antigens. Using Wilcoxon’s signed-rank test we found that the S.jIexneri serotype 1b LPS titres differed significantly
Shigella jlexneri
O-antigen
179
enzyme immunoassay
(PC 0.01 or less) in all immunoglobulin classes at all time periods and against all four LPS antigens. The specificity of the S.j?exneri serotype 1b LPS EIA was further studied by making a comparison of the 1b LPS titres in sera from S. jkxneri serotype 1b and S. Jlexneri serotype 2a infected patients at St. Paul’s Hospital. Because of the low number of 2a infected individuals these patients were combined into one group. Using the Kolmogorov-Smirnov test we found no difference at any time for the IgA and IgM titres (data not shown). For the IgG titres significant differences were found only in the 1- to 2-yearold group in samples collected 6C-160 days after infection (P= 0.01) and in the 3- to 4year-old group in samples collected 60-160 (P~O.001) and 161-250 days (P=O.Ol) after start of the dysentery. Specificity analyses of the S.Jlexneri serotype 2a LPS EIA were done as for the 1b LPS EIA: first a comparison between 2a and 1b, 3a, X and Y LPS titres in the same serum samples and, second, a comparison between the 2a LPS titres in sera from S. JEexneri serotype 1b and from S.jlexneri serotype 2a infected patients. No statistically significant difference could be observed for any comparison in any of the immunoglobulin classes at any time. Spec!jicit.v of the S. flexneri LPS EIA
The S.,flexneri EIAs based on LPSs extracted from the various serotypes apparently do not permit a serotype-specific diagnosis. We next tested the hypothesis that the use of S. flexneri LPS will allow a species-specific EIA. The hypothesis was tested in comparisons between IgA, IgM and IgG titres seen in sera from St. Paul’s patients with S.Jexneri serotype 1b or 2a infections using S. dysenteriae serotype 1, S. sonnei-specific and Salmonella serogroup A and B antigens and titres in sera from the Tu Liem age-matched groups. In none of the instances were titres significantly elevated (P> 0.05) against the S. dj-senteriueserotype 1, S. sonnei or Salmonella LPS antigens (data not shown).
lb
+ 0
(I.61
3Ahc.l
-,
I
cl
3GlcNAcl
+ 44
Q
I 208~
20 (II;
3,4)
+ a
I
GIG
3Rhol
T 4
a
p
+ a
3GlcNACI
+ 13
GiC
y (-;3,4)
Figure
(Rho1
3. 0-Antlgenic
+ a
2Rhol
epitopes
-, a
3Rhol
in serolypes
+ a
3GlcNAcl)
lb. Za and Y
-) P
180
E. Ekwall et al. Discussion
Previous investigations with a S. flexneri EIA and defined LPSs from the different serotypes as antigens showed that in two healthy Vietnamese populations there was an age-dependent increase in IgA, IgM and IgG titres4. For IgM and IgG the titres increased 5- to IO-fold from 6 months up to the 3- to 4-year-old age group where they reached plateaux which remained steady throughout life. For IgA the plateau was not reached until the children were 10-14 years old4. The titres in all the immunoglobulin classes were significantly higher against the S. Jlexneri LPS antigens in the Vietnamese populations than in a Swedish popu1ation4. Since titres against LPS antigens from Salmonella serogroups A and B were not different the results made us conclude that S. flexneri is endemic in the areas studied4. Since the bacteriological isolation of shigellae can be difficult, unless direct plating of or short transportation periods for faecal specimens are possible, we have evaluated the use of the S.Jlexneri LPS-based EIAs for a serological instead of a bacteriological diagnosis. The two populations studied, children below 5 years of age and admitted to the Department of Infectious Diseases at St. Paul’s Hospital, and adults aged 2c-24 years falling ill in an epidemic outbreak, were sampled for up to 9 months of their dysentery and the titres compared to age-matched groups in the healthy Tu Liem population. The youngest children, 0.5-l .O and l-2 years old, who had a bacteriologically verified S. jfexneri serotype 1b infection. responded with significantly elevated IgA titres in samples collected within 2 months after falling ill [Figure l(a) and (b)]. The most pronounced differences in the comparisons with healthy children were seen in the I- to 2year old group. In children below 1 year old, approximately one-quarter were IgA nonresponders, presumably an age-dependent effect, whereas in the uppermost quartile high IgA titres were seen [ > 200, Figure l(a)]. Children in the 3- to 4-year-old group and adults did not respond with elevated IgA titres. The IgM responses against the S. fiexneri serotype lb LPS antigen were less pronounced [Figure l(e)-(h)]. In particular, increases were seen in children below 1 year old, but the titre increases were not statistically significant. At least two factors may account for this. Firstly, it is not unreasonable to assume that B cells in the Peyer’s patches have been committed and switched to IgA production before migration, and hence IgA titre increases are seen. Secondly, in the EIA, low affinity IgM antibodies are at a selective disadvantage compared with IgA and IgG antibodies, and incubation at 22°C instead of at 37°C also discriminates against IgM.8. The IgG responses against the lb LPS antigen were highly significant [P
Shigellafixneri
O-antigenenzymeimmunoassay
181
revealed that the children responded with elevated IgA responses,particularly in samples collected within 2 months of the onset of disease[Figure 2(a)]. For IgM, small titre increases,or no increases,were seen. IgG titre increaseswere also seen[Figure 2(c)]. The antibody patterns seen with significant IgA and IgG (and to some extent IgM) responses in children 0.5-2.0 years old suggest that they experienced a primary S. jkxneri infection. Older children and adults showed only IgG titre increases which suggesteda secondary response. The fact that we saw differences in the antibody responsesin children who had been infected with S. JIexneri serotype 1b compared with children infected with S. Jlexneri serotype 2a (the 1b infected responded better) raised the question of whether S.Jlexneri serotype 1b infections were more virulent. Several observations suggest that this may indeed have been the case: (i) Of the children who initially entered the study at St. Paul’s Hospital, three out of four who died were infected with S. JEexneri serotype lb strains (none of these were included in the study). (ii) Of the patients enrolled in this study 80% with lb infection had bloody stools compared with 45% with 2a infection (Table 1). (iii) Two-thirds of the patients admitted for shigellosiswere infected with S. Jexneri serotype 1b. (iv) Most Shigelfu isolates in the Tu Liem district (population of 160,000inhabitants) between 1980 and 1982 were S.,ftexneri serotype 1b (unpublished data). The results showed that in the youngest age groups S.Jiexneri serotype 1b LPS EIAs estimates of IgA titres were diagnostic in the first 2 months after the onset of disease (Table 3). When measuring specific IgG titres the EIA was of diagnostic value from 4 weeks and onwards after the infection started and in children up to 3 years of age. IgM titre determinations were of little diagnostic value. Lipopolysaccharides cannot be used to produce a S. fiesneri serotype-specific diagnosis. The antibody responsesagainst epitopes present in the repeating unit and common to all S. jlexneri of serotypes l--5 prevent this. The result obtained so far suggest that the LPS-based ElA is suitable for a species-specificS. jlexneri immunoassay.
Acknowledgements This work was supported by the Swedish Agency for Research Co-operation with Developing Countries (SAREC) and the Swedish Medical Research Council (grant no. 16X-656). The skilled technical assistanceof MS Kerstin Forsell, Kerstin Karlsson and Gunilla Lindberg is gratefully acknowledged. We also thank the phage-section at the National Bacteriological Laboratory, Stockholm, Sweden, for the phage-typing of the S. ,fiexneri serotype lb isolates. We are indebted to Assistant Professor Adam Taube for fruitful statistical discussions.
References 1. Black RE, Merson MH, Rahman ASMM, Yunus M, Alim ARMA, Huq I, Yolken RH, Curlin GT. A two-year study of bacterial, viral, and parasiticagentsassociatedwith diarrhea in rural Bangladesh.J Infect Dis 1980;142:660-4.
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2. Appendix D-i 5. The prospectsfor immunizingagainstShigellaspp.In: Institute of Medicine. New Vaccine Department: Establishing Priorities. Vol. II. Diseasesof Importance in DevelopingCountries.WashingtonD.C.: National Academy Press,1986. 3. Lindberg AA, Ekwall E, Carlin NIA. S. jlexneri O-antigen specificenzyme immunoassay: lipopolysaccharidesand synthetic glycoconjugatesas antigens.SerodiagImmunother Infect Dis 1988;2: 63-78. 4. Ekwall E, Dac Cam P, Due Trach D, Taube A, Lindberg AA. S.flexneri O-antigen specific enzyme immunoassay:class-specificantibody titres against lipopolysaccharideantigensin healthy Vietnameseand Swedishpopulations.SerodiagImmunother Infect Dis 1988;2: 477 61. 5. EdwardsPR, Ewing WH. Identification of Enterobacteriaceae.Minneapolis: Burgess,1972. 6. SlopekS. Epidemiologieund Laboratoriumsdiagnostik.In: SedlakJ, RischeH, eds.Enterobacteriaceae-infektionen.Leipzig: VEB Georg Thieme, 1968:3754. 7. Ewing WH, Lindberg AA. Serology of Shigella. In: Methods in microbiology, Vol. 14. London: AcademicPress,1984:11342. 8. Karlsson K, Granstriim M, Lindberg AA. Salmonellasp. antibodies. XI. Antigens and Antibodies 2. In: BergmeyerHW, ed. Methods of enzymaticanalysis.Weinheim,FRG: VCH, Verlagsgesellschaft, 1986:85-98. 9. Formal SB, Levine MM. Shigellosis.In: Germanier R, ed. Bacterial vaccines.New York: Academic Press,1984:167-86. 10. Shaugnessy HJ, OlssonRC, BassK, Friewer F, LevinsonSO. Experimentalhumanbacillary dysentery:polyvalent dysenteryvaccinein its prevention.J Am Med Assoc1946;132:352-68. 11. Higgins AR, Floyd TM, Kader MA. Studiesin shigellosis.III. A controlled evaluation of a monovalentShigella vaccinein a highly endemicenvironment.Am J Trop Med Hyg 1955;4: 281-8. (Manuscript
accepted
22nd February
1988)