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Studies on diarrheal diseases
TROPICAL PEDIATRICS
Studies on diarrheal diseases The multiplicity of infectious agents in the intestinal flora o[ Puerto Rican children with gastroenteritis
A total o[ 214 children with diarrhea and 73 control children were studied [or the presence o[ enteric microorganisms. The percentage o[ bacterial and viral agents was higher in the acute than in the chronic diarrhea cases, and very low in the control group. Enteropathogenic Escherichia coli was the most [requently [ound bacterial agent in the patients with diarrhea. Shigellosis was absent in the control group. A serologic response against the viral agents isolated occurred in three [ourths o[ the diarrhea cases. A causal relationship o[ the viral agents isolated with the clinical syndrome is suggested. The prevalence o[ protozoan and helminthic injections was higher in the group with chronic diarrhea, and a direct relationship between egg load and chronicity was obtained. Multiplicity o[ possible in[ectious agents was obtained in about one [ourth o[ the patients. The possible role o[ combined viral, bacterial, and~or parasitic injection in diarrhea is discussed.
Aida Guardiola-Rotger, Ph.D., ~ Etervina Figueroa de Gonzalez, M.D., Emanuel Kauder, Captain, MC, USA, Aurea Munoz, M.D., Victor A. Lopez, Diana E. Gadea, B.S., and Merlyn J. Funkenbuseh, SP5 T I-I E etiology of diarrheal diseases in infants and children has been the subject of considerable debate during the past 20 years.
Much of this controversy has centered in the suspected etiologic role of certain serotypes of Escherichia coli and enteroviruses in gastroenteritis. A review of the literature reveals the efforts taken by a great number of workers in an attempt to delineate the problem of acute gastroenteritis. The causal role of a given number of enteroviruses and enteropatho-
From the Microbiology Division, United States Army. Tropical Research Medical Laboratory, APO 851, New York, N. Y. (San Juan, Puerto Rico). ~Address, United States Army Tropical Research Medical Laboratory, APO 851, New York, N. Y.
81
g2
Guardiola-Rotger et al.
genic E. coli serotypes has been recently s h o w n . 1-4 Ho~vever, the factor or factors which endow these microorganisms with enteropathogenicity have not been identified. Neither have the conditions which render the infant so extraordinarily susceptible to these enteropathogens, and conversely the older child and adult rather resistant, been elucidated. Limited information is available on the problem of mixed infections. Dual infections undoubtedly exist, s' a but it is difficult to interpret the findings on the cultural examinations alone for it is possible that the disease in some of these patients is due to one pathogen and the subject is a carrier of the other. In this report, data are presented on the nature of the multiplicity of infectious agents associated with .diarrheal diseases and the correlation of these findings with the clinical manifestations. M A T E R I A L S AND M E T H O D S Clinical material. A total of 214 pediatric patients, varying in age from 25 days to 12 years, and whose chief complaint was diarrhea, were studied at the Guaynabo Health Center and the Barrio Obrero outpatient department clinics. A patient was considered to have diarrhea if the passage of stool was more frequent than usual for a given child, and the consistency of the stool was soft or watery. A bout of diarrhea reported to have lasted 4 weeks or longer at the time of admission was arbitrarily classified as chronic diarrhea. Anyone who had received recent antimicrobial therapy was not included in the studies. A group of 73 children without diarrhea, at least 3 weeks before and 3 weeks after admission, were studied at the Puerta de Tierra Outpatient Clinic. On the day of admission a complete history and physical examination was done, and fecal samples, blood, and throat swabs were collected. Three to five weeks later, convalescent serum was obtained. Additional visits were based on need as determined by the clinical course. Specimens. 1. Stool samples. Fecal samples and/or rectal swabs were collected from each pa-
J,dy 1964
tient. Immediately after collection, bacteriologic media, namely SS, MacConkey and Staph 110 agar plates, and Selenite F enrichment medium, were inoculated. An aliquot of the sample was placed in screw-cap tubes containing 1 ml. of Hank's balanced salt solution (BSS), sealed with adhesive tape, and placed immediately in dry ice. The remaining portion was kept at room temperature for parasitologic examination. 2. Throat swabs. Two swabs were obtained from each patient. One was placed immediately in a screw-cap tube containing 1 ml. of Hank's BSS, sealed with adhesive tape, and placed in dry ice. T h e other was used for the inoculation of blood agar and Staph 110 plates. 3. Blood samples. On the day of admission, acute phase sera was obtained and a complete blood count done on each patient. Convalescent sera were collected 3 to 5 weeks later. Laboratory techniques. 1. Bacteriologic methods. On the average, there was a lapse of 3 to 4 hours from the time of inoculation of plates at the clinic and its arrival at the laboratory. The isolated species of Salmonella, Shigella, AlkalescensDispar group, and enteropathogenic E. coli were identified biochemically and typed according to Edwards and Ewing's 7 methods. 2. Parasitologic methods. The identification of all helminths and protozoans was done on each stool sample. The specimen arrived at the laboratory approximately 3 to 4 hours after collection. A direct saline smear was prepared and examined for the identification of protozoan trophozoites with special attention given to motility and quality of the unstained nucleus. A quantitative egg count examination was also performed on each stool according to the method described by Ritchie and Frick. s 3. Viral methods. a . I S O L A T I O N O F V I R U S E S . T h e throat and rectal swabs and formed stool specimens were kept frozen at -70 ~ C. until ready to be processed. At this time, the swabs were removed after thawing; 2.0 ml. of Hank's BSS were added to each tube and the tubes were
Volume 65
Number 1
Studies on diarrheal diseases
8 3
[---] - ACUTE [ 14Z CASES )
I-CHRONIC [~
(7E CASES) -- C O N T R O L ( 73 CASES )
E P E C - EN'i'EROPATHI~GENIC EI COLI bj o
C P S - - C O A G U L A S E pOSITIVE STAPHYLOCOCCI NI--NOT
> tO( ,O
C- 80
Fig. 1. agents children chronic control Rico.
Distribution of recovered from with acute and diarrhea and cases in Puerto
<0 6 0
~HELMINTHS
r~-
--1
~
PROTOZOA--'~
IDENTIFIED
COX--COXSACKIE A . I . - - A LUMBRICOIDES s HISTOLYTICA
BACTERIA ---1
,0,,~ ,,,j,o.,
VIRUSES
I
u.
o w 40 1.9
I00, i
CHRONIC
i
I 6g CASES )
90,
[[~[] <[ 2 5 0 0
2500- 5OOO 5000-1DO00
8 0 84
IO,O00 - 25,000 2 5 D 0 0 - 50,000
o
70
I
> 5o, ooo
60,
F-
z
ACUTE
I
( 131 CASES )
50-
5 LU I
u_ 4 0 0 r-CONTROL-~ 6 9 CASES
~< .50~J
20-
Fig. 2. The relationship of the chronicity of diarrhea to the helminths egg count.
I0-
r.
fr~chiu~
A
T
lumbriceides Hookworm
trichTura lumbrAcoidesHookworm
A.
frichiuro lumbr!coides
centrifuged at 3,000 r.p.m, for 30 minutes at 4 ~ C. O n e thousand units of penicillin a n d streptomycin p e r milliliter were a d d e d to the s u p e r n a t a n t fluid. F o r the f o r m e d stool specimens, a 10 per cent suspension was p r e p a r e d within H a n k ' s BSS a n d processed in a similar m a n n e r . This p r e p a r a t i o n was used for the inoculation of tissue cultures. ~ Both monkey kidney ( M K ) a n d H e p - 2 cells were used in the studies. T w o M K a n d H e p - 2 tubes were inoculated with 0.25 ml. of the specimen suspension a n d i n c u b a t e d for 1 h o u r at room temperatm'e. T h e inoculum was then rem o v e d a n d replaced with 1.0 ml. of u n d i l u t e d beef amniotic fluid for the M K cells and
Ginsberg m e d i a w i t h 4 p e r cent horse serum for the H e p - 2 cells, each containing 100 units of penicillin a n d streptomycin per milliliter. T h e tubes were held for 7 days at 37 ~ C. a n d observed daily for cytopathogenic effect ( C P E ) . Tubes in which C P E was observed were r e m o v e d from the incubator when a p p r o x i m a t e l y 50 p e r cent of the cell sheet h a d been affected a n d kept at - 7 0 ~ C. for identification a n d serologic studies. Enh a n c e m e n t passages were carried out on those samples which showed slow or slight CPE. b , I D E N T I F I C A T I O N OF V I R U S E S . T h e isolated viruses were identified b y neutralization tests by means of pools of antisera ~" contain-
*All the cells and antisera were purchased from Microbiological Associates, Inc., Bethesda, Md.
*All tile cells and antisera were purchased fi'om Microbiological Associates, Inc., Bethesda, Md.
84
Guardiola-Rotger et al
]uly 1964
Table I. Age group distribution of bacterial agents recovered from children with acute and chronic
No. of cases EPEC* 0126:B16 055:B5 086:B7 0124:B17 0127:B8 0119:B14 0128:B12 0111:B4 0112:Bll 026:B6 0125:B15 % EPEC
6 months Acute [ Chronic l Control 27 0 5 7 0 1 2 0 0 1 0 0 2 0 0 1 0 0 0 0 0 l 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 A--22.4%
< 2 years 6 months < 1 year Acute [Chronic I Control 19 1 11 3 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 Ch.--33.3 %
1-< 2years Acute [ Chronic[ Control 30 11 23~ 7 4 0 3 0 0 0 2 0 0 0 0 0 0 0 0 1 0 1 0 0 1 0 0 2 0 0 0 0 0 0 1 0 0 0 0 C--2.5%
Shigella
1
0
0
l
0
0
1
0
0
sonnei I flexneri 2 % Shigella CPSt % CPS
1 0
0 0 A--3.9% 0 A--10.5%
0 0
0 1
0 0
0 1
22
0
1
0 0 C--0% 0 C--10.2%
0 0
2
0 0 Ch.--0% 0 Ch.--0%
5
* E P E C ~ Enteropathogenic E. coll. t C P S = Coagulase positive staphylococci.
ing 20 units of each specific antibody. T h e composition of the pools used was as follows: polio pool (I, I I , I I I ) ; Coxsackie pool (Ag, B1, B2, Ba, B~, B~); E C H O pool I (1, 2, 3, 5) ; E C H O pool I I (6, 7, 8, 9, 12) ; E C H O pool I I I (11, 13, 14, 1 5 ) ; E C H O pool I V (10, 16, 17, 18, 19). If C P E was inhibited by any given pool, the virus was identified by doing neutralization tests against each individual virus type of the pool. Viruses not neutralized by any of the pools were tested against individual sera of E C H O types 4, 21, 27, and Coxsackie A7 and B6. I n the neutralization test, approximately 100 TCD~0 of virus was used, the exact dose being determined by a concurrent titration with virus dilution ranging from 10 -a'3 t h r o u g h 10-~'3. e . S E R O L O G I C S T U D I E S . Neutralization tests were carried out with the patient's acute and convalescent sera and the homologous virus to determine rises in antibody titer. RESULTS
O f the 214 patients studied, 142 were classified as having acute and 72 as having
chronic cases of diarrhea. Seventy-three children without diarrhea were also studied. Agents of possible etiologic significance were found in 79.2 per cent of the acute, in 95.7 per cent of the chronic, and in 54.8 per cent of the control cases. T h e distribution of the agents recovered are shown in Fig. 1. Bacteria which m a y be considered as possible etiologic agents in diarrhea were isolated in 25.9 per cent of the acute, 18.9 per cent of the chronic, and 16.4 per cent of the control cases. T h e incidence of enteropathogenic E. coil ( E P E C ) serotypes, as would be expected, was higher in the acute (14.8 per cent) than in the chronic (11.1 per cent) cases. Enteropathogenic E. coli were recovered in only 5.4 per cent of the control cases studied. T h e age group distribution of E. coli serotypes isolated is shown i n Table I. T h e highest incidence of E. coli serotypes was obtained in patients under 2 years of age in both acute (22.4 per cent) and chronic (33.3 per cent) cases. However, in the control group the highest incidence of enteropathogenic E. coli (8.8 per cent)
2
Volume 65 Number 1
Studies on diarrheal diseases
85
diarrhea a n d in control cases in Puerto Rico
2 years 2-~ 3 years Acuf~te [Chronic l Control
Acute
~ 3 years [Chronic I Control
Acute
Total t Chronic
I Control
15 1 0 0 0
16 0 0 0 0
10 1 0 0 0
51 3 1 0 0
44 4 1 0 0
24 2 0 0 0
142 21 6 2 2
72 8 1 2 0
73 4 0 0 0
1
0
0
0
0
0
3
0
0
0
0
0
1
2
0
1
3
0
0 0 0 0 0 0
0 0 0 0 0 0
1 0 0 0 0 0
0 1 0 0 0 0
0 0 0 0 0 1
0 1 1 0 0 0
2 2 2 1 0 0
0 0 0 0 1 1
1 2 1 0 0 0
1 1 0
0 0 0
2 2 0
3 3 0
1
1
2
3
A~6.1% 0 0 0
Ch.--5.6%
A--3.0% 1
A--4.5%
C--8.8%
Ch.--6.6%
0 0 0
14.8% 5 3 2 3.5%
11.1% 4 4 0 5.5%
5.4% 0 0 0 0%
3
i1
4
8
7.8%
2.7%
10.9%
C--0%
Ch.--6.6%
was observed in children over 2 years of age. T w o of the 4 E. cell serotypes isolated f r o m this group were 0.128:B12. N i n e serotypes of enteropathogenic E. coli were isolated from the acute cases, 0126:B16 being the most frequently found, followed by 0124: B17; for the chronic cases, 5 serotypes were isolated, 0127:B8 a n d 055:B5 being the most c o m m o n l y found. These d a t a are comp a r a b l e to t h a t obtained by C h e r r y and colleagues, 9 a l t h o u g h in their r e p o r t only acute cases were included. Shigellae were more frequently isolated from the chronic (5.5 p e r cent) t h a n from the acute cases (3.5 p e r c e n t ) , as shown in Fig. 1. N o Shigellae were isolated from the control group. D a t a on the age group distribution of Shigella serotypes are presented in T a b l e I. Shigella sonnei occurred most frequently in b o t h acute a n d chronic cases; in the latter group, being the only species isolated. T h e incidence of Shigellae in the acute cases was very similar in children u n d e r a n d over 2 years of age, a n d for the chronic cases it was higher in child r e n over 2 years of age. Coagulase positive
C--11.7%
staphylococci were isolated in 7.8 p e r cent of the acute cases a n d in 9.7 p e r cent of the chronic cases (Fig. 1 ). T h e highest incidence of coagulase positive staphylococci was obt a i n e d in the control group (10.9 p e r c e n t ) . T h e age distribution of this m i c r o o r g a n i s m in the chronic group was similar to t h a t of Shigellae ( T a b l e I ) . All of the coagulase positive staphylococci isolated from fecal specimens of chronic cases were also obtained from the throat cultures of the patient. F o r the acute, in 7 of the 11 cases w i t h positive findings in the fecal specimens, the eoagulase positive staphylococci were also isolated from the t h r o a t specimens. V i r a l agents, w h i c h were cytopathogenic for m o n k e y kidney tissue cultures, were obt a i n e d in 14.1 p e r cent of the acute, in 8.3 p e r cent of the chronic, a n d in 4.1 p e r cent of the control cases. As shown in T a b l e I I the age group distribution of the viral agents isolated followed a p a t t e r n similar to t h a t of the b a c t e r i a l isolates, i.e., the incidence of viral agents in b o t h the acute a n d chronic groups was higher in children u n d e r 2 years
86
GuardioIa-Rotger et al.
July 1964
T a b l e II. Age group distribution of viral agents recovered from children with acute a n d chronic diarrhea a n d in control cases in Puerto Rico
2 years -6 <
months ~6 months 2 years _. ~ 1 year
2-~ 3 years
1-<2 years
AIChlC A IChlC No. of cases
Viruses Coxsackie B~ Coxsackie Ba Coxsackie A~ ECHO 9 ECHO 7 ECHO 8 ECHO 12 ECHO 14 ECHO 1 Polio 3 N.I.w % Viruses
27
0
5
5 0 1 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 A-19.7%
19
1
11
4 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 Ch-16.7%
30 11 6 2 3 0 0 0
23 2 0 0
15 16 1 3 1 0 0 0
1
0
0
0 0 0 0
0 0 0 0
0 1 0 1
1
0
0
0 0
1 1
0 0
1
0
0
C-7.6%
10 0 0 0
Total
~ 3 years
IChlC A ICh I c
51 44 24 4 1 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 A-7.6% Ch-6.7 % C-0%
142
72
73
20 6 3 8 0 0 1 0 0 1 0 0 0 0 1 0 0 1 4 2 0 0 0 1 1 0 0 0 1 0 0 3 0 5 0 0 14.1% 8.3% 4.1%
e A - - A c u t e cases. ~Ch
-:
Chronic
cases.
:~C ~
C o n t r o l cases.
w
=
Unidentified
cytopathogenic
agents under
study.
of age. I n the control group, the 3 viral isolates were recovered from children u n d e r 2 years of age. T h e results of the neutralization tests carried out with the patient's acute a n d convalescent sera to determine the extent of a n t i b o d y f o r m a t i o n are shown in T a b l e I I I . Coxsackie B4 a n d E C H O 8 were the most c o m m o n l y f o u n d viruses in the acute cases. Eleven of the 13 k n o w n viruses isolated from these patients, a n d from which paired sera were available, elicited a rise in a n t i b o d y titer d u r i n g the diarrheal episode. Of the 8 Coxsackie B4 viruses recovered, all b u t one showed a n increase in a n t i b o d y titer. P a t i e n t G-58 h a d the same a n t i b o d y titer in both the acute a n d convalescent sera, therefore, one can assume t h a t the p a t i e n t was seen in the late acute phase. T w o of the three u n k n o w n cytopathogenic agents tested serologieally elicited some degree of host response , thus they are p r o b a b l y not tissue contaminants. T h r e e of the four viral agents recovered from chronic cases from which paired sera were available showed a rise in neutralizing a n t i b o d y titer d u r i n g the course of disease. No Coxsackie viruses were isolated in this group. T h r e e of the seventythree control cases h a d cytopathogenic
agents. I n 2 of the cases, E C H O 3 and E C H O 7, a rise in neutralizing a n t i b o d y titer was obtained. T h e results obtained in the parasitologic studies showed (Fig. 1) a striking difference in the degree of helminthic a n d protozoan infections between the acute a n d chronic cases. Protozoan infections were observed in 18.3 per cent of the acute a n d 52.1 per cent of the chronic cases. Giardia larnblia was the most c o m m o n l y found protozoan in both groups. E. histolytica a n d E. coli were present with almost similar frequency, although in both cases the degree of infestation was higher in the chronic group. T h e age distribution ( T a b l e I V ) showed that the protozoan infections were present at a higher rate in children over 2 years of age in the three groups, acute, chronic, a n d control cases. Helminths were recovered (Fig. 1) from 54.9 per cent of the acute a n d 89.8 per cent of the chronic a n d 34.2 per cent of the control cases. Trichiurls trichiura was f o u n d in about half of the acute cases studied a n d one fourth of the control group; parasitization by this worm was almost universal in the chronic group. Ascaris lumbricoides was f o u n d in a b o u t one fourth of the acute cases
Volume 65
Number 1
Studies
and in one third of the chronic cases. Hookworm was the least commonly found helminth. The age distribution (Table IV) showed, as expected, that helminthiases were present at a higher rate in children over 2 years of age in acute, chronic, and controI cases. The relationship of the trichiuris and ascaris egg load to the chronicity of diarrhea is shown in Fig. 2. The incidence of trichiuriasis with egg counts over 25,000 was observed in 3.8 per cent of the acute and in 37.7 per cent of the chronic cases. For ascaris, 7.6 per cent of the acute and 17.3 per cent of the chronic cases had egg counts ov~er 10,000. None of the control cases had trichiuris egg
o n d i a r r h e a l diseases
87
counts over 25,000 and in only 4 of the 73 controls, the ascaris egg counts were over 10,000. The number of cases with hookworm infections in the acute, chronic, and control groups constituted a small per cent of the total helminthiasis, therefore, it does not seem feasible to attempt a correlation between chronicity and egg load as done for the other two parasites. Of 110 acute cases with positive findings, 23.6 per cent had mixed viral, bacterial, or parasitic agents. Similar findings (24.4 per cent) were obtained for the 67 chronic cases with positive isolations. The distribution of the agents recovered are
Table III. Viral agents recovered and homologous neutralization tests from 142 acute and 72 chronic cases of diarrhea and 73 nondiarrheic children in Puerto Rico Neutralization tests (100 TCDso) ~
I Convalescent
Patient No.
Virus
Acute serum
serufn
Acute cases G-12 G-22 G-36 G-47 G-58 G-64 B-4 B-6 G-19 B-113 G-71 G-80 G-49 B-129 B-73-A B-123 B-38 B-68 B-75 B~124
Coxsackie B4 Coxsackie B~ Coxsackie B4 Coxsackie B4 Coxsackie B4 Coxsackie B~ Coxsackie B4 Coxsackie B4 Coxsackie B~ Coxsackie A9 ECHO 8 ECHO 8 ECHO 8 ECHO 8 E C H O 14 N.I.$ N.I. N.I. N.I. N.I.
4 4 64 ~ 4 64 < 4 < 4 ~ 4 < 4 N.D.+ ~ 4 8 N.D. < 4 ~ 4 16 N.D. N.D, ~ 4 ~ 4
32 64 128 128 64 16 32 128 32 N,D. 16 128 N.D. <4 8 16 N.D. N.D. < 4 8
Chronic cases B-14 G-78 G-85 Bd38 B-136 B-141
ECHO 1 ECHO 8 ECHO 8 Polio 3 Polio 3 Polio 3
~ 4 ~ 4 ~ 4 N.D. ~ 4 N.D.
128 64 < 4 N.D. 256 N.D,
Control cases 10-C 17-C 42-C
< <
ECHO 3 ~ 4 16 ECHO 7 < 4 64 E C H O 12 0 0 ~Acute sera drawn on day of admission, Convalescent sera drawlx 3 to 5 weeks after the acute sera. ?ND = Not done; no sera available. SNI = Unidentified cytopatbogenic agents under study.
Increase in titer
> 8 > 16 2 > 32 0 > 4 > 8 > 32 > 8 >
>
>
4 16 0 2 0
0 2
>32 ~ 16 0 > 64
> 4 > 16 0
8 8
G u a r d i o l a - R o t g e r et al.
July 1964
T a b l e I V . A g e g r o u p d i s t r i b u t i o n of h e l m i n t h s a n d p r o t o z o a r e c o v e r e d f r o m c h i l d r e n w i t h a c u t e and < 2 years < 6 months No. of cases No. of cases with helminths T. trichiura A. lumbricoides Hookworm
A
ch
27 0 0 0 0
0 0 0 0 0
% Helminths
6 months < 1 year
I
c
A
I
5 1 1 0 0
16 6 6 3 0
1 1 0 0
0 0 0 0
A-32.8%
No. of cases with protozoa Giardia lamblia E. histoIytica E. coli
0 0 0 0
0 0 0 0
% Protozoa
Ch
I
1 0 0 0 0
11 2 1 1 0
Ch-72.7%
A-2.8%
0 0 0 0
1 1 0 0
Ch-36.3 %
T a b l e V . D i s t r i b u t i o n o f m u l t i p l e a g e n t s r e c o v e r e d f r o m single f e c a l s a m p l e s in Puerto Rican children with acute diarrhea Patient No. G-71 B-123 B-117 G-19 B-129 G-45 B-131 G-96 B-115
Age (years)
Bacteria
25 (days) 35 (days) 6/12 6/12 11/12
Sh. sonnei I-CPS* 0126:B16-CPS 055 :B5-CPS* 0112:Bll
1 2/12 5/12 6/12
0111 : B4 0126:B16 0119LB14-ADt-01 0128:B12
1 1 1
Helminths
A. lumbricoides, T. trichiura T. trichiura T. trichiura
T. trichiura A. lumbricoides B-132 1 6/12 0111:B4 A. lumbricoides B-4 1 6/12 A. lumbricoides G-36 T. trichiura B-73-A 1 7/12 A. lumbrieoides G-12 1 1O~12 T. trichiura B-99 2 9/12 CPS T. trichiura, Hookworm, A. lumbricoides B-95 3 Sh. sonnei T. trichiura G-80 4 AD-02 T. trichiura, A. lumbricoides G-24 4 CPS T. trichiura, Hookworm B-69 4 Sh. sonnei T. trichiura, A. lumbricoides G-64 5 T. trichiura G-94 6 0128 9B 12 T. trichiura, Hookworm G-90 6 AD-01 T. trichiura, Hookworm B-39 6 9/12 0126"B16 T. trichiura G-58 8 T. trichiura B-2 10 0127 : B8 T. trichiura, A. lumbrieoides G-47 11 T. trichiura~ *CPS = Isolated from both fecal samples and throat swabs. tAD = Alkalescens--Dispargroup. ~+ = Moderate-to-severehelminthic infections.
c
Protozoa
Virus
Increase in titer
ECHO 8 N.I. Coxsackie Ba ECHO 8
>4x 0 >8x 0
Coxsackie B4 Coxsackle B4 E C H O 14 Coxsackle B4
>8x 2x >2x > 8x
G. larnblia
G. lamblia
G. lamblia
ECHO 8
16x
En. coli G. lamblia G. lamblia
Coxsackie B~
>4x
Coxsackie B4 En. coli En. coli
Coxsackie B4
<32x
Volume 65
Number 1
S t u d i e s on diarrheal diseases
8 9
chronic d i a r r h e a and control cases in P u e r t o Rico 2 years 1 ~ 2 years
a 27 17 12 9 0 2 2 0 0
I
ch
l
10 8 8 3 2 C-20.5% 4 4 0 0 C-12.8%
2 ~
3 years
c
A
ch
23 5 5 3 0
11 9 8 5
15 13 13 6
1
1
3 3 0 0
A-80.3% 5 11 3 7 1 3 1 3 A-36.1%
l
3 years
I
c 10 4 3 2
50 40 37 16 0 6 Ch-54.9% 2 17 2 10 0 3 0 2 Ch-55.2%
shown in Tables V a n d V I . H e l m i n t h s were a c o m m o n factor in all b u t 1 of the chronic cases. Helminthiasis was present in 21 of the 26 acute cases with m u l t i p l e agents. Giardia lamblia a n d E n t a m o e b a coli were the only Protozoa recovered from m i x e d infections, the incidence being higher in the chronic cases. M i x e d p r o t o z o a n a n d helminthic infections were observed in 41.1 p e r cent of the chronic cases a n d in 30.7 per cent of the acute cases. T h e incidence of m i x e d helminthic infections was higher in the chronic (52.9 per cent) t h a n in the acute (34.6 per cent) cases. D u a l bacterial infections were m o r e p r e v a l e n t in the acute t h a n in the chronic cases. Infections with 3 a n d / o r 4 different agents were observed in both groups of patients. I n the control group, only 5 cases h a d m i x e d infections. I n all cases, helminths were the c o m m o n factor. N o k n o w n p a t h o g e n i c agents were identified in 20.8 p e r cent of the acute, 4.3 p e r cent of the chronic, a n d 54.8 p e r cent of the control cases. DISCUSSION A group of 214 Puerto R i c a n children with acute a n d chronic gastroenteritis a n d 73 n o n d i a r r h e i c children were studied. Bacteria, viruses, a n d parasites of possible etiologic significance were f o u n d in all three groups of patients. T h e percentage of b a c t e r i a l a n d viral agents was higher in the acute t h a n in the chronic cases, and very low in the con-.
I
"
c
43 24 41 13 41 11 17 5 12 1 C-50.0% 21 5 12 4 5 0 7 2 C-20.5%
Total
I 131 72 63 33 ~7 54.9% 24 15 4 3 18.3%
ch 69 62 62 26 15 89.8% 36 23 8 10 52.1%
I c 73 25 21 11 1 34.2% 12 11 0 2 16.4%
trol group. E n t e r o p a t h o g e n i c E. coli was the most frequently f o u n d bacterial agent in both acute a n d chronic groups, the incidence being higher in the group of a c u t e cases. Coagulase positive staphylococci were the most p r e v a l e n t bacterial agents recovered from the control group of patients. Shigellosis was observed with similar frequency in b o t h groups; however, S. sonnei was the only species isolated from the chronic cases. These d a t a are c o m p a r a b l e to t h a t o b t a i n e d by other investigators 6, 9 in a relatively similar group of Puerto R i c a n children with gastroenteritis. Coagulase positive staphylococci were isolated from the stools only, in 4 of the 11 acute cases with positive findings. T h e r e m a i n i n g 7 a n d those recovered from the chronic cases were also present in the throat cultures. I t is possible t h a t in these cases the presence of the b a c t e r i a in the stool sample was incidental. T h e absence of Salmonella infections in these children was striking. N o reasonable e x p l a n a t i o n for this finding can be given at present. V i r a l enteritis has been described p r e viously by some investigators?, 2, 5, ~, 10 I n the present study Coxsackie virus B3 a n d B4, E C H O virus types 8 a n d 14, elicited a rise in neutralizing antibodies d u r i n g the diarrheal episode in acute patients. Similar results were obtained in the chronic cases b u t in a m u c h l o w e r p e r c e n t a g e with E C H O virus types 1 a n d 8 a n d Polio virus type 3. I n only 1 of these cases was there serologic evi-
90
July 1964
G u a r d i o l a - R o t g e r et al.
Table VI. Distribution of multiple agents recovered from single fecal samples in Puerto Rican children with chronic diarrhea Age
Patient No.
(years)
B-56
1
B-138
1
Bacteria
(055:B5) (026: B6) 0127:B8
Helrainths
Protozoa
T. trichiura%
G. larablia
Virus
lncrease in titer
Hookworm T. trichiura,
Polio 3
N.D.
ECHO 1
~32x
Polio 3 ECHO 8
> 64x 0
Polio 3
N.D.
Hookworm, B-89 B-14
1 6/12 1 9/12
055:B5
B-136 G-85 B-71 B-141
2 2
2 6/12 2 6/12
Sh. sonnei I
B-98 B-44 G-79 B-101 B-59
3 3 4 4 5
0127 : B8
G-78
5
B-107
6
0126:B16
G-62
8
0125 : B 15
B-82
9
0127:B8
A. lumbricoides T. trichiura ~ T. trichiura, A. lumbricoides T. trichiura r . trichiura T. trichiura, A. lumbricoides,
G. lamblia
G. lamblia G. lamblia
Hookworm
*zModerate-to-severe tCPS
~
Sh. sonnei I Sh. sonnei I
CPSt CPS
T. T. T. T. T.
trichiura trichiura trichiura trichiura trichiura, A. lumbricoides T. trichiura% A. lurabricoides T. trichiura ~, A. lumbricoides T. trichiura% A. lumbricoid,s T. trichiura% A. lumbricoides
En. coli
G. lamblia
ECHO 8
16x
En. coli
En. coli
helminthic infections.
Isolated from both fecal samples and throat swabs.
dence of previous host experience with the virus. Only 3 of the control cases had viral agents, 2 of which elicited host response. The three cytopathogenic agents belong to the E C H O group of viruses, and the types recovered in these groups were not found in the patients with diarrhea. These data strongly suggest the causal relationship of the viral agent with the clinical syndrome. The incidence of helminthic and protozoan infections was strikingly high in the chronic group. A direct relationship between chronicity and the trichiuris egg load, and to some extent, to the ascaris egg load, was observed in the chronic cases. In the acute cases the incidence of helminthic and protozoan infections was lower than in the chronic cases. However, it was high enough when compared with the control group to be seriously considered as possible etiologic agents, especially those cases with protozoan infection, or synergistic agents, in those cases with
helminthiasis. A concomitant helminthic and/or protozoan infection was observed in about 90 per cent of the cases from which multiple agents were recovered. These findings suggest the possibility that some type of synergism could exist between special groups of parasites a n d / o r bacteria and viruses. The damage done by the parasite to the linings of the intestinal wall with associated functional and chemical changes could alter the local environment, making it a suitable ecologic niche for any of a variety of pathogenic bacteria a n d / o r viruses. This would depend on the nature and number of organisms present, and the host's age, nutritional status, and susceptibility to the invader. The multiplicity of possible infectious agents which may cause acute diarrhea, plus the fact that often (20.8 per cent) no known pathogenic agent can be identified, points to the importance of the host's physiologic status and the host-parasite relationship in
Volume 65
Number 1
the causation of such disease. Studies are being carried out to e x p a n d the scope of the present investigation. SUMMARY
T h e microbiologic aspects of acute a n d chronic gastroenteritis in Puerto R i c a n children with d i a r r h e a and n o n d i a r r h e i c children are presented. Bacteria which m i g h t be considered causal agents of gastroenteritis, namely, e n t e r o p a t h o g e n i c E. coli, Shigella, and coagulase positive staphylococci, were recovered from 25.9 p e r cent of the acute, 18.0 p e r cent of the chronic, a n d 16.4 p e r cent of the control cases. I n the l a t t e r group, 75 per cent of the bacteria isolated were coagulase positive staphylococci. V i r a l agents were recovered from 14.1 p e r cent of the acute a n d 8.3 p e r cent of the chronic p a tients. A serologic response against these agents occurred in three fourths of the cases in which viruses were isolated a n d p a i r e d sera were available. T h e n u m b e r s of viral agents isolated in the control group was very low (4.1 per cent) a n d the types were different from those obtained in the patients with diarrhea. Protozoan infestations were observed in 18.3 p e r cent of the acute a n d 52.1 p e r cent of the chronic cases. H e l m i n t h s were recovered f r o m 54.9 p e r cent of the a c u t e a n d 89.8 p e r cent of the chronic cases. A direct relationship between egg load a n d c h r o n i c i t y was shown with some of the helminths recovered. M u l t i p l i c i t y of possible infectious agents was obtained in about 24 p e r cent of the patients. N o agents of possible significance in acute d i a r r h e a could be recovered from 20.8 per cent of the patients. This study points out the i m p o r t a n c e of investigating the possible role of c o m b i n e d viral, bacterial, a n d / o r parasitic infections in d i a r r h e a in order to attain a t h o r o u g h evalution of the disease. We wish to express our gratitude to Dr. Antonio Ortiz and Dr. Milton Alvarez, from the Department of Pediatrics, University of Puerto
Studies on diarrheal diseases
9 1
Rico School of Medicine; Dr. Egidio Colon and Dr. Osvaldo Gonzalez, from the Department of Pediatrics, San Juan City Hospital; and to the staff of the Barrio Obrero and Guaynabo and Puerta de Tierra Out-Patient Department Clinics for their assistance and cooperation in the clinical aspects of these studies. We are also indebted to Lt. Colonel Lyman P. Frick and Dr. Lawrence S. Ritchie from the Medical Zoology Division, United States Army Tropical Research Medical Laboratory, for their indispensable contributions in the parasitologic studies of these investigations.
REFERENCES 1. Ramos-Alvarez, M., and Sabin, A.: Enteropathogenic viruses and bacteria. Role in summer diarrheal disease of infancy and early childhood, J. A. M. A. 167" 147, 1958. 2. Eichenwald, H. F., Abadio, A., Arky, A. M., and Hartman, A. P.: Epidemic diarrhea in premature and older infants caused by ECHO virus type 18, J. A. M. A. 166: 1563, 1958. 3. Neter, E.: Enteritis due to enteropathogenic Escherichia coli, J. PEDIAT. 55" 223, 1959. 4. Ewing, W. H., Tatum, H. W., and David, B. R.: The occurrence of Escherichia coli serotype associated with diarrheal disease in the United States, Pub. Health Lab. 15" 118, 1957. 5. Gardner, P. S., McGregor, C. B., and Dick, K.: Association between diarrhea and adenovirus type 7, Brit. M. J. 1: 91, 1960. 6. Young, V. M., Lindberg, R. B., Ortiz, A., Jahiel, D., Sochard, M. E., and Hemphill, J. J." Studies of infectious agents in infant diarrhea. III. Bacteriologic, virologic and parasitologic flora of fecal samples collected from Puerto Rican children, Am. J. Trop. Med. 2 (3): 380, 1962. 7. Edwards, P. R., and Ewing, W. tI.: Identification of enterobacteriacease, Minneapolis, 1955, Burgess Publishing Company. 8. Ritchie, L. S., and Frick, L. P.: Unpublished, see Frick, L. P., Moon, A. P., and Lin, S. S.: Parasitologic studies in the Far East. XV. A preliminary survey for parasitism in southern Formosa, Metabolism 5: 302, 1956. 9. Cherry, William B., Thomassen, B. M., Pomales-Lebron, A., and Ewing, W. H.: Rapid presumptive identification of enteropathogenic Escherichia coli in fecal smears by means of fluorescent antibody, Bull. World Health Organization 25: 159, 1961. 10. Syverton, S.: An appraisal of enterovirus problem, Am. J. Trop. Med. 8: I01, 1959.
Studies on diarrheal diseases The multiplicity of infectious agents in the intestinal flora o[ Puerto Rican children with gastroenteritis
A total o[ 214 children with diarrhea and 73 control children were studied [or the presence o[ enteric microorganisms. The percentage o[ bacterial and viral agents was higher in the acute than in the chronic diarrhea cases, and very low in the control group. Enteropathogenic Escherichia coli was the most [requently [ound bacterial agent in the patients with diarrhea. Shigellosis was absent in the control group. A serologic response against the viral agents isolated occurred in three [ourths o[ the diarrhea cases. A causal relationship o[ the viral agents isolated with the clinical syndrome is suggested. The prevalence o[ protozoan and helminthic injections was higher in the group with chronic diarrhea, and a direct relationship between egg load and chronicity was obtained. Multiplicity o[ possible in[ectious agents was obtained in about one [ourth o[ the patients. The possible role o[ combined viral, bacterial, and~or parasitic injection in diarrhea is discussed.
Aida Guardiola-Rotger, Ph.D., ~ Etervina Figueroa de Gonzalez, M.D., Emanuel Kauder, Captain, MC, USA, Aurea Munoz, M.D., Victor A. Lopez, Diana E. Gadea, B.S., and Merlyn J. Funkenbuseh, SP5 T I-I E etiology of diarrheal diseases in infants and children has been the subject of considerable debate during the past 20 years.
Much of this controversy has centered in the suspected etiologic role of certain serotypes of Escherichia coli and enteroviruses in gastroenteritis. A review of the literature reveals the efforts taken by a great number of workers in an attempt to delineate the problem of acute gastroenteritis. The causal role of a given number of enteroviruses and enteropatho-
From the Microbiology Division, United States Army. Tropical Research Medical Laboratory, APO 851, New York, N. Y. (San Juan, Puerto Rico). ~Address, United States Army Tropical Research Medical Laboratory, APO 851, New York, N. Y.
81
g2
Guardiola-Rotger et al.
genic E. coli serotypes has been recently s h o w n . 1-4 Ho~vever, the factor or factors which endow these microorganisms with enteropathogenicity have not been identified. Neither have the conditions which render the infant so extraordinarily susceptible to these enteropathogens, and conversely the older child and adult rather resistant, been elucidated. Limited information is available on the problem of mixed infections. Dual infections undoubtedly exist, s' a but it is difficult to interpret the findings on the cultural examinations alone for it is possible that the disease in some of these patients is due to one pathogen and the subject is a carrier of the other. In this report, data are presented on the nature of the multiplicity of infectious agents associated with .diarrheal diseases and the correlation of these findings with the clinical manifestations. M A T E R I A L S AND M E T H O D S Clinical material. A total of 214 pediatric patients, varying in age from 25 days to 12 years, and whose chief complaint was diarrhea, were studied at the Guaynabo Health Center and the Barrio Obrero outpatient department clinics. A patient was considered to have diarrhea if the passage of stool was more frequent than usual for a given child, and the consistency of the stool was soft or watery. A bout of diarrhea reported to have lasted 4 weeks or longer at the time of admission was arbitrarily classified as chronic diarrhea. Anyone who had received recent antimicrobial therapy was not included in the studies. A group of 73 children without diarrhea, at least 3 weeks before and 3 weeks after admission, were studied at the Puerta de Tierra Outpatient Clinic. On the day of admission a complete history and physical examination was done, and fecal samples, blood, and throat swabs were collected. Three to five weeks later, convalescent serum was obtained. Additional visits were based on need as determined by the clinical course. Specimens. 1. Stool samples. Fecal samples and/or rectal swabs were collected from each pa-
J,dy 1964
tient. Immediately after collection, bacteriologic media, namely SS, MacConkey and Staph 110 agar plates, and Selenite F enrichment medium, were inoculated. An aliquot of the sample was placed in screw-cap tubes containing 1 ml. of Hank's balanced salt solution (BSS), sealed with adhesive tape, and placed immediately in dry ice. The remaining portion was kept at room temperature for parasitologic examination. 2. Throat swabs. Two swabs were obtained from each patient. One was placed immediately in a screw-cap tube containing 1 ml. of Hank's BSS, sealed with adhesive tape, and placed in dry ice. T h e other was used for the inoculation of blood agar and Staph 110 plates. 3. Blood samples. On the day of admission, acute phase sera was obtained and a complete blood count done on each patient. Convalescent sera were collected 3 to 5 weeks later. Laboratory techniques. 1. Bacteriologic methods. On the average, there was a lapse of 3 to 4 hours from the time of inoculation of plates at the clinic and its arrival at the laboratory. The isolated species of Salmonella, Shigella, AlkalescensDispar group, and enteropathogenic E. coli were identified biochemically and typed according to Edwards and Ewing's 7 methods. 2. Parasitologic methods. The identification of all helminths and protozoans was done on each stool sample. The specimen arrived at the laboratory approximately 3 to 4 hours after collection. A direct saline smear was prepared and examined for the identification of protozoan trophozoites with special attention given to motility and quality of the unstained nucleus. A quantitative egg count examination was also performed on each stool according to the method described by Ritchie and Frick. s 3. Viral methods. a . I S O L A T I O N O F V I R U S E S . T h e throat and rectal swabs and formed stool specimens were kept frozen at -70 ~ C. until ready to be processed. At this time, the swabs were removed after thawing; 2.0 ml. of Hank's BSS were added to each tube and the tubes were
Volume 65
Number 1
Studies on diarrheal diseases
8 3
[---] - ACUTE [ 14Z CASES )
I-CHRONIC [~
(7E CASES) -- C O N T R O L ( 73 CASES )
E P E C - EN'i'EROPATHI~GENIC EI COLI bj o
C P S - - C O A G U L A S E pOSITIVE STAPHYLOCOCCI NI--NOT
> tO( ,O
C- 80
Fig. 1. agents children chronic control Rico.
Distribution of recovered from with acute and diarrhea and cases in Puerto
<0 6 0
~HELMINTHS
r~-
--1
~
PROTOZOA--'~
IDENTIFIED
COX--COXSACKIE A . I . - - A LUMBRICOIDES s HISTOLYTICA
BACTERIA ---1
,0,,~ ,,,j,o.,
VIRUSES
I
u.
o w 40 1.9
I00, i
CHRONIC
i
I 6g CASES )
90,
[[~[] <[ 2 5 0 0
2500- 5OOO 5000-1DO00
8 0 84
IO,O00 - 25,000 2 5 D 0 0 - 50,000
o
70
I
> 5o, ooo
60,
F-
z
ACUTE
I
( 131 CASES )
50-
5 LU I
u_ 4 0 0 r-CONTROL-~ 6 9 CASES
~< .50~J
20-
Fig. 2. The relationship of the chronicity of diarrhea to the helminths egg count.
I0-
r.
fr~chiu~
A
T
lumbriceides Hookworm
trichTura lumbrAcoidesHookworm
A.
frichiuro lumbr!coides
centrifuged at 3,000 r.p.m, for 30 minutes at 4 ~ C. O n e thousand units of penicillin a n d streptomycin p e r milliliter were a d d e d to the s u p e r n a t a n t fluid. F o r the f o r m e d stool specimens, a 10 per cent suspension was p r e p a r e d within H a n k ' s BSS a n d processed in a similar m a n n e r . This p r e p a r a t i o n was used for the inoculation of tissue cultures. ~ Both monkey kidney ( M K ) a n d H e p - 2 cells were used in the studies. T w o M K a n d H e p - 2 tubes were inoculated with 0.25 ml. of the specimen suspension a n d i n c u b a t e d for 1 h o u r at room temperatm'e. T h e inoculum was then rem o v e d a n d replaced with 1.0 ml. of u n d i l u t e d beef amniotic fluid for the M K cells and
Ginsberg m e d i a w i t h 4 p e r cent horse serum for the H e p - 2 cells, each containing 100 units of penicillin a n d streptomycin per milliliter. T h e tubes were held for 7 days at 37 ~ C. a n d observed daily for cytopathogenic effect ( C P E ) . Tubes in which C P E was observed were r e m o v e d from the incubator when a p p r o x i m a t e l y 50 p e r cent of the cell sheet h a d been affected a n d kept at - 7 0 ~ C. for identification a n d serologic studies. Enh a n c e m e n t passages were carried out on those samples which showed slow or slight CPE. b , I D E N T I F I C A T I O N OF V I R U S E S . T h e isolated viruses were identified b y neutralization tests by means of pools of antisera ~" contain-
*All the cells and antisera were purchased from Microbiological Associates, Inc., Bethesda, Md.
*All tile cells and antisera were purchased fi'om Microbiological Associates, Inc., Bethesda, Md.
84
Guardiola-Rotger et al
]uly 1964
Table I. Age group distribution of bacterial agents recovered from children with acute and chronic
No. of cases EPEC* 0126:B16 055:B5 086:B7 0124:B17 0127:B8 0119:B14 0128:B12 0111:B4 0112:Bll 026:B6 0125:B15 % EPEC
6 months Acute [ Chronic l Control 27 0 5 7 0 1 2 0 0 1 0 0 2 0 0 1 0 0 0 0 0 l 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 A--22.4%
< 2 years 6 months < 1 year Acute [Chronic I Control 19 1 11 3 0 0 0 0 0 1 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 Ch.--33.3 %
1-< 2years Acute [ Chronic[ Control 30 11 23~ 7 4 0 3 0 0 0 2 0 0 0 0 0 0 0 0 1 0 1 0 0 1 0 0 2 0 0 0 0 0 0 1 0 0 0 0 C--2.5%
Shigella
1
0
0
l
0
0
1
0
0
sonnei I flexneri 2 % Shigella CPSt % CPS
1 0
0 0 A--3.9% 0 A--10.5%
0 0
0 1
0 0
0 1
22
0
1
0 0 C--0% 0 C--10.2%
0 0
2
0 0 Ch.--0% 0 Ch.--0%
5
* E P E C ~ Enteropathogenic E. coll. t C P S = Coagulase positive staphylococci.
ing 20 units of each specific antibody. T h e composition of the pools used was as follows: polio pool (I, I I , I I I ) ; Coxsackie pool (Ag, B1, B2, Ba, B~, B~); E C H O pool I (1, 2, 3, 5) ; E C H O pool I I (6, 7, 8, 9, 12) ; E C H O pool I I I (11, 13, 14, 1 5 ) ; E C H O pool I V (10, 16, 17, 18, 19). If C P E was inhibited by any given pool, the virus was identified by doing neutralization tests against each individual virus type of the pool. Viruses not neutralized by any of the pools were tested against individual sera of E C H O types 4, 21, 27, and Coxsackie A7 and B6. I n the neutralization test, approximately 100 TCD~0 of virus was used, the exact dose being determined by a concurrent titration with virus dilution ranging from 10 -a'3 t h r o u g h 10-~'3. e . S E R O L O G I C S T U D I E S . Neutralization tests were carried out with the patient's acute and convalescent sera and the homologous virus to determine rises in antibody titer. RESULTS
O f the 214 patients studied, 142 were classified as having acute and 72 as having
chronic cases of diarrhea. Seventy-three children without diarrhea were also studied. Agents of possible etiologic significance were found in 79.2 per cent of the acute, in 95.7 per cent of the chronic, and in 54.8 per cent of the control cases. T h e distribution of the agents recovered are shown in Fig. 1. Bacteria which m a y be considered as possible etiologic agents in diarrhea were isolated in 25.9 per cent of the acute, 18.9 per cent of the chronic, and 16.4 per cent of the control cases. T h e incidence of enteropathogenic E. coil ( E P E C ) serotypes, as would be expected, was higher in the acute (14.8 per cent) than in the chronic (11.1 per cent) cases. Enteropathogenic E. coli were recovered in only 5.4 per cent of the control cases studied. T h e age group distribution of E. coli serotypes isolated is shown i n Table I. T h e highest incidence of E. coli serotypes was obtained in patients under 2 years of age in both acute (22.4 per cent) and chronic (33.3 per cent) cases. However, in the control group the highest incidence of enteropathogenic E. coli (8.8 per cent)
2
Volume 65 Number 1
Studies on diarrheal diseases
85
diarrhea a n d in control cases in Puerto Rico
2 years 2-~ 3 years Acuf~te [Chronic l Control
Acute
~ 3 years [Chronic I Control
Acute
Total t Chronic
I Control
15 1 0 0 0
16 0 0 0 0
10 1 0 0 0
51 3 1 0 0
44 4 1 0 0
24 2 0 0 0
142 21 6 2 2
72 8 1 2 0
73 4 0 0 0
1
0
0
0
0
0
3
0
0
0
0
0
1
2
0
1
3
0
0 0 0 0 0 0
0 0 0 0 0 0
1 0 0 0 0 0
0 1 0 0 0 0
0 0 0 0 0 1
0 1 1 0 0 0
2 2 2 1 0 0
0 0 0 0 1 1
1 2 1 0 0 0
1 1 0
0 0 0
2 2 0
3 3 0
1
1
2
3
A~6.1% 0 0 0
Ch.--5.6%
A--3.0% 1
A--4.5%
C--8.8%
Ch.--6.6%
0 0 0
14.8% 5 3 2 3.5%
11.1% 4 4 0 5.5%
5.4% 0 0 0 0%
3
i1
4
8
7.8%
2.7%
10.9%
C--0%
Ch.--6.6%
was observed in children over 2 years of age. T w o of the 4 E. cell serotypes isolated f r o m this group were 0.128:B12. N i n e serotypes of enteropathogenic E. coli were isolated from the acute cases, 0126:B16 being the most frequently found, followed by 0124: B17; for the chronic cases, 5 serotypes were isolated, 0127:B8 a n d 055:B5 being the most c o m m o n l y found. These d a t a are comp a r a b l e to t h a t obtained by C h e r r y and colleagues, 9 a l t h o u g h in their r e p o r t only acute cases were included. Shigellae were more frequently isolated from the chronic (5.5 p e r cent) t h a n from the acute cases (3.5 p e r c e n t ) , as shown in Fig. 1. N o Shigellae were isolated from the control group. D a t a on the age group distribution of Shigella serotypes are presented in T a b l e I. Shigella sonnei occurred most frequently in b o t h acute a n d chronic cases; in the latter group, being the only species isolated. T h e incidence of Shigellae in the acute cases was very similar in children u n d e r a n d over 2 years of age, a n d for the chronic cases it was higher in child r e n over 2 years of age. Coagulase positive
C--11.7%
staphylococci were isolated in 7.8 p e r cent of the acute cases a n d in 9.7 p e r cent of the chronic cases (Fig. 1 ). T h e highest incidence of coagulase positive staphylococci was obt a i n e d in the control group (10.9 p e r c e n t ) . T h e age distribution of this m i c r o o r g a n i s m in the chronic group was similar to t h a t of Shigellae ( T a b l e I ) . All of the coagulase positive staphylococci isolated from fecal specimens of chronic cases were also obtained from the throat cultures of the patient. F o r the acute, in 7 of the 11 cases w i t h positive findings in the fecal specimens, the eoagulase positive staphylococci were also isolated from the t h r o a t specimens. V i r a l agents, w h i c h were cytopathogenic for m o n k e y kidney tissue cultures, were obt a i n e d in 14.1 p e r cent of the acute, in 8.3 p e r cent of the chronic, a n d in 4.1 p e r cent of the control cases. As shown in T a b l e I I the age group distribution of the viral agents isolated followed a p a t t e r n similar to t h a t of the b a c t e r i a l isolates, i.e., the incidence of viral agents in b o t h the acute a n d chronic groups was higher in children u n d e r 2 years
86
GuardioIa-Rotger et al.
July 1964
T a b l e II. Age group distribution of viral agents recovered from children with acute a n d chronic diarrhea a n d in control cases in Puerto Rico
2 years -6 <
months ~6 months 2 years _. ~ 1 year
2-~ 3 years
1-<2 years
AIChlC A IChlC No. of cases
Viruses Coxsackie B~ Coxsackie Ba Coxsackie A~ ECHO 9 ECHO 7 ECHO 8 ECHO 12 ECHO 14 ECHO 1 Polio 3 N.I.w % Viruses
27
0
5
5 0 1 1 0 0 0 0 0 0 0 0 0 0 1 0 0 0 2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 A-19.7%
19
1
11
4 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 Ch-16.7%
30 11 6 2 3 0 0 0
23 2 0 0
15 16 1 3 1 0 0 0
1
0
0
0 0 0 0
0 0 0 0
0 1 0 1
1
0
0
0 0
1 1
0 0
1
0
0
C-7.6%
10 0 0 0
Total
~ 3 years
IChlC A ICh I c
51 44 24 4 1 0 3 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 1 0 1 1 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2 0 0 0 0 0 0 0 0 0 0 A-7.6% Ch-6.7 % C-0%
142
72
73
20 6 3 8 0 0 1 0 0 1 0 0 0 0 1 0 0 1 4 2 0 0 0 1 1 0 0 0 1 0 0 3 0 5 0 0 14.1% 8.3% 4.1%
e A - - A c u t e cases. ~Ch
-:
Chronic
cases.
:~C ~
C o n t r o l cases.
w
=
Unidentified
cytopathogenic
agents under
study.
of age. I n the control group, the 3 viral isolates were recovered from children u n d e r 2 years of age. T h e results of the neutralization tests carried out with the patient's acute a n d convalescent sera to determine the extent of a n t i b o d y f o r m a t i o n are shown in T a b l e I I I . Coxsackie B4 a n d E C H O 8 were the most c o m m o n l y f o u n d viruses in the acute cases. Eleven of the 13 k n o w n viruses isolated from these patients, a n d from which paired sera were available, elicited a rise in a n t i b o d y titer d u r i n g the diarrheal episode. Of the 8 Coxsackie B4 viruses recovered, all b u t one showed a n increase in a n t i b o d y titer. P a t i e n t G-58 h a d the same a n t i b o d y titer in both the acute a n d convalescent sera, therefore, one can assume t h a t the p a t i e n t was seen in the late acute phase. T w o of the three u n k n o w n cytopathogenic agents tested serologieally elicited some degree of host response , thus they are p r o b a b l y not tissue contaminants. T h r e e of the four viral agents recovered from chronic cases from which paired sera were available showed a rise in neutralizing a n t i b o d y titer d u r i n g the course of disease. No Coxsackie viruses were isolated in this group. T h r e e of the seventythree control cases h a d cytopathogenic
agents. I n 2 of the cases, E C H O 3 and E C H O 7, a rise in neutralizing a n t i b o d y titer was obtained. T h e results obtained in the parasitologic studies showed (Fig. 1) a striking difference in the degree of helminthic a n d protozoan infections between the acute a n d chronic cases. Protozoan infections were observed in 18.3 per cent of the acute a n d 52.1 per cent of the chronic cases. Giardia larnblia was the most c o m m o n l y found protozoan in both groups. E. histolytica a n d E. coli were present with almost similar frequency, although in both cases the degree of infestation was higher in the chronic group. T h e age distribution ( T a b l e I V ) showed that the protozoan infections were present at a higher rate in children over 2 years of age in the three groups, acute, chronic, a n d control cases. Helminths were recovered (Fig. 1) from 54.9 per cent of the acute a n d 89.8 per cent of the chronic a n d 34.2 per cent of the control cases. Trichiurls trichiura was f o u n d in about half of the acute cases studied a n d one fourth of the control group; parasitization by this worm was almost universal in the chronic group. Ascaris lumbricoides was f o u n d in a b o u t one fourth of the acute cases
Volume 65
Number 1
Studies
and in one third of the chronic cases. Hookworm was the least commonly found helminth. The age distribution (Table IV) showed, as expected, that helminthiases were present at a higher rate in children over 2 years of age in acute, chronic, and controI cases. The relationship of the trichiuris and ascaris egg load to the chronicity of diarrhea is shown in Fig. 2. The incidence of trichiuriasis with egg counts over 25,000 was observed in 3.8 per cent of the acute and in 37.7 per cent of the chronic cases. For ascaris, 7.6 per cent of the acute and 17.3 per cent of the chronic cases had egg counts ov~er 10,000. None of the control cases had trichiuris egg
o n d i a r r h e a l diseases
87
counts over 25,000 and in only 4 of the 73 controls, the ascaris egg counts were over 10,000. The number of cases with hookworm infections in the acute, chronic, and control groups constituted a small per cent of the total helminthiasis, therefore, it does not seem feasible to attempt a correlation between chronicity and egg load as done for the other two parasites. Of 110 acute cases with positive findings, 23.6 per cent had mixed viral, bacterial, or parasitic agents. Similar findings (24.4 per cent) were obtained for the 67 chronic cases with positive isolations. The distribution of the agents recovered are
Table III. Viral agents recovered and homologous neutralization tests from 142 acute and 72 chronic cases of diarrhea and 73 nondiarrheic children in Puerto Rico Neutralization tests (100 TCDso) ~
I Convalescent
Patient No.
Virus
Acute serum
serufn
Acute cases G-12 G-22 G-36 G-47 G-58 G-64 B-4 B-6 G-19 B-113 G-71 G-80 G-49 B-129 B-73-A B-123 B-38 B-68 B-75 B~124
Coxsackie B4 Coxsackie B~ Coxsackie B4 Coxsackie B4 Coxsackie B4 Coxsackie B~ Coxsackie B4 Coxsackie B4 Coxsackie B~ Coxsackie A9 ECHO 8 ECHO 8 ECHO 8 ECHO 8 E C H O 14 N.I.$ N.I. N.I. N.I. N.I.
4 4 64 ~ 4 64 < 4 < 4 ~ 4 < 4 N.D.+ ~ 4 8 N.D. < 4 ~ 4 16 N.D. N.D, ~ 4 ~ 4
32 64 128 128 64 16 32 128 32 N,D. 16 128 N.D. <4 8 16 N.D. N.D. < 4 8
Chronic cases B-14 G-78 G-85 Bd38 B-136 B-141
ECHO 1 ECHO 8 ECHO 8 Polio 3 Polio 3 Polio 3
~ 4 ~ 4 ~ 4 N.D. ~ 4 N.D.
128 64 < 4 N.D. 256 N.D,
Control cases 10-C 17-C 42-C
< <
ECHO 3 ~ 4 16 ECHO 7 < 4 64 E C H O 12 0 0 ~Acute sera drawn on day of admission, Convalescent sera drawlx 3 to 5 weeks after the acute sera. ?ND = Not done; no sera available. SNI = Unidentified cytopatbogenic agents under study.
Increase in titer
> 8 > 16 2 > 32 0 > 4 > 8 > 32 > 8 >
>
>
4 16 0 2 0
0 2
>32 ~ 16 0 > 64
> 4 > 16 0
8 8
G u a r d i o l a - R o t g e r et al.
July 1964
T a b l e I V . A g e g r o u p d i s t r i b u t i o n of h e l m i n t h s a n d p r o t o z o a r e c o v e r e d f r o m c h i l d r e n w i t h a c u t e and < 2 years < 6 months No. of cases No. of cases with helminths T. trichiura A. lumbricoides Hookworm
A
ch
27 0 0 0 0
0 0 0 0 0
% Helminths
6 months < 1 year
I
c
A
I
5 1 1 0 0
16 6 6 3 0
1 1 0 0
0 0 0 0
A-32.8%
No. of cases with protozoa Giardia lamblia E. histoIytica E. coli
0 0 0 0
0 0 0 0
% Protozoa
Ch
I
1 0 0 0 0
11 2 1 1 0
Ch-72.7%
A-2.8%
0 0 0 0
1 1 0 0
Ch-36.3 %
T a b l e V . D i s t r i b u t i o n o f m u l t i p l e a g e n t s r e c o v e r e d f r o m single f e c a l s a m p l e s in Puerto Rican children with acute diarrhea Patient No. G-71 B-123 B-117 G-19 B-129 G-45 B-131 G-96 B-115
Age (years)
Bacteria
25 (days) 35 (days) 6/12 6/12 11/12
Sh. sonnei I-CPS* 0126:B16-CPS 055 :B5-CPS* 0112:Bll
1 2/12 5/12 6/12
0111 : B4 0126:B16 0119LB14-ADt-01 0128:B12
1 1 1
Helminths
A. lumbricoides, T. trichiura T. trichiura T. trichiura
T. trichiura A. lumbricoides B-132 1 6/12 0111:B4 A. lumbricoides B-4 1 6/12 A. lumbricoides G-36 T. trichiura B-73-A 1 7/12 A. lumbrieoides G-12 1 1O~12 T. trichiura B-99 2 9/12 CPS T. trichiura, Hookworm, A. lumbricoides B-95 3 Sh. sonnei T. trichiura G-80 4 AD-02 T. trichiura, A. lumbricoides G-24 4 CPS T. trichiura, Hookworm B-69 4 Sh. sonnei T. trichiura, A. lumbricoides G-64 5 T. trichiura G-94 6 0128 9B 12 T. trichiura, Hookworm G-90 6 AD-01 T. trichiura, Hookworm B-39 6 9/12 0126"B16 T. trichiura G-58 8 T. trichiura B-2 10 0127 : B8 T. trichiura, A. lumbrieoides G-47 11 T. trichiura~ *CPS = Isolated from both fecal samples and throat swabs. tAD = Alkalescens--Dispargroup. ~+ = Moderate-to-severehelminthic infections.
c
Protozoa
Virus
Increase in titer
ECHO 8 N.I. Coxsackie Ba ECHO 8
>4x 0 >8x 0
Coxsackie B4 Coxsackle B4 E C H O 14 Coxsackle B4
>8x 2x >2x > 8x
G. larnblia
G. lamblia
G. lamblia
ECHO 8
16x
En. coli G. lamblia G. lamblia
Coxsackie B~
>4x
Coxsackie B4 En. coli En. coli
Coxsackie B4
<32x
Volume 65
Number 1
S t u d i e s on diarrheal diseases
8 9
chronic d i a r r h e a and control cases in P u e r t o Rico 2 years 1 ~ 2 years
a 27 17 12 9 0 2 2 0 0
I
ch
l
10 8 8 3 2 C-20.5% 4 4 0 0 C-12.8%
2 ~
3 years
c
A
ch
23 5 5 3 0
11 9 8 5
15 13 13 6
1
1
3 3 0 0
A-80.3% 5 11 3 7 1 3 1 3 A-36.1%
l
3 years
I
c 10 4 3 2
50 40 37 16 0 6 Ch-54.9% 2 17 2 10 0 3 0 2 Ch-55.2%
shown in Tables V a n d V I . H e l m i n t h s were a c o m m o n factor in all b u t 1 of the chronic cases. Helminthiasis was present in 21 of the 26 acute cases with m u l t i p l e agents. Giardia lamblia a n d E n t a m o e b a coli were the only Protozoa recovered from m i x e d infections, the incidence being higher in the chronic cases. M i x e d p r o t o z o a n a n d helminthic infections were observed in 41.1 p e r cent of the chronic cases a n d in 30.7 per cent of the acute cases. T h e incidence of m i x e d helminthic infections was higher in the chronic (52.9 per cent) t h a n in the acute (34.6 per cent) cases. D u a l bacterial infections were m o r e p r e v a l e n t in the acute t h a n in the chronic cases. Infections with 3 a n d / o r 4 different agents were observed in both groups of patients. I n the control group, only 5 cases h a d m i x e d infections. I n all cases, helminths were the c o m m o n factor. N o k n o w n p a t h o g e n i c agents were identified in 20.8 p e r cent of the acute, 4.3 p e r cent of the chronic, a n d 54.8 p e r cent of the control cases. DISCUSSION A group of 214 Puerto R i c a n children with acute a n d chronic gastroenteritis a n d 73 n o n d i a r r h e i c children were studied. Bacteria, viruses, a n d parasites of possible etiologic significance were f o u n d in all three groups of patients. T h e percentage of b a c t e r i a l a n d viral agents was higher in the acute t h a n in the chronic cases, and very low in the con-.
I
"
c
43 24 41 13 41 11 17 5 12 1 C-50.0% 21 5 12 4 5 0 7 2 C-20.5%
Total
I 131 72 63 33 ~7 54.9% 24 15 4 3 18.3%
ch 69 62 62 26 15 89.8% 36 23 8 10 52.1%
I c 73 25 21 11 1 34.2% 12 11 0 2 16.4%
trol group. E n t e r o p a t h o g e n i c E. coli was the most frequently f o u n d bacterial agent in both acute a n d chronic groups, the incidence being higher in the group of a c u t e cases. Coagulase positive staphylococci were the most p r e v a l e n t bacterial agents recovered from the control group of patients. Shigellosis was observed with similar frequency in b o t h groups; however, S. sonnei was the only species isolated from the chronic cases. These d a t a are c o m p a r a b l e to t h a t o b t a i n e d by other investigators 6, 9 in a relatively similar group of Puerto R i c a n children with gastroenteritis. Coagulase positive staphylococci were isolated from the stools only, in 4 of the 11 acute cases with positive findings. T h e r e m a i n i n g 7 a n d those recovered from the chronic cases were also present in the throat cultures. I t is possible t h a t in these cases the presence of the b a c t e r i a in the stool sample was incidental. T h e absence of Salmonella infections in these children was striking. N o reasonable e x p l a n a t i o n for this finding can be given at present. V i r a l enteritis has been described p r e viously by some investigators?, 2, 5, ~, 10 I n the present study Coxsackie virus B3 a n d B4, E C H O virus types 8 a n d 14, elicited a rise in neutralizing antibodies d u r i n g the diarrheal episode in acute patients. Similar results were obtained in the chronic cases b u t in a m u c h l o w e r p e r c e n t a g e with E C H O virus types 1 a n d 8 a n d Polio virus type 3. I n only 1 of these cases was there serologic evi-
90
July 1964
G u a r d i o l a - R o t g e r et al.
Table VI. Distribution of multiple agents recovered from single fecal samples in Puerto Rican children with chronic diarrhea Age
Patient No.
(years)
B-56
1
B-138
1
Bacteria
(055:B5) (026: B6) 0127:B8
Helrainths
Protozoa
T. trichiura%
G. larablia
Virus
lncrease in titer
Hookworm T. trichiura,
Polio 3
N.D.
ECHO 1
~32x
Polio 3 ECHO 8
> 64x 0
Polio 3
N.D.
Hookworm, B-89 B-14
1 6/12 1 9/12
055:B5
B-136 G-85 B-71 B-141
2 2
2 6/12 2 6/12
Sh. sonnei I
B-98 B-44 G-79 B-101 B-59
3 3 4 4 5
0127 : B8
G-78
5
B-107
6
0126:B16
G-62
8
0125 : B 15
B-82
9
0127:B8
A. lumbricoides T. trichiura ~ T. trichiura, A. lumbricoides T. trichiura r . trichiura T. trichiura, A. lumbricoides,
G. lamblia
G. lamblia G. lamblia
Hookworm
*zModerate-to-severe tCPS
~
Sh. sonnei I Sh. sonnei I
CPSt CPS
T. T. T. T. T.
trichiura trichiura trichiura trichiura trichiura, A. lumbricoides T. trichiura% A. lurabricoides T. trichiura ~, A. lumbricoides T. trichiura% A. lumbricoid,s T. trichiura% A. lumbricoides
En. coli
G. lamblia
ECHO 8
16x
En. coli
En. coli
helminthic infections.
Isolated from both fecal samples and throat swabs.
dence of previous host experience with the virus. Only 3 of the control cases had viral agents, 2 of which elicited host response. The three cytopathogenic agents belong to the E C H O group of viruses, and the types recovered in these groups were not found in the patients with diarrhea. These data strongly suggest the causal relationship of the viral agent with the clinical syndrome. The incidence of helminthic and protozoan infections was strikingly high in the chronic group. A direct relationship between chronicity and the trichiuris egg load, and to some extent, to the ascaris egg load, was observed in the chronic cases. In the acute cases the incidence of helminthic and protozoan infections was lower than in the chronic cases. However, it was high enough when compared with the control group to be seriously considered as possible etiologic agents, especially those cases with protozoan infection, or synergistic agents, in those cases with
helminthiasis. A concomitant helminthic and/or protozoan infection was observed in about 90 per cent of the cases from which multiple agents were recovered. These findings suggest the possibility that some type of synergism could exist between special groups of parasites a n d / o r bacteria and viruses. The damage done by the parasite to the linings of the intestinal wall with associated functional and chemical changes could alter the local environment, making it a suitable ecologic niche for any of a variety of pathogenic bacteria a n d / o r viruses. This would depend on the nature and number of organisms present, and the host's age, nutritional status, and susceptibility to the invader. The multiplicity of possible infectious agents which may cause acute diarrhea, plus the fact that often (20.8 per cent) no known pathogenic agent can be identified, points to the importance of the host's physiologic status and the host-parasite relationship in
Volume 65
Number 1
the causation of such disease. Studies are being carried out to e x p a n d the scope of the present investigation. SUMMARY
T h e microbiologic aspects of acute a n d chronic gastroenteritis in Puerto R i c a n children with d i a r r h e a and n o n d i a r r h e i c children are presented. Bacteria which m i g h t be considered causal agents of gastroenteritis, namely, e n t e r o p a t h o g e n i c E. coli, Shigella, and coagulase positive staphylococci, were recovered from 25.9 p e r cent of the acute, 18.0 p e r cent of the chronic, a n d 16.4 p e r cent of the control cases. I n the l a t t e r group, 75 per cent of the bacteria isolated were coagulase positive staphylococci. V i r a l agents were recovered from 14.1 p e r cent of the acute a n d 8.3 p e r cent of the chronic p a tients. A serologic response against these agents occurred in three fourths of the cases in which viruses were isolated a n d p a i r e d sera were available. T h e n u m b e r s of viral agents isolated in the control group was very low (4.1 per cent) a n d the types were different from those obtained in the patients with diarrhea. Protozoan infestations were observed in 18.3 p e r cent of the acute a n d 52.1 p e r cent of the chronic cases. H e l m i n t h s were recovered f r o m 54.9 p e r cent of the a c u t e a n d 89.8 p e r cent of the chronic cases. A direct relationship between egg load a n d c h r o n i c i t y was shown with some of the helminths recovered. M u l t i p l i c i t y of possible infectious agents was obtained in about 24 p e r cent of the patients. N o agents of possible significance in acute d i a r r h e a could be recovered from 20.8 per cent of the patients. This study points out the i m p o r t a n c e of investigating the possible role of c o m b i n e d viral, bacterial, a n d / o r parasitic infections in d i a r r h e a in order to attain a t h o r o u g h evalution of the disease. We wish to express our gratitude to Dr. Antonio Ortiz and Dr. Milton Alvarez, from the Department of Pediatrics, University of Puerto
Studies on diarrheal diseases
9 1
Rico School of Medicine; Dr. Egidio Colon and Dr. Osvaldo Gonzalez, from the Department of Pediatrics, San Juan City Hospital; and to the staff of the Barrio Obrero and Guaynabo and Puerta de Tierra Out-Patient Department Clinics for their assistance and cooperation in the clinical aspects of these studies. We are also indebted to Lt. Colonel Lyman P. Frick and Dr. Lawrence S. Ritchie from the Medical Zoology Division, United States Army Tropical Research Medical Laboratory, for their indispensable contributions in the parasitologic studies of these investigations.
REFERENCES 1. Ramos-Alvarez, M., and Sabin, A.: Enteropathogenic viruses and bacteria. Role in summer diarrheal disease of infancy and early childhood, J. A. M. A. 167" 147, 1958. 2. Eichenwald, H. F., Abadio, A., Arky, A. M., and Hartman, A. P.: Epidemic diarrhea in premature and older infants caused by ECHO virus type 18, J. A. M. A. 166: 1563, 1958. 3. Neter, E.: Enteritis due to enteropathogenic Escherichia coli, J. PEDIAT. 55" 223, 1959. 4. Ewing, W. H., Tatum, H. W., and David, B. R.: The occurrence of Escherichia coli serotype associated with diarrheal disease in the United States, Pub. Health Lab. 15" 118, 1957. 5. Gardner, P. S., McGregor, C. B., and Dick, K.: Association between diarrhea and adenovirus type 7, Brit. M. J. 1: 91, 1960. 6. Young, V. M., Lindberg, R. B., Ortiz, A., Jahiel, D., Sochard, M. E., and Hemphill, J. J." Studies of infectious agents in infant diarrhea. III. Bacteriologic, virologic and parasitologic flora of fecal samples collected from Puerto Rican children, Am. J. Trop. Med. 2 (3): 380, 1962. 7. Edwards, P. R., and Ewing, W. tI.: Identification of enterobacteriacease, Minneapolis, 1955, Burgess Publishing Company. 8. Ritchie, L. S., and Frick, L. P.: Unpublished, see Frick, L. P., Moon, A. P., and Lin, S. S.: Parasitologic studies in the Far East. XV. A preliminary survey for parasitism in southern Formosa, Metabolism 5: 302, 1956. 9. Cherry, William B., Thomassen, B. M., Pomales-Lebron, A., and Ewing, W. H.: Rapid presumptive identification of enteropathogenic Escherichia coli in fecal smears by means of fluorescent antibody, Bull. World Health Organization 25: 159, 1961. 10. Syverton, S.: An appraisal of enterovirus problem, Am. J. Trop. Med. 8: I01, 1959.