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Some observations on the pathogenicity of strains of Entamoeba histolytica
606 TRANSACTIONS OF THE ROYAL SOCIETY OF TROPICAL MEDICINE AND HYGIENE.
Vol. 65.
No. 5.
1971.
SOME OBSERVATIONS ON THE PATHOGENICITY OF STRAINS OF ENTAMOEBA HISTOL YTICA V. GOPAL RAO AND M. C. PADMA From The Amoebiasis Research Unit, Department of Pathology, Institute of Medical Sciences, Osmania Medical College, Hyderabad A.P., India Introduction Although amoebiasis is widely prevalent all over the world, its clinical manifestations namely dysentery, liver abscess and other metastatic lesions are common only in tropical and subtropical areas. The incidence of such conditions is negligible in temperate climates (FAtaLlY et al., 1961), although asymptomatic infections seem to be as common as in the tropics ( H o ~ , 1950, 1952; CRAm, 1944; WRIGHT, 1950). The existence of asymptomatic infection was first demonstrated by WALKER and SEI.L~S in 1913, who termed this condition the "carrier state" and divided it into two classes: the convalescents and the contacts. The latter are those who have never suffered from clinical amoebiasis but pass cysts in faeces. The question is whether these amoebae are pathogenic or not; this is of importance from both clinical and academic points of view. At present divergent views are held by workers in different parts of the world (Ho~u~, 1952; CRAIG, 1944; FAUST, 1961). The existence of non-pathogenic strains of amoeba has been recognised since the early part of the century. ELMASSIAN (1909) termed such strains Entamoeba rninuta, and BRUMPT(1925) named them Entamoeba dispar. HOA~ (1947, 1949, 1950, 1952, 1957 and 1958) considered such non-invasive amoebae as being in a commensal phase and called them 'minuta' forms of Entamoeba histolytica. On the other hand, Cram (1944) and FAUST (1961) held the view that E. histolytica was an obligatory tissue parasite although the severity of the lesions might vary. The WHO Expert Committee (1969) in its report recommends the term 'asymptomatic infection' be substituted for the term 'carrier' on the grounds that "while infection without tissue invasion is recognised to be a common occurrence it cannot yet be distinguished from asymptomatic tissue invasion." On clinical grounds alone such a fine distinction between invasive and non-invasive amoebae appears almost impossible. Studies carried out in this Department over the last few years have revealed wide prevalence of various types of clinical amoebiasis as well as a high rate of asymptomatic infection. As the disease is endemic in this part of India, it was essential to know whether the amoebae found in asymptomatic infections were potentially pathogenic and thus contribute towards the morbidity, or existed as harmless commensals. Therefore, in the course of the present investigation 6 strains of E. histolytica isolated from asymptomatic patients were studied for their pathogenic effect on albino rats along with 4 strains isolated from patients with amoebiasis, namely acute dysentery and liver abscess. This investigation was a part of the collaborative research project between the Osmania Medical College, Institute of Medical Sciences, Hyderabad A.P. (India) and the National Institutes of Health, U.S.A. Funds were provided under Foreign Research Agreement No. 354308.
V. GOPAL RAO AND M. C. PADMA
607
M a t e r i a l s and m e t h o d s S t r a i n s o f E. h i s t o l y t i c a A total number of 139 strains of E. histolytica and 47 strains of E. hartmanni (PRowAzEK, 1912) were isolated in culture. The two species were distinguished by the authors on the basis of average cyst diameter, namely a diameter of 10 ~t or above as E. histolytica, and less than 10 ~ as E. hartmanni. This paper presents observations on pathogenicity of E. histolytica (large race) only. The routine medium employed in the cultivation of E. histolytica was the N I H modification (USPHS, 1948) of BOEeK and DRBOHLAV (1925), consisting of whole egg slant in Ringer's solution overlaid with modified Locke's solution with a pH of 7.0, to which a loopful of sterile rice starch was added before inoculation. T o prevent excessive growth of bacteria, the addition of 1.0 ml. of 1 : 500 sterile aqueous solution of acriflavin to each tube was found satisfactory. For inoculation into animals a 48 hour-old culture was harvested, pooled and concentrated by centrifugation at 1000 r.p.m, in the international centrifuge using head No. 240, for 5 minutes. The final inoculum thus prepared showed 3-4 × 106/ml. actively motile trophozoites. The amoebae were suspended in phosphate-buffered saline (pH 7.2) and kept at 37°C. before inoculation. All the amoebae cultures used for inoculation had mixed bacterial flora derived from human faeces. Experimental animals Weanling albino rats were used throughout. A stock of these rats was obtained from Nutrition Research Laboratories, Hyderabad, where they have been inbred over the past 35 years. The precise origin of the strain is not known. The animal colony was maintained on a stock diet consisting of the following ingredients: Wheat flour 40 g. Skim milk powder 5 g. Casein 7 g. Roasted Bengal gram 39 g. Ground nut oil 5 ml. Balanced salt mixture 4 g. Vitamin and choline mixture 0.2 g. Lucerne grass 2 g. T h e diet and water were given ad libitum. T h e litters were weaned at 4 weeks and placed on the stock diet or diet A or B, the composition of which is shown below (RAo, 1951):
Diet A 1. 2. 3.
Diet B
Protein (Casein) 20 g. 10 g. Dried yeast 5g. 2.5g. 1.0 ml. 0-5 ml. Fat soluble vitamin mixture consisting of: Vitamin A: 2,000 units Vitamin D : 10,000 units Vitamin E: 340 mg. Oleic acid: 100 ml. 4. Choline chloride 0"2g. 0"lg. 5. Vitamin K 0 "2 mg. 0" 1 mg. 6. Vitamin E 0.0 17 "0 mg. 7. Groundnut oil . 5.0g. 2"5g. 8. Balanced salt mixture 4"0g. 4.0g. 9. Potato starch 64.8 g. 80"4 g. Diet A is well balanced as regards protein and vitamins, while diet B is deficient in both. The animals reared on diet A showed healthy fur and normal growth curves, whereas those reared on diet B showed coarse and relatively scanty fur. The A group animals were alert and agile while the B group were sluggish, and weighed less (RAo, 1951). T h e weanlings were reared on these diets for 1-4 weeks prior to inoculation with E. histolytica. The age at inoculation varied from 5-8 weeks. T h e weight of the animals at the time of inoculation with E. histolytica was usually from 50.0 to 60.0 g. Both sexes were used in approximately equal numbers.
608
SOME OBSERVATIONS ON THE PATHOGENICITY OF STRAINS OF E N T A M O E B A H I S T O L Y T I C A
Method o f inoculation A method modified from GO0DWI~ et al. (1948) described earlier by one of us (RAo, 1951) was followed. The animals were anaesthetized with ether and laparotomy done under aseptic conditions with a right paramedian incision in the lower abdomen. The caecum was exposed and 1.0 ml. of culture suspension containing about 3-4 × 10e/m1. amoebae was inoculated with 22 gauge needle towards the blind end of the caecum. The abdominal wound was closed by suturing in layers and a light dressing applied. Pre-operatively, the animals were kept fasting overnight and were given 0-8 units of penicillin and 1 mg. of streptomycin intramuscularly to prevent wound sepsis. After operation, only glucose water was given for 24 hours. Thereafter, the usual diet was resumed. The animals were weighed twice a week and any change in weight was recorded. The animals dying within 72 hours of intracaecal inoculation were not taken into account. T h e survivors were killed between 8-10 days after inoculation. A complete autopsy was performed on each animal. T h e whole of the large gut from caecum downwards was slit open and the nature of the wall and the contents of the lumen were noted. The scoring of the lesions of the caecum was recorded in the following way (GogDM~'q, 1965): Grade 0.0 = No amoebae, no lesions. ,, 0.5 = Amoebae present, no lesions. ,, 1.0 : 1 to 10 discrete lesions, normal mucosa in between. ,, 2.0 = More than 10 lesions, less than 25% of the caecum involved. ,, 3.0 = Confluent lesions, 25-50% of caecum involved. ,, 4.0 = Confluent lesions, 50-100% of caecum involved. The amoebae were always present both in scrapings and in the contents, in the last four grades. For each strain of E. histolytica tested, the pathogenicity index was calculated by the authors as below : - Pathogenicity index
Actual score obtained on total number of animals infected Expected maximum score on the same n u m b e r of animals
For example, if the number of animals infected surviving 3 days or more is 5, and the sum total score of lesions on these animals is actually 15, then the expected maximum score would be 5 × 4.0 = 20. The pathogenicity index would be 15/20 = 0.75. I n addition to this, the average caecal score with the standard error (S.E.) was calculated for each strain. Immediately after opening the animal, smears in warm saline were made of the caecal contents and also of scrapings from the caecal wall for microscopic examination. As a rule, apart from the wet preparations, these smears were wet-fixed in Schaudirm's fluid and stained by trichrome procedure (USPHS, Loc. cir.) to confirm the morphology of amoebae observed. I n addition to microscopic examination of caecal contents and mucosal scraping-s, cultures were put up in the usual medium. Occasionally, in low grade infections, blind cultures were positive when the direct microscopy had failed to reveal the amoebae. The caecum was fixed in 10% formol-saline and representative blocks of tissue were processed for histology. Routinely, serial sections were stained with Ehrlich's haematoxylln and aqueous eosin. Heidenhain's iron haematoxylin staining was done where necessary (A.F.I.P., 1960).
Case histories (1) Strain No. 2567/66:--Case No. P 258, male aged 40 years, admitted for painful swelling in the right hypochondrium of 10 days duration. No preceding history of dysentery or diarrhoea; patient poorly nourished; tender liver palpable up to 3½ in. below the costal margin. A clinical diagnosis of amoebic liver abscess was made and aspiration done which yielded typical anchovy sauce pus. Microscopically, the pus showed large trophozoites of E. histolytica with ingested red cells. Culture of this pus yielded growth of the above strain. T h e age of the culture at the time of animal inoculation was 4-5 months. (2) Strain 324/65 :---Case No. P 33, male patient aged 20 years, admitted with signs and symptoms of acute dysentery. He gave a history of recurrent attacks of dysentery during the last 6 years. Faeces showed trophozoites of E. histolytica with ingested red cells. The strain isolated from the dysenteric stool was used for inoculation after in vitro cultivation for 3-5 months.
V. GOPAL RAO AND M. C. PADMA
609
(3) Strain 1713/66:pCase No. P 216, male aged 20 years, admitted for acute dysentery of 8 days duration. Faeces showed numerous trophozoites with ingested red cells. The strain was used for animal inoculation after 2-3 months after isolation. (4) Strain 1444/66:--Case No. P 201, male aged 25 years, admitted with signs and symptoms of subacute dysentery of 30 days duration. Faeces were typically dark reddishbrown in colour and showed numerous haematophagous trophozoites. The strain was used 5-6 months after isolation. (5) Strain 3/64:--Case No. SM 60, male child aged 5 years, admitted for hepatomegaly and ascites. A clinical diagnosis of Indian child_hood cirrhosis was made. The patient had no diarrhoea or other intestinal disturbances. The strain was isolated from faeces showing trophozoites without ingested red cells. Animal inoculation done after 1-2 months of cultivation. (6) Strain 2246/65:--Case No. SF 400, female aged 55 years, admitted for diabetic nephropathy; there were no signs of gastro-intestinal disturbances but cysts of E. histolytica were found in faeces. The average diameter of the cysts was 11"2 ~t. The strain was isolated from these cysts and inoculated into animals 1-3 months after isolation. (7) Strain 1797165 :---Case SF 345, female aged 45 years, admitted for benign essential hypertension. No history of diarrhoea or dysentery. The patient did not have any gastrointestinal disturbances. Cysts of E. histolytica measuring an average diameter of 14-4 were found in faeces. Animal inoculation was done 2 years after isolation. (8) Strain 686/66 :--Case No. SM 750, male aged 55 years, admitted for haemoptysis and cough. Investigations revealed bilateral pulmonary tuberculosis. There was no history of dysentery or diarrhoea nor any gastro-imestinal disturbances. The faeces showed cysts measuring 11.68 lz in average diameter. Experiments were performed one year after isolation of the strain from the faeces. (9) Strain 2060/65:--Case No. SF 383, female aged 30 years, admitted for severe anaemia. There was moderate splenomegaly which was attributed to chronic malaria. The patient had no gastro-intestinal disturbances nor any history of diarrhoea or dysentery. Faeces showed trophozoites of E. histolytica without ingested red cells. Animal inoculation was done 2 years after isolation. (10) Strain 500/68 :--Case No. SF 914, student nurse physically fit, with no history of dysentery or diarrhoea. Cysts of E. histolytica measuring an average diameter of 11.02 ~t were found in faeces. Animal inoculation was done 1-4 months after isolation. As a rule, control rats were inoculated with the bacterial flora of the respective amoebic strains.
Results T h e results of experiments performed on weanling rats are presented in Table I. O f the 10 strains of E. histolytica, No. 1 was from a patient with liver abscess, Nos. 2, 3 and 4 were from acute or subacute amoebic dysentery and the remaining 6 were from asymptomatic infections. T h e Table shows that there is considerable variation in infectivity as well as in pathogenicity amongst these strains. T h e infectivity ranged from 34-8% to 85.7% and the average caecal score varied from 0.75 to 3.25. T h e standard error for each strain was calculated on the number of infected animals. T h e pathogenicity index which runs closely parallel to the average caecal score, ranged from 0-20 to 0-81. T h e strain isolated from the liver abscess (No. 2567/66) gave the highest infectivity rate as well as highest caecal score and pathogenicity index. T h e 3 strains isolated from acute or subacute dysentery also gave high pathogenicity indices although their infectivity varied from 41% to 66%. O f the 6 strains isolated from asymptomatic infections, (serial Nos. 5 to 10) the infectivity ranged from 34.8% to 75%, the average caecal score from 0.75 to 2.5 and the pathogenicity index from 0.20 to 0-63. Amongst these 6 strains from asymptomatic cases, 2 showed the lowest pathogenicity index (strains 1444/66 and 3/64), the strain (1797/65) a moderate figure, but the remaining 3 strains showed pathogenicity indices comparable to or even higher than those from acute
610
SOME O B S E R V A T I O N S O N T H E P A T H O G E N I C I T Y O F S T R A I N S O F E N T A M O E B A H I S T O L Y T I G A
dysentery strains. The control rats inoculated with bacterial flora did not show any lesions in any of the cases.
Morbid anatomy of amoebic lesions The gross appearance of the caecum varied according to the degree of infection. It could be categorized into 5 types according to the extent and severity of the lesions. (1) Caecum normal in appearance externally, but on opening the lumen showed semisolid or liquid and occasionally frothy faeces; the mucous membrane showed on clearing the contents mild hyperaemia only. (2) Caecum similar to the above condition but mucosa showed fine discrete ulcers usually at the blind end. (3) Caecal contents frankly mucoid but usually associated with liquid faecal material. The mucosa showed more extensive ulceration involving the whole caecum but ulcers were still discrete and fine. (4) Caecum usually shrunken and thickened; ulcers seen from the serous surface. On opening abundant mucoid material with little or no faeces, ulcers usually confluent. (5) Caecum markedly shrunken, often thickened in places, and adherent to surrounding loops of bowel and parietal peritoneum. On opening no normal mucous membrane found; instead diffuse and deep ulceration was observed. In some instances, perforation with gangrene was also seen. Such ulcers were filled with black sloughs. The caecal wall in these cases was very soft and friable and adherent to surrounding structures. In all the above 5 categories, scrapings and contents showed haematophagous amoebae with active motility. Histopathology A wide range of lesions was observed from an early pre-ulcerative stage to small superficial ulcers. More extensive ulcers involving only the mucosa; deeper ulcers with the destruction of the muscular layers often associated with intense granulomatous reaction and finally, acute or chronic perforations. A brief account of these will now be given. (a) Pre-ulcerative stage. The mucosa showed hyperaemia, oedema and catarrhal desquamation of the surface epithelium with numerous amoebae in the crypts of Leiberldihn. No necrosis or ulceration seen at this stage nor any inflammatory exudate. This corresponds to grade 1 of caecal score. (b) Early superficial ulcer. Necrosis of the crypts and lamina propria of the mucosa with shedding of the necrotic material into the lumen. Amoebae confined to the mucous membrane both in the ulcer as well as in the adjoining healthy part with no inflammatory exudate. This corresponds to grade 2 of naked eye score. (c) Extensive superfidal ulceration of the mucosa. The mucous membrane was markedly hyperaemic and showed extensive necrosis with ulceration, the process stopping short at the muscularis mucosae. N o inflammatory response. Amoebae found both in the ulcers as well as in the adjoining mucous membrane. This condition corresponds to grade 3 of the caecal gross picture. (d) Deep ulcers. Ulceration involved the entire thickness of the mucous membrane and the submucosa; abundant amoebae were found in the necrotic slough as well as in the wall of the ulcer formed by intact mucous membrane. The submucosal layer at this
V. GOPAL RAO AND M. C. PADMA
611
stage showed intense granulomatous reaction, the cells being mainly histiocytes, large mononuclear cells, lymphocytes and few eosinophils. There was no neutrophilic response. In some cases there was marked hyperplasia of lymphoid follicles. (e) Deeper ulcers with chronic perforations. In these the muscular coat was replaced by granulation tissue heavily infiltrated with chronic inflammatory cells described in the preceding stage. In these cases the serosa was also granulomatous with adhesions to surrounding structures. Amoebae were found in the ulcers as well as in the adjoining mucous membrane. Histological descriptions of (d) and (e) correspond with grade 4 of the naked eye score. (f) Acute perforations with gangrenous change. Sections of the ulcer revealed the base of the ulcer formed by a thin ribbon of serous layer with extensive necrosis and gangrene of all the layers above. The blood vessels in the adjoining part showed thrombi. Amoebae were found both in the gangrenous slough although scanty in number as well as in the adjoining mucosa. (g) Healed ulcers. In addition to the above, in milder cases of caecal ulceration of grade 2 or 3, there were healed ulcers. Here the mucosa was replaced by granulation tissue with a thin ribbon of columnar epithelium covering it on the surface. Such ulcers did not show amoebae. Such healing lesions were usually observed in animals autopsied 12-15 days after inoculation. On analysing the histopathological findings observed with the 4 strains isolated from clinical cases of amoebiasis, namely, serial Nos. 1-4, it was found that the No. 1 strain, which gave the highest caecal score and highest pathogenicity index (Table I), produced in rats widespread caecal lesions, often deep and penetrating, and in one rat chronic perforation. The 3 strains isolated from cases of dysentery also produced fairly widespread lesions but the ulcers were not deep or penetrating. The strains isolated from asymptomatic injections, on the other hand, showed a wide spectrum of lesions corresponding to the gross lesions as shown in Table i. Of the 6 strains from asymptomatic infections 3 strains, serial Nos. 5, 6 and 9 showed only lesions of mild to moderate severity. The ulcers in general were very superficial and confined to the mucosa only. Two strains, serial Nos. 7 and 8 gave deeper lesions extending into the submucosa with marked granulomatous reaction. However, most extensive lesions were produced by a strain isolated from a student nurse who was normal clinically and gave no history of diarrhoea or dysentery at any time. This strain (500/68), although showing low infectivity, produced very widespread and deep ulcers including gangrenous ulcers with perforations in 2 out of 8 infected animals. These gangrenous ulcers contained jet-black sloughs comparable to "dyak hair" ulcers seen in humans with gangrenous ulcers. It is noteworthy that such lesions were produced by a strain of amoeba isolated from an asymptomatic infection. As described earlier none of the amoebic ulcers whether superficial or deep, showed any neutrophilic exudate or other features of bacterial invasion such as suppuration. The experiments on the r61e of host nutrition in the pathogenesis of intestinal amoebic lesions are still incomplete. The data obtained on limited number of animals reared on balanced diet A as compared to those reared on deficient diet B are presented in Table II. As the number in each dietary group is small it is premature to draw any conclusions on a quantitative basis. Therefore, in Table I the overall results have been shown for purposes of assessment of pathogenicity of individual strains. However, it may be mentioned that in general, deeper and widespread lesions were observed in group B animals with strains obtained both from pathogenic and symptomless infections as compared to those seen in group A animals. The two rats which showed gangrenous
612
SOME OBSERVATIONS O N T H E P A T H O G E N I C I T Y O F STRAINS OF E N T A M O E B A
HISTOLYTICA
ulcers with strain No. 500/68 obtained from asymptomatic infection were indeed reared on diet B. However, experiments on larger series of animals are required to establish a quantitative relationship between host nutrition and the severity of the caecal lesions. "ABLE I. Results of intracaecal inoculation of E. histolytica cultures into y o u n g albino rats.
SI. No.
Strain No.
Clinical category of pati-~nts f r o m w h o m steams of E. hist :lytica isolated
No. surviving for 72 No. infected hours or No. of rats with inoculated more* percentage
Average caecal score with S.E.
Pathogenicit~ index
1
2567/66
Amoebic liver abscess
12
7
6 (85' 7)
3'25±0"52
0"81
2
324/65
Acute dysentery
22
18
12 (66"6)
1"8
±0.462
0"45
±0.61
O' 50
3
1713/66
I Acute dysentery
15
12
5 (41.6)
2"0
4
1444/66
Acute dysentery
15
7
3 (42.9)
2"33±0.54
5
3/64
A s y m p t o m a t i e infection
14
11
6 (54" 5)
0"75±0'102
O" 20
6
2246/65
do
8
7
4 (57.1)
0'75±0"125
0"20
7
1797/65
do
8
4
3 (75- O)
1'16±0.121
O" 30
8
686/66
do
12
11
7 (63.6)
2'14±0"47
0"54
9
2060/65
do
11
8
5 (62.5)
2"3
±0"65
0"58
do
42
23
8 (34.8)
2"5
±0-641
0"63
10
500/68
0"56
# T h e animals dying in the first 72 hours after inoculation were not taken into account. T h e deaths were due m a i a l to post-operative shock or other causes unrelated to amoebic infection. TABLE I I . Results of intracaecai inoculation of cultures of E. histolytica into y o u n g albino rats.
$1. No.
Strain No.
Clinical category of patients f r o m w h o m strain was isolated Amoebic liver abscess
N o . of rats Proportion of inoculated and animals infected survived 72 hours Stock D i e t D i e t or longer diet A B
Pathogenicity index
Average caecal score of lesions in infected animais with S.E. Stock diet
Diet A
Di~ B
Stock D i e t diet A
Diet B
i
2567/66
--
0-63
0.75
2
324/65
Acute amoebic dysentery
18
12/18
:1.8
±0.462
0'45
--
--
3
1713/66
Acute amoebic dysentery
12
5/12
2"0
±0-61
0" 50
4
1444/66
Acute amoebic dysentery
7
3/7
2-33±0"54
0"56
5
3/64
A s y m p t o m a t i c infection
11
6/il
0"75±0-102
0 ' 20
6
2246/65
do
7
4/7
7
1797/65
do
4
--
7
3/3
--
3/4
2.5±0"85
4'0
±0
0"75±0'125 1/2
2/2
2"0
0'75±0"25
0'20
--
--
--
0.50
0.19
8
686/66
do
11
--
4/6
3/5
2.37±0.851.83±0.62
--
0'59
0"45 i
9
2060/65
do
8
--
4/4
1/4
2.37±0"822"0
--
0'59
0"50
10
500/68
do
23
--
4/14
4/9
1-25±0'503'75±0'10
--
0"32
0"93
Discussion Many studies have been carried out in different parts of the world on pathogenicity of strains of E. histolytica. As mentioned earlier, there is divergence of views regarding its pathogenic potential. It is essential at this stage to make a distinction between pathogenicity and virulence. The former refers to a general attribute of micro-organisms with regard to their capacity to produce disease while the latter generally indicates "the degree of pathogenicity of a given type, strain or a cldone as observed in a particular host species under defined conditions." (MILES, 1955). The results of the experiments reported here on 4 strains of E. histolytica isolated from patients with dysentery or liver abscess and 6 from asymptomatic infections showed
V. GOPAL RAO AND M. C. PADMA
613
that there was a considerable variation in virulence and pathogenicity. The strain from the liver abscess gave the highest caecal score and pathogenicity index (3.25 and 0.81 respectively), while the dysentery strains gave moderate degrees of virulence and pathogenicity index (Table I). All the 4 strains produced deep-seated lesions and the one from the liver abscess produced chronic perforation. The 6 strains isolated from asymptomatic infections gave a range of virulence and pathogenicity indices from 0.75 to 2.5, and 0.20 to 0-63 respectively. Of the 3 strains showing low virulence (Serial Nos. 4, 5 and 6) one strain No. 1797/65 produced deep-seated lesions involving the submucosa and part of the muscular layer. Moreover 3 other strains giving high caecal score and high pathogenicity index (0.54 to 0.63) produced penetrating lesions involving deeper layers, while one strain 500/68 produced classical gangrenous ulcers with perforations. Only 2 strains serial Nos. 4 and 5 gave low caecal score and showed only superficial mucosal erosions microscopically. These may be caUed relatively low virulent strains. Nevertheless, it is clear that 4 of the 6 strains isolated from asymptomatic infections produced lesions qualitatively and quantitatively comparable to those produced by strains isolated from dysenteric stools or liver abscess material. It is thus obvious that these strains could not be dismissed as commensals simply because at the time of isolation no lesions were clinically detected in the human host. A perusal of available literature on pathogenicity of strains of E. histolytica isolated from asymptomatic infections shows that a number of workers from East European countries and United States have found such strains to be pathogenic generally, while NEAL (1951, 1957, 1958, 1960 and 1961) in England and HUNNINEN and BOONE(1957) in America found "carrier" strains to be avirulent or non-invasive. On the other hand ROGOVA(1956), on the basis of her experiments with strains isolated from clinical as well as non-clinical cases, found the infectivity rate for guinea-pigs was the same with both categories, but the severity of lesions varied. However, she observed that strains from asymptomatic infections had produced deep-seated ulcers in over 20% of the test animals. SWELLENGREBELand RIJI'STRA (1958) pointed out that E. histolytica found in the commensal phase was capable of producing infection with clinical manifestations including acute dysentery. KRUPP and FAUST (1959) reported tissue invasion with ulceration in guinea-pigs with the 14 strains isolated from asymptomatic infections. KASPRZAK(1961) reported that 2 strains isolated from symptomless "carders" both of local and Vietnamese origin were found to produce caecal ulceration in guinea-pigs. MIZGIREVA(1966) experimented with 78 strains of E. histolytica isolated from different clinical categories of amoebiasis including 10 strains from "symptomless carriers." Although the virulence was variable with individual strains all the strains including those from "symptomless carriers" were pathogenic. She emphasizes the importance of host factors in determining the virulence of the parasite. NEAL in a series of papers (1951, 1955, 1956, 1957 and 1961) reported his observations on experiments on rats with different strains of large race E. histolytica. The rSle of associated bacterial flora was examined and also the effects of serial passages into the livers of hamsters on the virulence of amoebae. He has based his conclusions as regards the virulence of a given strain on the basis of average caecal score. Any strain giving a caecai score of 2.0 or less was considered as avirulent and the one giving above 2.0 was considered virulent. The papers do not report the histopathological observations, nor was there any attempt to classify the lesions on that basis. His conclusions were: (1) The amoebae and not the associated bacterial flora were responsible for tissue invasion and virulence of the strain; (2) There was a radical difference as regards virulence between the strains found in asymptomatic "carriers" and those isolated from cases of clinical amoebiasis. The former were avirulent giving a caecal score of less than 2.0, while the latter were
614
SOME OBSERVATIONS ON THE P A T H O G E N I C I T Y OF STRAINS OF E N T A M O E B . 4 H I S T O L Y T I C A
virulent giving scores well over 2.0. However, there were exceptions to this generalization in as much as certain strains isolated from asymptomatic "carriers" gave a high caecal score. NEAL (1957) explained this by postulating that "infection of man with a virulent type must pass through a quiescent phase when disease does not occur, in order to ensure survival by the production of cysts. This might explain the isolation of virulent strains from "carriers"." Such an explanation could hardly lead one to an understanding of the rather complex problem of the host-parasite balance in amoebiasis. It should be emphasized that no morphological differences were pointed out between virulent and avirulent amoebae. Therefore, the question arises as to how to decide whether a given strain of E. histolytica in a given patient is of clinical significance or not. In such cases the physician gets no guidance from the laboratory as to whether a patient should be treated by specific drugs or not. More important still is the public health implication of the so-called asymptomatic infections particularly with reference to their r61e in spreading clinical amoebiasis in the community. NEAL (1957) has answered this question by stating that "apart from inoculation of rats there is no way of d e c i d i n g . . . " It may be further noted that avirulent strains isolated by Neal were not only of local origin but some were derived from the tropics also. Conversely, strains isolated from asymptomatic infections in the tropics were found by him to be virulent to rats (BIRD and NEAL, 1962). While discussing the variations in virulence NEAL (1958) suggested that "invasive amoebae may arise by adaptation from the normal avirulent amoebae." From the results of experiments reported here and also from the review of literature it is clear that there can be no sharp and clear-cut distinction between E. histolytica found in asymptomatic infections and the haematophagous amoebae found in clinical cases in so far as the potential capacity for production of disease is concerned. As regards the r61e of bacteria the histological studies presented here revealed even at the earliest stage of tissue invasion by E. histolytica, the lesions consisted of coagulation necrosis of mucous membrane followed by liquefaction and shedding. The host's tissue response was mainly that of exudation of mononuclear cells and lymphocytes. In some there was marked lymphoid hyperplasia. No nentrophilic response or fibrinous exudate was observed to suggest bacterial intervention at any stage. Mention may be made here of the observations of NEAL and VINCENT(1955 and 1956) who showed.that the virulence of E. histolytica was not dependent on the associated bacterial flora but was due to the amoeba itself. Very little is known about the precise pathogenefic mechanisms involved in the production of lesions by E. histolytica. CRAIG (1927) demonstrated cytolytic, haemolytic and complement-fixing activity in alcoholic extracts of amoebae by in vitro experiments. The cytolytic activity of such an extract was not confirmed in vivo. NEAL (1960) demonstrated proteolytic activity in amoebic extracts, but this was not correlated with invasivehess (BIRD and NEAL, 1962). MAEGRAITH(1963) in his exhaustive review on pattlogenefic mechanisms in amoebiasis reported his own observations on the proteolytic enzymes and hyaluronidase activity in strains of E. histolytica. However, these observations have not led to a clear understanding of the mechanism of tissue invasion. In this connection the observations of one of the authors (RAO, 1951) may be mentioned. It was shown that filtrates of cultures of E. histolytica grown with a single bacterial associate, when inoculated into the portal vein of young rats resulted in the production of coagulation necrosis of hepatic parenchyma. The lesions were self-limiting and healed in about 4 days. The control animals inoculated with bacterial flora did not show any such necrotic lesions. It appears as though the amoebae when growing in culture produce a necrotizing factor the nature of which has yet to be ascertained. It has been observed in the present experiments that amoebae multiply in the crypts and necrosis occurs subsequently. This
V. GOPAL RAO AND M. C. PADMA
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initial multiplication of amoebae with subsequent production of necrotizing factor(s) may well be responsible for production of mucosal lesions. There are not many critical studies on the r61e of host nutrition in the pathogenesis of amoebiasis. ANN (1958) experimenting on kittens found that diets supplemented with meat and milk produced milder infection with E. histolytica as compared to animals not receiving these supplements. FAUST (1961) reviewed the various host factors influencing the course of infection with E. histolytica and considered that nutrition was an important factor. The results of our preliminary study on the r61e of host nutrition have dearly indicated that the weanling rats reared on low protein-low vitamin diet (diet B) developed more severe and extensive lesions including gangrenous ulcers than the controls reared on a balanced diet. However, more studies to elucidate the exact role of nutrition in enhancing the severity of the lesions and the mechanisms involved therein are needed.
Summary 1. Results of animal inoculation experiments on young albino rats with different strains of E. histolytica isolated from different patients with clinical amoebiasis, such as acute dysentery and liver abscess, as well as those isolated from asymptomatic infections have been presented and discussed. 2. Virulence and pathogenicity showed considerable variation both among the strains isolated from clinical cases as well as from asymptomatic infections. In the former group the liver abscess strain gave highest caecal score and pathogenicity index while the dysentery strains gave moderate figures. Of the 6 strains from asymptomatic infections 3 showed virulence and pathogenicity indices as high as strains from clinical cases. One of these strains showed moderate virulence and the remaining two strains showed lowvirulence of less than 1.0. 3. The morbid anatomy and histology of the lesions have been described in detail. The lesions varied from superficial ulcers involving only the mucosa to deep ulcers penetrating the submucous and muscular coats. In some, even perforations associated with gangrene were observed. It is to be noted that the gangrenous lesions were caused by a strain isolated from a healthy nurse without any symptoms or history of amoebiasis. Further, even the strains showing low virulence showed necrosis with ulceration of the mucosa and one strain produced deep lesions even though the caecal score was low. 4. The pathogenesis of the amoebic lesions in the gut have been discussed. It has been shown that amoebae multiply in the crypts initially with subsequent production of necrosis followed by ulceration. The possible existence of a necrotizing factor produced by amoebae has been raised. 5. Limited observations on the r61e of nutrition in the course of experimental amoebic infection in rats have shown that low protein-low vitamin diets aggravate the lesions as compared to the reaction seen in animals reared on a balanced diet. REFERENCES ANN, J. M. (1958). Am. ft. trop. Med. Hyg., 7, 158. ARMED FORCES INSTITUTE OF PATHOLOGY (A.F.I.P.), Washington D.C. (Mary Francis Gridley Memorial Fund), 1960. Manual of Histologic and Special Staining Technics. 2nd Ed. The Blakingston Division, McGraw-Hill Book Company Inc. BIRD, R. G. & NEAL, R. A. (1962). Trans. R. Soc. trop. Med. Hyg., 56, 87. BOECK, W. C. & DRBOHI.AV,J. (1925). Am. ft. Hyg., 5, 371. BI~UMPT, E. (1925). Bull. Acad. Med., 94, 943. CRAIG, C. F. (1927). Am. ft. trop. Med., 7, 225.
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(1944). The Etiology, Diagnosis and Treatment of Amoebiasis. 1st Ed. Baltimore: Williams and Wilkins Co. ELMASSIAlq,M. (1909). Centralb. Bakt. Abt. (1 Orig.), 52, 335. FAIRLEY,N. H., WOODRUFF,A. W. & WALTERS,J. H. (1961). In: Recent Advances in Tropical Medicine. 3rd Ed. London: J. & A. Churchill Ltd. FAUST, E. C. (1961). Clinical Parasitology. 7th Ed. Philadelphia: Lea and Febiger. GOODWlN, L. G., HOARE,C. A. & SHARP,T. M. (1958). Br. ft. PharmacoL Chemother., 3, 44. HOARE, C. A. (1947). Trans. R. Soc. trop. Med. Hyg., 41, 87. (1949). In: Handbook of Medical Protozoology. London: Bailliere, Tindall and Cox. (1950). Br. med. J., 2, 238. (1952). Exp. Parasit., 1, 411. (1957). Trans. R. Soc. trop. Med. Hyg., 51, 303. • (1958). Rice lnst. Pamphl., 4,5, 23. HrYNNINEN,A. V. & BOONE, H. A. (1957). Am. J. trop. Med. Hyg., 6, 32. KASPRAZAK,W. (1961). Acta Parasit. Polonica, 9, 211. KRUPP, I. & FAUST, E. C. (1959). J. Parasit., 45, 449. MAEGRAITH, B. G. (1963). In: Immunity to Protozoa. Oxford: Blackwell Scientific Publications. MILES, A. A. (1955). In: Mechanisms of Microbial Pathogenicity. New York: Cambridge University Press. MIZGIREVA,M. F. (1966). Med. Parasitol. Parasitic. Dis., Moscow, 35, 202. NEAL, R. A. (1951). Trans. R. Soc. trop. Med. Hyg., 44, 439. (1956). Parasitology, 46, 183. (1957). Trans. R. Soc. trop. Med. Hyg., 51, 313. (1958). Proc. 6th lnt. Congr. trop. Med. Malar., 3, 350. (1960). Parasitology, 50, 531. (1961). Trans. R. Soc. trop. Med. Hyg., 44, 439. & VINCENT, P. (1955). Parasitology, 4.5, 152. & (1956). Ibid., 46, 173. PROWAZEK, S. V. (1912). Weiterer Beitrag Zur Kenntnis der Entamoben Arch. F. Protistenk. X X V I , 241. RAO, V. G. (1951). Thesis submitted to the University of London for the degree of Ph.D. ROGOVA, L. I. (1956). Med. Parasit. Parasitic Dis., Moscow~ 2.5, 330. SWELLENGREBEL, N. H. & RIJPSTRA, A. C. (1958). Ned. Tijdschr. U. Geneesk., 102. UNITED STATES PUBLIC HEALTH SERVICE (USPHS) publication C.D.C. Bulletin (1948). Laboratory Diagnosis of Parasitic Diseases. WALKER, E. L. & SELLARDS,A. W. (1913). Philipp. J. Sci., 6, 259. WORLD HEALTHORGANISATIOt;(1969). Techn. Rep. Ser., 49.1, p. 8. WRIGHT, W. H. (1950). Am. ft. trop. Med., 30, 123.
Vol. 65.
No. 5.
1971.
SOME OBSERVATIONS ON THE PATHOGENICITY OF STRAINS OF ENTAMOEBA HISTOL YTICA V. GOPAL RAO AND M. C. PADMA From The Amoebiasis Research Unit, Department of Pathology, Institute of Medical Sciences, Osmania Medical College, Hyderabad A.P., India Introduction Although amoebiasis is widely prevalent all over the world, its clinical manifestations namely dysentery, liver abscess and other metastatic lesions are common only in tropical and subtropical areas. The incidence of such conditions is negligible in temperate climates (FAtaLlY et al., 1961), although asymptomatic infections seem to be as common as in the tropics ( H o ~ , 1950, 1952; CRAm, 1944; WRIGHT, 1950). The existence of asymptomatic infection was first demonstrated by WALKER and SEI.L~S in 1913, who termed this condition the "carrier state" and divided it into two classes: the convalescents and the contacts. The latter are those who have never suffered from clinical amoebiasis but pass cysts in faeces. The question is whether these amoebae are pathogenic or not; this is of importance from both clinical and academic points of view. At present divergent views are held by workers in different parts of the world (Ho~u~, 1952; CRAIG, 1944; FAUST, 1961). The existence of non-pathogenic strains of amoeba has been recognised since the early part of the century. ELMASSIAN (1909) termed such strains Entamoeba rninuta, and BRUMPT(1925) named them Entamoeba dispar. HOA~ (1947, 1949, 1950, 1952, 1957 and 1958) considered such non-invasive amoebae as being in a commensal phase and called them 'minuta' forms of Entamoeba histolytica. On the other hand, Cram (1944) and FAUST (1961) held the view that E. histolytica was an obligatory tissue parasite although the severity of the lesions might vary. The WHO Expert Committee (1969) in its report recommends the term 'asymptomatic infection' be substituted for the term 'carrier' on the grounds that "while infection without tissue invasion is recognised to be a common occurrence it cannot yet be distinguished from asymptomatic tissue invasion." On clinical grounds alone such a fine distinction between invasive and non-invasive amoebae appears almost impossible. Studies carried out in this Department over the last few years have revealed wide prevalence of various types of clinical amoebiasis as well as a high rate of asymptomatic infection. As the disease is endemic in this part of India, it was essential to know whether the amoebae found in asymptomatic infections were potentially pathogenic and thus contribute towards the morbidity, or existed as harmless commensals. Therefore, in the course of the present investigation 6 strains of E. histolytica isolated from asymptomatic patients were studied for their pathogenic effect on albino rats along with 4 strains isolated from patients with amoebiasis, namely acute dysentery and liver abscess. This investigation was a part of the collaborative research project between the Osmania Medical College, Institute of Medical Sciences, Hyderabad A.P. (India) and the National Institutes of Health, U.S.A. Funds were provided under Foreign Research Agreement No. 354308.
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M a t e r i a l s and m e t h o d s S t r a i n s o f E. h i s t o l y t i c a A total number of 139 strains of E. histolytica and 47 strains of E. hartmanni (PRowAzEK, 1912) were isolated in culture. The two species were distinguished by the authors on the basis of average cyst diameter, namely a diameter of 10 ~t or above as E. histolytica, and less than 10 ~ as E. hartmanni. This paper presents observations on pathogenicity of E. histolytica (large race) only. The routine medium employed in the cultivation of E. histolytica was the N I H modification (USPHS, 1948) of BOEeK and DRBOHLAV (1925), consisting of whole egg slant in Ringer's solution overlaid with modified Locke's solution with a pH of 7.0, to which a loopful of sterile rice starch was added before inoculation. T o prevent excessive growth of bacteria, the addition of 1.0 ml. of 1 : 500 sterile aqueous solution of acriflavin to each tube was found satisfactory. For inoculation into animals a 48 hour-old culture was harvested, pooled and concentrated by centrifugation at 1000 r.p.m, in the international centrifuge using head No. 240, for 5 minutes. The final inoculum thus prepared showed 3-4 × 106/ml. actively motile trophozoites. The amoebae were suspended in phosphate-buffered saline (pH 7.2) and kept at 37°C. before inoculation. All the amoebae cultures used for inoculation had mixed bacterial flora derived from human faeces. Experimental animals Weanling albino rats were used throughout. A stock of these rats was obtained from Nutrition Research Laboratories, Hyderabad, where they have been inbred over the past 35 years. The precise origin of the strain is not known. The animal colony was maintained on a stock diet consisting of the following ingredients: Wheat flour 40 g. Skim milk powder 5 g. Casein 7 g. Roasted Bengal gram 39 g. Ground nut oil 5 ml. Balanced salt mixture 4 g. Vitamin and choline mixture 0.2 g. Lucerne grass 2 g. T h e diet and water were given ad libitum. T h e litters were weaned at 4 weeks and placed on the stock diet or diet A or B, the composition of which is shown below (RAo, 1951):
Diet A 1. 2. 3.
Diet B
Protein (Casein) 20 g. 10 g. Dried yeast 5g. 2.5g. 1.0 ml. 0-5 ml. Fat soluble vitamin mixture consisting of: Vitamin A: 2,000 units Vitamin D : 10,000 units Vitamin E: 340 mg. Oleic acid: 100 ml. 4. Choline chloride 0"2g. 0"lg. 5. Vitamin K 0 "2 mg. 0" 1 mg. 6. Vitamin E 0.0 17 "0 mg. 7. Groundnut oil . 5.0g. 2"5g. 8. Balanced salt mixture 4"0g. 4.0g. 9. Potato starch 64.8 g. 80"4 g. Diet A is well balanced as regards protein and vitamins, while diet B is deficient in both. The animals reared on diet A showed healthy fur and normal growth curves, whereas those reared on diet B showed coarse and relatively scanty fur. The A group animals were alert and agile while the B group were sluggish, and weighed less (RAo, 1951). T h e weanlings were reared on these diets for 1-4 weeks prior to inoculation with E. histolytica. The age at inoculation varied from 5-8 weeks. T h e weight of the animals at the time of inoculation with E. histolytica was usually from 50.0 to 60.0 g. Both sexes were used in approximately equal numbers.
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SOME OBSERVATIONS ON THE PATHOGENICITY OF STRAINS OF E N T A M O E B A H I S T O L Y T I C A
Method o f inoculation A method modified from GO0DWI~ et al. (1948) described earlier by one of us (RAo, 1951) was followed. The animals were anaesthetized with ether and laparotomy done under aseptic conditions with a right paramedian incision in the lower abdomen. The caecum was exposed and 1.0 ml. of culture suspension containing about 3-4 × 10e/m1. amoebae was inoculated with 22 gauge needle towards the blind end of the caecum. The abdominal wound was closed by suturing in layers and a light dressing applied. Pre-operatively, the animals were kept fasting overnight and were given 0-8 units of penicillin and 1 mg. of streptomycin intramuscularly to prevent wound sepsis. After operation, only glucose water was given for 24 hours. Thereafter, the usual diet was resumed. The animals were weighed twice a week and any change in weight was recorded. The animals dying within 72 hours of intracaecal inoculation were not taken into account. T h e survivors were killed between 8-10 days after inoculation. A complete autopsy was performed on each animal. T h e whole of the large gut from caecum downwards was slit open and the nature of the wall and the contents of the lumen were noted. The scoring of the lesions of the caecum was recorded in the following way (GogDM~'q, 1965): Grade 0.0 = No amoebae, no lesions. ,, 0.5 = Amoebae present, no lesions. ,, 1.0 : 1 to 10 discrete lesions, normal mucosa in between. ,, 2.0 = More than 10 lesions, less than 25% of the caecum involved. ,, 3.0 = Confluent lesions, 25-50% of caecum involved. ,, 4.0 = Confluent lesions, 50-100% of caecum involved. The amoebae were always present both in scrapings and in the contents, in the last four grades. For each strain of E. histolytica tested, the pathogenicity index was calculated by the authors as below : - Pathogenicity index
Actual score obtained on total number of animals infected Expected maximum score on the same n u m b e r of animals
For example, if the number of animals infected surviving 3 days or more is 5, and the sum total score of lesions on these animals is actually 15, then the expected maximum score would be 5 × 4.0 = 20. The pathogenicity index would be 15/20 = 0.75. I n addition to this, the average caecal score with the standard error (S.E.) was calculated for each strain. Immediately after opening the animal, smears in warm saline were made of the caecal contents and also of scrapings from the caecal wall for microscopic examination. As a rule, apart from the wet preparations, these smears were wet-fixed in Schaudirm's fluid and stained by trichrome procedure (USPHS, Loc. cir.) to confirm the morphology of amoebae observed. I n addition to microscopic examination of caecal contents and mucosal scraping-s, cultures were put up in the usual medium. Occasionally, in low grade infections, blind cultures were positive when the direct microscopy had failed to reveal the amoebae. The caecum was fixed in 10% formol-saline and representative blocks of tissue were processed for histology. Routinely, serial sections were stained with Ehrlich's haematoxylln and aqueous eosin. Heidenhain's iron haematoxylin staining was done where necessary (A.F.I.P., 1960).
Case histories (1) Strain No. 2567/66:--Case No. P 258, male aged 40 years, admitted for painful swelling in the right hypochondrium of 10 days duration. No preceding history of dysentery or diarrhoea; patient poorly nourished; tender liver palpable up to 3½ in. below the costal margin. A clinical diagnosis of amoebic liver abscess was made and aspiration done which yielded typical anchovy sauce pus. Microscopically, the pus showed large trophozoites of E. histolytica with ingested red cells. Culture of this pus yielded growth of the above strain. T h e age of the culture at the time of animal inoculation was 4-5 months. (2) Strain 324/65 :---Case No. P 33, male patient aged 20 years, admitted with signs and symptoms of acute dysentery. He gave a history of recurrent attacks of dysentery during the last 6 years. Faeces showed trophozoites of E. histolytica with ingested red cells. The strain isolated from the dysenteric stool was used for inoculation after in vitro cultivation for 3-5 months.
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(3) Strain 1713/66:pCase No. P 216, male aged 20 years, admitted for acute dysentery of 8 days duration. Faeces showed numerous trophozoites with ingested red cells. The strain was used for animal inoculation after 2-3 months after isolation. (4) Strain 1444/66:--Case No. P 201, male aged 25 years, admitted with signs and symptoms of subacute dysentery of 30 days duration. Faeces were typically dark reddishbrown in colour and showed numerous haematophagous trophozoites. The strain was used 5-6 months after isolation. (5) Strain 3/64:--Case No. SM 60, male child aged 5 years, admitted for hepatomegaly and ascites. A clinical diagnosis of Indian child_hood cirrhosis was made. The patient had no diarrhoea or other intestinal disturbances. The strain was isolated from faeces showing trophozoites without ingested red cells. Animal inoculation done after 1-2 months of cultivation. (6) Strain 2246/65:--Case No. SF 400, female aged 55 years, admitted for diabetic nephropathy; there were no signs of gastro-intestinal disturbances but cysts of E. histolytica were found in faeces. The average diameter of the cysts was 11"2 ~t. The strain was isolated from these cysts and inoculated into animals 1-3 months after isolation. (7) Strain 1797165 :---Case SF 345, female aged 45 years, admitted for benign essential hypertension. No history of diarrhoea or dysentery. The patient did not have any gastrointestinal disturbances. Cysts of E. histolytica measuring an average diameter of 14-4 were found in faeces. Animal inoculation was done 2 years after isolation. (8) Strain 686/66 :--Case No. SM 750, male aged 55 years, admitted for haemoptysis and cough. Investigations revealed bilateral pulmonary tuberculosis. There was no history of dysentery or diarrhoea nor any gastro-imestinal disturbances. The faeces showed cysts measuring 11.68 lz in average diameter. Experiments were performed one year after isolation of the strain from the faeces. (9) Strain 2060/65:--Case No. SF 383, female aged 30 years, admitted for severe anaemia. There was moderate splenomegaly which was attributed to chronic malaria. The patient had no gastro-intestinal disturbances nor any history of diarrhoea or dysentery. Faeces showed trophozoites of E. histolytica without ingested red cells. Animal inoculation was done 2 years after isolation. (10) Strain 500/68 :--Case No. SF 914, student nurse physically fit, with no history of dysentery or diarrhoea. Cysts of E. histolytica measuring an average diameter of 11.02 ~t were found in faeces. Animal inoculation was done 1-4 months after isolation. As a rule, control rats were inoculated with the bacterial flora of the respective amoebic strains.
Results T h e results of experiments performed on weanling rats are presented in Table I. O f the 10 strains of E. histolytica, No. 1 was from a patient with liver abscess, Nos. 2, 3 and 4 were from acute or subacute amoebic dysentery and the remaining 6 were from asymptomatic infections. T h e Table shows that there is considerable variation in infectivity as well as in pathogenicity amongst these strains. T h e infectivity ranged from 34-8% to 85.7% and the average caecal score varied from 0.75 to 3.25. T h e standard error for each strain was calculated on the number of infected animals. T h e pathogenicity index which runs closely parallel to the average caecal score, ranged from 0-20 to 0-81. T h e strain isolated from the liver abscess (No. 2567/66) gave the highest infectivity rate as well as highest caecal score and pathogenicity index. T h e 3 strains isolated from acute or subacute dysentery also gave high pathogenicity indices although their infectivity varied from 41% to 66%. O f the 6 strains isolated from asymptomatic infections, (serial Nos. 5 to 10) the infectivity ranged from 34.8% to 75%, the average caecal score from 0.75 to 2.5 and the pathogenicity index from 0.20 to 0-63. Amongst these 6 strains from asymptomatic cases, 2 showed the lowest pathogenicity index (strains 1444/66 and 3/64), the strain (1797/65) a moderate figure, but the remaining 3 strains showed pathogenicity indices comparable to or even higher than those from acute
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SOME O B S E R V A T I O N S O N T H E P A T H O G E N I C I T Y O F S T R A I N S O F E N T A M O E B A H I S T O L Y T I G A
dysentery strains. The control rats inoculated with bacterial flora did not show any lesions in any of the cases.
Morbid anatomy of amoebic lesions The gross appearance of the caecum varied according to the degree of infection. It could be categorized into 5 types according to the extent and severity of the lesions. (1) Caecum normal in appearance externally, but on opening the lumen showed semisolid or liquid and occasionally frothy faeces; the mucous membrane showed on clearing the contents mild hyperaemia only. (2) Caecum similar to the above condition but mucosa showed fine discrete ulcers usually at the blind end. (3) Caecal contents frankly mucoid but usually associated with liquid faecal material. The mucosa showed more extensive ulceration involving the whole caecum but ulcers were still discrete and fine. (4) Caecum usually shrunken and thickened; ulcers seen from the serous surface. On opening abundant mucoid material with little or no faeces, ulcers usually confluent. (5) Caecum markedly shrunken, often thickened in places, and adherent to surrounding loops of bowel and parietal peritoneum. On opening no normal mucous membrane found; instead diffuse and deep ulceration was observed. In some instances, perforation with gangrene was also seen. Such ulcers were filled with black sloughs. The caecal wall in these cases was very soft and friable and adherent to surrounding structures. In all the above 5 categories, scrapings and contents showed haematophagous amoebae with active motility. Histopathology A wide range of lesions was observed from an early pre-ulcerative stage to small superficial ulcers. More extensive ulcers involving only the mucosa; deeper ulcers with the destruction of the muscular layers often associated with intense granulomatous reaction and finally, acute or chronic perforations. A brief account of these will now be given. (a) Pre-ulcerative stage. The mucosa showed hyperaemia, oedema and catarrhal desquamation of the surface epithelium with numerous amoebae in the crypts of Leiberldihn. No necrosis or ulceration seen at this stage nor any inflammatory exudate. This corresponds to grade 1 of caecal score. (b) Early superficial ulcer. Necrosis of the crypts and lamina propria of the mucosa with shedding of the necrotic material into the lumen. Amoebae confined to the mucous membrane both in the ulcer as well as in the adjoining healthy part with no inflammatory exudate. This corresponds to grade 2 of naked eye score. (c) Extensive superfidal ulceration of the mucosa. The mucous membrane was markedly hyperaemic and showed extensive necrosis with ulceration, the process stopping short at the muscularis mucosae. N o inflammatory response. Amoebae found both in the ulcers as well as in the adjoining mucous membrane. This condition corresponds to grade 3 of the caecal gross picture. (d) Deep ulcers. Ulceration involved the entire thickness of the mucous membrane and the submucosa; abundant amoebae were found in the necrotic slough as well as in the wall of the ulcer formed by intact mucous membrane. The submucosal layer at this
V. GOPAL RAO AND M. C. PADMA
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stage showed intense granulomatous reaction, the cells being mainly histiocytes, large mononuclear cells, lymphocytes and few eosinophils. There was no neutrophilic response. In some cases there was marked hyperplasia of lymphoid follicles. (e) Deeper ulcers with chronic perforations. In these the muscular coat was replaced by granulation tissue heavily infiltrated with chronic inflammatory cells described in the preceding stage. In these cases the serosa was also granulomatous with adhesions to surrounding structures. Amoebae were found in the ulcers as well as in the adjoining mucous membrane. Histological descriptions of (d) and (e) correspond with grade 4 of the naked eye score. (f) Acute perforations with gangrenous change. Sections of the ulcer revealed the base of the ulcer formed by a thin ribbon of serous layer with extensive necrosis and gangrene of all the layers above. The blood vessels in the adjoining part showed thrombi. Amoebae were found both in the gangrenous slough although scanty in number as well as in the adjoining mucosa. (g) Healed ulcers. In addition to the above, in milder cases of caecal ulceration of grade 2 or 3, there were healed ulcers. Here the mucosa was replaced by granulation tissue with a thin ribbon of columnar epithelium covering it on the surface. Such ulcers did not show amoebae. Such healing lesions were usually observed in animals autopsied 12-15 days after inoculation. On analysing the histopathological findings observed with the 4 strains isolated from clinical cases of amoebiasis, namely, serial Nos. 1-4, it was found that the No. 1 strain, which gave the highest caecal score and highest pathogenicity index (Table I), produced in rats widespread caecal lesions, often deep and penetrating, and in one rat chronic perforation. The 3 strains isolated from cases of dysentery also produced fairly widespread lesions but the ulcers were not deep or penetrating. The strains isolated from asymptomatic injections, on the other hand, showed a wide spectrum of lesions corresponding to the gross lesions as shown in Table i. Of the 6 strains from asymptomatic infections 3 strains, serial Nos. 5, 6 and 9 showed only lesions of mild to moderate severity. The ulcers in general were very superficial and confined to the mucosa only. Two strains, serial Nos. 7 and 8 gave deeper lesions extending into the submucosa with marked granulomatous reaction. However, most extensive lesions were produced by a strain isolated from a student nurse who was normal clinically and gave no history of diarrhoea or dysentery at any time. This strain (500/68), although showing low infectivity, produced very widespread and deep ulcers including gangrenous ulcers with perforations in 2 out of 8 infected animals. These gangrenous ulcers contained jet-black sloughs comparable to "dyak hair" ulcers seen in humans with gangrenous ulcers. It is noteworthy that such lesions were produced by a strain of amoeba isolated from an asymptomatic infection. As described earlier none of the amoebic ulcers whether superficial or deep, showed any neutrophilic exudate or other features of bacterial invasion such as suppuration. The experiments on the r61e of host nutrition in the pathogenesis of intestinal amoebic lesions are still incomplete. The data obtained on limited number of animals reared on balanced diet A as compared to those reared on deficient diet B are presented in Table II. As the number in each dietary group is small it is premature to draw any conclusions on a quantitative basis. Therefore, in Table I the overall results have been shown for purposes of assessment of pathogenicity of individual strains. However, it may be mentioned that in general, deeper and widespread lesions were observed in group B animals with strains obtained both from pathogenic and symptomless infections as compared to those seen in group A animals. The two rats which showed gangrenous
612
SOME OBSERVATIONS O N T H E P A T H O G E N I C I T Y O F STRAINS OF E N T A M O E B A
HISTOLYTICA
ulcers with strain No. 500/68 obtained from asymptomatic infection were indeed reared on diet B. However, experiments on larger series of animals are required to establish a quantitative relationship between host nutrition and the severity of the caecal lesions. "ABLE I. Results of intracaecal inoculation of E. histolytica cultures into y o u n g albino rats.
SI. No.
Strain No.
Clinical category of pati-~nts f r o m w h o m steams of E. hist :lytica isolated
No. surviving for 72 No. infected hours or No. of rats with inoculated more* percentage
Average caecal score with S.E.
Pathogenicit~ index
1
2567/66
Amoebic liver abscess
12
7
6 (85' 7)
3'25±0"52
0"81
2
324/65
Acute dysentery
22
18
12 (66"6)
1"8
±0.462
0"45
±0.61
O' 50
3
1713/66
I Acute dysentery
15
12
5 (41.6)
2"0
4
1444/66
Acute dysentery
15
7
3 (42.9)
2"33±0.54
5
3/64
A s y m p t o m a t i e infection
14
11
6 (54" 5)
0"75±0'102
O" 20
6
2246/65
do
8
7
4 (57.1)
0'75±0"125
0"20
7
1797/65
do
8
4
3 (75- O)
1'16±0.121
O" 30
8
686/66
do
12
11
7 (63.6)
2'14±0"47
0"54
9
2060/65
do
11
8
5 (62.5)
2"3
±0"65
0"58
do
42
23
8 (34.8)
2"5
±0-641
0"63
10
500/68
0"56
# T h e animals dying in the first 72 hours after inoculation were not taken into account. T h e deaths were due m a i a l to post-operative shock or other causes unrelated to amoebic infection. TABLE I I . Results of intracaecai inoculation of cultures of E. histolytica into y o u n g albino rats.
$1. No.
Strain No.
Clinical category of patients f r o m w h o m strain was isolated Amoebic liver abscess
N o . of rats Proportion of inoculated and animals infected survived 72 hours Stock D i e t D i e t or longer diet A B
Pathogenicity index
Average caecal score of lesions in infected animais with S.E. Stock diet
Diet A
Di~ B
Stock D i e t diet A
Diet B
i
2567/66
--
0-63
0.75
2
324/65
Acute amoebic dysentery
18
12/18
:1.8
±0.462
0'45
--
--
3
1713/66
Acute amoebic dysentery
12
5/12
2"0
±0-61
0" 50
4
1444/66
Acute amoebic dysentery
7
3/7
2-33±0"54
0"56
5
3/64
A s y m p t o m a t i c infection
11
6/il
0"75±0-102
0 ' 20
6
2246/65
do
7
4/7
7
1797/65
do
4
--
7
3/3
--
3/4
2.5±0"85
4'0
±0
0"75±0'125 1/2
2/2
2"0
0'75±0"25
0'20
--
--
--
0.50
0.19
8
686/66
do
11
--
4/6
3/5
2.37±0.851.83±0.62
--
0'59
0"45 i
9
2060/65
do
8
--
4/4
1/4
2.37±0"822"0
--
0'59
0"50
10
500/68
do
23
--
4/14
4/9
1-25±0'503'75±0'10
--
0"32
0"93
Discussion Many studies have been carried out in different parts of the world on pathogenicity of strains of E. histolytica. As mentioned earlier, there is divergence of views regarding its pathogenic potential. It is essential at this stage to make a distinction between pathogenicity and virulence. The former refers to a general attribute of micro-organisms with regard to their capacity to produce disease while the latter generally indicates "the degree of pathogenicity of a given type, strain or a cldone as observed in a particular host species under defined conditions." (MILES, 1955). The results of the experiments reported here on 4 strains of E. histolytica isolated from patients with dysentery or liver abscess and 6 from asymptomatic infections showed
V. GOPAL RAO AND M. C. PADMA
613
that there was a considerable variation in virulence and pathogenicity. The strain from the liver abscess gave the highest caecal score and pathogenicity index (3.25 and 0.81 respectively), while the dysentery strains gave moderate degrees of virulence and pathogenicity index (Table I). All the 4 strains produced deep-seated lesions and the one from the liver abscess produced chronic perforation. The 6 strains isolated from asymptomatic infections gave a range of virulence and pathogenicity indices from 0.75 to 2.5, and 0.20 to 0-63 respectively. Of the 3 strains showing low virulence (Serial Nos. 4, 5 and 6) one strain No. 1797/65 produced deep-seated lesions involving the submucosa and part of the muscular layer. Moreover 3 other strains giving high caecal score and high pathogenicity index (0.54 to 0.63) produced penetrating lesions involving deeper layers, while one strain 500/68 produced classical gangrenous ulcers with perforations. Only 2 strains serial Nos. 4 and 5 gave low caecal score and showed only superficial mucosal erosions microscopically. These may be caUed relatively low virulent strains. Nevertheless, it is clear that 4 of the 6 strains isolated from asymptomatic infections produced lesions qualitatively and quantitatively comparable to those produced by strains isolated from dysenteric stools or liver abscess material. It is thus obvious that these strains could not be dismissed as commensals simply because at the time of isolation no lesions were clinically detected in the human host. A perusal of available literature on pathogenicity of strains of E. histolytica isolated from asymptomatic infections shows that a number of workers from East European countries and United States have found such strains to be pathogenic generally, while NEAL (1951, 1957, 1958, 1960 and 1961) in England and HUNNINEN and BOONE(1957) in America found "carrier" strains to be avirulent or non-invasive. On the other hand ROGOVA(1956), on the basis of her experiments with strains isolated from clinical as well as non-clinical cases, found the infectivity rate for guinea-pigs was the same with both categories, but the severity of lesions varied. However, she observed that strains from asymptomatic infections had produced deep-seated ulcers in over 20% of the test animals. SWELLENGREBELand RIJI'STRA (1958) pointed out that E. histolytica found in the commensal phase was capable of producing infection with clinical manifestations including acute dysentery. KRUPP and FAUST (1959) reported tissue invasion with ulceration in guinea-pigs with the 14 strains isolated from asymptomatic infections. KASPRZAK(1961) reported that 2 strains isolated from symptomless "carders" both of local and Vietnamese origin were found to produce caecal ulceration in guinea-pigs. MIZGIREVA(1966) experimented with 78 strains of E. histolytica isolated from different clinical categories of amoebiasis including 10 strains from "symptomless carriers." Although the virulence was variable with individual strains all the strains including those from "symptomless carriers" were pathogenic. She emphasizes the importance of host factors in determining the virulence of the parasite. NEAL in a series of papers (1951, 1955, 1956, 1957 and 1961) reported his observations on experiments on rats with different strains of large race E. histolytica. The rSle of associated bacterial flora was examined and also the effects of serial passages into the livers of hamsters on the virulence of amoebae. He has based his conclusions as regards the virulence of a given strain on the basis of average caecal score. Any strain giving a caecai score of 2.0 or less was considered as avirulent and the one giving above 2.0 was considered virulent. The papers do not report the histopathological observations, nor was there any attempt to classify the lesions on that basis. His conclusions were: (1) The amoebae and not the associated bacterial flora were responsible for tissue invasion and virulence of the strain; (2) There was a radical difference as regards virulence between the strains found in asymptomatic "carriers" and those isolated from cases of clinical amoebiasis. The former were avirulent giving a caecal score of less than 2.0, while the latter were
614
SOME OBSERVATIONS ON THE P A T H O G E N I C I T Y OF STRAINS OF E N T A M O E B . 4 H I S T O L Y T I C A
virulent giving scores well over 2.0. However, there were exceptions to this generalization in as much as certain strains isolated from asymptomatic "carriers" gave a high caecal score. NEAL (1957) explained this by postulating that "infection of man with a virulent type must pass through a quiescent phase when disease does not occur, in order to ensure survival by the production of cysts. This might explain the isolation of virulent strains from "carriers"." Such an explanation could hardly lead one to an understanding of the rather complex problem of the host-parasite balance in amoebiasis. It should be emphasized that no morphological differences were pointed out between virulent and avirulent amoebae. Therefore, the question arises as to how to decide whether a given strain of E. histolytica in a given patient is of clinical significance or not. In such cases the physician gets no guidance from the laboratory as to whether a patient should be treated by specific drugs or not. More important still is the public health implication of the so-called asymptomatic infections particularly with reference to their r61e in spreading clinical amoebiasis in the community. NEAL (1957) has answered this question by stating that "apart from inoculation of rats there is no way of d e c i d i n g . . . " It may be further noted that avirulent strains isolated by Neal were not only of local origin but some were derived from the tropics also. Conversely, strains isolated from asymptomatic infections in the tropics were found by him to be virulent to rats (BIRD and NEAL, 1962). While discussing the variations in virulence NEAL (1958) suggested that "invasive amoebae may arise by adaptation from the normal avirulent amoebae." From the results of experiments reported here and also from the review of literature it is clear that there can be no sharp and clear-cut distinction between E. histolytica found in asymptomatic infections and the haematophagous amoebae found in clinical cases in so far as the potential capacity for production of disease is concerned. As regards the r61e of bacteria the histological studies presented here revealed even at the earliest stage of tissue invasion by E. histolytica, the lesions consisted of coagulation necrosis of mucous membrane followed by liquefaction and shedding. The host's tissue response was mainly that of exudation of mononuclear cells and lymphocytes. In some there was marked lymphoid hyperplasia. No nentrophilic response or fibrinous exudate was observed to suggest bacterial intervention at any stage. Mention may be made here of the observations of NEAL and VINCENT(1955 and 1956) who showed.that the virulence of E. histolytica was not dependent on the associated bacterial flora but was due to the amoeba itself. Very little is known about the precise pathogenefic mechanisms involved in the production of lesions by E. histolytica. CRAIG (1927) demonstrated cytolytic, haemolytic and complement-fixing activity in alcoholic extracts of amoebae by in vitro experiments. The cytolytic activity of such an extract was not confirmed in vivo. NEAL (1960) demonstrated proteolytic activity in amoebic extracts, but this was not correlated with invasivehess (BIRD and NEAL, 1962). MAEGRAITH(1963) in his exhaustive review on pattlogenefic mechanisms in amoebiasis reported his own observations on the proteolytic enzymes and hyaluronidase activity in strains of E. histolytica. However, these observations have not led to a clear understanding of the mechanism of tissue invasion. In this connection the observations of one of the authors (RAO, 1951) may be mentioned. It was shown that filtrates of cultures of E. histolytica grown with a single bacterial associate, when inoculated into the portal vein of young rats resulted in the production of coagulation necrosis of hepatic parenchyma. The lesions were self-limiting and healed in about 4 days. The control animals inoculated with bacterial flora did not show any such necrotic lesions. It appears as though the amoebae when growing in culture produce a necrotizing factor the nature of which has yet to be ascertained. It has been observed in the present experiments that amoebae multiply in the crypts and necrosis occurs subsequently. This
V. GOPAL RAO AND M. C. PADMA
615
initial multiplication of amoebae with subsequent production of necrotizing factor(s) may well be responsible for production of mucosal lesions. There are not many critical studies on the r61e of host nutrition in the pathogenesis of amoebiasis. ANN (1958) experimenting on kittens found that diets supplemented with meat and milk produced milder infection with E. histolytica as compared to animals not receiving these supplements. FAUST (1961) reviewed the various host factors influencing the course of infection with E. histolytica and considered that nutrition was an important factor. The results of our preliminary study on the r61e of host nutrition have dearly indicated that the weanling rats reared on low protein-low vitamin diet (diet B) developed more severe and extensive lesions including gangrenous ulcers than the controls reared on a balanced diet. However, more studies to elucidate the exact role of nutrition in enhancing the severity of the lesions and the mechanisms involved therein are needed.
Summary 1. Results of animal inoculation experiments on young albino rats with different strains of E. histolytica isolated from different patients with clinical amoebiasis, such as acute dysentery and liver abscess, as well as those isolated from asymptomatic infections have been presented and discussed. 2. Virulence and pathogenicity showed considerable variation both among the strains isolated from clinical cases as well as from asymptomatic infections. In the former group the liver abscess strain gave highest caecal score and pathogenicity index while the dysentery strains gave moderate figures. Of the 6 strains from asymptomatic infections 3 showed virulence and pathogenicity indices as high as strains from clinical cases. One of these strains showed moderate virulence and the remaining two strains showed lowvirulence of less than 1.0. 3. The morbid anatomy and histology of the lesions have been described in detail. The lesions varied from superficial ulcers involving only the mucosa to deep ulcers penetrating the submucous and muscular coats. In some, even perforations associated with gangrene were observed. It is to be noted that the gangrenous lesions were caused by a strain isolated from a healthy nurse without any symptoms or history of amoebiasis. Further, even the strains showing low virulence showed necrosis with ulceration of the mucosa and one strain produced deep lesions even though the caecal score was low. 4. The pathogenesis of the amoebic lesions in the gut have been discussed. It has been shown that amoebae multiply in the crypts initially with subsequent production of necrosis followed by ulceration. The possible existence of a necrotizing factor produced by amoebae has been raised. 5. Limited observations on the r61e of nutrition in the course of experimental amoebic infection in rats have shown that low protein-low vitamin diets aggravate the lesions as compared to the reaction seen in animals reared on a balanced diet. REFERENCES ANN, J. M. (1958). Am. ft. trop. Med. Hyg., 7, 158. ARMED FORCES INSTITUTE OF PATHOLOGY (A.F.I.P.), Washington D.C. (Mary Francis Gridley Memorial Fund), 1960. Manual of Histologic and Special Staining Technics. 2nd Ed. The Blakingston Division, McGraw-Hill Book Company Inc. BIRD, R. G. & NEAL, R. A. (1962). Trans. R. Soc. trop. Med. Hyg., 56, 87. BOECK, W. C. & DRBOHI.AV,J. (1925). Am. ft. Hyg., 5, 371. BI~UMPT, E. (1925). Bull. Acad. Med., 94, 943. CRAIG, C. F. (1927). Am. ft. trop. Med., 7, 225.
616
SOME OBSERVATIONS ON THE PATHOGENICITY OF STRAINS OF E N T A M O E B A H I S T O L Y T I G A
(1944). The Etiology, Diagnosis and Treatment of Amoebiasis. 1st Ed. Baltimore: Williams and Wilkins Co. ELMASSIAlq,M. (1909). Centralb. Bakt. Abt. (1 Orig.), 52, 335. FAIRLEY,N. H., WOODRUFF,A. W. & WALTERS,J. H. (1961). In: Recent Advances in Tropical Medicine. 3rd Ed. London: J. & A. Churchill Ltd. FAUST, E. C. (1961). Clinical Parasitology. 7th Ed. Philadelphia: Lea and Febiger. GOODWlN, L. G., HOARE,C. A. & SHARP,T. M. (1958). Br. ft. PharmacoL Chemother., 3, 44. HOARE, C. A. (1947). Trans. R. Soc. trop. Med. Hyg., 41, 87. (1949). In: Handbook of Medical Protozoology. London: Bailliere, Tindall and Cox. (1950). Br. med. J., 2, 238. (1952). Exp. Parasit., 1, 411. (1957). Trans. R. Soc. trop. Med. Hyg., 51, 303. • (1958). Rice lnst. Pamphl., 4,5, 23. HrYNNINEN,A. V. & BOONE, H. A. (1957). Am. J. trop. Med. Hyg., 6, 32. KASPRAZAK,W. (1961). Acta Parasit. Polonica, 9, 211. KRUPP, I. & FAUST, E. C. (1959). J. Parasit., 45, 449. MAEGRAITH, B. G. (1963). In: Immunity to Protozoa. Oxford: Blackwell Scientific Publications. MILES, A. A. (1955). In: Mechanisms of Microbial Pathogenicity. New York: Cambridge University Press. MIZGIREVA,M. F. (1966). Med. Parasitol. Parasitic. Dis., Moscow, 35, 202. NEAL, R. A. (1951). Trans. R. Soc. trop. Med. Hyg., 44, 439. (1956). Parasitology, 46, 183. (1957). Trans. R. Soc. trop. Med. Hyg., 51, 313. (1958). Proc. 6th lnt. Congr. trop. Med. Malar., 3, 350. (1960). Parasitology, 50, 531. (1961). Trans. R. Soc. trop. Med. Hyg., 44, 439. & VINCENT, P. (1955). Parasitology, 4.5, 152. & (1956). Ibid., 46, 173. PROWAZEK, S. V. (1912). Weiterer Beitrag Zur Kenntnis der Entamoben Arch. F. Protistenk. X X V I , 241. RAO, V. G. (1951). Thesis submitted to the University of London for the degree of Ph.D. ROGOVA, L. I. (1956). Med. Parasit. Parasitic Dis., Moscow~ 2.5, 330. SWELLENGREBEL, N. H. & RIJPSTRA, A. C. (1958). Ned. Tijdschr. U. Geneesk., 102. UNITED STATES PUBLIC HEALTH SERVICE (USPHS) publication C.D.C. Bulletin (1948). Laboratory Diagnosis of Parasitic Diseases. WALKER, E. L. & SELLARDS,A. W. (1913). Philipp. J. Sci., 6, 259. WORLD HEALTHORGANISATIOt;(1969). Techn. Rep. Ser., 49.1, p. 8. WRIGHT, W. H. (1950). Am. ft. trop. Med., 30, 123.