IV THE EFFECTIVENESS AND PRACTICABILITY OF VARIOUS METHODS OF EXAMINATION

IV

THE EFFECTIVENESS AND PRACTICABILITY OF VARIOUS METHODS OF EXAMINATION.

1. INTRODUCTORY.

The choice of method or methods for protozoological examination must be determined by the kind of information that the worker expects to extract from his material. A parasitologist primarily interested in the biology of the several protozoal species, is likely to devote the chief part of his work to cultivation, which often enables him to control the protozoa and to perform various experiments fairly undisturbed by unknown factors. A parasitologist who wants to ascertain what species of protozoa are present in a population, and to what extent, will need methods by which all species can be demonstrated and which will give him, il not exhaustive, yet representative figures from which he is able to calculate with fair accuracy the real incidences of infections. The sanitarian or Public Health worker who wants to investigate how far different conditions of personal hygiene and general sanitation facilitate or prevent the spread of intestinal iiifections in general, using the frequency of protozoa as an index, may regard it as sufficient to consider a limited number of species only, provided the demonstrated incidences of infections with these are sufficiently representative of actual conditions to allow comparisons with similarly collected data from different populations. Finally, the clinician, suspecting a certain protozoon to be the cause of disease in his patient, will require an exhaustive examination of the intestinal fauna in order to detect or exclude this infection as far as possible.

30 RUTH SVENSSON

In the following discussion I shall use the term exhaustive examination to denote that every protozoal infection harboured by a certain individual is, with probability amounting almost to certainty, disclosed. A parasitologically representative survey will stand for a system of examinatiuns which can be expected to reveal all protozoal species present in a population in approximately constant proportions of actual incidences. Finally, for a system of examinations which leaves out of account species of rare occurrence or difficult demonstrability (though usable for collecting and presenting data regarding common and easily demonstrable species, which are comparable with similar figures from other populations), I propose the name endemiologically representafive survey, a name perhaps not complying with the most common usage, but defensible from an etymological point of view.

Before it can be determined how these 3 different kinds of survey can be accomplished with the greatest economy of time and labour, the advantages and drawbacks of the separate examination methods must be demonstrated and compared. As a general fact i t may first of all be pointed out that none of the methods used is sufficient and none superfluous for proving, or rather excluding, infections with every protozoal species. Thus the examination of formed stools fails to give information regarding those species which do not encyst, such as Dientamoeba fragilis and Trichontonas hominis, or of those which encyst very rarely, such as Balantidium coli. Enteronronas hominis also can hardly be recorded reliably from formed stools, as the cysts of this species are very small and inconspicuous and very easily overlooked or confused with yeasts in iodine preparations. Cultivation cannot be an exhaustive method until media suitable for growing Giardia infesfinalis are discovered. In loose stools no species will be systematically overlooked: but experience has shown that in some cases, where infections have been demonstrated in formed stools or by cultivation, loose stools have sometimes appeared negative. This especially applies to very light infections, as one or a few organisms may be practically impossible to detect in direct preparations, although the may give rise to good strains on cultivation.

STUDIES ON HUMAN INTESTINAL PROTOZOA 31

The indispensability and insufficiency of the separate methods for exhaustive examination are demonstrated in tables I and 11, where all figures refer to the same group of infected persons, who were all examined very carefully by purging, cultivation, and, in the majority of cases, the investigation of a t least 6 formed stools. Table I shows how many infections were discovered by one method only - and would clearly have been overlooked if this particular method had not been used. Table I1 shows how many infections escaped detection by each single method.

In the tables, for the sake of uniformity, the findings in one formed stool only are given, as several cases did not receive repeated examination. In those cases where-6 or more formed stools were examined the following additional infections were demonstrated which had escaped detection in loose stool, culture, and a single formed stool : E. hisfolytica 29 cases - 1 infection, E. coli 57 cases - 3 infections, E. nana 47 cases - 2 infections, I , biitschlii 26 cases - 1 infection, G. intestinalis 16 cases - 1 infection, Ch. mesnili 48 cases - 1 infection.

Table I . Known infections demonstrated by each single

Actual infecr Infections demonstrated : tions (discove- In one In one In

method exclusively. - red bv all formed loose culture

Species :

All

Enfamwba hisfolytica Enfamoeba coli . . . . . . Endolimax nana . . . . . lodanioeba bufschlii . . Dienfamoeba fragilis . Giardia intestinalis . . Chilomastix rnesnili , . Trichomonas hominis . Enteronionas hominis . Balanfidiurn coli . . . . . species . . . . . . . . . . . . .

meth’ods combined) Number

44 73 61 39 59 17 58 12 54 4

42 1

stool only

Number 0 1 2 1 0 4 1 0 0 0 9

stool only

Number 3 0

14 10 2 6

10 0 1 0

46

only

Number 4 0 5 3

23 0 5 1

20 0

61

32 RUTH SVENSSON

Table II, Known infections missed by each single method.

Species : Enfarnoeba histolyfica Entanioeba coli , , . . . . Endolimax nana . . . . . Iodanioeba biifschlii . Dienfamoeba fragilis Giardia intesfinalis . . Chiloniastix mesnili . . Trichomonas hominis . Enteromonas hominis Balantidium coli . . . . .,

All species . . . . . . . . . . . . . ,

Actual infections

Number 44 73 61 39 59 17 58 12 54 4

42 1

Infections not discovered : In one In one In cult- formed loose ure

stool stool Number Number Number

27 11 5 28 9 17 31 12 29 23 7 17 59 23 2 7 5 17

36 10 14 12 1 0 54 20 1

4 0 0 281 98 102

- c -

It is evident from tables I and I1 that the examination of formed stools adds little to the results obtainable by the other two methods and that more infections escape detection in formed stools than in loose stools and in cultures. If formed stools had been omitted altogether 18 out of 421 infections would have remained undiscovered, and, had only one formed stool from each case been examined, 9 infections would have escaped detection. The other two methods ate indispensable in a much higher degree, a s 46 and 61 infections respectively would not have been detected, had any of these methods been omitted.

2. DISCUSSION OF THE SEPARATE EXAMINATION METHODS.

After this demonstration of the shortcomings of single methods in general, I shall give an account of the advantages and drawbacks of each method separately, leaving their utility for particular purposes to the later sections of this chapter.

a. Examination of formed sfools. The greatest advantage of this method is that the encysted forms contained in formed stools keep unchanged for several days if prevented from drying. Faecal specimens can therefore be brought to the laboratory without haste, and can be examined at any time convenient.


The most conspicuous limitation of formed stools is that only cyst-forming species are discoverable: and even among these, as has already been pointed out, Balantidium and Enteromonas will seldom be recorded.

The figures obtained by examination of formed stools cannot be used directly for comparison with other surveys. It was generally realized already at an early stage of the survey work carried out during the Great W a r that single-specimen examinations revealed only a part of the protozoal infections actually present, and also that dissimilar proportions of the several species were demonstrated. The first estimation of the proportion of the actual infections demonstrable by a method of one examination per individual was made by D o b e I 1 (191 7) who from the figures at his disposal estimated that probably not more than one-third of the actual infections was discovered. Shortly after, C a r t e r, M a c k i n n on , Ma t t h e w s & S m i t h (1917) published figures from which approximately the same estimation could be made. An attempt to determine the demonstrability of the several species in different numbers of stools was made in Peking by Dr. J. F. K e s s e l and the writer ( K e s s e l €3 S v e n s s o n 1924). The estimations were, however, made on a presumption, which later investigation has shown to be erroneous, that a constant proportion (about 90 % ) of the actual infections was revealed by 6 examinations.1) The demonstrability in different numbers of examinations was calculated in percentages referring to the findings in 6 stools. The figures obtained (loc. cit. Table I ) have later been used by several authors as standard measures, by means of which surveys conducted on systems of varying numbers of examinations per case might be reduced to a common basis and thus be rendered directly comparable. Later investi- gations have conclusively shown such calculations to be mis- leading. A statistical analysis made on material collected by me ( S v e n s s o n with L i n d e r s 1934) has proved that the proportions of known infections demonstrable in one, as well

l ) Even earlier it had been shown by D o b e 1 1 (1917) and C a r t e r, M a c k i n n o n , M a t t h e w s 6 S m i t h (1917) regarding certain species that dissimilar proportions of actual incidences were discovered in 6 stools.

34 RUTH SVENSSON

as in 6 examinations per case (oide p. 39) may be significantly different, not only regarding the separate species, but also for different populations.

The figures for the analysis here referred to were obtained from heavily infected patients in the mental hospital of Vaster- vik, one group of men and one of women, These patients were very thoroughly examined by all three examination methods, the minimum number of formed stools being 6 in all cases but one. The demonstration of the infections was made in loose stools or culture and thus independently of the findings in formed stools. I t therefore seems justifiable to assume that such infections which seldom betray themselves by the presence of cysts were equally well represented as infections with frequent occurrence of cysts in the stools. In fact, many infections never revealed themselves in the formed stools and these would have passed as truly negative if the other methods had not been used. In such instances many negative formed stools from actually infected cases would have been left out of account in the calculations of demonstrability, and the estimation of the chances of demonstrating infections would consequently have been too high. - The men were more heavily infected than the women, the mean number of infections per individual being 6.5 for the former against 4.4 for the latter. It was found that the average demonstrability of E. coin', E, nana, Iodamoeba and Chilomasfix in one formed stool from any single individual was higher in the group of men than in the group of women. The differences were high enough to be practically certain. E. histolytica showed only a small difference in average demonstrability, the figures obtained being here somewhat higher for the women than the men (0,31 and 0.28 respectively). No estimation was made for Giardia, as infections in which the flagellates seldom encyst cannot be demonstrated by other methods as easily with this species as with the others. In order to ascertain whether sex could be responsible for the difference in demonstrability, comparisons were made with the two series of examinations on British soldiers just mentioned, made by D o b e l l , and by C a r t e r , M a c k i n n o n , M a t t h e w s G S m i t h on similar principles, only that the demonstration


of infections was made in their surveys by examining large numbers of spontaneously passed stools. In the British publica- tions figures were given only for E. histoolytica. E. coli, and Giardia. In order to make the estimations of the two British series comparable with ours, standard errors were computed with reference, not to the demonstrability in any one of the total number of stools, but in the stools from the single infected individuals. Even by this method it was found that the difference in demonstrability of E. coli between the men in Vastervik on the one hand, and the 2 groups of British soldiers on the other, was statistically established. With these significant differences in demonstrability of one protozoal species between different groups of men, no opinion of sex as such influencing the demonstrability could be advanced.

As this severe treatment and detailed analysis of the material has proved that the individual infections in the different groups do not influence the demonstrability to any practically appreciable degree, I have here allowed myself to treat the material in my further discussion as 4 in themselves homogeneous groups, leaving the numbers of infected individuals out of account and taking only the total numbers of stools into consi- deration. The average demonstrability for each group as well a s differences between the different groups are thus obtained as presented in tables 111 and IV. T h e standard errors of the percentages of positive stools (P) are here computed by the formula

where Q= the percentage of negative and N = the total number of stools. Regarding the material used for constructing table 111 it may be mentioned that in D o b e 11's series no case was included which had received less than 12 examinations: the mean number of stools examined from each case amounted to 19.9 for E. histolytica and 44.9 for E. coli. In the series of C a r t e r , M a c k i n n o n , M a t t h e w s fi S m i t h the minimum number of examinations was 10 and the mean number 23.6 per case for E. histolytica, the corresponding numbers for

36 RUTH SVENSSON

E. coli being 11 and 22.7. In my two Vastervik groups one man, harbouring all 5 species, had only 5 formed stools examined, while all the others had 6 or more, the mean number per case being in the men's group for E. histolytica 6.1, E. coli 6.4, E. nana 6.9, Iodamoeba 6.2, and Chilomastix 6.5 and in the womens group for E. histolytica 9.3, E. coli 10.4, E. nana 10.2, Iodamoeba 9.9, and Chilomastix 8.8. In all cases the infections were demonstrated in loose stool or culture, Further particulars regarding all four groups are given below:

SVENSSON : Men E. hisfolyfica . . , . . E. coli . . . . , . . . . . E, nana . . . . . . . . . . I . butschlii . . . , . . . . Ch, mesniii . . . . . . . .

Number Positive of infected stools

Women E. hisfolytics . . . . . . E. coli . . , . . . . . . . E. nana . . . . . . . . . . . I . butschlii . . . . . . . . Ch. mesnili . . . . . .

DOBELL: Men E. hisfolyfica , . . . . . E. coli . . . . . . . . . . .

CARTER, MACKINNON, MATTHEWS 6 SMITH : Men

E. histolytica E. coli . .. . . . , . . ..

. . . .

cases

13 23 18 86 15 61 16 64 18 54

15 48 29 165 25 87 8 22

25 73

9 71 46 646

10 1 05 18 133

Negative stools

63 41 68 42 57

101 164 178 57

180

108 1,419

131 276

Total number of stools

86 127 129 106 111

149 329 265 79

253

179 2,065

236 409

Percentages with standard errors of the positive stools recorded above are presented in table 111.


Table Ill. Percentages of known infections demonstrable in one formed stool in different populations.

Vastervik mental hospital British soldiers cases

SVENSSON DOBELL CARTER G all. Men Women Men Men

E. histolytica . . . 2 7 ~ 4 . 8 32 i 3.8 40+ 3.7 445 3.2 E. coli . . . . . . . . 6 8 ~ 4 . 1 50k2.8 31 k1.0 3 3 t 2 . 3 E. nana . . . . . . . . 4 7 + 4 . 4 33+2.9 I. biitschlii . . . . . . 605 4.8 2 8 2 5.0 Ch. mesnili . . . . . 49 i 4.7 29 ~ 2 . 8

Table IV. Differences in average demonsfrabilify hetween the populations of tabEe 111.1)

Difference men-women Difference men-men E. histolyfica . . -5k 6.1 yo E. histolytica . . --13i6.1 ?A E. coli . . . . . . . . 1855.0 .. E. coli . . . . . . . . 3614.2 .. E. nana . . . . . . . . 14k5.3 .. Difference women-men I. butschlii . . . . . 33 5 6.9 .. E. histolytica . . -7 25.3 yo Ch. mesnili . . . . 202 5.5 .. E. coli . . . . . . . . 1922.9 ..

SVENSSON SVENSSON-DOBELL

SVENSSON-CARTER G all. Difference men-men Difference women-men

E. histolyfica . . -1825.8 yo E. histolytica . . -12 i 5.0 7; E. coli . . . . . . . . 3554.8 .. E. coli . . . . . . . . 18L3.6 ..

In the two Vastervik series the average demonstrability is higher in the group of men than in the women's group for all species except E. histolytica. The difference is most pronounced and statistically well established for E. coli, lodamoeba, and Chilomasfix, but also E. nana shows as high a difference as 14 o/o and the probability that this difference is significant is very great. Both men and women in Vastervik show much higher demonstrability of E. coli than the two groups of British men (the smallest of the differences amounts to 5 times its standard error ) .

') In computing the differences decimals have been considered, which explains some discrepancies in comparison with the figures in table 111 and some following tables.

38 RUTH SVENSSON

The factors which determine the demonstrability in formed stools seem to be entirely different for E, histolytica and the other species. In the Vastervik groups the women show somewhat higher demonstrability of E. histolytica than the men, but the two British groups give considerably higher figures than any of the Vastervik groups, and these differences are high enough to seem statistically significant. This is, however, possibly only apparent, as the authors of both British publica- tions point out that the figures for positive examinations are probably too high, as they were derived from cases not chosen at random, but selected because E. histolytica was discovered already in the first stools examined. In the Peking survey 40 out of 91 infections discovered in 6 stools from each of 340 persons were detected in the 1st examination. In the present exhaustive survey 72 % of the actual infections with E. histolytica were detected in 6 formed stools, the same percentage being recorded for men and women (vide infra), If the same proportion of the actual infections is assumed to have been detected in 6 stools in the Peking survey, then the recorded 40 infections in the series of 1st examinations in reality mean 32 5% of the real incidence. All these observations seem to indicate that D o b e 11's original estimation that not more than one- third of the existing infections is discoverable by a method of examining one stool from each person in a survey has general validity for E. histolytica in different kinds of populations,

Regarding the other species no survey has, as far as I know, been published which would offer possibilities for direct comparison of demonstrability with mine: as the requirement that all existing infections should have been discovered either in culture and loose stools, or in a large number of formed stools, has not been fulfilled. In the Peking survey, however, 340 persons had as many as 6 examinations of spontaneously passed stools each, and with some prudence the recorded figures might be used for calculation of demonstrability of the different species in single stools. This could be done safely if approximately constant proportions of the actual infections were detected in only 6 stools. This is not so, however, as appears from table V, the figures in which are taken from my publication with


L i n d e r s (loc. cit. Table XII. ilities for demonstrating infections in [ormed stools, p. 32 1 ) .

Graduated cumulative probab-

Table V . Demonstrability of known infections in 6 formed stools.1)

Men Women Difference . . . . . . . . . E. hisfolytica 72 % 72 % 0 %

E. coli . . . . . . . . . . . . . . . 96 ,, 85 9 , 11 t ,

E. nana . . . . . . . . . . . . . . 94 ,, 75 r , 19 I ,

I . biitschlii . . . . . . . . . . . . 90 ,, 69 ,. 21 I ,

Ch. niesnili . . . . . . . . . . . 91 ,, 51 ), 40 ,,

As shown in table V E. histolytica is demonstrable in 6 stools in equal proportions of the actual incidence. The other species still show considerable preponderance for the men. As will be explained later, the higher demonstrability seems to be connected with the intensity of infections; so that a group with a higher mean number of infections shows higher demonstrability, for each species, than a group with fewer infections in each individual. Now one examination of the 340 individuals in Peking revealed a mean number of only 0,5 infections per case, whereas 0,9 were found in the 714 mental hospital women in Sweden, while the 51 1 male patients in the mental hospitals showed a mean number of 1.6 infections - all found by a single examination. If the demonstrability in 6 examinations is presumed to be equal for the Peking series and the mental hospital women, any error will certainly consist in giving too high a value to the former and consequently also to the percentages of actual incidences demonstrated in the series of 1st examinations. O n the assumption of equality the following table is computed.

I ) In Do b e 1 1’s work cited above 78 yo of the ultimately found infections with E. c 0 1 i were detected in the 6 first examinations, while the corresponding figures found in the work of C a r t e r , M a c k i n n o n , M a t t h e w s 8 S m i t h are 83 yo for E. c o l i and 40 yo for C h i l o - m a s t i x.

YH] RUTH SVENSSON

Table VI. Actual numbers found, estimated real incidences, and percentages of the latter discovered in the series of 1st

examinations on 340 persons in Peking. Numher of infections

found in 1st examination

E. hiatolyfica . . . . . . . . . 40 E. coli . . . . . . . . . . . . . . . 48 E. nana . . . . . . . . . . . . . . 58 I. bufschlii . . . . . . . . . . . 12 Ch. mesnili . . . . . . . . . . . 6

Numher of infections found in 6

examinations 91

105 122 40 21

Estimated real

incidence

126 124 163 58 41

% of estimated real incidence found in 1st examination

32 39 36 21 15

This table must, of course, be regarded with certain reserv- ation. The general tendency for lower demonstrability to be correlated with lower incidence of infections is, however, very striking, and stands out clearly in comparison with the two Vastervik series, as shown in table VII.

Table VII . Comparison of percentages of infections demonstrable by single examinations.

Mental hospital case8 in Vaotervik Peking Men Women Men and women

E. hislolytica . . . . . . . . . . . 27 32 32 E. coli . . . . . . . . . . . . . . . . 68 50 39 E. nana . . . . . . . . . . . . . . . 47 33 36 I. butschlii . . . . . . . . . . . . . 60 28 21 Ch. mesnili . . . . . . . . . . . . . 49 29 15

It is of special interest to note that the high demonstrability of E. nana by single examinations in the Peking series coincides with the unusually high incidence of this particular species: 36 "/o found by 6 examinations, compared with 40 "/o found in arbitra- rily chosen female patients in Vastervik by the far more effective diagnostic method of examining one formed and one loose stool from each.

From the figures already presented I feel justified in con- cluding that there is a definite relation between demonstrability of cyst-forming intestinal protozoa and incidence of infections: and another observation may be recorded as confirming this


correlation. If the women's group from Vastervik is further divided in two, one including all cases with 4 and more infections and one with 1 to 3 infections per case: then we get in the group with higher incidence of infections 24 cases of E. coli giving 130 positive examinations out of a total number of 232, in the group of lower frequency 8 E. coli carriers giving only 36 positive examinations in a total of 90. Too few carriers of the other species are found in the group with lower incidence for any reliable calculations to be made. The highest number is found for Chilomastix with 6 infections giving a total number of 53 examinations, but considering the very fitful appearance observed in this species such a small series is likely to be too much influenced by peculiarities in the individual infections to allow any reliable conclusions. The figures below therefore refer to E. coli alone.

Table V I I I . Demonstrability of E. cdi by single examinations in 2 groups of female mental hospital patients.

Number Number Positive examinations : of of

Women with: cases stools Number Per cent

A/ 1-3 infectians per case 8 90 36 40 t: 5.2 B/ 4-8 infections per case 24 232 130 56 2 3.3

Difference /A-B/ = 16k6.1 yo. The calculated demonstrability of E. coli infections in one

stool in different populations, based upon actual observations, can now be presented together in a rising scale:

Table IX. Demonstrahility of E. coli by single examinations in 5 different populations.

British soldiers Vastervik mental hosp. patients SVENSSON

Women with Women with Men with 1-3 infectlons 4-8 intrctlons 3-8 infections')

b-- - CARTER G all. - DOBELL

31tl.0y0 33+2.3% 40+ 5.20,; 5 6 3 3 % 6824.1%

In my publication with L i n d e r s I have given some plausible explanations why demonstrability of infections should be so high in mental hospital patients of long residence in the insti-

I) Among the men there was none infected with less than 3 species.

42 RUTH SVENSSON

tution. Great importance is there ascribed to the large variety of intestinal bacteria which obviously have equally good chances of spreading from patient to patient as the several protozoal species. It is well known by now, especially through D o b e 1 1's recent works, that a suitable concomitant bacterial flora is one of the most important requirements for encystation in oitro. Although less easy to control or demonstrate, similar conditions must be expected in vivo. Consequently, where people of unsatisfactory hygienic habits live in close contact, they will transmit to each other not only diverse kinds of protozoa, but also a larger variety of bacteria favourable for the encystation of various protozoa than more isolated people can be expected to harbour. That such general distribution really occurs is shown by the fact that 90 mental hospital patients from whom I have made cultures were all found harbouring starch-splitting bacteria, which are not constantly present in populations living under normal conditions.

Another more direct explanation may be ventured here, The demonstrability of infections in formed stools requires that cysts should be present in fairly large numbers, as even in a most careful examination only a very small portion of the stool can be searched. Even infections, found in loose stools to be quite heavy, may sometimes be very difficult to discover in formed stools - no cysts, or only very few, being found on careful examination. O n the other hand, light infections can never show great numbers of cysts and will therefore always be difficult to demonstrate in formed stools. Infections in people in sanitary surroundings, and comparatively isolated living conditions, may well become slight and therefore difficult to disd cover; whereas people in mental hospitals are usually exposed to repeated re-infections with the same species, and this tends to keep infections heavy. Moreover, what is recorded as one infection (with E. histolytica, E. coIi etc.) may in reality represent several infections with the same species. W e know, indeed, that various species of protozoa consist of distinctly different races, characterized inter alia by size and structure of the cysts: and this may sometimes enable us to demonstrate the existence of multiple infections, as in D o b e l l tj J e p p s (1918).


Now it goes without saying that 3 infections of E. coli in one person will be more easily discovered than one single strain in another individual. The evidently very good chances that mental hospital patients have to pick up all species and strains of protozoa can with great likelihood be considered as one of the factors responsible for the high demonstrability of their infections. This leads to a question of great interest, viz., how far a mental hospital population is typical in regard to demonstrability of protQzoa1 infections, so that the demonstrability, a t least with E. coii. may be expected to be approximately the same in any population with similar frequency of infections. No actual observations are known to me from which this problem could be solved. From purely theoretical considerations I am inclined to believe that the demonstrability found in my mental hospital groups is exceptionally high. Protozoal infections may be acquired either by close personal contact or from contaminated food or water. In the first case, the concomitant bacterial flora of the donor is likely to be partially acquired at the same time as the protozoa. If, on the other hand, the majority of infections are acquired by a more indirect way with contaminated food or drinking-water, a fairly long time (days or even weeks) may have passed from the moment when cysts were voided in the stool of an infected person till they were ingested by the new host. Meantime many of the bacterial strains which originally accompanied the cysts may have been separated from them, or have succumbed because unable to withstand the conditions outside the human bowels as long as the fairly resistant protozoal cysts. Chronic mental hospital patients who live under conditions of strictly controlled foodhandling and water-supply, but in close contact with fellow patients of unsatisfactory personal hygiene, are likely to acquire the majority of their infections in a very direct way and together with a variety of intestinal bacteria, thus developing conditions especially favourable for encystment of the protozoa. Where most infections are acquired in a more indirect way, such convergence of factors facilitating infections as well as encystment cannot a priori be expected to occur.

The conclusions which can be drawn from the observations

44 RUTH SVENSSON

and considerations stated above are that figures obtained by examination of formed stools are not directly comparable in different surveys, as the demonstrability of protozoal infections in single, or even 6, formed stools from each case may be very different in different populations, yet with an exception for E. histolytica, of which probably about one-third is discovered by single examinations and slightly more than two-thirds in 6 examinations in any kind of population.

6. Examination of loose stools, The great advantage of loose stools obtained after administration of a saline purgative is that no protozoal species is systematically overlooked and that, in addition to the trophic forms, cysts are about as likely to occur in these stools as in formed samples. Several workers have therefore in their surveys used this examination method, either solely, as B a r r o w (1924), S mi t h i e s (1926), L y n c h ( 1926), and R i 1 e y ( 1929), or in combination with other methods - examination of formed stools or cultivation or both - as C r a g g (1917), W i g h t (1926), and M a g a t h 6 W a r d (1927). There are hardly any theoretical limitations to the diagnostic value of this examination method, What insufficiency may have been observed in this method, as demonstrated in the 3rd column of table I, from the figures of which can be calculated that 23 % of known infections - 98 out of 421 - escaped detection in one loose stool per case, may well be referred to practical difficulties. B a r r o w, who made 9 examinations on each of 575 cases, found that 78 % of the ultimately demonstrated infections were disclosed in the first examination, which is in very good agreement with my findings (table XII, p. 50). A n d r e w s (1934) who examined 6 formed and 1 loose stool from each of 50 individuals found 32 or 89 % of the total of 36 demonstrated infections in the loose stools. This result is also in good agreement with mine, as it is very probable that the total number of infections would have increased if cultivation had also been employed in A n d r e w s’ survey. In my survey here referred to, only one sample from each patient was brought to the laboratory. In some cases this sample may have been derived from an early defaecation before the contents of the higher parts of the bowels had joined the current; in other cases


the sample perhaps was taken on a late stage of the provoked diarrhoea with the risk of lower situated protozoa already having been swept away. Probably very few of the species present would escape detection if samples of every single stool on the day of purging were examined. The practical difficulty is, however, that the examiner and the patient must then be within easy reach of each other for several hours - in many cases the whole day - as the stools must, for identification of trophic forms, be examined very shortly after they have been voided. This requirement might be easily fulfilled in single instances, but is almost impossible to meet in extensive surveys. The identification of all species requires, moreover, the examination of stained as well as fresh preparations. In the latter flagellates are easily recognizable on account of their characteristic motility, arrangement of flagella and presence or absence of an undula- ting membrane. In many cases amoebae can also be identified from the shape of their pseudopodia and their type of movement, 'hough exact diagnosis of amoebic species must be foun- ded on the nuclear structure in stained preparations. A careful technician may well be trusted with the preparation and fixing of films in Schaudinn's solution, after which the staining in Heidenhain may be postponed to any convenient time. But for mere diagnostic purposes Heidenhain-stained films are much less suitable than Quensel-stained preparations, as many amoebae are lost in the manipulations required for fixing, staining, destaining and dehydrating, and infections represented by only a few individuals may be impossible to recover in the finally mounted preparation. The Quensel- preparation, requiring only a suspension of the stool specimen in the staining solution, retains every protozoal individual originally present and thus lessens the risks of very light infections being lost. The rapid overstaining with this method forbids, however, delay in examination and thus necessitates the same prompt attention to the faecal samples as does direct examination.

After these practical difficulties have been pointed out, the diagnostic superiority of the examination of loose stools over formed stools. however numerous, may be summed up as follows:

46 RUTH SVENSSON

(1 ) The chances of finding cysts are almost as good in artificially loose stools as in formed. ( 2 ) Trophic forms are always present in the loose contents of the bowels and are therefore very likely to be found when these are examined in a fresh state. ( 3 ) No protozoal ,species will be systematically overlooked.

The chief advantage of cultivation is that multiplication of the protozoa occurs so that even light infections, where the original inoculum contains only a single or very few individuals, easily overlooked in direct preparation, may give rise to flourishing cultures after 12 and 24 hours’ incubation. Preparations made from cultures are also easier to examine than direct preparations from stools, as in the former the protozoa are surrounded by much less faecal material than in the latter and therefore easier to detect. As a diagnostic working-method cultivation has, however, more drawbacks than advantages. The results are uneven and unreliable, as a concomitant harmful bacterial flora may entirely check the growth of certain protozoal species, Giardia is not demonstrable at all by cultural methods. Moreover, what is called cultivation in my tables does not simply mean incubation and examination of one single culture tube from each case, but mostly implies that primary cultures have been made in at least two different media and most cultures have been subcultivated for one or more generations, - Cultivation as a diagnostic method has also been discussed by C r a i g 8 S t . J o h n (1927), M a g a t h G W a r d (1928) and K e s s e l 6 M a s o n (1930), but as in their surveys only egg-media and liquid Locke-serum media were used, their figures are not comparable with those obtained in my work by the additional use of a more suitable medium and by subcultivation. M a g a t h S W a r d present figures for 7 species and find cultivation inferior to loose stools for every species. C r a i g S S t, J o h n present figures for amoebae only and report better results of cultivation than of direct examination, K e s s e 1 8 M a s o n find cultivation inferior to “smear” for detecting infections with E , histolyfica, but equal or superior for Chilomasfix and Trichomonas.

In tables X and XI the merits of the two chiefly used culture media are demonstrated. In each of the cases recorded here

c . Culfiuation.


subcultivation has been made of all negative primary cultures - and the majority of positive also.

Table X. Known infections demonstrated by cultivation in DL + S + F medium.

Species : E. hisfolyfica . . . . . . . . . . . . . . . . . . E. coli . . . . . . . . . . . . . . . . . . . . . . . E. nana ........................ I , biifschlii ..................... D. fragilis ..................... Ch. mesnili . . . . . . . . . . . . . . . . . . . . . Tr, hominis .................... Es. hominis ....................

Infections present

Number 40 65 49 30 62 48 13 59

Infections demonstrated

Number 010 38 95 54 83 26 53 19 63 61 98 23 48 10 77 55 93

Table XI . Known infections demonstrated by cultivation in BD-medium.

Spcdes :

E. histolytica . . . . . . . . . . . . . . . . . . E. coli . . . . . . . . . . . . . . . . . . . . . . . . E. nana . . . . . . . . . . . . . . . . . . . . . . . I . biitschlii ..................... D. [ragilis ...................... Ch. mesnili .................... Tr. hominis .................... Es. hominis ....................

Ink c ti o n I present

Number 20 28 28 13 36 46 14 49

Infections demonstrated

Number 010 13 65 10 36 11 39 3 23

22 61 33 72 10 71 43 88

It is evident from the tables that DL + S + F is on the whole a much more suitable medium than BD, although the latter is superior for Chilomastix and almost equally good for flagellates in general. E. nana and Zodamoeba grow badly in both kinds of media. The use of both media has, of course, given better results than either of them separately. Yet, even by combination of cultivation methods as much as 24 % of known infections has escaped detection, as can be calculated from the last column in table I1 (p. 32). Table I (p. 31) shows that

48 RUTH SVENSSON

Dientamoeba and Enteromonas are the species for which cultivation is least dispensable, 39 and 37 % respectively having been demonstrated only by cultivation, As just these two species are most easy to cultivate in DL + S + F, and usually appear already in the primary cultures, cultivation in this medium without subcultivation may be regarded as a good complement to the other diagnostic methods and fairly well justifying the labour spent.

Now it may be remarked that my material has been exceptionally difficult to cultivate on account of the constant presence of vigorous starch-splitting bacteria and Blastocystis. Better results might be obtainable in populations with a different intestinal flora. But the very fact that the success or failure of protozoal cultures depends so essentially upon the concomitant bacteria makes cultivation as a general diagnostic method very fallacious in spite of its obvious advantages for certain species. The necessity to start the cultures from very fresh loose stools is also an additional difficulty, although a trustworthy assistant may be relied upon for this part of the work.

3. EXHAUSTIVE EXAMINATION.

It is, of course, impossible to prove that any system of examinations has been effective for disclosing every protozoal species present in a human host. Every single method used in my work has proved insufficient for exhaustive examination. It must always remain a matter of personal opinion how far the combination of several methods can be regarded as having a fair chance to detect all infections. The diagnostic methods are undoubtedly most uncertain regarding Giardia: this species cannot be demonstrated by cultivation and the appearance of cysts may be very irregular. ( 191 7) reported two cases followed over a long period of time. In one case cysts were found only once in 128 examinations, whereas another case was found positive on each of 50 successive days. As has already been pointed out in a previous section of this chapter the theoretically best chances of finding all infections

Thus e. 9.. D o b e 1 1


would be to examine every stool passed as the result of an effective dose of a purgative (magnesium sulphate in repeated doses of 15 gm or in a single dose of 40 gm has served very well in my work). Even Giardia is likely to appear as trophic forms in some of the later defaecations and thus be diagnosed also during cystless periods. Cultivation from a fresh loose stool in DL + S + F and BD with subcultivation for one or two generations is a very good supplementary method, as the multiplication in vitro will give light infections a chance to show up. I am personally satisfied that very few - if any - infections would escape detection by this combination of examination methods. Formed stools may safely be omitted, as any cysts present at the time of examination will probably have as good a chance of being found in the first stools passed after the administration of the salt as in a spontaneously voided sample.

4. PARASITOLOGICALLY REPRESENTATIVE SURVEYS.

W e shall now analyze the material already referred to in order to ascertain how a parasitologically representative survey can be conducted with the best economy of time and labour. Examination of formed stools alone can be ruled out at once, as several species cannot be discovered at all in such specimens, In two tables below the results obtained by the two remaining single methods (table XII) and by combination of one formed with one loose stool as well as combination of loose stool and culture (table XI11 ) are recorded. The theoretically possible combination of one formed stool and culture is not considered as a practical system of examinations, as cultures, for giving uniform results, should be started from very fresh, loose stools, and the same specimens from which the inoculums are taken will naturally be subjected to microscopic examination.

50 RUTH SVENSSON

Table XU. Known infections demonstrated by single examination methods.

Infections present

species : Numbet

E. histolytica 44 E. coli 73 E. nana 61 I . biitschlii 39 D. fragilis 59 G. intestinalis 17 Ch. mesnili 58 Tr. hominis 12 Es. hominis 54

Infections demonstrated : - In one loose stool In culture

Number O/O Number Of0

33 75 k 6.5 39 89 ? 4.8 64 882 3.8 56 77 2 4.9 49 802 5.1 32 52 L 6.4 32 82+ 6.1 22 56 2 7.9 36 61 -1- 6.3 57 97 L 2.4 12 7 1 ~ 1 1 . 0 0 0 48 83- 5.0 44 76 t 5.6 11 92+ 8.0 12 100 34 63+ 6.6 53 982 1.9

Table XlIl. Known infections demonstrated by combinations of examination methods.

Infections present

Species: Number

E. hisfolytica 44 E. coli 73 E. nana 61 I . bdtschlii 39 D. fragilis 59 G. intestinalis 17 Ch. mesnili 58 Tr. hominis 12 Es. hominis 54

Infections demonstrated : c- --4

In one formed and In one loose stool one loose stool and culture

Number 010 Number Of0

39 89 & 4.8 43 98 t 2.3 70 96 k 2.3 69 95 t 2.7 54 89 k 4.1 57 93 ?r 3.2 35 90 Lt: 4.9 37 95 2 3.5 36 61 + 6.3 59 100 16 94 i. 5.7 12 71 2 1.1 52 90 k 4.0 56 97 t 2.4 11 92 J- 8.0 12 100 34 63+ 6.6 54 100

If the cultivation methods are considered first in combination with the examination of one loose stool, as shown in the last column of table XIII, it is found that by this system of examination are obtained not only very representative but nearly exhaustive results: between 90 and 100 510 of the infections with all species except Giardia being detected. The omission of formed stools in a parasitologically representative survey might be fully justified but for the fact that it is much easier to diagnose infections with E, histolytica and E. coli from cysts in formed stools, than from amoebae in loose stools or cultures, In the present


series the examination of, in most cases, a minimum of 6 formed stools from each individual did not add more than 18 infections to those detected in loose stools and cultures. Out of these 18 as many as 5 were Giardia, and if, as seems inevitable, one must generally abstain from estimating the true incidence of this species, a few more or less Giardia infections discovered will make little difference to the representativeness of a survey. Thus only 13 elucidating discoveries were made in the formed stools, a negligible number out of the 421 infections present.

Culture methods alone give very good results with some species, but rather poor with others. The omission of loose stools would therefore very considerably lessen the representativeness of the survey. It would not even Save much time, as cultures, as already stated, are preferably started from fresh loose stools, which can conveniently be examined immediately after the inoculation of the cultures tubes, and do not require more time than about 20 minutes.

In table XI1 we find that examination of one loose stool per case will reveal roughly 3/4 of the infections present. The minimum demonstrability is found for Dientarnoeba and Entero- monas which show as low percentages of discovery as 61 and 63 respectively, while E. coli and Trichomonas, for the detection of which the method has proved most effective, show as high percentages as 88 and 92. It is evident from a comparison of both tables that although examination of loose stools alone gives less varying and for some species better results than cultivation alone, it is decidedly inferior to the combination of both methods.

Although it may theoretically be considered very desirable to demonstrate as high and, above all, as constant proportions of the actual infections as the combination of loose stools and culture renders possible, it is an important fact that cultivation requires much time, skill, and equipment, and may be practically impossible to carry out where facilities for preparation of media and incubation of cultures are lacking. Moreover, the amount of labour required for comparatively complete cultivation is out of proportion to the information gained where an exhaustive examination is not intended. - Where cultivation is easy to do,

52 RUTH SVENSSON

I should recommend making a primary culture, without subcultivation, in DL + S + F from each case in order to detect the infections with Dientamoeba and Enteromonas in proportions about equal to the other species. But even without any cultivation the examination of loose stools gives sufficiently representative results: one has only to remember that the figures obtained for Dientamoeba and Enteromonas represent comparatively low proportions of actual incidence.

If comparison be made between examination of one loose stool only, and the combination of this method with one formed stool, as presented in tables XII and XIII, we find again that the latter method adds nothing to the representativeness of the survey. as only the cyst-forming species are discovered in higher percentages. The less laborious method of examining one loose stool only must therefore be regarded as more even and therefore more representative than examination of one formed as well as one loose stool per case. It can also be expected to be representative to the same degree in any population, as there is no reason to suppose that an artificially provoked diarrhoea should, in different surveys, bring out different proportions of the trophic forms living in the liquid contents of the bowels. Only where trophic amoebae, but no cysts, of E. h$tolytica and E. coli have been found in the loose stool, a confirmation of the diagnosis from cysts in formed stools may sometimes be necessary.

5. ENDEMIOLOCICALLY REPRESENTATIVE SURVEYS.

In the foregoing section we have seen that representative and nearly exhaustive figures for the different protozoal species are obtained by examination of fresh loose stools and cultivation from such specimens. The general use of these methods in more extensive survey work meets, however, with practically unsurmountable difficulties. especially if the requirement is upheld that the conductor of the surveys should personally identify thle species. The necessity of attending to the stools immediately after they have been passed implies that the worker as well as the persons who have to supply the mate-


rial must stay in or near the laboratory for a considerable length of time, as the effect of a purgative may either be very prompt or much delayed. The examination of the specimen itself can be made in a relatively short time, but cultivation is, as already expounded, a very laborious process, while the necessary equipment may not be available when a survey is desirable. The only practicable method in all cases and circum- stances will therefore remain the examination of spontaneously passed stools, Moreover, if loose stools and cultivation were the examination methods solely acceptable, the whole continuity of survey work all over the world would be broken, and extensive and valuable surveys conducted prior to the discovery of cultivation methods in 1924 would be unfit for comparisons with recent surveys.

In my definition of an endemiologically representative survey the stipulation has been made that only such figures should be used as are comparable in different surveys. The number of protozoal species considered is unimportant. As it has been shown that E. histolytica is the only species with a fairly constant demonstrability in formed stools, it may seem practical to use the records for this species only. E. histolytica is, however, a rare species in many populations and very large numbers of people would have to be examined in order to get usable figures. A better scheme might be to determine the demonstrability of different species separately for each population by examining, in addition to the formed stools, one loose stool from a number of people representative of the population investigated. This method will disclose, as shown in table XIII, roughly 90 % of the existing infections with cyst-forming species. From the figures thus obtained the chances of detecting infections in formed stools might then easily be calculated with sufficient accuracy to make comparisons with other surveys possible. This method will, however, be reliable only where a series of single-specimen examinations is representative of the population from which it is a sample. In order to test whether this is the case, the records of the first 6 examinations of the exhaustively examined mental-hospital patients are treated as 6 separate series of single-specimen examinations on the same population

54 RUTH SVENSSON

of infected individuals. Again the men and women are treated separately. The whole material is presented case by case in table XIV and in a concentrated form in tables XV and XVI,

In table XIV the following abbreviations are used:

E. h. = Entamoeba histolytica E . c, = Entamoeba coli E . n. = Endolirnax nana I . b. = Iadamoeba butschlii G. i. = Giardia intestinalis Ch. m. = (Chilomastix mesnili

A positive examination is always marked by a +. A negative examination is marked by a 0 if the case has been found infected with the species in question by some method, and with a - if the case has been negative to all methods of examination.


Table XN. Results of each single examination in a series of 6 formed stools per case. M E N.

Case Species

9V 40

9V 42

9V 45

9V 73

9v 74

9V 76

Serial nr. of stool:

1 2 3 4 5 6 - E. h. E. c. E. n. I . b. G. i. Ch. m.

E. h. E. c. E. n. I , b. G. i. Ch. m.

E. h. E. c. E. n. I . b. G. i. Ch. m.

E. h. E. c. E. n. I . b. G. i. Ch. m.

E. c. E. n. I.. b. G. i. Ch. m.

E.h- E. c. E. n. I . b. G. i . ICh. m.

E. h.

_ _ _ --- + + + o + + o + o + + + 0 0 0 o o + + + + + + + + o o 0 0 0

V 77

V 78

J 237

J 278

r 282

I 291

+ o + + + + 0 0 0 + + +

E. h.

E. n. I . b. G. i. Ch. m.

E. h. E. c. E. n. 1. b. G. i. Ch. m.

E. h. E. c. E. n. I . b. G. i. Ch. m.

E.h. E. c. E. n. I . b. G. i. Ch. m.

E. c. E. n. I . b. G. i. Ch. m.

E. c. E. n. I . b. G. i. Ch. m

E. h.

E. h*

--- --- 0 0 0 0 0 0

o + o 0 0 0 + + + + + + + + o + + + + + + + + + + + + + + + + + + o + + 0 0 0 + o o + + + 0 o + + + + + + + + + + + o + o o + 0 0 0 + o 0 + + + o + + + + o o + + + + o

o o + + o +

_ - - --- _ _ _ ---

--- ---

Case 1 Species Serial nr. of stool :

I 2 3 + S 6

- _ - --- $ 0 0 o + + 0 0 0 0 0 0 + o o o + + + + + + + o + o o o + o

0 0 0 o o +

0 0 0 o + + + + o + + + 0 0 0 + o o + + o + o o + + + + o + + o + + + + + + + + + + + o + o + +

- _ - --- _ _ _ ---

- _ - ---

--- --- + + o o + + o o + + o + o o + 0 0 0

0 0 0 0 0 0

+ + o o + o + + o + + + + + o + o o + + + + + + + o + + + + 0 0 0 0 0 0 o + + + + + 0 0 0 0 0 0 + + o 0 0 0 + o + + + +

_ _ - ---

- _ - ---

- _ - I--

56 RUTH SVENSSON

Serial nr. of stool : Serial nr. of stool : Care 1 Species 1

1 2 3 4 6 6 1 2 3 4 5 6

V 304 E.h . 0 0 0 0 0 0 V 381 E . h . 0 0 0 0 0 + E . c . + + + + 0 0 E.c . 0 + + + + + E.# . 0 + 0 0 0 + E.n . 0 + 0 0 0 0

1.6. + + + 0 0 + fob. + + + + 0 + G. i. Ch.m. + 0 0 + 0 0 Ch,m, 0 + 0 0 + 0

G.i, - - - - - - _ _ - ---

V 3 0 7 E . h . 0 0 0 0 0 0 V 3 8 5 E . h . --- --- E . c . + 0 0 0 + + 8 . c . 0 0 0 0 + + E.n. + + + 0 + + e.n. 0 0 0 0 0 + 1.b. + + + + + f I.b. 0 0 0 0 + +

0.1. 0 0 0 + + + G. i, Ch.m. + + 0 0 0 0 Ch.m, 0 0 0 + 0 0

_ _ - - - - -

v 323 E.h . 0 0 0 0 0 0 V - y 2 2 E . h . --- --- E.cc. - - - - - - E , n , _ _ - --- E . c . + + + 0 + +

E.n. + 0 0 0 + 0 1.b. - - - - - - 1.b. + 0 + + + + G. i. Ch.m. + + + 0 0 0 Ch.m. --- ---

~ 3 4 6 E . h . 0 + + 0 + + V - y 2 6 E . h . --- --- E . c . 0 0 + + + + E.C. 0 0 + 0 0 0 E . n . + 0 + 0 0 0 E.n. 0 0 + + + 0 1.6. 0 0 0 0 0 + G. i. Ch.m. 0 + + + 0 0 Ch.m. --- ---

~ 3 4 9 E . h . + + + + + V - y 2 8 E . h . 0 0 0 0 + + E.c. + + + + + + E . c . 0 + + + + +

E.n . 0 + 0 0 0 0 1.b. 0 + 4- + + + G.i. - - - - - - 1.b. + + 0 + + +

G. i. Ch.m, 0 0 0 + + 0 Ch.m. + 0 + + 0 t-

E . C . 0 + + + + 0

_ _ - --- G. i. _ - - ---

1.b. _ _ _ _ _ _ - - - - - - _ _ - ---

E a n . - - - - - -

_ _ _ ---

~ 3 5 4 E . h , 0 0 + 0 0 0

E.n . 0 0 4- -k 4- 0 1.b . 0 + + + + + G.i. 0 0 0 4- 0 0 Ch.m, 0 4- + 0 0 0


Serial ar. of stool : Senal nr. of stool :

1 2 3 4 5 6 1 2 5 4 5 6

9Vt 1 E.h. --- --- 9V 13 E.h . --- --- E.c . + 0 0 0 + 0 E.C. + + + + + + E. n'" 0 0 0 .O 0 0 E.n . 0 0 0 + 0 0 I ,b . 0 0 0 0 + 0 1.b. - _ - - - _

G.i. + + + + + 3. Ch.m. 0 0 0 0 0 0 Ch.m. 0 0 0 0 0 0

9V 4 E , h . --- --- 9 v 15 E . h . + -k + + + 0 E.c . 0 0 0 0 0 0 E . c . + + 4- + + + E.n. + 0 0 0 0 + E.n. 0 + + + + + G.i. + + + + + + G.i. + 0 0 0 0 0 Ch.m. 0 0 0 0 0 0 Ch.m. --- ---

G.i. - _ - - - -

1. b. I - - - - - 1.b. --- - _ _

9V 5 E . h . 0 0 0 0 0 0 9V 19 E.h . 0 + 0 + + 0 E . C . + + + + + 0 E . c . 0 0 0 0 0 0

E , t t 0 0 0 0 0 0 E.n. --- --- l . b . 0 0 0 0 0 0 1.b- --- ---

--- _ - _ G.i. 0 0 0 0 0 0 G. i. Ch.m. 0 0 0 0 0 0 Ch.m. 0 0 0 0 0 0

9 v 9 E.h . 0 0 + 0 0 0 9V 20 E.,.h. 0 0 0 0 0 0 E.c. + + + + 4- f E.c . + + + + 4- 0

E.n . 0 + 0 + -I- + 1.b. 0 + + + + 0 I , b . 0 0 0 + 0 0 G.i. 0 + 0 0 + 0 G. i. Ch.m. 0 + + + + 0 Ch.m. 0 0 + + 0 0

9V11 E.h . --- --- 9V24 E , h . 0 0 0 0 + + E.c . 0 + 0 0 0 0 E.c. 0 0 0 + 0 + E,n. u 0 0 0 0 0 E.n. + 0 0 0 + 0 1.b. - - - - - - I . & 0 0 0 0 0 0

G.i. + + 0 + + + G. i. Ch.m. 0 0 0 0 0 0 Ch.m. 0 0 0 0 0 0

E . n . - - - - - - --- - - _

--- ---

9V 12 E , h . 0 0 0 0 0 0 9V25 E . h . --- --- E.c . 0 0 + + 0 0 E . n , --- --- E . C . 0 + + + + +

E.n . + 0 + + 0 4- I.b. 0 0 0 0 0 0 1.b. - - - - - - G.i. 0 0 0 0 0 0 G.i. - - - - - - Ch.m. 0 0 0 -I- + + - Ch. m. - - - _ _

W O M E N .

58 RUTH SWNSSON

9V 71

9V 72

Case

- E, h.. E. c. E. n. I . b. G. i. Ch. m.

E. h.

r 31

9V 79

9v 34

9v. 38

9V 64

9V 66

G. i. Ch. m.

E. h., E. c.

9V 70

9V 83

v 2

&rial nr, of stool:

1 2 3 4 5 6 species

E. h. E. c. E, n. I . b. G. 1. Ch. m.

E. h. E. c. E, n. I . 6. G. i. Ch. m.

E. h. E. c. E. n. I . b. G. i. Ch. m.

v 3

E. h. E. c. E. n. I . b. G. i. Ch. m.

E. h. E. c. E. n. I . b. G. i. Ch. m.

E. h. E. c. E. n. I . b. G. i. Ch. m.

E. h. E. c. E. n. I . b. G, i. Ch. m.

1E. h. E. c.

11. b. IG. i.

‘ E . n.

C h , m.

IE. h. 1 E. c. 1E.n. 11. b. G. i. k h . m.

0 0 0 0 0 0 --- --- - - _ --- --- --- _ _ _ o + o + o o

--- _ _ _ + o o 0 0 0 0 0 0 0 0 0

--- - _ _ + + + ++-I-

--- - - _ + + o 0 0 0 0 0 0 o + + --- ---

--- _ _ _ 0 0 0 0 0 0 0 0 0 0 0 0

Serial nr. ot stool: Case I Species I

l a 3 4 5 6

o + o + + + 0 0 0 0 0 0 0 0 0 0 0 0

+ Q O o + + + o + o + + + o o o o +

--- _ _ _ 0 0 0 0 0 0

0 0 0 + o o

0 0 0 0 0 0

--- - - _ --- - - _

--- 0 0 0 o + o o + o 0 0 0


60 RUTH SVENSSON

V 143 E.h. E. c. E. n. I . b. G. i. Ch. m.

V-y 6 E.h. E. c. E. n. 1. b. G. i. Ch. m.

V-y 12 E. h. E. c. E. n. I . b. G. 1.

I Ch. m.

Selial nr. of stool :

1 2 5 4 5 6

E. c. E. n. 1. b. G. i. ‘Ch. m.

+ + + + + o s o + + o 0

%rial nr. of stool : j 1 2 3 4 5 6 1

o + o 0 0 0 o + o + + + _ - - ---

Tab

le X

V.

Infe

ctio

ns d

isco

vere

d in

6 s

erie

s of s

ingl

e- s

peci

men

exa

min

atio

ns o

n th

e sa

me

mat

eria

l. 2

I. b

irts

chlii

G

. inte

stin

alis

18

9 N

umbe

r!

O/o

N

umbe

r1

o/t~

10

56

6 1 6

7

Ch.

mes

nili

19

__

__

_

Num

ber\

o/o

11

~ 58

In

fect

ions

fou

nd i

n:

1st

exam

inat

ion

. .

. .

. 2n

d ,,

..

..

.

3rd

,, .

..

..

4t

h ,.

..

..

.

5th

,, .

..

..

6t

h .,

..

..

.

8 44

11

7 1

78

42

~ M

ean

in o

ne

exam

inat

ion

. .

1 Sta

ndar

d er

ror

in o

ne e

xam

inat

ion

~~

Num

ber

of

infe

ctio

ns p

rese

nt

M E

N.

__

_

~

E. h

isto

lytic

c

14

Num

ber/

o/

,

E. c

oli

2 4

21

14

29

10

1 10

I

9.3 E. n

ana 53

53

49

11.5

19

16

13.8

76

66

10.4

Table X

VI.

Infections discovered in 6 series of single-specim

en examinations on the same m

aterial.

Infections found in:

1st examination . . . . .

2nd ..

..

..

.

3rd ,.

..

..

.

4th ..

..

..

.

5th ..

..

..

.

6th ,,

..

..

.

Mean in one exam

ination. - . . Standard error in one exam

ination

WO

ME

N.

4 4 4 5 7 3

4.5

E. h

istolyth

14 N

umber of iD

fedions present 1 29 29 29 36 50 21

32 12.5

E. coli

32 _

_

Jum

h

-

14 17 15 15 17 13

15.2

~

o/o -

44 53 47 47 53 41

47 8.8

E. nana

25 rlum

be

7 5 6 10 8 10

7.7

28 20 24 40 32 40

31 9.2

I. biitschlii

rlumbe

1 1 2 4 2 0

1.7 8 -

010 -

12.5 12.5 25 50 25 0

21 14.5

i. intestinalis

rlumbe -

4 4 2 3 4 4

3.5

7 01, -

57 57 29 43 57 57

50 18.9

Ch. rnesnili

-

riumbe -

3 5 7 10 4 5

5.7 27 O

h -

11 19 26 37 15 19

21 7.9


Regarding the different species the following observations can be made:

E, histolytica. The numbers of infections detected in the different series of examinations are very small and therefore too much affected by fluctuations of sampling for any reliable calculations to be made from this material. As it has been shown elsewhere the average demonstrability of this species is, however, fairly constant (oide esp. pp. 39-40), and it may therefore be assumed that the findings in single-specimen examinations are representative, if the series is sufficiently large.

E. coli. Both in the men and the women very similar percentages of infections are discovered in the separate series of examinations, the deviations from the respective means in no case exceeding the standard error in one examination.

E. nana. Of this species also it can be said that no great variations occur in the different series.

I. biifschlii. The number of infections in the women's group is so small, that conclusions can hardly be drawn from the seemingly great variations in the different series. As regards the group of men, the deviations of the different series from the mean are much larger than in the foregoing species. amounting to nearly twice the standard error. As Zodamoeba has already been proved to possess a very variable average demonstrabili,ty in different populations and now is found to give results in different series of single-specimen examinations which are very poor representatives of actual conditions, it seems advisable not to use records of this species in comparisons between different surveys,

Giardia is present in numbers too small, in both sex groups, for basing any conclusions on the observations.

Chilomasfix. In the women's group the plain figures show so much dissimilarity that it seems very probable that large variations, due not only to chance, occur in the different series. In the men's group the recorded figures are more uniform, yet deviations from the mean exceeding the standard error occur. With this species also it seems advisable not to use figures derived from single-specimen examinations in comparisons between different surveys.

64 RUTH SVENSSON

From what has been said, only E. histolytica, E. coli, and E , nana can be relied upon to give results of single-specimen examinations fairly representative -of the actual conditions ip the population in which the survey is made. Instead of using them separately (after a determination of their demonstrability in each population) it is expedient to consider the several species together, This will to some extent neutralize the irregularities in demonstrability from day to day of each species separately, and at the same time diminish the standard errors in the percentages of infections found - which is important when one has to ascertain whether any population is more infected than another, regardless of the infecting species.

In table XVII the total numbers of infections with (A+) E. histolytica, E, coli, and E. nana, and ( B . ) with all species, are considered simply as protozoal infections, without distinction of species, and the 6 series of single-specimen examinations are again presented for men and women separately. The mean percentages of infections found in any single series are also given together with the standard errors in one examination.

Tab

le X

VII

. D

emon

stra

tion

of

know

n in

fect

ions

in

6 se

ries

of

sing

le-s

peci

men

exa

mm

atia

m.

A:c

ompo

und

figur

es f

or E

. his

toly

tica,

E. c

oli, a

nd E

. nan

a.

B:c

ompo

und

figur

es f

or a

ll sp

ecie

s.

v,

I 2 B

-~

8

~ ._

~_

_~

A

. B

.

v,

All

spec

ies

~~~

E.

hist

olyt

ica,

E.

coli

and

E. n

ana

Wom

en

Men

W

omen

71

10

0 11

3

X

C

1 I

52

I 52

33

29

1s

t ex

amin

atio

n .

. .

. .

. 25

'

46

1 25

1

35

3rd

49

1 49

,

36

4th

_.

..

_.

1

25

46

5th

..

..

. .

I 27

i 5

0 ~

45

53

53

~ 42

I

Num

ber

of i

nfec

tions

pre

sent

1 In

fect

ions

dem

onst

rate

d in

:

1 I

2nd

,, .

..

..

.

27

50

26

37

50

, 50

,

36

......

I 25

~

46

~

55

' 55

1

35

-1

~ 6t

h .

..

..

38.2

,

33.8

1

0

I 1 M

ean

dem

onst

ratio

n in

one

exa

mrn

a-

tion

. .

. .

. .

. .

. .

. .

. ~ 26

.5

~ 49

.1

I 27

.3

I 38

.5

51.7

1

51.7

Stan

dard

err

or i

n on

e ex

amin

atio

n .

. 1 6.

80

1 I 5

.77

I 5.

00

1

Diff

eren

ce

in

mea

n de

mon

stra

tion

I

men

-wom

en

(one

exa

min

atio

n)

10.6

8.

92 O

fo

17.9 f 6.6

9 O;

o I

OI

ul

66 RUTH SVENSSON

From this table it is evident that the combined figures for all 6 species are not representative of the actual infections in the group from which they are derived, as single series of examinations may show deviations from the mean approaching twice the standard error in one examination.

More uniform results of the separate series of single-specimen examinations are obtained if only E. histolytica, E. coli, and E. nana are chosen as compound incidences. The deviations of the single series are by this presentation of findings small, the largest deviation from the mean demonstration being of the magnitude of the standard error. There is, however, a much more important advantage in using the compound figures for the 3 species rather than for all 6 species. At the bottom of table XVII the differences in mean demonstration between the two sex groups are given. If all species are included, the difference is still as pronounced as when the species are considered separately, whereas compound figures for the 3 species in the two groups show a much smaller difference, which, moreover, only slightly exceed8 its standard error. The practically important deduction from this fact is that single-specimen surveys where the average demonstrability of the separate protozoal species is unknown or even known to be different, can be compared with a fairly satisfactory degree of accuracy if the figures for E. histolytica, E. coli, and E. nana are presented simply as so many infections, regardless of species.

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IV THE EFFECTIVENESS AND PRACTICABILITY OF VARIOUS METHODS OF EXAMINATION