The American Journal of Tropical Medicine and Hygiene - Volume s1-30, Issue 2, March 1950

By way of introduction to this panel on amebiasis we would like to recall the statement of the eminent English protozoologist, Dr. C. M. Wenyon (1947), who recently stated that interest in amebiasis seems to be stimulated by great wars. That was true after the First World War and it is occurring again at the present time. We know that groups of our servicemen were exposed to Endamoeba histolytica during their time in the services; and from the work of Becker (1946), Jacobs et al. (1946), and others, we realize that many of these servicemen must have returned to civilian life harboring the parasite, but—what has been the effect of these service-acquired infections upon the amebiasis problem in the United States? In view of the pre-existing prevalence of E. histolytica, it is well to remember the statement of Simmonds (1943):

Inquiry indicates that there are many variable factors in the reporting of amebiasis/amebic dysentery in the United States. In some States, cases of amebic dysentery only are reported while in other States reports include cases of amebiasis. In one State, reports are based on clinical evidence alone, while in 16 States diagnosis is confirmed by laboratory examination before the case is reported. In other States, some of the reported cases are confirmed by laboratory examination. Replies indicate that in 13 States, cases of infection disclosed by routine laboratory examination of stools are included in reports without clinical examination or reference of the report to the physician. Information on these various points is not available for certain States either because replies were equivocal or because of failure to answer the specific questions.

Over the past 15 years, there has been a gradual increase in the number of reported cases of amebiasis/amebic dysentery, with a more marked increase since 1945. Admissions to Veterans Administration Facilities have also increased since that year but it is not believed that such admissions account entirely for the increase in reported cases.

During the above-mentioned period, a gradual decrease has taken place in reported deaths from amebiasis/amebic dysentery. This decrease has not been as spectacular as has the decrease in deaths from bacillary dysentery. Deaths from unspecified dysentery have been out of proportion to reported cases and some of these deaths may have been due to amebic dysentery.

On the basis of cases reported, morbidity rates, and deaths reported, it would appear that amebiasis is more prevalent in the West South Central States, comprising Arkansas, Louisiana. Oklahoma, and Texas, than in any other part of the United States. There is evidence that the disease is more widespread in the Southern States as a whole than in any other section, with the possible exception of the Pacific Coast States, where the morbidity rate is comparatively high. On the other hand, New England ranks lowest in all of the above-mentioned categories.

It would appear that up to the present time the return of infected military personnel to the United States has influenced very little the amount of reported amebiasis in this country.

Dr. D'Antoni told you at lunch today that he has seen in his personal practice here in New Orleans more cases than are reported for the entire State of Louisiana. I can cite similar experience in Winston-Salem, N. C. We have found the incidence to be approximately 20 per cent in a miscellaneous group of some 2,500 individuals examined in the course of the last 2 years. In the recent past I have had the opportunity of discussing this problem with officials of the State Health department of North Carolina. I was informed that practically no cases were reported and that the infection was regarded as essentially non-existent within the state.

Dr. Wright's figures conclusively demonstrate the fallacy of health department statistics concerning the incidence of infection by E. histolytica. When an efficient reporting system is combined with accurate diagnosis such statistics have great value. This combination is rarely if ever achieved with respect to amebiasis.

Any discussion of amebiasis certainly ought to include what we know about our water treatment processes, insofar as their ability to destroy or remove the cysts is concerned. What I would like to do is present a general review of what is known about most of the processes that are used presently, and at the same time about other procedures that were tested, particularly during the war, in the attempt to find a more suitable and effective cysticidal treatment. I shall attempt to credit only some of the various sources.

I would like to discuss this subject from two approaches; namely, large-scale or municipal water supplies, and the small-scale, portable, or individual water supplies. With the former, the municipal supplies, one of the first treatments that is usually used is that of sedimentation. This is essentially a mechanical procedure in which it is anticipated that, with an adequate detention period, some of the larger particles may settle out.

In the absence of Mr. Mark Hollis, is there any other sanitary engineer present who would care to discuss the subject of water treatment measures in control of amebiasis that was presented by Mr. Newton?

There is one thing that should be pointed out. Mr. Newton may wish to comment further on this later on at this meeting. Even though as he indicated, most modern purification systems may be adequate to produce water free of histolytica cysts, we must recognize that poor plumbing may contaminate the water supply again. The experiences in Chicago have been adequate to impress the possibility that back-siphoning and cross-connections can result in the contamination of supposedly pure water (McCoy, et al., 1936).

I am not going to try to discuss the total problem of hepatic amebiasis in this period of time, of course. I thought it might be well to pick out three aspects of the clinical picture which are of current importance.

I want to say something first about the evolution of our knowledge of the clinical picture. When the relationship of solitary abscess of the liver to amebiasis was first recognized, it became perfectly clear that a clinical entity in the liver resulting from E. histolytica occurred and that abscess developed. The picture of abscess is an advanced picture. The process should be recognized long before it reaches this stage, although there are individuals who have so few symptoms that they may not consult a physician until they have an abscess dangerously close to rupture. But those patients are few and far between.

Let me begin by agreeing with Dr. Sodeman that hepatomegaly is an unfortunate name for the condition we are discussing. In effect, we are describing a clinical condition under the nomenclature of one of its manifestations, which is undesirable, even though this particular finding is the most prominent manifestation. None of us, however, have been able to think of anything better, so the name must remain hepatomegaly for the present.

The first point I would like to make is that in children who harbor E. histolytica—and let me emphasize that in these cases there is laboratory proof of the presence of parasites—the status of the liver as respects size depends upon the age at which the child is seen. Hepatomegaly is a prominent finding in children with amebiasis under the age of 6 years. It becomes manifest early in the infection and in untreated cases it continues up to this age.

Using the commercial antigen now available and the method described above, the complement fixation test appears to be of diagnostic value in hepatic amebiasis. A positive test is of considerable significance although negative results may occur in clinically proven cases. The complement fixation test as performed was of no value in the diagnosis of intestinal amebiasis; the sera of only 2.4 per cent of persons harboring E. histolytica in their stools gave a positive reaction. This may be an advantage, however, for the common occurrence of intestinal amebiasis does not confuse the diagnosis of amebic hepatitis by a positive complement fixation test.

Sera from patients with the following diseases gave negative amebiasis complement fixation tests: malaria, visceral leishmaniasis, Echinococcus cyst of the liver, infectious hepatitis, cirrhosis of the liver, carcinoma of the liver, cholecystitis, infectious mononucleosis, intestinal carcinoma, rickettsialpox, sprue, syphilis, ascariasis, and trichiniasis. Sera from single patients with clonorchiasis, malaria, chromoblastomycosis, and proctitis-sigmoiditis gave false positive reactions which in general were weak.

I came to this meeting to support the work of Drs. Hussey and Brown, but now I find that I am going to change my talk and violently oppose Hussey's and Brown's conclusions as to the value of the complement fixation test for the diagnosis of intestinal amebiasis. We have been conducting this test since about 1942, and have run over 13,000 tests. I want to point out that there probably is a little discrepancy in the technic and the antigens employed which is really responsible for the difference in the results between the two laboratories. In the first place, they are using the three 50 per cent units of complement for the titration and a more concentrated antigen as prescribed by Kent and Rein (1946). Now the original work instituted in the Army Medical Center was done at the instigation of General Callender. He was interested in amebiasis and began all the work at Walter Reed.

I have been asked to discuss the chemotherapy of amebiasis, a subject which I must necessarily approach from the viewpoint of a laboratory worker who is concerned with the search for more effective therapeutic amebacidal agents. Discussion of the chemotherapy of hepatic amebiasis might seem superfluous after Dr. Conan's presentation of the excellent results obtained in the treatment of hepatic amebiasis with chloroquine, but there are certain aspects of the disease which deserve further consideration or emphasis. Since the development, evaluation, and successful clinical application of amebacidal drugs will be affected by factors which are inherent in the pathogenesis of the disease and in the various properties of a drug, I wish to direct your attention particularly to those aspects of pathogenesis and pharmacological behavior which are determining factors in successful chemotherapy.

The etiology and pathology of amebic lesions appear to be clearly defined and uncomplicated.

John Bozicevich, National Institutes of Health, Bethesda, Md. The complement fixation test is a tool and guide to assist the physician in diagnosing amebiasis. The serological test should be conducted concomitantly with the stool examinations. Both of these tests should be conducted prior to specific therapy. Unfortunately, the stool examinations for the identification of E. histolytica are difficult. It requires a carefully trained individual. The impression gleaned from some of the medical courses is dangerous, because the impression conveyed is that the stool examination for proper identification of E. histolytica is a simple task. Prior to this panel discussion, I talked with Dr. Faust concerning the stool examination and he remarked that in his opinion it was necessary for an individual to study the morphology of E. histolytica 8 hours a day for a period of 6 months and then to continue the study at intervals before an individual could become proficient in identification of the amebae.

• 1.  Eleven hundred and ten students at the University of Wisconsin, most of whom were service veterans of World War II, have been examined for E. histolytica: 4.5 per cent of overseas veterans, 2.5 per cent of those with domestic service, and 1.6 per cent of those with no war service, were found infected.
• 2.  Studies on the incidence of amebiasis in over 50,000 service personnel during the late war are summarized and the average incidence of 14.7 per cent has been computed.
• 3.  Studies on the incidence of amebiasis in college student populations prior to the war are summarized and the average incidence of 4.9 per cent has been computed.
• 4.  The incidence of amebiasis among service personnel during the war is comparable to the estimated incidence in the general population for the United States. When compared to similar groups three years after the war this rate seems to be significantly higher.
• 5.  The incidence in a postwar student population composed of men with military service is no greater than before the war in student populations.
• 6.  It seems probable on the basis of the present findings that there has been little change in general incidence of amebiasis as a result of the war.

This study was designed to assess by selected criteria the capacity of amebic antigen to influence the resistance to infection and the gross pathology in young rats on subsequent exposure to Endamoeba histolytica. Whereas 37 per cent of 90 antigen-injected rats did not develop amebic lesions, only 8 per cent of 83 control animals, which received no antigen prior to inoculation failed to develop lesions. The infection rate and average degree of infection in antigen-injected animals were 63 per cent and 2.6 respectively. In contrast, the control rats had an infection rate of 92 per cent and an average degree of infection of 4.6. The data obtained appear to indicate a definite influence of the amebic antigen on the infection rate and on the average degree of infection in rats.

• 1.  Bacteria-free cultures of E. histolytica with active T. cruzi have been maintained through 50 serial transfers and are in use for the production of antigens and for the inoculation of experimental animals with the ameba.
• 2.  Similar cultures, with T. cruzi heat-treated at 48°C. for 10 minutes, have been maintained through 25 serial transfers.
• 3.  Some growth of amebas was demonstrated in microtubes with T. cruzi heat-treated at 48°C. for 10 minutes; no growth of amebas could be demonstrated with T. cruzi treated at 52°C.
• 4.  Rich suspensions of T. cruzi were required for good growth of E. histolytica in microcultures.
• 5.  E. histolytica failed to grow with rich suspensions of T. cruzi from a dialysate medium in routine use for the cultivation of the trypanosome.
• 6.  A new experimental method of approach has been provided for problems of amebiasis.

• 1.  Ten antibiotics have been studied in vitro by Hansen and Bradin et al., and among these agents, only antibiotic “S” and NA7M10 appear to act against E. histolytica, and not indirectly by inhibition of associated bacteria.
• 2.  Thirty-one naturally infected macaques were given orally maximum tolerated amounts of antibiotic “S”, aureomycin, actidione, lupulon, subtilin, and a mixture of bacitracin, polymyxin B and streptomycin, as well as NA7M10 subcutaneously.
• 3.  Aureomycin, in amounts which were eventually lethal, provided temporary clearance of amebas for periods up to 25 days. The mixture of three antibiotics, (bacitracin, polymyxin B and streptomycin), provided immediate cessation of diarrhea, prompt gain in weight and at least temporary clearance of E. histolytica in three of six monkeys given tolerated amounts.
• 4.  Antibiotic NA7M10, while most active in vitro (at 1:5 million dilution) proved to be lethal on subcutaneous injection in four of eight animals when given in doses of 8 mgm. per kilo or more. Lower doses given over 7 days, cleared 2 of 4 macaques of E. histolytica for periods up to 30 days after therapy.
• 5.  The advantage of the tri-antibiotic mixture, as a possible substitute for emetine, is that it may afford prompt symptomatic relief of diarrhea or dysentery in tolerated dosage. NA7M10 deserves consideration, provided a suitable form for oral use is made available.
• 6.  The necessity for interpretation of these results, except with aureomycin and NA7M10, as “contact” antibiotic therapy without appreciable systemic effects is emphasized.
• 7.  Should these studies be pursued in man, it is imperative that a systemically active amebacide be employed subsequently.

Chloramphenicol was effective against amebae in vitro. Higher concentrations were required in the presence of coagulated whole egg than in egg yolk infusion. It exerted only transient suppressive action against amebic colitis in dogs but was effective against cecal infections in rats.

Although penicillin G was not amebacidal in vitro, when given orally it exerted some therapeutic effect in dogs and was highly effective in rats. Limited observations pointed to a similar discrepancy between in vitro action and therapeutic action in rats by streptomycin.

Bacitracin, subtilin and methylated subtilin did not exert encouraging antiamebic action in vitro, and bacitracin did not eradicate infections in dogs.

In vitro and experimental chemotherapeutic data are presented for carbarsone, acetarsone, diiodo-oxyquinoline, chiniofon, Vioform and emetine hydrochloride.

Phthalyl sulfacetimide was ineffective in rats and dogs.

Follow-up observations are given for periods up to 14 months on a group of Guianese patients infected with Wuchereria bancrofti and treated with various doses of Hetrazan. Negative microfilarial counts were maintained in 53 out of 83 patients (63.9 per cent) for 14 months, and with few exceptions the remainder exhibited counts far below the original. The reduction in total microfilariaemia within the entire group was greater than 90 per cent, including all dose ranges of Hetrazan. With the exception of total doses less than 50 mg. per kg. no significant relationship could be determined between the amount of Hetrazan administered and the presence or absence of microfilariae during the follow up period. The greatest number of recurrences occurred at the 6th month follow up examination and declined thereafter. No new recurrences of small numbers of circulating microfilariae occurred during the 12th, 13th or 14th months.

Clinical symptoms after treatment varied considerably. Some patients who showed complete or partial relief from pretreatment during therapy remained free of symptoms throughout the follow-up period and are apparently cured. Others revealed recurrences of pretreatment symptoms with various degrees of frequency after treatment. The number of microfilariae present after treatment seemed to bear no relationship to the presence or absence of symptoms after treatment.

It is believed that the sustained absence of microfilariae in a large proportion of the patients treated, together with the complete absence of symptoms after treatment in many cases, demonstrates indirectly that mature worms are permanently affected by treatment with Hetrazan.

• 1.  Water centrifugal sedimentation is a simple rapid method of stool examination for detecting eggs of Schistosoma japonicum in the stools of infected patients.
• 2.  This method requires no chemical reagents and only a minimum amount of standard laboratory equipment.
• 3.  In our experience it was more efficient than direct smear examination of stool material free of grossly visible bloody mucus, or examination by acid-ether, zinc sulfate, or brine flotation. Viability of eggs found after treatment can be determined.
• 4.  Direct smear examination of mucus present in some stools revealed a high percentage of positives for S. japonicum in such material. Occasionally, trophozoites of E. histolytica were also found in this material.
• 5.  Water centrifugal sedimentation done repeatedly in the course of the diagnosis of suspected schistosomiasis or for evaluation of specific therapy will frequently detect the presence of other protozoa and helminths in addition to S. japonicum.
• 6.  In our experience in these studies the stool method of choice in the study of schistosomiasis japonica patients was as follows:
  • (a)  Careful visual examination of the gross stool for the presence of bloody mucus. Direct smear examination of this material if present.
  • (b)  Water centrifugal sedimentation of all specimens not containing grossly visible bloody mucus and specimens negative by direct smear of bloody mucus if present.
  • (c)  Routine zinc sulfate and brine flotation for the detection of protozoa and helminths other than S. japonicum which may or may not be found by water centrifugal sedimentation, and which may be present in a high percentage of patients under observation for schistosomiasis japonica.
• 7.  From 20 to 30 stool examinations during a period of 4 to 6 weeks were required to detect light asymptomatic infections in a group of patients exposed on Leyte. Eggs had not been detected previously in any of these individuals and none had received treatment.
• 8.  Stool examinations should be begun 30 days after completion of treatment and continued for 8 to 10 weeks, 3 stools weekly. Most treatment failures are discovered from 5 to 12 weeks after completion of therapy with fuadin or tartar emetic.

In the interval between these studies at Moore General Hospital and the present, numerous techniques have been introduced which were designed to increase the probability of detecting eggs of schistosomes. These methods are critically evaluated in a recent review by Stoll (37). In follow-up studies of schistosomiasis japonica in veterans and prisoners of war subsequently to be reported we have carried out most of these techniques. The method we now use is a combination of alcohol sedimentation, (38) hatching, and treatment of the alcohol sediment with ether, hydrochloric acid, sodium sulfate and Triton NE. (39).

• (1)  An analysis was made of 315 electrocardiograms taken on 100 patients during various stages of treatment with tartar emetic and fuadin for schistosomiasis infection.
• (2)  Eleven per cent of the patients showed an increased amplitude of P-waves in leads II and III.
• (3)  Forty-five per cent of the patients showed a fusion of S-T segment and T-waves.
• (4)  Ninety-nine per cent of the patients showed varying degrees of decrease in amplitude of T-waves in all leads resulting in deep inversion in many cases. This change was more pronounced during tartar emetic treatment than during fuadin treatment.
• (5)  The Q-T interval was prolonged beyond the limits of normal in 27 per cent of the patients in this study.
• (6)  The etiology and significance of these changes is unknown. It is our opinion that they represent a transient side action of antimony not indicative of cardiac damage or serious impairment of cardiac function.
• (7)  Recent antimony therapy must be considered in evaluating abnormal EKG's found in veterans.

C) Effect of Treatment on the Intradermal Antigen Test. As previously noted, one of the main purposes in conducting tests with the intradermal cercarial antigen was an attempt to evaluate cases which had been previously diagnosed as having schistosomiasis and who had been treated before they reached this hospital. In a group of 57 men who had been reported to have positive stools overseas, but in whom repeated stool examinations were negative, 23 or 40 per cent had a positive skin test. In examining these cases an attempt was made to correlate this observation with the amount of treatment each group had received. It was found that the group with positive skin tests had had an average of 61 cc. of fuadin, while the negative group had an average of 48 cc., a difference of no statistical significance. The length of time which had elapsed between the last date of treatment and the skin test was of no significant difference either, it being an average of 6.6 months in the positives and 6.1 months in the negatives. This observation was of considerable interest in view of the statement made by Culbertson (78) that “positive skin reactions persist for years after the need for treatment is ended.” In our experience, the skin test with 1:5000 dilution became negative in at least 60 per cent of proven cases within 6 months after completion of successful therapy. It is of interest in this connection that Alves and Blair (58) recently reported that 85 per cent of a group of 53 patients treated intensively for S. haematobium and S. mansoni infections developed negative skin tests to cercarial antigen two to three months after completion of successful treatment.

The proper evaluation of the treatment given patients with schistosomiasis japonica presents many difficulties in view of the fact that a single course o treatment is frequently found to be insufficient, and because of the necessity for repeated stool examinations to determine whether or not therapy has been adequate. It would, therefore, be of considerable value if a simple means could be found to determine the effectiveness of therapy. A number of patients with schistosomiasis were skin tested 30 days or more following completion of treatment in whom stools and skin tests were positive prior to therapy. Of 49 patients treated with 1.8 to 2.08 grams of tartar emetic, 25 still had positive skin reactions 30 to 90 days after completion of treatment, and 24 were found negative. Stools were examined at least 3 times weekly for 90 days or more after completion of treatment. No treatment failures, i.e. positive stools, were discovered in the 24 patients whose skin reactions had become negative, while in the 25 patients whose skin reactions remained positive, 4 treatment failures were subsequently discovered. Of 31 patients treated with 100 cc. of fuadin during 14 days, 20 still had a positive skin test 30 days or more after completion of treatment. Three treatment failures occurred in this group.

Further studies of the dispersion of sylvan mosquitoes marked with bronzing powders confirm and extend the observations previously reported. Using the same technique, but searching over a wider area, Haemagogus spegazzinii, perhaps the most important transmitter of jungle yellow fever in Brazil, was recaptured as far as 11.5 kilometers from the point of release, and Aedes leucocelaenus, another efficient vector, was recovered up to 5.7 kilometers away. Out of a total of 7,624 marked H. spegazzinii released, 98 or 1.3 per cent, were recaptured. Other species of mosquitoes recovered at long distances were A. serratus at 11.5 kilometers, Psorophora ferox at 10.8 kilometers, Wyeomyia sp. at 5.7 kilometers, A. terrens at 5.6 kilometers, and Chagasia sp. at 2.3 kilometers. The longest survival so far recorded is that of P. ferox, a specimen of which was found 55 days after release. In view of these findings, it seems logical to suspect that yellow fever virus may be disseminated by the movement of insect vectors in regions of small residual forests, surrounded by open pasture land and cultivated fields, as typified by the country in western Minas Gerais.

An additional series of experiments to determine the influence of wind on this movement showed that forest mosquitoes travel in the directions of the prevailing winds during the hours of daylight. On no occasion was a marked specimen recaptured in a forest which could have been reached only by flight against the wind.

Following the successful eradication of an invading malaria vector, Anopheles gambiae, in Brazil and in Egypt, the next logical step in the use of this technique was an attempt to eradicate an indigenous vector species. Three such projects are at present under way, two of them in British colonial possessions, the islands of Cyprus and Mauritius, and the third in Sardinia. As the work on this latter project is still proceeding, no attempt will be made in this report to analyze the enormous amount of data collected, to break down finances or to draw final conclusions. This present statement will be confined to a brief history of the project, including both the progress made and the difficulties encountered.

Sardinia is the second largest island in the Mediterranean, having an area of about 23,896 square kilometers. For comparative purposes the following figures will be of interest:

Territory Sise in square kilometers