The American Journal of Tropical Medicine and Hygiene - Volume 18, Issue 4, 1969

Three 7-chlorinated lincomycin derivatives exhibited curative activity against blood-inoculated Plasmodium cynomolgi infection in rhesus monkeys. Acute infections were treated by oral or subcutaneous administration for 5 consecutive days with doses ranging from 6.25 to 100 mg per kg body weight. Cures were obtained when U-21,251F (7-chlorolincomycin) and U-24,729A (7-chloro N-demethyl-4′-pentyllincomycin) were given orally at 50 mg per kg and when U-26,285A (7-chloro N-demethyllincomycin) was given at 100 mg per kg. U-24,729A and U-26,285A were also curative when given subcutaneously at 25 and 50 mg per kg, respectively. With all dosage regimens the blood was cleared of parasites 3 to 6 days after cessation of treatment. The rate of clearance of parasites did not appear to be related to dose. No side-effects due to the antibiotics were observed.

Reports that Trimethoprim® plus Sulfalene® are an effective cure for Plasmodium falciparum malaria in man suggested a laboratory study of these drugs to aid in evaluating tests with animals in the search for new antimalarials. Rhesus monkeys with trophozoite-induced P. knowlesi infections were treated with Trimethoprim and Sulfalene, alone or in combination, and followed clinically for 65 days. The infection was lethal for all untreated animals within 5 to 7 days after inoculation. Seven daily oral doses of 100 mg per kilogram of body weight (mpk) of Trimethoprim or 0.5 mpk of Sulfalene were effective in curing P. knowlesi, clearing parasitemia within 3 to 4 days. Single doses of 25 mpk Trimethoprim or 0.5 mpk Sulfalene alone were not curative; but as a combined treatment, these drug levels cured all animals, clearing parasitemia in an average of 4.6 days. The antimalarial efficacy of Trimethoprim and Sulfalene against P. knowlesi infection appear to be at least additive. These results parallel the findings in the radical cure of falciparum malaria in man and support the continued use of simian models in the search for new antimalarials.

In addition to the high incidence of malaria among the American millitary in Vietnam, there has been a progressive increase in the number of imported cases occurring in such persons following their return to the United States. Most of these cases are due to Plasmodium vivax infection. The combined chloroquine-primaquine tablet, taken once a week for 8 consecutive weeks, is effective for radical cure for vivax infection, and all returnees supposedly complete this course of therapy. This study was made to assess the effectiveness of this prophylactic program. A questionnaire survey was made of 671 Army officers and enlisted men regarding malaria prophylaxis. Of the total group, 70% failed to complete the full course of therapy. The rate of failure among officers and enlisted men was nearly equal. There was no predominant cause for failure, but common reasons given included 1) forgetting to take medication, 2) loss of pills, 3) not receiving sufficient number of pills, and 4) untoward side-effects of therapy. Persons stationed in “high-risk” malaria areas were more likely to complete therapy successfully, although previous clinical malaria did not improve the success rate in such affected persons. This extremely high failure rate of the current malaria chemoprophylaxis program would appear to be a significant factor contributing to the high incidence of imported malaria occurring in the United States.

As a practical and sensitive technique for the demonstration of serum antibodies against Trypanosoma and Leishmania, the antiglobulin fluorescence test could be useful in epidemiologic studies of flagellate-produced diseases. However, cross-reactivity of serum from patients with Chagas' disease or leishmaniasis against Leishmania braziliensis and Trypanosoma cruzi has been the rule. Absoprtions with flagellate antigens could remove homologous or heterologous reactivity, or both. To differentiate between blood samples from patients with trypanosomiasis and leishmaniasis, I developed a simple fluorescenceinhibition procedure. Easy to perform, it can be used as a complement to the fluorescent-antibody test in serologic surveys when overlapping of these diseases occurs.

A study was undertaken to determine if gold sodium thiomalate had antileishmanial activity. Thirty hamsters were infected with Leishmania donovani obtained from the spleen of an infected hamster, and one-half were treated with gold sodium thiomalate by weekly intramuscular injection. Both groups were observed for as long as 106 days, and parasitosis was estimated from imprint smears obtained by biopsy on the 12th and 43rd day after infection and at autopsy. Gold treatment resulted in complete suppression of growth of parasites in 13 of 15 hamsters and was in sharp contrast to the untreated group, in which all animals became moribund with extreme parasitosis and were killed before the 106th day. Histologic examination at autopsy revealed amyloidosis in both groups but significantly less in the gold-treated group. The effect of gold sodium thiomalate on leishmanial growth in culture was also measured and compared with control cultures. The results indicated a suppression of growth of Leishman-Donovan bodies in the cultures containing gold sodium thiomalate. It is concluded that gold sodium thiomalate has anti-leishmanial properties. This was demonstrated by the inhibition of growth of Leishman-Donovan bodies in vivo in hamsters, and in vitro.

When Toxoplasma gondii is grown in the peritoneal cavity of white Swiss Webster mice, an exudate forms that is rich both in parasites and white cells. After the removal of all of the cells from the exudate, the resulting fluid produces lethal convulsions when injected into the tail veins of mice. Our studies have demonstrated that these lethal effects can be positively correlated with the viscosity and surface-tension characteristics of this fluid. Our studies also suggest that the observed lethal effects of supernatant fluids from Toxoplasma-induced mouse peritoneal exudates may be attributed to protein-mucopolysaccharide complexes in the exudate, rather than to a specific toxin elaborated by the Toxoplasma organisms.

We sought a drug for the killing of Dracunculus medinensis, infection with which is endemic to more than two-thirds of India. Patients with blebs and palpable worms were treated, as we wished to kill the worm before it discharged larvae. Niridazole (Ambilhar®)—25 mg per kg body weight daily—was administered to 36 Indian villagers and to 20 hospitalized persons. Remission of symptoms of dracunculiasis, with extrusion of the worm, occurred in all but six patients. Side-effects, principally gastrointestinal disturbances and headache, did not warrant discontinuation of the drug. Many villagers, however, stopped treatment, owing to suspicion and refusal to tolerate side-effects. Niridazole killed adult worms, resulting in the healing of the acute lesions of dracunculiasis; protected water supplies must, however, accompany chemotherapy for eradication of the disease.

More than 3,000 persons were studied in a survey on the prevalence of nematode infections in Indonesia and Belgium. In Indonesia, ascariasis and hookworm infection were more prevalent than expected (at least 67% and 15%, respectively), and in the Belgian children studied enterobiasis was surprisingly widespread (at least 65%). A single dose (2.5 mg per kilogram) of L-tetramisole was about 90% effective against ascariasis and about 80% effective against hookworm infection, but ineffective against trichuriasis and almost ineffective against enterobiasis. L-tetramisole was well-tolerated even at several times the recommended dose (2.5 mg per kilogram).

In order to develop a logical method of purifying antigenically active fractions of Ascaris lumbricoides, we devised a hemagglutination-inhibition technique. This test measures the ability of a particular fraction to inhibit the activity of a standard antiserum in the hemagglutination test. A homogenate of adult A. lumbricoides obtained by treatment of infected persons was extracted with phosphate-buffered saline solution. The resulting extract was subjected to treatment with 95% ethanol, saturated ammonium sulfate, and chloroform-normal butanol; the resulting fractions were compared. Attempted chromatographic separation of the fractions was unsuccessful. The effect of digestion with papain and amylase on antigenic activity was also noted. The hemagglutination-inhibition test identified three active precipitates of A. lumbricoides obtained with ethanol with or without papain digestion and with ammonium sulfate. Papain digestion destroyed the ability of the fraction to sensitize erythrocytes but not its activity in the hemagglutination-inhibition test. Amylase digestion destroyed antigenic activity. Consequently, we concluded that antigenicity of the polysaccharide-protein moiety obtained by ethanol precipitation resides in the polysaccharide portion, while the protein portion is necessary to allow the antigen to coat the erythrocytes.

A method has been devised for the separation and subsequent perfusion of the liver from cadavers, the hemodynamics of four normal and six schistosomal fibrotic livers being studied. The mean arterial hepatic flow to the total hepatic flow was 36% in normal, and 66% in schistosomal, livers. The portal-vein outflow ratio (PVOR) in the normal and schistosomal livers was 38% and 72% respectively. Histamine had no effect on the hepaticarterial flow, but it increased the resistance of both hepatic and portal veins. Histamine increased the PVOR as well as the ratio of hepatic-arterial flow. We suggest that histamine can reproduce in the perfused normal human liver most of the hepatic hemodynamic changes observed in the schistosomal liver, and that histamine may play a role in the physiopathology of portal hypertension.

Since the mature egg is the pathogenic unit in schistosomiasis, sterilizing agents. by inhibiting egg-laying, should be therapeutic. The effects on Schistosoma mansoni of sterilization by X-radiation were studied in vivo, in mice, and in vitro. After the hepatic shift of adult S. mansoni produced by potassium antimony tartrate (PAT), the livers of mice were irradiated with 1,500 r. The worm and egg burdens, the survival times of the mice, and the morphology of the recovered worms, indicated that X-radiation at this dose had no effect. Groups of adult worms were exposed to various doses of X-ray in vitro and subsequently maintained in a medium consisting of 90% Medium No. 199 and 10% calf serum. Death from irradiation began 2 weeks after exposure. The LD50 was 68,000 r for males, and 59,000 r for females. As compared with nonirradiated controls, the rate of oviposition was inhibited 50% by 14,400 r and 90% by 100,000 r. The dose used in vivo inhibited egg-laying in vitro by only 4%. Sterilization of adults was measured indirectly by comparing the number of fertilized eggs (those maturing to the miracidial stage) laid after different X-ray doses with those laid by nonirradiated worms. Adults were sterilized 50, 90, and 100%, by 350, 1,000, and 5,000 r, respectively. The dose used in vivo was 92.5% sterilizing in vitro. The dose of X-rays used in vivo could neither sterilize adults nor inhibit egg-laying. However, by inhibiting the maturation of eggs, it should have extended the lives of the treated animals. Lack of efficacy in vivo may have been due to administering X-radiation too late in the course of the disease.

A specific schistosomal antigen in the serum of animals heavily infected with Schistosoma mansoni has recently been detected by immunodiffusion and immunoelectrophoresis. Evidence now obtained by radial diffusion methods indicates a direct relation between the number of worms in the hamster and the amount of antigen in its serum. Preliminary characterization of this antigen was undertaken by concentrating immunologically identical materials from worm extracts with preparative starch-block electrophoresis. The antigen was heat-stable, dialyzable, and had a molecular weight of less than 10,000. Despite an ultraviolet absorption peak of 260 mµ, the antigen showed no change in absorption pattern or behavior on a Sephadex column after incubation with deoxyribonuclease or ribonuclease; neither ribose nor deoxyribose was detectable by paper chromatography. Immunodiffusion studies with urine specimens from infected animals indicated the occasional presence of small amounts of specific antigen identical to that in the serum. When urine from infected animals was reacted with serum from the same or from other infected hamsters, additional precipitin lines were revealed. The latter were not obtained with serum from normal hamsters nor with antiserum prepared in rabbits by injection of worm extracts, nor could the responsible antigens be identified with those in extracts of schistosomes.

We have designed a volumetric device to estimate the weight of stool in the cellophane thick-smear technique; this is a 1-ml plastic syringe sectioned 3 mm from its tip, with a calibrator to ensure a constant displacement of the plunger and hence a constant volume of stool. Statistical analysis showed no significant variation among the weights of stool samples measured in syringes of the same size. There was no significant difference in the weight of 20 random samples when the same syringe and the same calibrator were used. The device is recommended for use in public-health programs on helminthiasis, due to its ease of manufacture, low cost, and reproducibility.

The cases of three patients with Shigella sonnei infection and skin lesions, two of whom also had Shigella bacteremia, were studied for a viral etiology of the skin lesions, but the findings were negative. Shigella bacteremia is rare and cannot be diagnosed clinically. Published reports of six other patients with skin lesions and shigellemia were found. We suggest that skin manifestations may be related to the bacteremia and serve as a clinical clue that shigellemia has occurred.

The neutralization of Rickettsia typhi toxin observed in preliminary studies with normal human serum prompted further investigations on the prevalence and nature of the neutralizing activity. As shown by toxin neutralization (TN) tests in mice with toxic suspensions of rickettsiae, serum of 50% of 207 normal persons had titers of 1:32 or greater against R. typhi toxin, regardless of age or residence in the United States. Within this group were 35 persons less than 20 years old who had never received typhus vaccine, and who resided in a typhus-free area (Montana). Serum from all of 25 normal monkeys had titers of 1:512 or greater. With three exceptions, serum specimens positive in murine TN tests did not neutralize toxin of epidemic typhus or react in complement-fixation or microagglutination tests with murine or epidemic typhus antigens. Serum of normal burros, horses, sheep, goats, rabbits, and chickens did not neutralize toxins of either murine or epidemic typhus. The neutralizing factor in serum from normal monkeys was not affected by treatment with potassium periodate or trypsin, but could be completely precipitated by 50% saturation with ammonium sulfate or by acetone. These findings suggest that the murine TN substance detected in the primate serum was of nonspecific origin, not the result of exposure to R. typhi or Rickettsia prowazekii, and must be considered in evaluating the significance of the TN test as a diagnostic procedure.

Experimental trachoma in nonhuman primates has been handicapped because trachoma (as opposed to inclusion conjunctivitis) produces very mild clinical disease and minimal microbiologic changes. In a direct comparison between the Old World Taiwan monkey, Macaca cyclopis, and the New World owl monkey, Aotus trivirgatus, the latter was found to be much more susceptible. Owl monkeys manifested severe disease for a period of 2 or more weeks, while none was observed in Taiwan monkeys during the same period of time. Given identical inoculations of 2,000 EID50 in the left eye and 20 EID50 in the right eye, six Taiwan monkeys on the 7th day averaged 21 inclusions in the left eye and one in the right. Comparable figures for six owl monkeys were 641 and 1,396, respectively.

Epidemic dengue hemorrhagic fever recurred in 1967 on an island in the Gulf of Thailand. All cases seen were categorized by clinical syndrome as shock, hemorrhagic fever, dengue fever, or undifferentiated fever. Paired serum samples were examined for HI, CF, or neutralizing antibody, or both, to dengue, for classification, where possible, into primary or secondary antibody response. There were 68 cases of dengue or probable dengue seen. Among them were five cases of shock syndrome and 10 of hemorrhagic fever; in none of these 15 was there a primary type of antibody response. Dengue virus type 4 was recovered from seven cases in man, while dengue types 1, 2, and 3 had been reported there previously. Cases occurred from mid-July through November; most patients were between 2 and 10 years of age; no sex selection was seen. Areas of the island that had high attack rates in 1966 were relatively spared in 1967, possibly indicating some degree of long-lasting cross-immunity. These observations continue to support the second-infection hypothesis.

Attempts were made to recover viruses from patients and mosquitoes during an epidemic of dengue hemorrhagic fever on an island in the Gulf of Thailand in 1967. LLC-MK2 cell cultures were used for isolation, and identification was done by plaque-reduction neutralization tests. Aedes mosquitoes were collected resting indoors and biting outdoors. Ten strains of dengue-4 were recovered from patients' serum. Nineteen strains of dengue-4 were recovered from pools of Aedes aegypti and 10 strains from pools of Aedes albopictus. The results suggest that both species were acting as vectors of dengue-4.

A strain of dengue type 3 virus was isolated from the blood of an Antiguan patient in cell cultures of African green-monkey kidney during studies on a dengue outbreak that occurred in the Caribbean region in 1963 and 1964. The virus was adapted to mice by blind intracerebral passage. Illness appeared in mice at the 11th mouse-brain passage. Four other agents, presumed to be dengue type 3 strains, were isolated from Antiguan patients. Serologic studies on paired serum samples from patients of five Caribbean territories showed that the outbreak was due to a group B arbovirus.

The precipitating effect of polyethylene glycol 6000 (PEG) on arboviruses propagated in Vero cell cultures was studied. Efficiency of precipitation depended on the concentration of the polymer, of hydrogen ions, and of sodium chloride. Optimum recovery of Venezuelan equine encephalomyelitis virus infectivity was found at a PEG concentration of 10% at pH 7.6 and 0.25 m NaCl, the mixture having stood at 4°C for 1 hour. Concentration of four serologically unrelated arboviruses with this method showed that recoveries compared well with those obtained after ultracentrifugation. The method is recommended as an initial step in the concentration of arboviruses propagated in tissue culture, as it is simple, rapid, and inexpensive.