The American Journal of Tropical Medicine and Hygiene - Volume 23, Issue 6, 1974

A method applied previously to Plasmodium falciparum was applied to P. vivax to study the maturation of asexual erythrocytic forms of P. vivax in vitro and the effects exerted by chloroquine upon such maturation. Detailed studies were carried out in an effort to translate morphological observations into quantitative data and to identify particular morphological features that might be useful as indices of maturation and inhibition. With this experimental system, 1) asexual erythrocytic forms of P. vivax underwent maturation in vitro; 2) chloroquine added in vitro inhibited this maturation; 3) findings after incubation depended in substantial part upon the maturity of the asexual parasites present at the outset of incubation; 4) no single morphological feature or endpoint served as a consistently satisfactory indicator of maturation and inhibition; and 5) relationships between concentrations and effects of chloroquine were at times inconsistent or erratic, particularly when the asexual parasites present at the outset of incubation consisted mainly of late ameboid forms rather than early ameboid forms or ring forms. Although this experimental system may be of value as a means for assessing the effects of chloroquine upon P. vivax in vitro under some circumstances, it appears that—in terms of simplicity of quantitative assessment and interpretation, reliability, and validity—this system may be considerably less useful or satisfactory when appled to P. vivax than when applied to P. falciparum from man.

For the routine in vitro cultivation of Plasmodium falciparum, the initial parasitemia is usually adjusted to 1% to 2%. Large quantities of parasites in mature stages are desirable for most biochemical studies. Since only ring stages of the parasites normally appear in the peripheral blood of P. falciparum-infected Aotus monkeys, we have attempted to culture large quantities of parasites to older stages by a modification of the usual in vitro culture methods employed in this laboratory. This modification involves: 1) using moderately high parasitemia in the starting material; 2) addition of TES (N-tris[hydroxymethyl]methyl-2-aminoethansulfonic acid) as a supplementary buffer in our modified Harvard medium; 3) increasing the ratio of blood to culture medium from 1:9 to 1:18; and 4) using 250- and 500-ml flasks as culture vessels. The results obtained in a series of experiments using the modified technique indicate that we can produce mature stages of P. falciparum with parasitemia as high as 75%.

Total serum lipids, cholesterol, and protein bound iodine levels became strikingly altered during the course of Trypanosoma gambiense infection in rabbits. Serum lipid and cholesterol values increased progressively with time, attaining a 3- to 4-fold increase over control values nearing the terminal stage of infection. Electrophoresis of serum lipids revealed increased beta and pre-beta lipoprotein and the appearance of a chylomicron band. Serum protein bound iodine values were decreased in infected rabbits, indicating possible thyroid involvement. Despite widespread tissue necrosis, no changes in serum lactic dehydrognase activity or isozyme patterns were observed.

Human eye infections with filariae of man and animals have been reported from many countries, but there are no previous records of the recovery of such worms in malaya. In the present case, a live, mature but unfertilized female filaria, identified as most probably a Brugia sp., was recovered from the conjunctiva of a patient in an area where human and animal infections with species of this genus are known to be common.

Exposure to infection with Wuchereria bancrofti was studied for 1 year in a suburb of Calcutta, India, and correlated with parasitological and clinical observations on the human study population. Blood surveys at the beginning and end of the study showed microfilaremia rates of 12.4% and 14.8%, respectively. The median microfilarial density remained constant at 18 per 20 mm3 of blood. A clinical survey at the end of the study revealed little severe filarial disease. However, between 30% and 40% of the males in the age groups above 20 years had milder symptoms suggestive of filariasis, mostly small hydroceles and cord abnormalities. Exposure to infection with stage III larvae was very heavy and continuous throughout the year. It was estimated that a person sleeing indoors during the year of this study would have been bitten by 1,850 “infective” mosquitoes carrying a total of 5,904 infective larvae. A person sleeping outdoors during the same period would have been bitten by 1,352 “infective” mosquitoes carrying 4,056 larvae. This heavy exposure was shown to be primarily due to the extremely high mosquito biting density of over 115,000 bites per person per year in the study area. The reasons for the discrepancy between the relatively moderate microfilaremia rates and lack of severe filarial disease which suggest low exposure to infection and the epidemiological data which suggests very high exposure to infection are discussed.

Fluorescent precipitates were demonstrated on the cuticle and at the anal pore of Dirofilaria immitis microfilariae by using fluorescein isothiocyanate-labelled canine and human anti-D. immitis sera. Using fluorescein conjugated rabbit anti-human globulin, these reactions were shown to be antibody mediated. The precipitating antibodies were heat and 2-mercaptoethanol stable and induced specific passive Arthus reactions in guinea pigs when D. immitis antigens were injected intravenously. The titer of canine anti-D. immitis antibodies correlated with the microfilaremia of the serum donor, and the prevalence of human anti-D. immitis reactors in a community related to the numbers of infected dogs in the locality. Sera from people with pulmonary dirofilariasis gave high titers of anti-D. immitis antibody; rabbits injected with D. immitis antigens gave similar reactions, but both groups of sera cross-reacted with Toxocara canis larvae. Sera from individuals with T. canis visceral larva migrans, diagnosed from biopsied worms, did not react with D. immitis larvae. Concurrent gastrointestinal nematode infections in Aborigines did not affect the anti-D. immitis fluorescent antibody test.

Trauma to the gut resulting from feeding activities of hookworm was investigated in dogs infected via duodenal puncture or per os with adult worms in gelatine capsules and killed at intervals ranging from 2 to 24 hours and at daily intervals of 1 to 17 days. Mechanical trauma is a local phenomenon involving about nine villi in maximally developed lesions. Formation of a microabscess deep in the lamina propria with overlying confluent villi and profusely sloughing epithelium and stroma signal the end of an attachment period. While each worm may produce as many as six deep lacerations or numerous shallow ones per day, repair of these sites occurs quickly, precluding ulceration with persistent bleeding. Hemorrhage associated with attached hookworms when numerous may be of considerable importance; upon detachment of the parasites, however, the transience of the lesions renders blood loss and impaired absorptive function of relatively secondary importance in the pathogenesis of hookworm anemia.

Urinary schistosomiasis was analyzed quantitatively by gross and microscopic pathologic, bacteriologic, tissue digestion, and epidemiologic techniques in 190 consecutive autopsies from the Cairo University Faculty of Medicine Hospitals between December 1970 and April 1971. Urinary schistosomiasis was associated with obstructive uropathy (hydroureter and hydronephrosis), lower urinary tract infection, urothelial changes (hyperplasia, metaplasia and dysplasia), urolithiasis, and a high frequency of renal parenchymal disease. Schistosomal obstructive uropathy was directly related to infection intensity as measured by tissue egg burden. It was also associated with a higher frequency of pyelonephritis than found in all other cases. A close association between lower urinary tract infection and pyelonephritis was observed in patients with schistosomal obstructive uropathy, but not in other patients. Urinary schistosomiasis accounted directly or indirectly for 6.4% of all of the deaths in the series, and for 10% of deaths among all patients infected with Schistosoma haematobium. In those with high intensity infections with S. haematobium, urinary schistosomiasis resulted in or contributed to death in 42.7%. These findings, correlated with clinical and pathological reports from other endemic foci, conclusively show that urinary schistosomiasis is a life threatening disease in areas where high intensity infections are prevalent.

Frozen sections of Schistosoma mansoni adults and cercariae were prepared for use as slide antigens in the indirect immunofluorescence test for schistosomiasis. Unfixed adults embedded in Tissue-Tek O.C.T. medium for frozen sectioning gave the best combination of sensitivity and specificity. Cercarial antigens reacted intensely with Trichinella anti-sera. Extensive evaluation of the frozen S. mansoni and S. haematobium adult antigens indicated that the sensitivities were 91.7% and 85.4%, respectively; specificities were 95.8% and 96.9%, respectively. Within-run reproducibility within one twofold dilution was 93.7% and 97.9%, respectively. Determination of infecting species by serology was not possible.

Circulating antigen of schistosomal origin (Schistosoma mansoni) can be detected in sera from infected animals by complement fixation (CF). Rabbit antisera employed in the CF reaction were prepared by immunization with antigen extracted from whole worms which was purified and concentrated by starch-block-electrophoresis and ultrafiltration. Concentrated antigen was complexed with methylated bovine serum albumin before emulsification with adjuvant and use as an immunogen. By gel diffusion, the rabbit antisera detected a single antigen in extracts of adult worms and in sera from hamsters heavily infected with Schistosoma mansoni. The sensitivity of detection of circulating antigen by CF as compared to gel-diffusion was increased many-fold. Circulating antigen could be detected in the sera of some hamsters infected with 800 cercariae as early as the 18th day after infection. Titers of circulating antigen were assayed by CF during the 6-week period after infection. Two patterns were revealed. In 75% of infected animals titers rose steadily from time of detection; in 25% titers rose and then plateaued after the 32nd to 35th day. A direct relationship was demonstrated between the titer of antigen and the worm burden in infected hamsters between the 4th and the 7th weeks of infection. Using the CF reaction, antigen with properties of circulating antigen was not detected in egg extracts.

Sections of adult Schistosoma mansoni and of infected mouse liver were incubated with antiserum monospecific for the antigen found in the circulation of schistosome-infected animals. By indirect immunofluorescence, antigen was found only in epithelial cells of the gut and the border of these cells with the lumen. This corresponded to an area of diastase-fast PAS-positive staining, which is consistent with previous data indicating that this antigen was a polysaccharide. The antigen was not found elsewhere in the worms or in the liver of infected mice.

Immunofluorescent staining of schistosome worms with specific anti-circulating schistosome antigen (CSA) rabbit immunoglobulin plus goat anti-rabbit immunoglobulin-fluorescein conjugate strongly suggests that the gut, and not the integument, is the site of production of circulating schistosome antigen. Plans for further studies of CSA and of its role in schistosomiasis are outlined.

The antigens for the Echinococcus granulosus-specific arc 5 were found in 40 of the 42 hydatid cyst fluid (HCF) pools obtained from the lungs and liver of sheep by the immunoelectrophoresis (IEP) test using sheep and rabbit antisera to HCF. The criteria for the selection of antigens for the diagnosis of human hydatidosis by the IEP test are reviewed in the light of this finding. Sheep antisera to ovine HCF did not show antibody activity to the host contaminants in HCF. The value of such an antiserum for the isolation of the E. granulosus antigens in HCF, and in immunologic and phylogenetic studies of parasites and its application as a reference serum for the IEP test for human hydatid disease are discussed.

A pharmacologic study of clofazimine (B663) was carried out in mice and human subjects to provide information needed for interpretation of data from studies of drug action in the two species. The quantities of B663 in mouse carcasses were measured to determine the half-time of disappearance (t ½) of the drug and the absorption of the drug after oral administration. The antimicrobial activity of orally-administered B663 was studied in mice infected with Mycobacterium leprae and correlated with the quantity of B663 in the carcass. Published studies of the effect of the drug in murine infections with M. leprae and M. tuberculosis were reinterpreted. The t ½ of B663 is about 1 week in male BALB/c mice. The antimicrobial effect of the drug appears to require a concentration in the mouse carcass smaller than 1 mg/kg for M. leprae and greater than 5 to 10 mg/kg for M. tuberculosis. The concentration of B663 was also measured in the plasma, urine and feces of volunteers and leprosy patients. The t ½ of the drug is at least 70 days in man. The disposition of the drug thus differs greatly between the two species, making difficult the transfer of information from laboratory to clinic.

Chemical methods are described for the determination of clofazimine in serum, urine, and feces, and in homogenates of liver and spleen. Feeding clofazimine to mice resulted in a large accumulation of crystalline drug in the liver and spleen. When dosage with clofazimine was terminated tissue and serum concentrations fell extremely slowly, at rates over a period of 4 months equivalent to a half-life of about 70 days. The concentrations of clofazimine were also measured in the serum, urine, or feces of leprosy patients and a healthy volunteer. Clofazimine appeared to be incompletly absorbed in man. The relevance of these findings to the treatment of leprosy with clofazimine is discussed.

Clofazimine (B663) has been the subject of considerable study in the experimental animal because of its antimycobacterial activity. This is the first study of the tissue distribution of B663 in man. Three leprosy patients were studied at autopsy; a skin biopsy was studied in a fourth patient. Tissue concentrations were analyzed by a simple chemical method. Tissue coloration was observed in the internal organs. Initially, the subcutaneous tissues appeared gray to opaque, but within minutes of exposure to the air began turning a yellow-orange color; after some ten minutes, tissues high in fat content were orange-red. Highest concentrations of B663 were observed in tissues with high fat content and in the bile. Tissues with a reticuloendothelial component or high vascularity also showed relatively high concentrations. High levels were present also in the liver and in the gall bladder.

The effect of rifampin therapy in leprosy was studied in two clinical short-term trials in which skin punch biopsy specimens were taken at regular intervals for the inoculation of mice in order to monitor the decrease in proportion of viable Mycobacterium leprae in the patients' lesions. In a trial of rifampin in a dosage of 600 mg daily, the bacterial viability fell to undetectable levels in the first specimen taken after the start of therapy (at 3–4 days in 4 patients, 7–8 days in 9, and 14 days in 2). Dapsone-treated controls required 20 to more than 112 days for the same change. In a trial of a single dose of 1,500 mg rifampin, the viability fell to undetectable levels in the first specimen taken after the start of therapy also (at 3–5 days in all 14 patients).

The distribution of Vibrio parahaemolyticus in Korea was studied. Isolation rates were high in sea mud (37.2%), followed by shellfish, fish, and sea water in decreasing order. The rise of the level of this organism in marine environments coincided with the rise of water temperature. About 59.4% of isolatd strains were of known K types, and a majority of marine specimens were contaminated with multiple K types including nontypables. A large proportion of strains showed positive Kanagawa hemolysis. An outbreak of food poisoning associated with types K3 and K57 of V. parahaemolyticus was observed in the summer of 1972. The rate of isolation from apparently healthy persons was very low even in warm months.

In an epidemic of Lassa fever in the Eastern Province, Sierra Leone, 63 suspected cases were identified in patients admitted to two hospitals over a 2-year period. Although hospital workers were at high risk of infection, most cases were acquired outside the hospital. There appeared to be intrafamilial outbreaks around several cases. The clustering of seropositivity in families suggested person-to-person spread. Six percent of the population surveyed had complement-fixing antibody against Lassa virus, while only 0.2% had recognized disease. This suggests that the fatality from Lassa virus infection is considerably less than the 38% observed among cases.

Twelve patients hospitalized with a confirmed diagnosis of Lassa fever were studied during an epidemic of this disease in Sierra Leone. Clinical observations confirmed and extended those made in previous outbreaks in Liberia and Nigeria. Two patients were treated with plasma containing antibodies to Lassa virus; both had a favorable response. Lassa virus was isolated from serum, throat swabs, or urine from all patients sampled during the first 15 days of illness. Virus was recovered from the pharynx of patients with circulating complement-fixing (CF) antibodies. CF antibodies were detectable by the 3rd week of illness. A strain of Lassa virus from one of the patients was compared with strains recovered in Liberia and Nigeria by CF test. Serum from convalescent patients infected with Nigerian virus did not fix complement in the presence of strains from Liberia or Sierra Leone; this result may indicate antigenic differences between Lassa virus strains which must be confirmed by neutralization test.