Observations on early and late post-sporozoite tissue stages in primate malaria. I. Discovery of a new latent form of Plasmodium cynomolgi (the hypnozoite), and failure to detect hepatic forms within the first 24 hours after infection.

Previous work in this laboratory has demonstrated the ability of the immunofluorescence technique to detect pre-erythrocytic stages of the primate malaria parasite, Plasmodium cynomolgi bastianellii, in hepatic tissue obtained as early as 48 hours after sporozoite inoculation. In an attempt to visualize still earlier post-sporozoite stages, hepatic tissue obtained from a rhesus monkey infected with 12,000,000 sporozoites was examined at 2, 12, 24, and 48 hours after inoculation, employing antisera reactive with both invertebrate and vertebrate stages of the parasite. Tissue was also obtained at 7, 50, 102, and 105 days after sporozoite inoculation, and was examined for adequacy of the hepatic infection and for the presence of late exoerythrocytic schizonts. Although a new, previously unrecognized, uninucleate latent stage of 5 micrometer diameter (the "hypnozoite") was detected among large maturing schizonts in the 7-day and later biopsies, no intrahepatic parasites were found in tissue taken at 24 hours or earlier, despite the presence of up to 61 7-day schizonts and eight hypnozoites per 5 X 8 mm section. Pre-erythrocytic forms again were detected at 48 hours, although in far smaller numbers than expected on the basis of the density of parasites at 7 days after infection. The significance of these observations is discussed in the context of previous negative findings.

Observations on early and late post-sporozoite tissue stages in primate malaria. IV. Pre-erythrocytic schizonts and/or hypnozoites of Chesson and North Korean strains of Plasmodium vivax in the chimpanzee.

In a continuing reexamination of plasmodial tissue stages within the context of the hypnozoite theory of malarial relapse, 2 strains of Plasmodium vivax with distinct and disparate relapse characteristics in humans were studied in chimpanzees. Following intravenous inoculation of massive numbers of salivary gland sporozoites, both the frequently relapsing Chesson strain and a North Korean strain characterized by predominantly delayed relapses exhibited relapse patterns and antimalarial sensitivity in the splenectomized chimpanzee essentially indistinguishable from those seen in humans. Examination of hepatic biopsies obtained at 7 and 10 days after infection revealed both pre-erythrocytic (pre-e) schizonts and hypnozoites in tissue obtained from the animal infected with the Chesson strain, but only rare hypnozoites (no pre-e schizonts) at 7 days in the animal infected with the North Korean strain. These findings, combined with the comparability of relapse behavior--which indicates the suitability of the chimpanzee as a model for the natural (human) host-parasite relationship--are essentially as predicted by the hypnozoite theory, despite the small numbers of tissue forms seen. Pre-erythrocytic schizogony of the Chesson strain in the liver was essentially indistinguishable from that of other strains studied, also underlining the suitability of this model system for tissue stage studies of P. vivax.

Demonstration of hypnozoites in sporozoite-transmitted Plasmodium vivax infection.

Hypnozoites of two strains of the human relapsing malaria parasite, Plasmodium vivax, have been detected among maturing 7- and 10-day pre-erythrocytic schizonts in liver biopsies of chimpanzees infected by intravenous inoculation of sporozoites obtained from dissected salivary glands of heavily infected anopheline mosquitoes. As in the simian relapsing species, P. cynomolgi, the hypnozoites of P. vivax at 7 and 10 days are uninucleate forms of approximately 4-5 micrometers diameter, lying within the cytoplasm of individual hepatocytes. Their presence in this relapsing human species is added support for the hypnozoite theory of malarial relapse.

Observations on early and late post-sporozoite tissue stages in primate malaria. II. The hypnozoite of Plasmodium cynomolgi bastianellii from 3 to 105 days after infection, and detection of 36- to 40-hour pre-erythrocytic forms.

Confirmation of the existence of a persistent, uninucleate, dormant pre-erythrocytic stage, the hypnozoite, of the relapsing simian malaria parasite, Plasmodium cynomolgi bastianellii, has been obtained by means of experiments involving the intravenous injection into susceptible monkeys of 48 to 85 x 10(6) sporozoites derived from mosquitoes of a different species and source than employed previously. The development of these hypnozoites was traced from 3 days until 105 days after sporozoite inoculation, employing a sensitive immunofluorescence technique followed by restaining with Giemsa. From an average mean diameter of 4 micrometers at 3 and 5 days, uninucleate hypnozoites grow to 5 micrometers at 7 days, then persist with little change until at least 105 days after infection. Strong evidence for the viability of these persistent forms was obtained by treatment of a host monkey with primaquine, which eliminated all trace of hypnozoites present 2 weeks before. Examination of hepatic tissue from a monkey injected with sporozoites 36 and 40 hours earlier revealed rare uninucleate pre-erythrocytic forms of 2.5-micrometers diameter. These early forms were present in hepatocytes in a density only approximately 1/30th of that expected on the basis of numbers of pre-erythrocytic stages found in the same animal's liver 7 days after infection. Nevertheless, subinoculation experiments appeared to rule out the circulation as a vehicle for dissemination of any putative early intermediate hepatotropic forms from another site.

The epidemiology of infection with Entamoeba histolytica in the Gambia, West Africa.

A country-wide stool survey of The Gambia showed the prevalence of Entamoeba histolytica cysts to range from 13.7% up-country in the dry season to 52.3% near the coast. A longitudinal survey showed a near 100% infection rate over one year and a sharp rise in prevalence as the rains commence with an equally sharp fall as the rains progress. Specific antibody levels are elevated and reasonably constant through the year. Carriers generally show no specific lymphocyte reactivity to amoebic antigen but consistently parasite-negative individuals tended to show elevated lymphocyte reactivity. Attempts to discover the presence of cysts in the environment of villages by cultivation of specimens of water, soil, food, flies and washing from clothes and hands generally failed though E. histolytica was recovered once from a well.

Falciparum malaria and pregnancy.

During pregnancy an increase occurs in the prevalence and density of Plasmodium falciparum malaria. The increase relative to non-pregnant women remains fairly constant with age though there is an over-all decrease in prevalence and density in both groups with age. The increase is at a height relatively early in pregnancy and declines after mid-term. At the height of the malaria season in The Gambia less antimalarial antibody appears in cord bloods than in the early dry season when transmission is lower. Infants show an increase in malaria prevalence and density in the early dry season as compared to the mid-wet season while the remainder of the population shows a decrease.

The human materno-foetal relationship in malaria: I. Identification of pigment and parasites in the placenta.

To facilitate investigations of the consequences of malarial infection during human pregnancy, several methods for the recognition of pigment and parasites in the placenta were evaluated. Pigment was visualized in infected blood smears and placental tissue using both white light and modified fluorescence microscopy. However, the characteristic pigment dots observed with fluorescent light were also apparent in unstained cryostat and deparaffinized placental sections, and following reaction with immunohistological reagents. Intact parasites were recognized immunohistologically in placental sections and blood smears using rabbit antisera to Plasmodium falciparum and P. berghei. Using these procedures, numerous erythrocytes containing parasites associated with small pigment dots were seen in intervillous spaces in heavily infected placentae. In these organs, larger irregular pigment aggregates were present within maternal cells which were shown to be monocytes by esterase staining. Pigment was also observed in the cytoplasm of the trophoblast and not infrequently in the mesenchymal stroma, but no intact parasites were observed within chorionic villi. These simple and sensitive methods thus confirm placental localization of parasites and pigment. Furthermore, the finding of pigment in all Gambian placentae examined, of which seven were thought initially to be uninfected, indicates that malaria may complicate pregnancy more frequently than hiterto anticipated.

The human materno-foetal relationship in malaria. II. Histological, ultrastructural and immunopathological studies of the placenta.

Histological and ultrastructural studies of four placentae heavily infectd with Plasmodium falciparum revealed large intervillous accumulations of erythrocytes containing parasites together with monocytes which had ingested pigment. These appearances were associated with focal syncytial necrosis, loss of syncytial microvilli and proliferation of cytotrophoblastic cells. In addition, marked irregular thickening of trophoblastic basement membranes and protrusion of tongue-like projections of syncytiotrophoblast into the basement membrane were observed. In six other placentae which contained scanty amounts of pigment but no parasites, representing past or inactive infection, no large collections of monocytes or abnormalities of trophoblast were apparent but basement membrane thickening was evident. Immunohistological studies revealed no significant differences between placentae positive for parasites and those containing pigment only, although the amount of certain immunoproteins and clotting factors was clearly increased above normal. These findings establish that P. falciparum infection in the placenta may result in substantial damage although lesions within the villus are rare. Furthermore, previous infection, although adequately controlled, may leave a heritage of pigment deposition, basement membrane thickening and immunopathological lesions. These results may thus account for both the high frequency of intra-uterine growth retardation and the rarity of congenital malaria in the presence of P. falciparum malaria.

Zoonotic cutaneous leishmaniasis in Saudi Arabia: the incrimination of Phlebotomus papatasi as the vector in the Al-Hassa oasis.

Surveys of the phlebotomine fauna in a focus of zoonotic cutaneous leishmaniasis (ZCL) in the Al-Hassa oasis, Eastern Province, Saudi Arabia, revealed only one species of Phlebotomus (P. papatasi) and three of Sergentomyia (S. antennata, S. clydei and S. fallax). 11 specimens of P. papatasi from six sites in the oasis were found with promastigotes in the midgut. An isolate from one of the sandflies was typed by the examination of isoenzymes and identified as Leishmania major, zymodeme LON-4 (= Montpellier zymodeme 26), the principal zymodeme of L. major isolated from patients with ZCL in the oasis. Three isolates from leishmanial lesions at sites of the bites of wild caught specimens of P. papatasi were also identified as the same zymodeme of L. major as the isolate from the sandfly. The findings show that P. papatasi is the vector of ZCL in the Al-Hassa oasis and probably in other ecologically similar foci in the Kingdom.

Observations on early and late post-sporozoite tissue stages in primate malaria. V. The effect of pyrimethamine and proguanil upon tissue hypnozoites and schizonts of Plasmodium cynomolgi bastianellii.

Two rhesus monkeys were each infected with 2.1 x 10(6) sporozoites of Plasmodium cynomolgi bastianellii; one was treated with 1.0 mg of pyrimethamine base per kg body weight for 5 d after sporozoite inoculation. A further 2 monkeys were each infected with 9.75 x 10(6) sporozoites of the same parasite; one was treated with 10 mg of proguanil per kg body weight for 4 out of 5 d after inoculation. The treated monkeys showed a delayed primary parasitaemia and relapses. In sections of liver biopsies taken 7.5 d after sporozoite inoculation, all monkeys showed numerous hypnozoites. However, there were no full grown schizonts and only rare retarded schizonts in the treated monkeys, in contrast to the untreated monkeys which had many mature or nearly mature schizonts. Later biopsies confirmed the continued presence of hypnozoites in all monkeys.

Observations on early and late post-sporozoite tissue stages in primate malaria. III. Further attempts to find early forms and to correlate hypnozoites with growing exo-erythrocytic schizonts and parasitaemic relapses in Plasmodium cynomolgi bastianellii infections.

Rhesus monkeys were heavily infected with sporozoites of Plasmodium cynomolgi bastianellii in an attempt to demonstrate the site of invasion of sporozoites into tissue cells and their growth there. Further attempts were made to correlate the appearance and loss of hypnozoites with parasitaemic relapses. Hypnozoites were demonstrated and once again shown to decrease in numbers over 229 days during which time the infection showed parasitaemic relapses. Liver biopsies taken at two-day intervals for 12 days showed that hypnozoites decreased in numbers over-all and growing schizonts were demonstrated in the liver. At this time a parasite the size of a hypnozoite was seen with two nuclei and another was seen with an elongate, possibly dividing nucleus in one monkey. an attempt to find the location of the early intracellular exoerythrocytic forms in the liver at various times less than 40 hours after infection using smears and immunological staining with newly prepared anti-sera failed. Large numbers of sporozoites of P. knowlesi were also injected into a rhesus monkey the liver of which on the fifth day after infection showed no hypnozoites among 157 sections of growing schizonts and no parasites at all on the 42nd day after infection. In P. cynomolgi bastianellii infections parasites, mostly hypnozoites, were found in the liver up to 229 days after infection.

The effect of parasitization by Leishmania mexicana mexicana on macrophage function in vitro.

Macrophages infected with amastigotes of Leishmania mexicana mexicana as compared to normal macrophages show decreased migration both randomly and through a 5 microns pore in response to a known chemotaxin, an increased ability to pinocytose and an increased bactericidal ability. Unless very heavily parasitized their ability to phagocytose is unaltered. Parasitized macrophages are unaltered in their ability to secrete extracellularly lysosomal enzymes, prostaglandins and lysozyme in response to known stimuli, or to kill target cells in an antibody dependent cell mediated cytotoxicity assay.

An ultrastructural study of the exoerythrocytic schizonts of Plasmodium cynomolgi and P. knowlesi in Macaca mulatta.

Exoerythrocytic schizonts of Plasmodium cynomolgi and P. knowlesi were examined by electron microscopy in biopsy samples of primate livers. With maturity the parasitophorous vacuole membrane becomes highly sculptured by the addition of a discontinuous dense thickening, the distribution of which can be a distinguishing character between these two species. The parasitophorous vacuole membrane follows the contours of the parasite faithfully with a minimal surrounding vacuole. The marked destruction of the cytoplasm of the host hepatocyte by most of the parasites studied however gave the distinct, but erroneous, appearance of a large parasitophorous vacuole at the light microscope level. The mature parasite often exhibited a highly invaginated surface contour with the result that the cytoplasm of the host cell and parasite became intimately interdigitated, this interweaving is unlikely to be recognized in light microscopic studies.

Clinical experience with a continuous monitor of intracranial compliance.

Intracranial compliance, as estimated from a computerized frequency analysis of the intracranial pressure (ICP) waveform, was continuously monitored during the acute postinjury phase in 55 head-injured patients. In previous studies, the high-frequency centroid (HFC), which was defined as the power-weighted average frequency within the 4- to 15-Hz band of the ICP power density spectrum, was found to inversely correlate with the pressure-volume index (PVI). An HFC of 6.5 to 7.0 Hz was normal, while an increase in the HFC to 9.0 Hz coincided with a reduction in the PVI to 13 ml and indicated exhaustion of intracranial volume-buffering capacity. The mean HFC for individual patients in the present study ranged from 6.8 to 9.0 Hz, and the length of time that the HFC was greater than 9.0 Hz ranged from 0 to 104.8 hours. The mortality rate increased concomitantly with the mean HFC, from 7% when the mean HFC was less than 7.5 Hz to 46% when the mean HFC was 8.5 Hz or greater. The length of time that the HFC was 9.0 Hz or greater was also associated with an increased mortality rate, which ranged from 16% if the HFC was never above 9.0 Hz to 60% if the HFC was 9.0 Hz or greater for more than 12 hours. In 12 patients who developed uncontrollable intracranial hypertension or clinical signs of tentorial herniation during the monitoring period, 75% were observed to have had an increase in the HFC to 9.0 Hz or more 1 to 36 hours prior to the clinical decompensation. The more rapid the increase in the HFC, the more likely the deterioration was to be caused by an intracranial hematoma. Continuous monitoring of intracranial compliance by computerized analysis of the ICP waveform may provide an earlier warning of neurological decompensation than ICP per se and, unlike PVI, does not require volumetric manipulation of intracranial volume.

Management of neurosurgical problems in pregnancy.

The effects of pregnancy on neurosurgical disease are complex and require a detailed knowledge of physiology, anatomy, and pharmacology. The following article details the pathophysiology and treatment of the more common disorders of the nervous system seen during pregnancy.

Note on the history of cutaneous leishmaniasis in the Mediterranean and Middle East area.

There exist records of what seems to be cutaneous leishmaniasis at least as far back as 650 BC, and possibly much earlier in the Tigris/Euphrates basin. It was described by Avicenna in the 10th century AD, and was well-known in Aleppo and Baghdad by the 18th century AD. Cutaneous leishmaniasis due to Leishmania infantum may have occurred in Crete in the 18th century. Artificial transmission was effected in Algeria and Aleppo in the 18th century.

Blood parasites of Ethiopian birds. 1. General survey.

Five thousand and forty-six smears from 352 species of birds in Ethiopia were examined for blood parasites in an attempt to provide base-line data, to indicate fruitful areas for further study, on avian hematozoa. The prevalence of infection and the parasites found, with particular reference to Plasmodium, are discussed. At least 22 parasite species were recognized.