Ehrlichiosis mimicking thrombotic thrombocytopenic purpura. Case report and pathological correlation.

Human ehrlichiosis is a tick-borne zoonosis caused by the newly described human hematotropic rickettsiae, Ehrlichia chaffeensis. The pathology and pathogenesis of human ehrlichiosis have not been adequately studied. Even with immunoperoxidase, the only previously known method to detect these organisms in tissue, ehrlichae are difficult or impossible to identify. This led many investigators to speculate that the pathogenesis of ehrlichiosis was not caused directly by the organism but could be caused by host-mediated injury. In this case study, a patient presented with rapidly progressive central nervous system symptoms and severe thrombocytopenia, prompting a presumptive diagnosis of thrombotic thrombocytopenic purpura (TTP). Despite corticosteroids, and later, antibiotics, the patient rapidly deteriorated and died. Postmortem examination showed hemorrhages in multiple organs and mononuclear inclusions of infection with a monocytic ehrlichia. Other findings included widespread lymphohistiocytic perivascular infiltrates, focal hepatic necroses, interstitial pneumonitis, interstitial nephritis, mononuclear phagocyte invasion and proliferation in splenic, liver, and bone marrow, and hemophagocytosis. The diagnosis was proven by serology, immunohistology with both polyclonal and monoclonal anti E chaffeensis, and polymerase chain reaction on paraffin-embedded tissues using E chaffeensis-specific oligonucleotide primers. The presence of numerous ehrlichia with notable tissue and cellular injury but without a marked host response indicate that unlike other cases of documented human ehrlichiosis, this patient died after significant direct ehrlichia-mediated injury, and that immune mechanisms initiated after ehrlichiosis played little if any role in the pathogenesis.

Studies of resistance to chloroquine, quinine, amodiaquine and mefloquine among Philippine strains of Plasmodium falciparum.

One hundred cases of slide-confirmed Plasmodium falciparum malaria admitted to the San Lazaro Hospital, Manila, Philippines were screened for in vitro resistance to chloroquine, quinine, amodiaquine and mefloquine using the microtechnique. 59 of the 100 primary parasite isolates produced schizonts, whereas the remaining 41 isolates did not. 51 of the 59 isolates tested were resistant in vitro to chloroquine and eight were sensitive. In contrast, three of the primary isolates were resistant to quinine, three showed resistance to amodiaquine and four were mefloquine-resistant. 43 of the strains judged chloroquine-resistant in vitro were fully in vitro sensitive to amodiaquine, quinine and mefloquine. One chloroquine-resistant isolate was also resistant to quinine alone. Three isolates that were resistant to chloroquine were also resistant to amodiaquine. An additional three were cross-resistant to chloroquine and mefloquine. A single isolate was found to be resistant to chloroquine, quinine and mefloquine and another was cross-resistant to chloroquine, quinine and amodiaquine. All strains demonstrating in vitro resistance to amodiaquine, quinine or mefloquine also showed in vitro resistance to chloroquine. The parasites in 22 patients showed in vivo resistance to chloroquine therapy. 86% were of the R1 type, 9% were R2 and 5% R3. All 22 patients demonstrating in vivo resistance to chloroquine showed in vitro resistance.

Absence of malaria mortality in villagers with chloroquine-resistant Plasmodium falciparum treated with chloroquine.

We carried out a series of malaria studies in Robek , Flores, Indonesia, a coastal village of 900 farmers and fishermen where malaria is hyperendemic by parasite rate and holoendemic by spleen rate. The studies showed that: (i) 28 of 31 isolates (90%) of Plasmodium falciparum were resistant to chloroquine in vitro, (ii) 3 of 12 isolates (25%) were resistant at the R-11 level in vivo, (iii) 376 P. falciparum infections occurred in 301 individuals during one year, (iv) no villagers who were treated with chloroquine for P. falciparum infections during the year died, and (v) increasing the dosage of chloroquine base from 15 to 25 to 37.5 mg/kg led to improved clearing of parasitaemia. We conclude that chloroquine can still be used as the primary antimalarial in Robek , but the dosage may have to be increased to clear parasitaemia.

Effect of 60Co-irradiation on the development and immunogenicity of Plasmodium berghei sporozoites in Anopheles stephensi mosquitoes.

Protection conferred to mice by Plasmodium berghei sporozoites increased significantly when the time interval between 60Co-irradiation of the infected mosquitoes and harvest of sporozoites increased. One thousand sporozoites conferred no protection against challenge if harvested on the day of irradiation, but protected 60% of recipient mice when harvested 28 days postirradiation. When the time between feeding of mosquitoes and irradiation was varied, sporozoites from mosquitoes irradiated 3 days after feeding were infective for mice. Sporozoites from mosquitoes irradiated on day 10 postfeeding were not infective, but were immunogenic. In all experiments a decline occurred in the number of recoverable sporozoites over a 28-day period postirradiation to less than 10% of the yield on the day of irradiation.

Chloroquine resistant Plasmodium falciparum: effect of rabbit serum and incubation time on the in vitro (microtechnique) prediction of in vivo resistance.

A study of chloroquine resistance of 54 isolates of Plasmodium falciparum is reported. Sixty-four percent of the isolates tested produced schizonts in vitro (micro-technique), whereas the remaining 36 percent did not. The accuracy of the in vitro test to predict in vivo resistance was increased when the primary parasite isolates were cultured in the presence of rabbit serum and when the cultures were allowed to incubate for more than 48 hours. Thirteen isolates of P. falciparum that showed in vitro resistance were confirmed in vivo resistant. Eleven of these cases were identified as R-I and two as R-II. Only one case of in vivo resistance (R-II) was observed among the 19 isolates that failed to produce schizonts in vitro.

Chloroquine resistant Plasmodium falciparum on the island of Flores, Indonesia.

The in vitro sensitivity of Plasmodium falciparum to chloroquine was studied in parasites from 45 children on the island of Flores, Indonesia. The micro in vitro culture technique and a 12-volt battery-operated field incubator were found to be well suited to the field situation encountered. Parasites from seven children (15.6%) were resistant to chloroquine in vitro: two at 8 pico-moles of chloroquine, two at 16 pico-moles, and three at 32 pico-moles. This is the first report of chloroquine resistant from Flores.

Chloroquine and quinine resistant Plasmodium falciparum on the island of Mindoro, Philippines, 1982.

A field study was conducted on the island of Mindoro, Republic of the Philippines in which over 800 persons were screened for malaria and approximately 8% were found positive. The in vitro microtechnique was used to test for sensitivity to chloroquine, amodiaquine, mefloquine and quinine in 20 slide-confirmed P. falciparum cases. Sixteen of these cases were also followed for in vivo chloroquine sensitivity. Four cases showed in vitro resistance to chloroquine; 2 also showed resistance to quinine. All showed in vitro sensitivity to mefloquine and amodiaquine. The results of in vivo test were consistent with either a sensitive (S) or R-1, resistant response to chloroquine. Taken together, the in vitro and in vivo chloroquine tests indicate 4 cases of chloroquine resistance at the R1 level.

Kinetics of immunosuppression of sporozoite-induced immunity by Mycobacterium bovis BCG.

The data reported in this study demonstrate that the vaccination of NIH/Nmri mice with viable Mycobacterium bovis BCG organisms induces a state of immunosuppression that renders the recipient animals incapable of a protective immune response to the malaria sporozoite vaccine. The expression of this altered protective immune response is dependent upon the dosage of the two live vaccines, as well as upon the sequence of their administration. Data presented here show that the skin test responses (Arthus and delayed type) of BCG-vaccinated mice do not correlate with the suppression of sporozoite immunity. Evidence is also presented to support the hypothesis that the abrogated immune response to sporozoite vaccination induced by BCG is a result of a loss of immunological memory.

A preliminary survey of malaria in Occidental, Mindoro, Philippines.

A survey of malaria in Northwest Mindoro, Occidental, Mindoro, Philippines is reported. Three species of human plasmodia were identified from 600 blood films examined. The overall prevalence of malaria was 7% (2.8% P. falciparum, 4.3% P. vivax, 0.7% P. malariae). The prevalence of malaria was highest (24%) among children 0 to 15 years of age and only 4 cases (12%) were found among persons over the age of 15. Males and females were equally infected. Study sites with the highest slide-positivity rate were located in the foothill regions which corresponded with the observed presence of two malaria vectors, Anopheles minimus flavirostris and An. maculatus. These sites appeared to be adequate for future studies of drug-resistance, although large numbers of suitable candidates would not be expected.

Effect of route of Mycobacterium bovis BCG administration on induction of suppression of sporozoite immunity in rodent malaria.

Intravenous immunization of mice with 16,000, 60Co--gamma-irradiated, attenuated sporozoites produced solid immunity to sporozoite-induced malaria when the mice were challenged 21 days after immunization. In contrast, mice injected by various routes with 10(7) viable units of Mycobacterium bovis (BCG) before immunization with irradiated sporozoites were not completely immune to challenge. The extent of reduced protection against viable sporozoites demonstrated with these animals was dependent upon the injection route mycobacteria. The intravenous administration of BCG induced the greatest degree of suppression, followed by the intraperitoneal and subcutaneous routes. BCG injected intramuscularly before sporozoite immunization did not suppress development of immunity. In contrast, mice injected with BCG after immunization with attenuated sporozoites exhibited a lesser degree of suppression. In these animals, only the intravenous injection of mycobacteria reduced immunity.

Effect of visceral leishmaniasis on congenitally athymic mice.

Congenitally athymic mice were more susceptible to challenge with amastigotes of Leishmania donovani than were their thymus-intact littermates. This increased susceptibility correlated with a lack of Arthus and delayed-type responses when animals were skin tested with leishmanial antigen.

The use of membrane screen filters in the isolation of Plasmodium berghei sporozoites from mosquitos.

An improved procedure is presented for the isolation of Plasmodium berghei sporozoites from host mosquitos. The method employs filtration through a series of Nuclepore membranes followed by two consecutive centrifugations of the filtrate layered over Renografin-60 solutions of different densities. A Coulter Counter was used to compare isolations prepared by this technique with those prepared by a routinely employed discontinuous gradient method. When the sporozoite concentration in each preparation was standardized at 300 sporozoites per ml, isolations prepared by the new technique were significantly cleaner than isolations prepared by the discontinuous gradient method, containing an average of 1706 total particles per ml compared with 46 107 total particles per ml. The latter procedure was more effective, however, in removing viable microorganisms. Sporozoites isolated by both techniques were similar in immunogenicity and virulence.

Rodent malaria: BCG-induced protection and immunosuppression.

One dose of 10(7) viable units of Mycobacterium bovis, strain BCG, protected a significant number of Swiss mice from a primary challenge with 10(4) thoracic sporozoites of Plasmodium berghei. Immunization with irradiated sporozoites induced greater protection than that observed in BCG-treated with BCG and surviving a primary sporozoite challenge were not protected from rechallenge, whereas mice immunized with irradiated sporozoites and surviving initial challenge of sporozoites were solidly immune to further challenge. Immunizing mice with BCG and irradiated sporozoites simultaneously resulted in a synergistic effect of increased protection against a primary challenge of sporozoites only if the two immunogens were administered on the same day and if the mice were challenged 1 to 3 days later. Mice given BCG and irradiated sporozoites and surviving a primary challenge of sporozoites were unable to survive rechallenge. BCG given to mice previously immunized with irradiated sporozoites suppressed their protective immunity against sporozoite challenge.

Antigenic cross-reactivity between Mycobacterium bovis (BCG) and Leishmania donovani.

Antigenic cross-reactivity was demonstrated between Mycobacterium bovis (BCG) and Leishmania donovani, using delayed hypersensitivity as a criterion.