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Safety Testing of Immunomodulatory Drugs in Primates. Difficulties in Differentiating Test Article Effects from Occult Diseases— MalariaEstuary Consultants, 340 Marlborough St., Boston, MA 02115 Immunomodulatory drugs have the potential to elicit clinical expression of latent diseases in nonhuman primates. Of particular concern are malaria, tuberculosis, and the various viral diseases, endemic in the primate population used for preclinical testing of new drug candidates. Malaria, particularly, presents a special case because no longer are blood smears evaluated by qualified medical technologists for differential white cell counts and cell morphologies. Instead, leukocytes, erythrocytes, and platelets are recognized mostly by their biochemical profile. Cell morphology has largely been ignored because automated equipment is not yet capable of interpreting morphology beyond size and granularity. Doehle bodies, toxic granulation, Heinz bodies, and other inclusions including malarial pigment and organisms that may occur at a low incidences are no longer identified. This is to be regretted as much important critical diagnostic information is now lost. This deficiency is not limited to veterinary medicine as the case reported by Bruckner et al. (Am J Clin Pathol 83(4) 520–521, 1985) illustrates. The authors report a serious misdiagnosis—Waldenstrom’s macroglobulinemia, and extensive, expensive laboratory testing over 2.5 years with automated analyzers that failed to reveal an obvious intracellular pathogen, Babesia sp., present on all the hematology slides subsequently examined. Malaria is not well characterized in nonhuman primates. The incidence of infections is unknown; however, based on the author’s experience it is probably quite high. Clinical behavior, especially severity of the various malarial strains producing infections, is poorly defined and the incidence of dual or multiple infections is not known. Although diagnosis is easily obtained when either malarial pigment or stages of the parasite are present in the peripheral blood smear, determining whether a monkey (or human) is infected is complex because positive and negative serological findings are difficult to interpret. Encysted organisms produce negative test results. Test methods for detecting P. falciparum based on the presence of HRP 2 or pLDH antigens in either sexual or asexual stages, may remain positive after the organisms have cleared (Bell et al., Am J Trop Med Hyg 73(1):199–203, 2005). In spite of recent developments in diagnostic tests for human strains including fluorescent microscopy of acridine orange-stained parasites, dipstick immunoassays for species-specific-(HRP 2 and pLDH) and organism specific-antigens (aldolase), and nucleic acid detection by highly sensitive PCR, the blood smear remains the gold standard for diagnosis of malaria, but no longer can morphology be used for speciation, because the traditional pictorial characteristics of plasmodia have changed (reported in man) (Moody, Clin Microbiol Rev. 15(1):66–78, 2002). To date there is no information for subhuman primates on how well the human diagnostic test kits perform in monkeys infected with human strains with respect to sensitivity or specificity, or whether aldolase even identifies the presence of subhuman primate plasmodia. Unfortunately the clinical expressions of malaria in subhuman primates including anemia, hyperglobulinemia, bilirubinemia, leukocytosis, and hyperthermia are not specific and may be caused by the new drug candidate itself. Many important marketed drugs, such as penicillins, cephalosporins, levodopa, quinidine and dapsone (in G6PD deficient patients) fall into this category. Thus, separation of drug-effects from disease-effects becomes a conundrum and attempting to differentiate the two based on dose-dependency is not helpful. There is no easy solution for protection of safety studies in primates from the effects of endemic disease, however, several steps or a combination thereof can enhance detection of infected primates and thus reduce the likelihood of rejection. Clearly the problem would not arise if there were an adequate supply of pathogen-free primates. Given the current situation, there are two means of reducing the chances of placing infected animals on safety studies. One means involves testing, which can take several forms: examination of blood smears both before and during the course of a study or use of serological tests or a combination of the two. Fortuitously, smears for detection of plasmodia (and their products) are best made from capillary blood and, serological tests are now automated. The other means of control is treatment, which may not be an option if it interferes with the effect of the test-article. If the decision is made to treat study animals then treatment and subsequent testing should be completed well before the study starts in order to increase the chances of obtaining enough negative animals for the study.
Toxicologic Pathology, Vol. 33, No. 7,
802 (2005)
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