Dane A. Mathiesen

Infection with a Babesia-Like Organism in Northern California

BACKGROUND Human babesiosis is a tick-transmitted zoonosis associated with two protozoa of the family Piroplasmorida: Babesia microti (in the United States) and B. divergens (in Europe). Recently, infection with an unusual babesia-like piroplasm (designated WA1) was described in a patient from Washington State. We studied four patients in California who were identified as being infected with a similar protozoal parasite. All four patients had undergone splenectomy, three because of trauma and one because of Hodgkins disease. Two of the patients had complicated courses, and one died. METHODS Piroplasm-specific nuclear small-subunit ribosomal DNA was recovered from the blood of the four patients by amplification with the polymerase chain reaction. The genetic sequences were compared with those of other known piroplasm species. Indirect immunofluorescent-antibody testing of serum from the four patients and from other potentially exposed persons was performed with WA1 and babesia antigens. RESULTS Genetic sequence analysis showed that the organisms from all four patients were nearly identical. Phylogenic analysis showed that this strain is more closely related to a known canine pathogen (B. gibsoni) and to theileria species than to some members of the genus babesia. Serum from three of the patients was reactive to WA1 but not to B. microti antigen. Serologic testing showed WA1-antibody seroprevalence rates of 16 percent (8 of 51 persons at risk) and 3.5 percent (4 of 115) in two geographically distinct areas of northern California. CONCLUSIONS A newly identified babesia-like organism causes infections in humans in the western United States. The clinical spectrum associated with infection with this protozoan ranges from asymptomatic infection or influenza-like illness to fulminant, fatal disease.

A Fatal Case of Babesiosis in Missouri: Identification of Another Piroplasm That Infects Humans

Human cases of the tick-borne disease babesiosis are caused by the bovine parasite Babesia divergens in Europe [1, 2], by the rodent parasite B. microti in the northeastern and upper midwestern United States [2, 3], and by WA1-type piroplasms in Washington and California [4-6]. We describe the first reported zoonotic case of babesiosis acquired in Missouri and provide evidence to show that this fatal case was caused by an intraerythrocytic piroplasm (MO1) that is probably distinct from but shares morphologic, antigenic, and molecular characteristics with B. divergens. Case Report A 73-year-old man was hospitalized on 1 July 1992 because of fever, a rigor, and thrombocytopenia. He had developed a dry cough, mild headache, sore throat, and joint pain 4 days before admission and had had a temperature of 38.9 C and a platelet count of 70 109/L (baseline count, 100 109/L to 150 109/L) 2 days before admission. He began receiving erythromycin therapy on an outpatient basis but did not improve. His medical history included systemic lupus erythematosus, which had been diagnosed in 1979 and for which he was taking prednisone (10 mg/d). He had had a splenectomy in 1979 because of hemolytic anemia and thrombocytopenia and had had an intracerebral hemorrhage in 1989 because of thrombocytopenia. Except for proteinuria due to membranous glomerulonephritis, his systemic lupus erythematosus had been quiescent since that time. His medical history was also notable for a myocardial infarction in 1986 and recurrent supraventricular tachycardia, for which he was taking digoxin. The patient lived with his wife on 1 acre of land (all mowed or gardened) in a rural area of southeastern Missouri (Cape Girardeau County). He primarily stayed indoors, but he mowed the lawn with a riding mower and did some gardening. He had not traveled outside Missouri in the previous 3 to 4 years, had not traveled outside the midwestern United States in the previous 10 years, and had never been in a country other than the United States. He had no pets or known tick exposures. He had intermittently been employed to feed dairy cattle, which neighbors kept about 1 mile from his home, until 8 years before his hospitalization on 1 July 1992. On admission to the hospital, the patient was febrile Table 1, had a small effusion in one knee joint, and had slight pain on shoulder rotation. He was thrombocytopenic Table 1, his total bilirubin and lactase dehydrogenase levels were elevated, a 24-hour urine specimen contained 11.4 g of protein, and his creatinine clearance was 0.95 mL/s (57 mL/min). Complement levels were normal, no anti-DNA antibody was detectable, and his antinuclear antibody titer was 80 (speckled pattern), suggesting that his systemic lupus erythematosus was quiescent. The patient tested negative for antibody to the human immunodeficiency virus, and blood and urine cultures obtained on 1 July and periodically thereafter also tested negative. He was treated with aztreonam, cefazolin, and an increased dosage of prednisone (80 mg/d). Table 1. Clinical Data for a Patient Who Acquired Babesiosis in Missouri On 2 July, babesiosis was diagnosed after intraerythrocytic ring forms were noted on the patients blood smear Table 1, Figure 1. The antibiotic regimen was changed to oral quinine sulfate, 650 mg three times daily, and intravenous clindamycin, 600 mg three times daily. The patient became afebrile on 3 July, but his parasitemia level continued to increase. His lactate dehydrogenase, total bilirubin, and creatinine levels also increased. Hemodialysis was instituted on 6 July and was provided periodically thereafter. By 11 July, the prednisone dosage had been reduced to 10 mg/day. By 13 July, the 12th day of therapy for babesiosis, the patients parasitemia level had markedly decreased. Figure 1. Giemsa-stained blood smear obtained on 2 July from a patient who acquired babesiosis in Missouri. On 15 July, the patient had a cardiopulmonary arrest that was attributed to hypoxemia; he was intubated and resuscitated. Diffuse pulmonary infiltrates were noted and were thought to be at least partly due to volume overload. After his arrest, the patient had a generalized seizure and never fully regained his baseline mental status. Intravenous methylprednisolone therapy (10 mg three times daily) was started. The quinine dosage was decreased to 650 mg twice daily because of high quinine levels (as high as 7.1 g/mL). On 16 July, the patient again became febrile, but no parasites were noted on his blood smear; ceftazidime therapy was started because of Enterobacter cloacae pneumonia. On 17 July, the patient developed ventricular tachycardia and was cardioverted. On 20 July, the 20th day of hospitalization, supportive therapy was discontinued, and the patient died. No autopsy was done. Methods Serologic Assays At the Centers for Disease Control and Prevention, indirect immunofluorescent antibody testing was used to assay serum specimens, in serial fourfold dilutions, for reactivity to B. microti and WA1 antigens [4, 7]. At the Laboratoire de Biologie Cellulaire, serum specimens were tested for antibody to B. divergens and B. canis (a canine piroplasm). Indirect immunofluorescent antibody testing and immunoprecipitation assays were done as previously described [8, 9]; however, the immunoprecipitation assays were done on long-term cultures of B. divergens (human isolate Rouen 1987) [9] but on short-term cultures of B. canis (isolate Gignac 1994) [10]. Babesia divergens had been obtained from a naturally infected human and maintained in jirds (Mongolian gerbils [Meriones unguiculatus]) by syringe passage twice weekly [11] or in long-term in vitro culture [9]. Babesia canis had been obtained from a naturally infected dog. At the U.S. Department of Agriculture, serum specimens were tested for antibody to bovine isolates of B. divergens (German isolate) and B. bovis (Mexican isolate); a Babesia species from desert bighorn sheep (Ovis canadensis nelsoni; California isolate) that is morphologically similar to B. divergens and serologically cross-reacts with it to some degree [12]; and B. odocoilei (Texas isolate), a parasite of white-tailed deer (Odocoileus virginianus) [13, 14]. The techniques for obtaining in vitro-derived antigens from these isolates have been described previously [12, 15-18]. Indirect immunofluorescent antibody testing was done as previously described [19], except that fluorescein-conjugated recombinant protein G was used to detect specific IgG [20]. When human serum specimens were tested, fluorescein-conjugated goat antihuman IgG (Kirkegaard and Perry Laboratories, Gaithersburg, Maryland) was used. Animal Inoculations Whole blood from the patient was inoculated into hamsters (Mesocricetus auratus), jirds (some of which were immunosuppressed with dexamethasone), and calves and bighorn sheep that had had splenectomy and were immunosuppressed (Table 2). Hamsters and jirds are suitable animal hosts for both B. microti and WA1 [4]; jirds and calves are suitable hosts for B. divergens [2, 11]; and bighorn sheep (or the bighorn sheep culture system) are suitable hosts for various Babesia species that infect wild ruminants [12, 23]. Sheep BHR-32 Table 2 was challenged intravenously on day 63 after inoculation with a stabilate of the bighorn Babesia species (2 108 merozoites) that had been cryopreserved in polyvinylpyrollidone-40 (Sigma, St. Louis, Missouri). On day 27, calf C-03 was challenged intravenously with a cryopreserved stabilate of B. bovis (2 108 merozoites), and sheep BHR-34 was challenged intravenously with a cryopreserved stabilate of the bighorn Babesia species (2 108 merozoites). Table 2. Inoculations of Animals with Blood from a Patient Who Acquired Babesiosis in Missouri* In Vitro Culturing At the Laboratoire de Biologie Cellulaire, 0.5-mL aliquots of whole blood obtained from the patient before treatment on 2 July and cryopreserved in 10% dimethyl sulfoxide were used for each of two in vitro culture systems: B. divergens [9] and B. canis [10]. Human erythrocytes were used for the former; canine erythrocytes were used for the latter. At the U.S. Department of Agriculture, in vitro culturing of blood from bighorn sheep (before and after inoculation with the patients blood; Table 2) was attempted as previously described [16, 17], except that bighorn sheep erythrocytes and medium supplemented with bighorn sheep serum were used. The sheep blood was cultured fresh, with the exception of the specimen taken before inoculation from the sheep inoculated in May (Table 2); this specimen had been cryopreserved in polyvinylpyrollidone-40. The cultures were monitored for 30 days. Molecular Studies At the Mayo Clinic, MO1 DNA was isolated [24] from whole blood that had been obtained from the patient on 2 July and cryopreserved in 10% dimethyl sulfoxide. Broad-range amplification with the polymerase chain reaction, to recover piroplasm-specific nuclear small-subunit ribosomal DNA, and DNA sequence analysis of a 144 base-pair region of the amplification product were done as previously described [5, 6]. Phylogenetic analysis was done by maximum parsimony analysis in PAUP (phylogenetic analysis using parsimony) version 3.1.1 [25]; the analysis included 119 alignable nucleotides and 28 phylogenetically informative positions. The sequences for the other pathogens included in the analysis were previously known [5, 6, 26]; the GenBank accession number for the nuclear small-subunit ribosomal RNA gene for B. odocoilei is U16369 [26]. Results Morphologic Analysis Most of the intraerythrocytic parasites noted on the patients blood smear Figure 1 were in a subcentral position; those in a subperipheral position did not protrude from the erythrocytes. Most erythrocytes were multiply infected. The parasites were polymorphic. Punctiform (< 1 m in diameter), annular (1 m to 2.5 m in diameter), piriform (1 m to 2.5 m in length), and tetrad (Maltese cross) forms were noted. T

Preclinical pharmacology of the anthrapyrazole analog oxantrazole (NSC-349174, piroxantrone).

SummaryOxantrazole (now designated as piroxantrone) is an anthrapyrazole analog under evaluation as a potentially useful anthracycline-like antitumor agent. In preparation for phase I clinical trials, we characterized certain aspects of oxantrazole preclinical pharmacology, including plasma stability, murine pharmacokinetics, in vitro/in vivo metabolism, and DNA damage following incubation with human tumor cells in culture. Oxantrazole was relatively unstable in fresh mouse and dog plasma and particularly unstable in fresh human plasma (t1/2 >5 min at 37°C). Its decomposition in plasma was prevented by the addition of ascorbic acid, suggesting oxidative degradation. following rapid i.v. administration of oxantrazole to mice, plasma elimination was best described by a two-compartment open model with an elimination-phase half-life, total body clearance, and steady-state volume of distribution of 330 min, 458 ml/min per m2, and 87.9 l/m2, respectively. The c x t value calculated following i.v. administration of 90 mg/m2 oxantrazole to mice was 177 μg-min/ml. This value was subsequently used in a pharmacologically guided dose-escalation scheme for the oxantrazole phase I clinical trial. Oxantrazole was converted to a polar conjugate, presumably a β-glucuronide, by rat but not mouse hepatic microsomal preparations and in vivo by the mouse. Oxantrazole introduced protein-associated DNA strand breaks following incubation with a human rhabdomyosarcoma cell line. Repair of the damage was complete by 15 h. Clinical pharmacologic studies are currently under way in conjunction with the phase I clinical trial of oxantrazole.

Differences in N-acetylation of the experimental antitumor agent batracylin in the mouse and the rat

SummaryBatracylin (NSC-320846) is a quinalzolineone recently evaluated as a potential antitumor agent by the National Cancer Institute. The analog was active against a number of murine tumors, including colon adenocarcinoma 38 and multidrug resistant sublines of P-388 leukemia. Preclinical toxicity studies revealed that batracylin was much more toxic when administered orally to rats than to mice. The combined sex LD10 in mice was 5,655 mg/m2 while 576 mg/m2 was lethal to all rats treated at that dose. We determined that following oral administration of batracylin, systemic exposure of parent drug to the rat was only 14.9% of that to the mouse. It was subsequently noted that systemic exposure of a relatively non-polar metabolite was approximately 9 times greater in the rat than in the mouse. The metabolite was identified as N-acetylbatracylin by TLC, HPLC and mass spectral analyses. Observations by the National Cancer Institute that N-acetylbatracylin was not toxic following oral administration to mice or rats prompted evaluation of systemic exposure following oral administration to rats. Following oral administration of N-acetylbatracylin to rats, systemic exposure was almost nil. Indeed, exposure of rats to N-acetylbatracylin was several orders of magnitude greater following oral administration of six-fold lower doses of the parent drug, batracylin. Thus, N-acetylation may play a role in the toxicity of batracylin despite the lack of toxicity observed following oral administration of N-acetylbatracylin. In addition, further metabolism of the N-acetyl conjugate, analogous to that of other aromataic amines, may be involved in the pharmacology of batracylin and similar analogs.

High-performance liquid chromatographic assay for the experimental anticancer agent oxantrazole.

Oxantrazole is an anthrapyrazole analogue developed as an anthracycline-like agent with potentially reduced cardiotoxicity. A reversed-phase high-performance liquid chromatographic assay was developed using a C2 column and mobile solvent system of dimethylformamide-acetonitrile-0.2 M ammonium acetate, pH 4.5 (20:5:75, v/v/v) at a flow-rate of 1 ml/min. Drug and internal standard were detected by ultraviolet absorbance at 514 nm. Isolation of drug and internal standard was afforded by elution from C18 disposable isolation columns with a mixture of methanol-glacial acetic acid-0.02 M sodium acetate, pH 4.0 (12:1:3, v/v/v). The assay was linear (r2 greater than 0.99) over concentrations of 0.025-2.5 micrograms/ml and the limit of detection was 10 ng/ml plasma. Oxantrazole was unstable in neutral and particularly alkaline aqueous solutions. Utilizing this assay, plasma pharmacokinetics were determined following intravenous infusion of oxantrazole to beagle dogs. Plasma elimination was rapid with elimination phase half-life values less than 45 min.