John M. Kobayashi

Babesiosis in Washington State: A New Species of Babesia?

Babesiosis is an intraerythrocytic protozoan infection, transmitted by ticks, and characterized by malaria-like symptoms and hemolytic anemia. The first reported zoonotic cases in Europe and the United States occurred in 1956 [1] and 1966 [2], respectively. In Europe, cases of this infection have generally been in splenectomized persons infected with the cattle parasites Babesia divergens and Babesia bovis [3, 4], which are thought to be maintained by Ixodes ricinus ticks. No national surveillance system for babesiosis exists in the United States, but hundreds of cases have been reported [4, 5]. Most have been attributed to infection with Babesia microti, a rodent parasite maintained by Ixodes dammini ticks, the primary vector of the agent of Lyme disease (Borrelia burgdorferi). Human babesiosis is endemic on various coastal islands in the northeastern United States, such as Nantucket Island and Marthas Vineyard, Block Island, Long Island, and Shelter Island, and in mainland Connecticut [6]. Cases acquired in Wisconsin have been reported as well [7, 8]. Asplenic, immunocompromised, and elderly persons infected with B. microti are at greatest risk for clinical illness [5, 7, 9-13], which may be severe, whereas other infected persons commonly are asymptomatic or only mildly symptomatic. Only three human cases of babesiosis acquired in the western United States have been reported previously, all of which occurred in splenectomized patients in California [2, 14, 15]; the infecting species was not definitively identified for any of these cases. In September 1991, the first recognized case of babesiosis acquired in Washington State was diagnosed. We present the clinical details of this case, which occurred in an apparently immunocompetent person, and provide evidence that it was not caused by B. microti. We also provide results of 1) serologic testing that was done in an attempt to identify the species of the patients Babesia isolate [referred to as WA1]; 2) experimental inoculations of various mammalian species to determine WA1s host specificity; 3) a comparison of the DNA hybridization patterns of WA1, B. microti, and B. gibsoni using a Babesia-specific, ribosomal-DNA (rDNA) probe [16]; 4) a serosurvey of the patients family members and neighbors for antibody to WA1; and 5) attempts to identify WA1s reservoir host and tick vector. Case Report A 41-year-old man from a rural forested area in south-central Washington State went to a local emergency room on 15 September 1991 because of a 1-week history of fever, rigors, anorexia, rhinorrhea, cough, and headache. He had previously been in good health, was not taking any medications, and had never had a blood transfusion. He had a dog, two cats, and two head of cattle and was exposed daily to tick habitats, but he did not recall any tick bites. He had not been outside the Washington-Oregon border region in many years and had never been in areas reported to be endemic for babesiosis or malaria. On evaluation, he had a few rales in his left-lung field and a platelet count of 48 109/L. He was treated for a presumed bronchopneumonia, with intravenous cefazolin to be followed with oral cefixime, and was sent home. He was hospitalized the next day (September 16) because of persistent fever, severe rigors, and dark-colored urine. He had a temperature of 39.5 C, tenderness in both upper quadrants of his abdomen without hepatosplenomegaly, a normal hematocrit (0.44) and leukocyte count, a platelet count of 46 109/L, 1+ occult blood on urine-dipstick analysis with no cells detected microscopically, a normal chest radiograph, and blood and urine culture results that were negative for bacteria. He was treated with cefazolin and gentamicin without improvement. On September 18, his temperature peaked at 40.4 C; his hematocrit was 0.36, his serum lactate dehydrogenase level was 21.34 kat/L (1280 U/L), and his total bilirubin level was 20.5 mol/L (1.2 mg/dL). Intraerythrocytic ring forms attributed to Plasmodium falciparum malaria were noted on his peripheral blood smear. He was treated with mefloquine and sent home at his request. He was rehospitalized the next day (September 19) with a temperature of 40 C, rigors, and vomiting. On re-examination of his blood smears from September 15 and 18, intraerythrocytic ring forms (in 1.2% and 3.0% of the erythrocytes) were noted as well as tetrad forms characteristic of Babesia (Figure 1). Therapy with oral quinine (650 mg, three times daily) and intravenous clindamycin (600 mg, four times daily) was begun on September 20, with symptomatic improvement by the next day. By September 23, he was afebrile, his hematocrit was 0.35, the parasitemia had decreased from 3.7% (September 20) to 0.4%, and his platelet count had increased to 143 109/L. On September 24, he was sent home on oral quinine and clindamycin (600 mg, three times daily) to complete a 10-day course of therapy. Figure 1. Photographs of Giemsa-stained peripheral blood smears from a patient who acquired babesiosis in Washington State. Left. Right. On September 30, he was evaluated because of a diffuse urticarial rash. His blood smear was normal, and he was treated with a 12-day tapering course of prednisone for a presumed hypersensitivity reaction. On November 3, when he felt feverish and had a headache, his temperature was 37.5 C, his hematocrit 0.34, and he had a detectable parasitemia of <1%. Urticaria recurred after a dose of quinine (650 mg). Although he appeared to be improving without further therapy for babesiosis, he was treated with intravenous clindamycin (1.2 grams, twice daily for 10 days). His hematocrit decreased to 0.28 on November 7; his anemia had resolved by December 12. Because of slowly resolving fatigue, he did not return to work until 6 January 1992. No parasites were detected on his blood smear in December 1991 or on smears in January, March, July, and September 1992, during which time he remained asymptomatic. Evaluation of his immunologic status with a Western-blot test for human immunodeficiency virus; a liver-spleen scan; quantitative immunoglobulins (including immunoglobulin-G subtypes); and serologic testing for antibody to tetanus toxoid, rubella virus, and streptolysin O, indicated normal results. Methods Serologic Studies Serum specimens from the patient were tested at the Centers for Disease Control and Prevention (CDC) in serial fourfold dilutions by indirect immunofluorescent antibody (IFA) testing [17] for antibody to B. microti and the patients isolate (WA1), which was propagated in hamsters inoculated with his blood (see below). A titer of at least 64 to B. microti was considered positive. Stored serum specimens from patients in the northeastern United States with B. microti-antibody titers ranging from 64 to greater than 4096 and blood smears with intraerythrocytic ring forms were assayed for IFA reactivity with WA1. In another laboratory (University of California at Davis [UCD]) with a different IFA protocol [18], serum from the patient was assayed in serial twofold dilutions for reactivity with various Babesia isolates maintained by passage in animals (Table 1). A titer of at least 320 to B. microti was considered positive. Fluorescein-labeled, affinity-purified antibody to human IgG (Kirkegaard & Perry, Gaithersburg, Maryland) was used as the secondary antibody. Blood smears from the patient were examined for B. bovis, Babesia equi, and Babesia bigemina antigens by direct immunofluorescence testing with monoclonal antibodies specific for these species [24-26]. Table 1. Indirect Immunofluorescent Antibody Reactivity of Serum from November 1991 from a Patient Who Acquired Babesiosis (Isolate WA1) in Washington State* Animal Inoculations Whole blood specimens from the patient were inoculated intraperitoneally (1-mL inocula) into at least two hamsters (Mesocricetus auratus) or jirds (Mongolian gerbils; Meriones unguiculatus). Giemsa-stained thin smears of blood from the inoculated animals were examined (at least 25 oil-immersion fields per slide) weekly for 6 to 8 weeks. Erythrocytes from hamsters infected with WA1 were washed in Pucks Saline G and were inoculated into a splenectomized 1-year-old female golden Labrador retriever (5.6 109 parasitized erythrocytes were administered intravenously and an equal number, subcutaneously) and into a hamster (9 106 parasitized erythrocytes, intraperitoneally). During the 34-day monitoring period, the dogs clinical status and hematocrit were checked daily for 20 days and then 3 times weekly, and thin smears were examined (>5000 erythrocytes/slide) daily through day 20 and then twice weekly. Pre-and postinoculation serum samples from the dog were assayed for IFA reactivity (UCD) with WA1, B. microti (GI [20] and P20 isolates), and B. gibsoni. Southern-Blot Analysis Babesia-infected erythrocytes (P1 pellets) were obtained as previously described [16]; erythrocytes infected with WA1, a human isolate of B. microti (2Bm) [16], and a canine isolate of B. gibsoni (6Bg) were used. Control mammalian leukocytes were separated from uninfected blood of a hamster and a dog by differential centrifugation (400 x g, 4 C, 20 min) on Ficoll-paque (Pharmacia LDB Biotechnology, Piscataway, New Jersey) gradients. After Babesia and leukocyte DNA samples were prepared [27], approximately 1 g of each DNA sample was digested with restriction endonucleases (HindIII or HaeIII; Boehringer Mannheim, Indianapolis, Indiana), as previously described [16]. DNA fragments were separated by electrophoresis in horizontal 0.8% (weight/volume) agarose gels in 45 mM Tris-borate and 1 mM ethylenediaminetetraacetic acid at 40 V for 16 to 18 hours. A Babesia-specific rDNA probe was hybridized to Southern blots of the restriction-endonuclease- digested DNAs; the probe had been produced by polymerase chain reaction amplification of sequences from B. microti DNA, with universal primers directed against highly conserved portions of the nuclear sma

Outbreak of coccidioidomycosis in Washington state residents returning from Mexico.

In July 1996 the Washington State Department of Health (Seattle) was notified of a cluster of a flulike, rash-associated illness in a 126-member church group, many of whom were adolescents. The group had recently returned from Tecate, Mexico, where members had assisted with construction projects at an orphanage. After 1 member was diagnosed with coccidioidomycosis, we initiated a study to identify further cases. We identified 21 serologically confirmed cases of coccidioidomycosis (minimum attack rate, 17%). Twenty cases (95%) occurred in adolescents, and 13 patients (62%) had rash. Sixteen symptomatic patients saw 19 health care providers; 1 health care provider correctly diagnosed coccidioidomycosis. Coccidioides immitis was isolated from soil samples from Tecate by use of the intraperitoneal mouse inoculation method. Trip organizers were unaware of the potential for C. immitis infection. Travelers visiting regions where C. immitis is endemic should be made aware of the risk of acquiring coccidioidomycosis, and health care providers should be familiar with coccidioidomycosis and its diagnosis.

Reactive Arthritis and Reiter's Syndrome Following an Outbreak of Gastroenteritis Caused by Salmonella enteritidis

Reactive arthritis and Reiters syndrome have been reported following gastroenteritis. Prevalence studies for these conditions are uncommon, and the prevalence of Reiters syndrome after Salmonella enteritidis infection has not been previously reported. After a large outbreak of S. enteritidis gastroenteritis, a survey of persons exposed to the implicated food source was conducted, and those with reactive arthritis were evaluated for possible risk factors. Among 481 persons responding to the questionnaire, 217 cases of S. enteritidis gastroenteritis were identified (31 confirmed and 186 clinical cases; attack rate, 45%). Twenty-nine percent of the cases had symptoms of reactive arthritis, 3% had symptoms of Reiters syndrome, and 10% had reactive arthritis with oral ulcers. Markers for severe illness (diarrhea > or =7 days, emergency room visit or hospitalization, and antibiotic treatment) were statistically significant but colinear factors associated with reactive arthritis. Increased awareness of postdysenteric reactive arthritis and Reiters syndrome is recommended.

Enhancing student practicum opportunities: the outbreak investigation option.

An outbreak investigation practicum option for graduate students at the University of Washington School of Public Health and Community Medicine has three goals of education, scientific investigation, and exposure to operations of public health agencies. Over a six-year period, 48 students performed 40 outbreak investigations in collaboration with adjunct faculty based in local public health agencies. An outbreak investigation practicum at Yale University had similar goals, but the rapid-response team approach there generally involved one regular faculty member and a team of graduate students. In either program, the level of faculty involvement necessary for outbreak investigations is sustainable only if the faculty value public health practice as part of the schools mission. Since public health students need to acquire many practical skills, a short outbreak investigation practicum experience should not be viewed in isolation, but rather should be one of a variety of practicum offerings available to students at schools of public health.