Barbara L. Herwaldt

An Outbreak in 1996 of Cyclosporiasis Associated with Imported Raspberries

BACKGROUNDnCyclospora cayetanensis is a parasite that causes gastroenteritis. Until last year most of the documented cases of cyclosporiasis in North America were in overseas travelers. In 1996, a large outbreak of cyclosporiasis occurred in North America. We investigated this outbreak.nnnMETHODSnHealth departments solicited information from clinicians and laboratories on cases of cyclosporiasis, which were then reported to the Centers for Disease Control and Prevention and to Health Canada. We conducted retrospective cohort studies for the cases associated with events (e.g., luncheons) and attempted to identify the sources of the implicated food.nnnRESULTSnA total of 1465 cases of cyclosporiasis were reported by 20 states, the District of Columbia, and 2 provinces. Of these cases, 978 (66.8 percent) were laboratory confirmed and 725 (49.5 percent) were associated with 55 events that were held from May 3 through June 14. Raspberries were definitely served at 50 events and may have been served at 4 events. For 27 of the 41 events for which adequate data were available (65.8 percent), the associations between the consumption of berries (raspberries with or without other berries) and cyclosporiasis were statistically significant (P<0.05). For all 29 events for which there were good data, the raspberries definitely came from Guatemala (21 events, 72.4 percent) or may have come from Guatemala (8 events, 27.6 percent). As few as five Guatemalan farms could have accounted for the 25 events for which the raspberries could be traced to a single exporter per event. The mode of contamination of the raspberries remains unclear.nnnCONCLUSIONSnThis large outbreak of cyclosporiasis in North America in 1996 was associated with the consumption of Guatemalan raspberries. The outbreak illustrates the need to consider that a local cluster of foodborne illness may be part of a widespread outbreak and to pursue investigations to the source of the implicated vehicle.

Molecular Characterization of a Non–Babesia divergens Organism Causing Zoonotic Babesiosis in Europe

In Europe, most reported human cases of babesiosis have been attributed, without strong molecular evidence, to infection with the bovine parasite Babesia divergens. We investigated the first known human cases of babesiosis in Italy and Austria, which occurred in two asplenic men. The complete 18S ribosomal RNA (18S rRNA) gene was amplified from specimens of their whole blood by polymerase chain reaction (PCR). With phylogenetic analysis, we compared the DNA sequences of the PCR products with those for other Babesia spp. The DNA sequences were identical for the organism from the two patients. In phylogenetic analysis, the organism clusters with B. odocoilei, a parasite of white-tailed deer; these two organisms form a sister group with B. divergens. This evidence indicates the patients were not infected with B. divergens but with an organism with previously unreported molecular characteristics for the 18S rRNA gene.

Cryptosporidiosis: An Outbreak Associated with Drinking Water Despite State-of-the-Art Water Treatment

Cryptosporidium parvum is transmitted through the ingestion of oocysts excreted in human or animal feces. Commonly recognized modes of spread include person-to-person and animal-to-person contact, exposure to contaminated objects, and ingestion of contaminated food or water [1-3]. The March 1993 outbreak of cryptosporidiosis in Milwaukee, Wisconsin, which affected more than 400 000 persons [4], heightened awareness about waterborne transmission of C. parvum. The Milwaukee water utility, like those associated with the previously recognized outbreaks caused by waterborne C. parvum in the United States [4-6], had met all existing state and federal standards for drinking water. We report an outbreak of cryptosporidiosis that occurred in 1994 in Clark County, Nevada, a county of about 1 million residents, most of whom live in Las Vegas. The outbreak was associated with drinking water, despite a state-of-the-art water treatment plant and water quality that was much better than that noted during the outbreak in Milwaukee. Cryptosporidium infection has been reportable in Nevada since 1992; physicians and laboratories are required to report stool specimens that test positive for the organism to their county health departments. Three cases of Cryptosporidium infection in residents of Clark County were reported by the state health department in 1992, and 23 were reported in 1993; 1 of the 1992 cases (33%) and 18 of the 1993 cases (78%) were known to have occurred in persons infected with the human immunodeficiency virus (HIV). In contrast, more than 70 cases of cryptosporidiosis were reported in the first 4 months of 1994, and most of these were in HIV-infected persons. We conducted an investigation to identify the magnitude and cause of this increase and to determine whether many cases had occurred in persons who were not infected with HIV. This outbreak (which was documented, in part, because the community was one of the few in the United States in 1994 that had a surveillance system for cryptosporidiosis) raises the question of how often outbreaks caused by waterborne C. parvum are unrecognized in the United States. Methods Case Ascertainment and Confirmation All testing of specimens from county residents for Cryptosporidium is done by two laboratories in Clark County and one at the Nevada state health department; testing is done only at the request of a physician. We reviewed the records from laboratory A, which does more than 95% of this testing, and the surveillance records at the county health department to identify all cases of Cryptosporidium infection newly diagnosed during the 4-month study period (1 January through 30 April 1994). We also reviewed the death certificates of persons who had had laboratory-confirmed cryptosporidiosis to determine whether cryptosporidiosis or Cryptosporidium was listed as an immediate or contributing cause of death. Case-Control Study of Adults with HIV Infection A case-patient was defined as an HIV-infected adult ( to 18 years of age) living in Clark County in whom laboratory-confirmed cryptosporidiosis was diagnosed for the first time during the study period. Each case-patient was matched by primary physician or clinic with three HIV-infected adult controls from the county; one control in each of three CD4+ cell count categories (< 100 cells/mm3, 100 to 199 cells/mm3, and more than or equals to 200 cells/mm3) was selected. Stool specimens from controls were not tested for Cryptosporidium. Telephone interviews were done using a standardized questionnaire in May and June 1994. The exposure period for case-patients was defined as the 4 weeks before the case-patients became ill; matched controls were asked about exposures during this period and about illness in the 4 months before their interviews. Study participants were asked whether they had been exposed to persons who may have been infected with Cryptosporidium (whether they lived in the same household with or visited or cared for a person who had diarrhea); whether they lived in a household in which someone attended or worked in a child-care setting or in which a child wore diapers; whether they had changed a childs diaper; and whether they had had any type of sexual activity or had engaged in high-risk sexual activity (anal-oral intercourse). Participants were asked about contact with newborn animals (< 4 months of age) and about visits to farms, pet stores, animal shows, animal pounds, petting zoos, and veterinarians; about restaurant patronage and consumption of uncooked and cold foods, unpasteurized dairy products, health foods, and dietary supplements; about exposure to recreational water (in a pool, whirlpool bath, hot tub, lake, or river); and about types of drinking water used at home and work (for example, tap water, tap water filtered at its point of use, bottled water, or well water). Study patients were asked whether they had immunosuppressive medical conditions other than HIV infection, whether they took immunosuppressive medications, and whether they had used nontraditional therapy for HIV infection; they were also asked about miscellaneous exposures, such as travel outside of Clark County and attendance at bars, clubs, and social functions. Case-Control Study of Immunocompetent Children In a casecontrol study of children that was similar to the study of adults described above, each case-patient who had laboratory-confirmed cryptosporidiosis was matched with three controls by age ( 3 years), primary physician or clinic, and week of medical evaluation; parents were interviewed. In contrast to the study of HIV-infected adults, no questions were asked about sexual activity and attendance at bars, and additional questions were included (for example, about attendance at child-care facilities, diaper use, and extracurricular activities). Community Health Survey A questionnaire was distributed in June 1994 to all employees at two Clark County agencies (agencies A and B) to determine whether they had had diarrheal illness during the study period and to identify their sources of drinking water. Employees were asked only about water drunk at home; however, the agencies did not provide bottled water, and tap water at the agencies was not filtered at its point of use. Water Quality Analysis and Environmental Survey Data on water quality for source water (Lake Mead) were reviewed for a 50-month period (1 March 1990 through 30 April 1994); data on water quality for finished (treated) water were reviewed for a 28-month period (1 January 1992 through 30 April 1994). The water treatment plant that serves all of Clark County was inspected. We reviewed the treatment procedures and the log of malfunctions and repairs at the plant and in the pipes that distribute water throughout the county. Statistical Analysis We used conditional logistic regression (SAS version 6.10 for Windows [PROC PHREG] [SAS Institute, Cary, North Carolina]) to calculate matched odds ratios for the casecontrol studies, and we used the chi-square test (Epi-Info version 5.1 [Centers for Disease Control and Prevention, Atlanta, Georgia, and the World Health Organization, Geneva, Switzerland]) to compare proportions for the community health survey. The Wilcoxon two-sample test was used to compare the ranked distributions of ordinal variables. We report two-tailed P values. Results Case Ascertainment and Confirmation We identified 78 persons in whom laboratory-confirmed Cryptosporidium infection was diagnosed during the study period (Figure 1). No procedural or personnel changes had been made that affected diagnosis or reporting. At laboratory A, the mean percentage of stool specimens per month that tested positive for Cryptosporidium had increased from 4% in 1993 to 21% in the first quarter of 1994. Figure 1. Number of cases of Cryptosporidium infection reported to the Clark County District Health Department by month of diagnosis, January 1993 to December 1994 (n = 148). Cryptosporidium Sixty-one of the 78 persons with cryptosporidiosis (78.2%) were HIV-infected adults Figure 2, and more than 90% of the 61 had CD4+ cell counts less than 100 cells/mm3 (Table 1). Four of the 78 (5.1%) were adults without HIV infection; 1 of these was receiving corticosteroid therapy for renal transplantation, and another was receiving chemotherapy for testicular cancer. Two of the 78 (2.6%) were HIV-infected children, and 11 (14.1%) were immunocompetent children. Figure 2. Flow diagram of the review of laboratory records, the casecontrol studies, and the community health survey. Table 1. CD4+ Cell Count Distribution of Adults with Human Immunodeficiency Virus (HIV) Infection and Laboratory-Confirmed Cryptosporidiosis and of All HIV-Infected Residents of Clark County Persons who had laboratory-confirmed cryptosporidiosis lived throughout Clark County (Figure 3, top), in four of the five geographic areas served by the water treatment plant (all except Nellis Air Force Base [Figure 3, bottom]). The epidemic curves of the dates of onset of illness for the HIV-infected adults and the immunocompetent children are similar (Figure 4). These epidemic curves and that for the month of diagnosis of the cases reported to the health department (Figure 1) show that the outbreak apparently began in December 1993 (when the first infected persons reported onset of illness) and extended through June 1994 (after which the number of reported cases notably decreased). The total number of laboratory-confirmed cases associated with the outbreak period was 103 (78 during the study period, 16 in May, and 9 in June). Figure 3. Maps of Clark County showing the residences of persons with cryptosporidiosis and the water distribution system. Top. n Bottom. Figure 4. Month of onset of diarrhea among adults with human immunodeficiency virus (HIV) infection and cryptosporidiosis, immunocompetent children with cryptosporidiosis, and county employees with diarrheal illness: Clark County, December 1993 to April 1994.

Infection with a Babesia-Like Organism in Northern California

BACKGROUNDnHuman 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.nnnMETHODSnPiroplasm-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.nnnRESULTSnGenetic 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.nnnCONCLUSIONSnA 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.

Transfusion-Associated Babesiosis in the United States: A Description of Cases

BACKGROUNDnBabesiosis is a potentially life-threatening disease caused by intraerythrocytic parasites, which usually are tickborne but also are transmissible by transfusion. Tickborne transmission of Babesia microti mainly occurs in 7 states in the Northeast and the upper Midwest of the United States. No Babesia test for screening blood donors has been licensed.nnnOBJECTIVEnTo ascertain and summarize data on U.S. transfusion-associated Babesia cases identified since the first described case in 1979.nnnDESIGNnCase series.nnnSETTINGnUnited States.nnnPATIENTSnCase patients were transfused during 1979-2009 and had posttransfusion Babesia infection diagnosed by 2010, without reported evidence that another transmission route was more likely than transfusion. Implicated donors had laboratory evidence of infection. Potential cases were excluded if all pertinent donors tested negative.nnnMEASUREMENTSnDistributions of ascertained cases according to Babesia species and period and state of transfusion.nnnRESULTSn159 transfusion-associated B. microti cases were included; donors were implicated for 136 (86%). The case patients median age was 65 years (range, <1 to 94 years). Most cases were associated with red blood cell components; 4 were linked to whole blood-derived platelets. Cases occurred in all 4 seasons and in 22 (of 31) years, but 77% (122 cases) occurred during 2000-2009. Cases occurred in 19 states, but 87% (138 cases) were in the 7 main B. microti-endemic states. In addition, 3 B. duncani cases were documented in western states.nnnLIMITATIONnThe extent to which cases were not diagnosed, investigated, reported, or ascertained is unknown.nnnCONCLUSIONnDonor-screening strategies that mitigate the risk for transfusion transmission are needed. Babesiosis should be included in the differential diagnosis of unexplained posttransfusion hemolytic anemia or fever, regardless of the season or U.S. region.nnnPRIMARY FUNDING SOURCEnNone.

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

The Return of Cyclospora in 1997: Another Outbreak of Cyclosporiasis in North America Associated with Imported Raspberries

In the spring of 1996, a multistate outbreak of cyclosporiasis linked to fresh Guatemalan raspberries occurred in the United States and Canada (1-3). Although the mode of contamination of the raspberries was not identified, the outbreak was probably caused by an attribute or practice shared by multiple, but possibly relatively few, Guatemalan farms (1, 2). During the 1996 fall and winter export season for Guatemalan raspberries, no outbreaks of cyclosporiasis were noted in North America. In anticipation of the 1997 spring export season, the Guatemalan Berry Commission voluntarily instituted various control measures on farms; these measures focused on improving sanitation, employee hygiene, and the quality of water used in agriculture (1, 4). The Commission stipulated that only farms that the Commission classified as low risk in such regards could export fresh raspberries to the United States in the spring of 1997 (beginning on 22 April 1997). Nonetheless, another multistate, multicluster outbreak of cyclosporiasis linked to Guatemalan raspberries occurred in the spring of 1997 (4-6). The Centers for Disease Control and Prevention (CDC) learned of this outbreak in early May, when several health departments reported clusters of cases of cyclosporiasis associated with events (such as banquets) held in April. The outbreak, which is described here, ended shortly after the Guatemalan government and the Guatemalan Berry Commission voluntarily suspended exports of fresh raspberries to the United States. No shipments occurred from 29 May through 14 August (4). The occurrence of raspberry-associated outbreaks of cyclosporiasis in consecutive years, as well as other outbreaks of cyclosporiasis in 1997 that were linked to mesclun lettuce and basil that did not come from Guatemala (4, 7), highlights the need for improved understanding of the biology and epidemiology of Cyclospora cayetanensis. Although much has been learned in recent years about this coccidian parasite (8-10), unresolved issues that are relevant to foodborne outbreaks include whether animal reservoirs of infection exist (11-18); the maximum rate at which excreted, noninfectious oocysts can sporulate (that is, develop two internal sporocysts, each with two internal sporozoites) and become infectious; the effects of environmental conditions on the rate of sporulation; the median infective dose of oocysts; the factors that make cyclosporiasis seasonal and influence whether infection is symptomatic (for example, causing protracted, relapsing gastroenteritis) (8, 19-21); and the therapeutic alternatives to trimethoprim-sulfamethoxazole (22) for persons intolerant toward sulfa drugs. Methods Epidemiologic Investigation A cluster of cases of cyclosporiasis was defined as two or more cases occurring among persons who 1) ate at an event during the period from 1 April through 15 June 1997, 2) developed at least one gastrointestinal symptom [such as loose stools] 12 hours to 14 days after the event, and 3) were not known to be associated with the outbreaks linked to mesclun lettuce (in March and early April in Florida [4]) or basil (from mid-June through mid-July in the metropolitan area including northern Virginia, the District of Columbia, and Baltimore [7]). At least one case per cluster had to be confirmed by laboratory testing (for example, with modified acid-fast or hot safranin staining, examination of wet mounts, or demonstration of autofluorescence or oocyst sporulation) (8-10, 23-25). Clinical case definitions for probable cases varied among clusters (Table 1). Health departments conducted retrospective cohort studies by using structured questionnaires about symptoms and event-related exposures. Table 1. Clusters of Cases of Cyclosporiasis and Laboratory-Confirmed and Probable Cases of Cyclosporiasis in the Raspberry-Associated Outbreak, by Likely Site of Acquisition of Infection, in the United States and Canada in 1997 Case-patients with sporadic cases had laboratory-confirmed cyclosporiasis, developed gastrointestinal symptoms from 1 March through 31 August, were not included in clusters or known to be associated with other outbreaks, and had not traveled outside of the United States or Canada during the 2 weeks before they became ill. The cases of persons who became ill during the period from 1 April through 15 June were defined as having occurred during the period of this outbreak. Traceback Investigation To identify sources of the implicated berries, case-patients and investigators identified establishments where the berries were bought or eaten and the dates of purchase or consumption. These establishments identified suppliers (such as distributors) and delivery dates, and suppliers provided shipping documents. A branch of a traceback corresponding to a particular supplier was considered well documented if each step from the consumers back to the country or state of origin was confirmed verbally and in writing (for example, by invoices). An entire traceback was considered well documented if all branches of the traceback were well documented. For Guatemalan berries, airway bill and invoice numbers were used to identify farms that contributed to shipments. The initials previously used to designate Guatemalan exporters (that is, A-G [1]) are used here. The U.S. Department of Agricultures Agricultural Marketing Service supplied weekly data for sources and amounts of domestic and imported raspberries shipped in the United States. Statistical Analysis We used Epi-Info, version 6.04a (CDC, Atlanta, Georgia), for analyses. Univariate relative risks were calculated for exposure variables. Two-tailed P values were computed by using the chi-square test or, if appropriate, the Fisher exact test. A P value less than 0.05 was considered to indicate statistical significance. A relative risk was defined as infinite if the attack rate was greater than 0 among exposed persons but was 0 among the unexposed and if no row or column total in the two-by-two table was 0. A relative risk or P value was considered undefined if a row or column total was 0. Results Epidemiologic Investigation Clusters Forty-one clusters of cases of cyclosporiasis were reported (Tables 1 and 2) in association with events held during the period from 1 April through 26 May (Figure 1, top). Of the 41 events, 20 (48.8%) occurred in private residences; 16 (39.0%) occurred in restaurants, clubs, hotels, inns, and resorts; and 5 (12.2%) occurred in other locations. An estimated total of 2541 persons attended the 41 events. Information was available for 1572 attendees (61.9%); 848 had event-associated illnesses and 762 (48.5% of interviewees) were classified as case-patients. Of these 762 persons, 192 (25.2%) had laboratory-confirmed cyclosporiasis. Table 2. Comparison of Clusters of Cases of Cyclosporiasis in the Berry-Associated Outbreaks in 1996 and 1997 Figure 1. Top. Dates of 41 events associated with clusters of cases of cyclosporiasis ( n =762 cases) in the United States and Canada in April and May 1997. Bottom. The incubation periods for the cases ranged from 1 to 14 days (by definition, the incubation period was<15 days); two cases with incubation periods of 1 day and one case with an incubation period of 2 days were laboratory confirmed. The median of the event-specific median incubation periods was 7 days (based on 38 events with available data). The median interval from symptom onset (the date associated with the events median incubation period) to notification of public health personnel of event-associated illness was 17 days (range, 5 to 59 days; based on 37 events). The index cases of clusters were brought to the attention of public health authorities in various ways. Index cases for at least 19 clusters (46.3%) were reported by laboratories. Ten of these 19 cases were reported because the laboratory was in a FoodNet site (26) and thus had enhanced laboratory surveillance for various emerging gastrointestinal pathogens (5 cases), cases of cyclosporiasis were officially reportable in the locale (3 cases), or both (2 cases). At least 6 clusters (14.6% of 41) were reported by an event attendee who learned of cyclosporiasis through the media, the Internet, or a medical journal. Fresh raspberries were the only food common to all 41 events (at 1 of these events, they quite possibly were served). For 28 (71.8%) of 39 events, the raspberries had reportedly been rinsed in water. Estimates of the numbers of raspberries per serving were available for 4 events; these estimates were 1, 4, 5, and 12 raspberries. At 16 events (39.0%), raspberries were the only type of berry served (9 events [22.0% of 41]) or were served separately from other berries (7 events [17.1%]). Fresh strawberries, blackberries, and blueberries were served at no more than 30 (73.2%), 22 (53.7%), and 20 (48.8%) events, respectively. At some events, mesclun lettuce (7 events), fresh basil (3 events), or both (2 events) probably were served; none of these events occurred in states known to have had outbreaks linked to these foods (4, 7). Consumption of raspberries was strongly associated with cyclosporiasis. For the 37 events for which information was available about more than 10% of attendees, the median event-specific attack rate, regardless of exposures, was 66.3% (range, 13.0% to 100%). The median attack rate was higher (91.7% [range, 32.5% to 100%]) among persons who ate items that contained raspberries, with or without other types of berries. Event-specific relative risks for the associations between raspberry-containing items and cyclosporiasis were elevated (>3.0 for 9 events [24.3% of 37 events]; median, 6.8 [range, 3.5 to 10.1]), infinite (16 events [43.2%]), or undefined (11 events [29.7%]) for all but 1 event (relative risk, 1.1). P values for the associations between raspberry-containing items and cyclosporiasis were undefined for 11 events (29.7%) and statistically significant for 15 events (40.5%), including 11 even

Outbreak of Cyclosporiasis Associated with Imported Raspberries, Philadelphia, Pennsylvania, 2000

An outbreak of cyclosporiasis occurred in attendees of a wedding reception held in Philadelphia, Pennsylvania, on June 10, 2000. In a retrospective cohort study, 54 (68.4%) of the 79 interviewed guests and members of the wedding party met the case definition. The wedding cake, which had a cream filling that included raspberries, was the food item most strongly associated with illness (multivariate relative risk, 5.9; 95% confidence interval, 3.6 to 10.5). Leftover cake was positive for Cyclospora DNA by polymerase chain reaction analyses. Sequencing of the amplified fragments confirmed that the organism was Cyclospora cayetanensis. The year 2000 was the fifth year since 1995 that outbreaks of cyclosporiasis definitely or probably associated with Guatemalan raspberries have occurred in the spring in North America. Additionally, this is the second documented U.S. outbreak, and the first associated with raspberries, for which Cyclospora has been detected in the epidemiologically implicated food item.

Epidemiologic studies of Cyclospora cayetanensis in Guatemala.

In 1996 and 1997, cyclosporiasis outbreaks in North America were linked to eating Guatemalan raspberries. We conducted a study in health-care facilities and among raspberry farm workers, as well as a case-control study, to assess risk factors for the disease in Guatemala. From April 6, 1997, to March 19, 1998, 126 (2.3%) of 5,552 surveillance specimens tested positive for Cyclospora; prevalence peaked in June (6.7%). Infection was most common among children 1.5 to 9 years old and among persons with gastroenteritis. Among 182 raspberry farm workers and family members monitored from April 6 to May 29, six had Cyclospora infection. In the case-control analysis, 62 (91%) of 68 persons with Cyclospora infection reported drinking untreated water in the 2 weeks before illness, compared with 88 (73%) of 120 controls (odds ratio [OR] 3.8, 95% confidence interval [CI] 1.4, 10.8 by univariate analysis). Other risk factors included water source, type of sewage drainage, ownership of chickens or other fowl, and contact with soil (among children younger than 2 years).

Epidemic Visceral Leishmaniasis in Southern Sudan: Treatment of Severely Debilitated Patients under Wartime Conditions and with Limited Resources

Southern Sudan is experiencing an epidemic of the classic visceral leishmaniasis syndrome, commonly called kala-azar. This syndrome is manifested by fever, cachexia, hepatosplenomegaly, and pancytopenia, and it is typically fatal if not appropriately treated. Although visceral leishmaniasis reportedly occurs in 47 countries, more than half of the recent cases have been in Sudan and India [1-3]. The epidemic in southern Sudan reportedly began in 1984 in the Western Upper Nile province but was first recognized by medical personnel in 1988 [4-6]. It has occurred in an area that was not previously considered a focus for visceral leishmaniasis [7]; the areas infrastructure and civilians have been severely affected by the civil war that erupted again in 1983. Medicins Sans Frontieres-Holland estimates that the excess mortality attributable to visceral leishmaniasis in Western Upper Nile is about 100,000 deaths among about 300,000 persons at risk for this syndrome [6, 8, 9]. The etiologic agent associated with the epidemic is Leishmania donovani, and the sand fly vector is Phlebotomus orientalis (whose habitat is Acacia-Balanites woodland) [10]. Vector-control strategies have not been feasible because of logistic and war-related constraints. It is not known whether humans are the main reservoirs of L. donovani in Western Upper Nile and whether treating patients has been an effective control measure. Medecins Sans Frontieres established visceral leishmaniasis treatment centers in Western Upper Nile in the village of Ler in May 1989 and in the village of Duar (80 km north of Ler and reportedly near the initial focus of the epidemic) in July 1990 (Figure 1). Approximately 19,000 patients were treated from 1989 to 1995, and about three quarters of these were treated in Duar. To identify patient characteristics associated with various treatment outcomes, we analyzed data for a cohort of 3076 patients who were treated in Duar with the pentavalent antimonial compound sodium stibogluconate (Pentostam, Wellcome Foundation Ltd., London, United Kingdom) [11] during the first year the center was operational (August 1990 to July 1991). Figure 1. Map of Sudan. The patient census peaked at about 800 patients at the end of the cohort period (July 1991) and thereafter increased to more than 1500 patients (August to November 1991). The treatment center was staffed by an average of two expatriates and about 25 Sudanese health auxiliaries trained by the expatriates. To reach Duar, patients typically walked or were carried for days. After admission, they stayed in crowded conditions, without access to latrines or adequate shelter, food, or water. The light aircraft that was the centers only means of transporting supplies could not accommodate bulky shipments of food or other provisions. In clinics held outdoors under shade trees, patients received daily parenteral therapy for about a month. Despite their severe debility and the exceptionally difficult circumstances under which they were treated, most fared remarkably well. Methods Diagnosis of Visceral Leishmaniasis and Study Cohort The diagnosis of visceral leishmaniasis was considered for persons who reportedly had been febrile for at least 2 weeks and had either palpable splenomegaly or wasting and lymphadenopathy. The diagnosis was supported serologically with a direct agglutination test [12, 13] by analysis of finger-prick blood specimens collected on filter paper. The definition of a positive serologic result varied depending on the time elapsed since the transmission season and the batch of antigen used. A Giemsa-stained smear of a specimen obtained by splenic aspiration (lymph node aspiration was used if splenic aspiration was considered too dangerous [for example, in young children]) was examined for parasites 1) if expeditious diagnosis was needed, 2) if the serologic result was negative or borderline but leishmaniasis was the likely diagnosis, or 3) if supplies for serologic testing were unavailable. Of the approximately 5000 persons evaluated for visceral leishmaniasis in Duar during the study period, about 3335 (66.7%) were admitted with this diagnosis and received at least some antimonial therapy. The study cohort consisted of 3076 patients (92.2% of 3335) previously untreated for visceral leishmaniasis whose medical records were found. Data were extracted from these records, clinic registration books, and monthly summary reports. Treatment of Visceral Leishmaniasis Patients received once-daily intramuscular injections of sodium stibogluconate (20 mg of pentavalent antimony/kg of body weight) for 30 consecutive days. By protocol, the minimum and maximum daily doses were 200 mg and 850 mg, respectively. Therefore, the 1383 adults ( 18 years of age) in the cohort (median weight, 47.0 kg) received a median daily dose of 18.1 mg of antimony/kg. Vitamin A was given on admission to nonpregnant patients; other vitamins, folic acid, and ferrous sulfate were provided daily; and chloroquine prophylaxis for malaria was given weekly. Supplemental nutrition could not be provided routinely. A test-of-cure specimen was obtained, typically on the 25th day of treatment, through aspiration of the spleen or a lymph node. If the specimen was positive for parasites, treatment was continued until two consecutive weekly specimens were negative. If parasites were still detectable after 60 days of therapy, the treatment regimen was changed from a once-daily intramuscular dose of 20 mg of antimony/kg (maximum daily dose, 850 mg) to twice-daily intravenous doses of 10 mg of antimony/kg (no maximum dose in mg). Treatment was continued until two consecutive specimens were negative. Discharged patients who reported recurrent symptoms (including fever for more than equals 2 weeks) and had a positive splenic or lymph node aspirate were retreated and classified as having relapsed; 15 such patients were readmitted on clinical grounds, most of them during periods when expatriate staff had been evacuated. Patients readmitted by the end of 1994 (3.5 years after the end of the study period) were included in the analyses. After treatment, patients could not be routinely monitored for relapse. Study Variables The following patient characteristics were assessed on admission (unless otherwise specified) and included in the analyses: age (usually estimated by the staff), sex, self-reported duration of illness, hemoglobin level, body mass index (measured in patients aged 18 years who did not have obvious signs of pregnancy; measured as weight in kg/height in m2 [14]), spleen size (Hackett classes 1 to 5 [15]), grade of parasite density in a splenic aspirate (negative = 0, positive = 1 to 6 [16, 17]), and symptoms during the treatment course. Patients were asked each day about vomiting, diarrhea ( 3 loose or watery stools in 24 hours), and bleeding (epistaxis, bleeding gums, and hematemesis). Data on diarrhea and bleeding were available only for patients admitted from March to July 1991. We assessed four mutually exclusive treatment outcomes: 1. Default: treatment stopped against medical advice without receipt of at least 25 doses of sodium stibogluconate, including, if indicated, the additional doses specified after a test-of-cure specimen was positive for parasites (number of patients who defaulted/number of patients admitted). 2. Death: death for any reason during the treatment course (number of patients who died/number of patients admitted). 3. Relapse: discharge after receipt of at least 25 drug doses (including, if indicated, the additional doses specified after a test-of-cure specimen was positive for parasites) and readmission by the end of 1994 because of relapsed visceral leishmaniasis (number of patients who relapsed/[number of patients admitted numbers of patients who defaulted or died during the first treatment course]). 4. Successful treatment: discharge after receipt of at least 25 drug doses (including, if indicated, the additional doses specified after a test-of-cure specimen was positive for parasites) and no readmission by the end of 1994 for retreatment (number of patients successfully treated/number of patients admitted). Statistical Analysis We explored the associations between patient characteristics and treatment outcomes by doing univariable analyses and calculating risks. To assess statistical significance, we used approximate 95% CIs and, when appropriate, the chi-square test for trend. We used the Wilcoxon two-sample test to compare the ranked distributions of ordinal variables. Associations between characteristics and outcomes were further assessed by multivariable analyses. We used MULTLR software for unconditional multiple logistic regression [18] with maximum likelihood fitting [19] and used adjusted odds ratios with 95% CIs to approximate risk ratios for death (see below for variables included in the models). The word significantly indicates that the CI excludes 1.0 or that the P value is less than 0.05. We studied nutritional status (body mass index) only for adults because children were not measured in a standardized way or with sufficient care to justify analysis of their nutritional data. Of all 1383 adults in the cohort, 1269 (91.8%) had their height and weight recorded (that is, their body mass index could be calculated), as well as age, sex, duration of illness, hemoglobin level, and spleen size. Of these, 1207 (95.1%) did not default or relapse and thus constitute the so-called adult cohort. The values (medians or proportions) of the patient characteristics for the adult cohort were not significantly different from the values for those patients of the 176 adults not included in the adult cohort for whom data about the given characteristic were available. The exception was that the adult cohort included patients who were somewhat more malnourished (median body mass index of 15.2 kg/m2 compared with 15.7 kg/m2; P = 0.04). We used this cohort and a subset (the so-called complet