James G. Olson

Epidemiology of Human Rabies in the United States, 1980 to 1996

One of the oldest recognized zoonotic diseases, rabies continues to plague humankind and causes more than 35 000 deaths annually [1]. These potentially preventable deaths occur primarily in Asia, Africa, and Latin America, where animal control, vaccination programs, and effective human postexposure prophylaxis are not widely available. In contrast, in the United States, deaths in humans caused by rabies totaled 99 in the 1950s, 15 in the 1960s, 23 in the 1970s, 10 in the 1980s, and 22 from 1990 through 1996 [2, 3]. The epidemiology of human rabies is ultimately linked to cycles of rabies virus transmission in animals. With the interruption of dog-to-dog transmission in most regions, the incidence of human rabies in the United States has reached a level that cannot be further reduced without targeting wildlife. An understanding of epidemiologic patterns of rabies virus maintenance in natural populations has emerged in the past 20 years, largely because of advances in immunology and molecular biology. Monoclonal antibody and genetic sequence analyses of rabies virus variants permit detailed descriptions of enzootic maintenance cycles of specific virus variants in the United States [4, 5]. These analyses have led to an understanding of how variants of rabies virus are maintained in natural reservoirs within geographic regions and have provided information on variability of the virus itself. Current epidemiologic patterns of rabies in the United States can be summarized as follows: The annual reports of rabies in wildlife exceed those of rabies in domestic animals [6]; rabies variants in bats are associated with a disproportionate number of infections in humans, although bats constitute only about 10% of all reported rabies cases in animals annually; most other cases of human rabies diagnosed in the United States can be attributed to infections acquired in areas of enzootic canine rabies outside of the United States; most persons with a case of rabies that originated in the United States have no history of an animal bite; and rabies is diagnosed after death in more than one third of the latter group. The last published summary of cases of human rabies in the United States covered the period from 1960 to 1979 [3]. This review discusses the clinical and epidemiologic features of cases of human rabies in the United States from 1980 to 1996. Methods Case Definition This report includes all laboratory-confirmed cases of human rabies in the United States or its territories from 1980 to 1996 [7-31]. All of the cases were reported to the Centers for Disease Control and Prevention (CDC) by health authorities as part of ongoing national surveillance. Variable Definitions Onset of illness was defined as either the first day of reported symptoms attributable to rabies or the date of initial presentation for medical care before confirmation of rabies. Clinical signs attributable to rabies included paresthesia, anxiety, agitation, confusion, disorientation, hydrophobia, aerophobia, hypersalivation, dysphagia, paresis, paralysis, and fluctuating levels of consciousness [32, 33]. The type of transmitting animal and the geographic location of exposure were listed if the case history included a definite animal bite. The reliability of information that linked rabies exposure to a human was assessed by subsequent laboratory typing of the rabies virus variant. All other exposures were defined as unknown. The diagnosis of rabies was considered antemortem if it was tentatively made and samples were obtained specifically for rabies testing before the patients death. Laboratory Tests The diagnosis of rabies was confirmed by using standard tests [34] conducted at the CDC or at a state laboratory. Serology Two tests were used to detect rabies antibody: the rapid fluorescent focus inhibition test and the indirect immunofluorescence assay. The rapid fluorescent focus inhibition test measures neutralizing antibody. An antibody titer of 1:5 or more, as defined by the reciprocal of the serum or cerebrospinal fluid dilution that reduces the challenge virus by 50%, was considered positive. An indirect immunofluorescence assay, using patient serum or cerebrospinal fluid diluted 1:4 or more, detects serum reactive with rabies antigen in infected cell cultures. The presence of antibody in serum was considered diagnostic if no vaccine or antirabies serum was given to the patient. Antibody in the cerebrospinal fluid, regardless of the rabies immunization history, was considered indicative of rabies virus infection. Virus Isolation Suspensions of brain or saliva specimens were added to mouse neuroblastoma cells and cultured for 24 and 48 hours. Culture slides were fixed and examined by direct immunofluorescence assay for antigen. Samples that were initially negative were maintained for an additional 3 to 4 days and retested. The negative result was considered definitive if it occurred both times. Antigen Detection Antigen detection was performed by direct immunofluorescence of assay serial frozen sections of nuchal skin biopsy specimens, touch impressions of corneal epithelial cells, or fresh brain matter. Paraffin-embedded fixed brain matter was sectioned and enzyme-digested before direct immunofluorescence. RNA Detection Standard extraction procedures and reagents were used to obtain nucleic acids from samples of undiluted saliva; from fresh or paraffin-embedded fixed samples of the brain; or, occasionally, from other tissues. Reverse transcription of RNA and complementary DNA amplification were performed by polymerase chain reaction (PCR) with primers derived from the sequence of the N protein gene. The nucleotide sequence of all PCR products was obtained by standard dideoxynucleotide sequencing methods. Rabies virus variants were identified by comparing samples of rabies virus obtained from all known reservoirs for rabies in the United States [5] with samples of rabies virus obtained from dogs in Asia, Africa, and Latin America [35]. Statistical Analysis Data analyses were performed by using EPI INFO 6 (Centers for Disease Control and Prevention, Atlanta, Georgia) or SPSS 6.0 for Windows (SPSS Inc., Chicago, Illinois) [36, 37]. Specific tests are identified in the text. Some variables were dichotomized before statistical comparisons for determination of odds ratios and 95% CIs. All reported P values are for two-tailed tests of significance. Results Demographic Information Thirty-two persons died of rabies in the United States from 1980 through 1996. Patients ranged in age from 4 to 82 years (median, 27 years) and 20 (63%) were male (Table 1). Cases were reported from 20 states; 7 cases (22%) were reported in California and 6 in Texas. Eleven patients were exposed to rabies in eight foreign countries on the basis of variant typing. The onset of illness occurred in all months and had no apparent seasonal pattern. Dates of exposure, based on the history of an animal bite, were obtained for 7 patients (22%) (Table 1). Table 1. Human Rabies in the United States, 1980-1996 Exposure History A definite history of animal exposure was identified in 7 of the 32 patients (22%), and 25 remained unknown or indefinite (Table 1). Of the 7 cases of definite exposure, 6 resulted from a dog bite received in a foreign country and 1 was from a bat bite received in the United States. Although rabies was not diagnosed in any of the animals that inflicted bites, in each case the rabies virus variant identified in the human sample was consistent with that in the animal species implicated as the source of infection (Table 1). Contact with an animal, thereby suggesting the source for infection, was identified in 12 persons (8 with a bat, 2 with a dog, 1 with a cow, and 1 with a cat). This human-animal contact, however, could not be linked to a bite or mucous membrane contact with the saliva of an animal potentially infected with rabies virus. The remaining 13 patients did not report animal contact; thus, a potential source of exposure was not identified. Histories were obtained before death from friends or relatives in 9 cases and from 4 children aged 11 to 13 years. Prophylaxis None of the 32 patients received a complete series of rabies prophylaxis after exposure; patient 7 reported receiving a single injection of an unknown type after a dog bite in Guatemala, and patients 15, 29, and 30 received human rabies immune globulin during the course of their clinical illness. Clinical Presentation For the 7 patients in which a definite animal bite occurred, the median incubation period was 85 days (range, 53 to 150 days). The first signs and symptoms of rabies were often nonspecific, including fever, sore throat, chills, malaise, anorexia, headache, nausea, vomiting, dyspnea, cough, and weakness. Specific symptoms, such as paresthesias at or near the presumed exposure site, were also reported early in the clinical course, and 19 of the 32 patients (59%) had three or more clinical findings suggestive of rabies during the course of their illness (Table 2). The 32 patients were seen by physicians on an outpatient basis a median of one time (range, 0 to 5 times) before hospitalization, and the median length of time from the onset of illness attributable to rabies to hospitalization was 4 days (range, 1 to 10 days). Table 2. Clinical Findings Suggestive of Rabies in 32 Patients* On admission, 21 of the 32 patients (66%) were febrile (oral temperature > 37.8C), including 12 patients with temperatures greater than 39.5C. Of the 11 patients who were afebrile on admission, 5 reported being febrile before admission, 2 became febrile within 2 days of admission, and 4 had no additional temperatures recorded. The antemortem diagnosis of rabies was first considered at the time of hospitalization in 5 patients, within 1 day of hospitalization in 5 patients, and after a median of 6 days of hospitalization (range, 2 to 12 days) in 10 patients. In 12 patients, rabies was diagnosed after death. Th

Chapare virus, a newly discovered arenavirus isolated from a fatal hemorrhagic fever case in Bolivia.

A small focus of hemorrhagic fever (HF) cases occurred near Cochabamba, Bolivia, in December 2003 and January 2004. Specimens were available from only one fatal case, which had a clinical course that included fever, headache, arthralgia, myalgia, and vomiting with subsequent deterioration and multiple hemorrhagic signs. A non-cytopathic virus was isolated from two of the patient serum samples, and identified as an arenavirus by IFA staining with a rabbit polyvalent antiserum raised against South American arenaviruses known to be associated with HF (Guanarito, Machupo, and Sabiá). RT-PCR analysis and subsequent analysis of the complete virus S and L RNA segment sequences identified the virus as a member of the New World Clade B arenaviruses, which includes all the pathogenic South American arenaviruses. The virus was shown to be most closely related to Sabiá virus, but with 26% and 30% nucleotide difference in the S and L segments, and 26%, 28%, 15% and 22% amino acid differences for the L, Z, N, and GP proteins, respectively, indicating the virus represents a newly discovered arenavirus, for which we propose the name Chapare virus. In conclusion, two different arenaviruses, Machupo and Chapare, can be associated with severe HF cases in Bolivia.

Temporal and Geographic Patterns of Aedes aegypti (Diptera: Culicidae) Production in Iquitos, Peru

Abstract Large-scale longitudinal cohort studies are necessary to characterize temporal and geographic variation in Aedes aegypti (L.) (Diptera: Culicidae) production patterns and to develop targeted dengue control strategies that will reduce disease. We carried out pupal/demographic surveys in a circuit of ≈6,000 houses, 10 separate times, between January 1999 and August 2002 in the Amazonian city of Iquitos, Peru. We quantified the number of containers positive for Ae. aegypti larvae and/or pupae, containers holding pupae, and the absolute number of pupae by 4-mo sampling circuits and spatially by geographic area by using a geographic information system developed for the city. A total of 289,941 water-holding containers were characterized, of which 7.3% were positive for Ae. aegypti. Temporal and geographic variations were detected for all variables examined, and the relative importance of different container types for production of Ae. aegypti was calculated. Ae. aegypti larvae and pupae were detected in 64 types of containers. Consistent production patterns were observed for the lid status (lids: 32% wet containers, 2% pupal production), container location (outdoor: 43% wet containers, 85% pupal production), and method by which the container was filled with water (rain filled: 15% wet containers, 88.3% pupal production); these patterns were consistent temporally and geographically. We describe a new container category (nontraditional) that includes transient puddles, which were rare but capable of producing large numbers of pupae. Because of high variable pupal counts, four container categories (large tank, medium storage, miscellaneous, and nontraditional) should be targeted in addition to outdoor rain-filled containers that are not covered by a lid. The utility of targeted Ae. aegypti control is discussed, as well as the ability to achieve control objectives based on published but untested threshold values.

Human Metapneumovirus, Peru

We retrospectively studied 420 pharyngeal swab specimens collected from Peruvian and Argentinean patients with influenzalike illness in 2002 and 2003 for evidence of human metapneumovirus (HMPV). Twelve specimens (2.3%) were positive by multiple assays. Six specimens yielded HMPV isolates. Four of the 6 isolates were of the uncommon B1 genotype.

Hidden Mortality Attributable to Rocky Mountain Spotted Fever: Immunohistochemical Detection of Fatal, Serologically Unconfirmed Disease

Rocky Mountain spotted fever (RMSF) is the most severe tickborne infection in the United States and is a nationally notifiable disease. Since 1981, the annual case-fatality ratio for RMSF has been determined from laboratory-confirmed cases reported to the Centers for Disease Control and Prevention (CDC). Herein, a description is given of patients with fatal, serologically unconfirmed RMSF for whom a diagnosis of RMSF was established by immunohistochemical (IHC) staining of tissues obtained at autopsy. During 1996-1997, acute-phase serum and tissue samples from patients with fatal disease compatible with RMSF were tested at the CDC. As determined by indirect immunofluorescence assay, no patient serum demonstrated IgG or IgM antibodies reactive with Rickettsia rickettsii at a diagnostic titer (i.e., >/=64); however, IHC staining confirmed diagnosis of RMSF in all patients. Polymerase chain reaction validated the IHC findings for 2 patients for whom appropriate samples were available for testing. These findings suggest that dependence on serologic assays and limited use of IHC staining for confirmation of fatal RMSF results in underestimates of mortality and of case-fatality ratios for this disease.

Arboviral etiologies of acute febrile illnesses in Western South America, 2000-2007.

Background Arthropod-borne viruses (arboviruses) are among the most common agents of human febrile illness worldwide and the most important emerging pathogens, causing multiple notable epidemics of human disease over recent decades. Despite the public health relevance, little is know about the geographic distribution, relative impact, and risk factors for arbovirus infection in many regions of the world. Our objectives were to describe the arboviruses associated with acute undifferentiated febrile illness in participating clinics in four countries in South America and to provide detailed epidemiological analysis of arbovirus infection in Iquitos, Peru, where more extensive monitoring was conducted. Methodology/Findings A clinic-based syndromic surveillance system was implemented in 13 locations in Ecuador, Peru, Bolivia, and Paraguay. Serum samples and demographic information were collected from febrile participants reporting to local health clinics or hospitals. Acute-phase sera were tested for viral infection by immunofluorescence assay or RT-PCR, while acute- and convalescent-phase sera were tested for pathogen-specific IgM by ELISA. Between May 2000 and December 2007, 20,880 participants were included in the study, with evidence for recent arbovirus infection detected for 6,793 (32.5%). Dengue viruses (Flavivirus) were the most common arbovirus infections, totaling 26.0% of febrile episodes, with DENV-3 as the most common serotype. Alphavirus (Venezuelan equine encephalitis virus [VEEV] and Mayaro virus [MAYV]) and Orthobunyavirus (Oropouche virus [OROV], Group C viruses, and Guaroa virus) infections were both observed in approximately 3% of febrile episodes. In Iquitos, risk factors for VEEV and MAYV infection included being male and reporting to a rural (vs urban) clinic. In contrast, OROV infection was similar between sexes and type of clinic. Conclusions/Significance Our data provide a better understanding of the geographic range of arboviruses in South America and highlight the diversity of pathogens in circulation. These arboviruses are currently significant causes of human illness in endemic regions but also have potential for further expansion. Our data provide a basis for analyzing changes in their ecology and epidemiology.

Epidemiology of Dengue Virus in Iquitos, Peru 1999 to 2005: Interepidemic and Epidemic Patterns of Transmission

Background Comprehensive, longitudinal field studies that monitor both disease and vector populations for dengue viruses are urgently needed as a pre-requisite for developing locally adaptable prevention programs or to appropriately test and license new vaccines. Methodology and Principal Findings We report the results from such a study spanning 5 years in the Amazonian city of Iquitos, Peru where DENV infection was monitored serologically among ∼2,400 members of a neighborhood-based cohort and through school-based absenteeism surveillance for active febrile illness among a subset of this cohort. At baseline, 80% of the study population had DENV antibodies, seroprevalence increased with age, and significant geographic variation was observed, with neighborhood-specific age-adjusted rates ranging from 67.1 to 89.9%. During the first 15 months, when DENV-1 and DENV-2 were co-circulating, population-based incidence rates ranged from 2–3 infections/100 person-years (p-years). The introduction of DENV-3 during the last half of 2001 was characterized by 3 distinct periods: amplification over at least 5–6 months, replacement of previously circulating serotypes, and epidemic transmission when incidence peaked at 89 infections/100 p-years. Conclusions/Significance Neighborhood-specific baseline seroprevalence rates were not predictive of geographic incidence patterns prior to the DENV-3 introduction, but were closely mirrored during the invasion of this serotype. Transmission varied geographically, with peak incidence occurring at different times among the 8 geographic zones in ∼16 km2 of the city. The lag from novel serotype introduction to epidemic transmission and knowledge of spatially explicit areas of elevated risk should be considered for more effective application of limited resources for dengue prevention.

Antibodies to Nipah-Like Virus in Bats (Pteropus lylei), Cambodia

Serum specimens from fruit bats were obtained at restaurants in Cambodia. We detected antibodies cross-reactive to Nipah virus by enzyme immunoassay in 11 (11.5%) of 96 Lyle’s flying foxes (Pteropus lylei). Our study suggests that viruses closely related to Nipah or Hendra viruses are more widespread in Southeast Asia than previously documented.

Characterization of Spotted Fever Group Rickettsiae in Flea and Tick Specimens From Northern Peru

ABSTRACT Evidence of spotted fever group (SFG) rickettsiae was obtained from flea pools and individual ticks collected at three sites in northwestern Peru within the focus of an outbreak of febrile disease in humans attributed, in part, to SFG rickettsia infections. Molecular identification of the etiologic agents from these samples was determined after partial sequencing of the 17-kDa common antigen gene (htrA) as well as pairwise nucleotide sequence homology with one or more of the following genes: gltA, ompA, and ompB. Amplification and sequencing of portions of the htrA and ompA genes in pooled samples (2 of 59) taken from fleas identified the pathogen Rickettsia felis. Four tick samples yielded molecular evidence of SFG rickettsiae. Fragments of the ompA (540-bp) and ompB (2,484-bp) genes were amplified from a single Amblyomma maculatum tick (tick 124) and an Ixodes boliviensis tick (tick 163). The phylogenetic relationships between the rickettsiae in these samples and other rickettsiae were determined after comparison of their ompB sequences by the neighbor-joining method. The dendrograms generated showed that the isolates exhibited close homology (97%) to R. aeschlimannii and R. rhipicephali. Significant bootstrap values supported clustering adjacent to this nodule of the SFG rickettsiae. While the agents identified in the flea and tick samples have not been linked to human cases in the area, these results demonstrate for the first time that at least two SFG rickettsia agents were circulating in northern Peru at the time of the outbreak. Furthermore, molecular analysis of sequences derived from the two separate species of hard ticks identified a possibly novel member of the SFG rickettsiae.

Nonviral Vector-Borne Zoonoses Associated with Mammals in the United States

Interest in vector-borne zoonoses has increased during the past few years as new disease agents have been identified and old ones have re-emerged due to important changes in their ecology or epidemiology. This article reviews nonviral vector-borne zoonoses that occur in the United States and are associated with mammals and their ectoparasites. The zoonoses discussed in this review include plague, tularemia, Lyme disease, tick-borne relapsing fevers, Rocky Mountain spotted fever, rickettsialpox, louse-borne typhus, flea-borne typhus, Q fever, and human ehrlichiosis.