**2.4.2 Serology**

RABV antibody testing for cases reported before 1973 utilized the mouse neutralization test (Jackson, 2003). After 1973, serology was determined using the rapid fluorescent focus inhibition test (RFFIT) or the indirect fluorescence assay (IFA), as described previously (Noah, 1998). The RFFIT measures RABV neutralizing antibodies while the IFA detects serum reactive with RABV antigen in infected cell cultures. Antibodies in serum were considered diagnostic if there was no history of rabies immunization prior to sample collection. Antibodies in cerebrospinal fluid (CSF) were considered diagnostic regardless of rabies immunization history.

#### **2.4.3 Virus isolation**

RABV was isolated through intracerebral inoculation of suckling mice or by addition of suspensions of brain or saliva specimens to cultured mouse neuroblastoma cells, as described previously (Noah, 1998).

### **2.4.4 RNA detection**

Viral nucleic acids were obtained using standard extraction procedures and reagents. Samples used for nucleic acid extraction included saliva, fresh brain, paraffin-embedded brain, and nuchal skin. Reverse transcription polymerase chain reaction (RT-PCR) was performed using primers targeting the sequence of the nucleoprotein gene. Standard dideoxynucleotide sequencing methods were utilized to determine the nucleotide sequences of all PCR products obtained, as described previously (Noah, 1998).

#### **2.4.5 Identification of rabies virus variants**

RABV variants were identified through antigenic and/or molecular typing. Antigenic typing uses a reference panel of monoclonal antibodies directed against the nucleoprotein to determine the variant of RABV isolates. Molecular typing methods identify the RABV variant by comparing the nucleotide sequence obtained by RT-PCR with a database of sequences from known reservoirs within the United States as well as foreign countries throughout the world.



Table 1. Human Rabies in the United States, 1960–2010


Table 1. Human Rabies in the United States, 1960–2010

Human Rabies Epidemiology and Diagnosis 255

The majority of human rabies cases (78/108; 72%) resulted from exposures that occurred within the United States and its territories. However, exposures in 12 foreign countries were responsible for a total of 30 imported cases. Exposures in Mexico were the most common and accounted for 13 imported cases. Animals were linked epidemiologically to 98 cases (91%) either by exposure history or RABV variant typing. Transplantation of infected organs or tissue was responsible for 5 cases (5%) and exposure to a laboratory RABV was implicated in two cases (2%). In 3 cases (3%) there was no history of exposure and no RABV variant identified to suggest a likely source of infection. The animal species linked epidemiologically to human rabies were bats (48/98; 49%), dogs (37/98; 38%), skunks (5/98; 5%), foxes (2/98; 2%), a cat (1/98; 1%), a bobcat (1/98; 1%), and a raccoon (1/98; 1%). The species responsible for infection was unclear in three cases in which the RABV variant identified did not match the exposure history; patient 24 reported a bite from a stray cat while a RABV variant associated with skunks was identified, patient 55 reported contact with a sick cow that later died while a RABV variant associated with bats was identified, and patient 101 had a history of exposure to a dog and a fox while the RABV variant identified was most closely related to viruses found in bats. These appear to have been spillover infection from a primary reservoir species to another animal. In all other cases where an animal exposure was reported the RABV variant identified matched the species of

A definite history of exposure to RABV was reported in 54 cases; 47 reported an animal bite, 2 involved exposures to laboratory RABV, and 5 had undergone transplantation of infected organs or tissue. A probable exposure was reported in 26 cases, 21 of which involved a probable exposure to a bat and 5 described a probable exposure to a dog. Of the 28 cases with an unknown exposure, a bat RABV variant was identified in 16 cases, a dog RABV variant was identified in 8 cases, and RABV variants associated with raccoons and skunks were identified in 1 case each. Among the definite exposures involving animal bites, 27 cases were indigenous cases involving bites from 12 bats, 7 dogs, 4 skunks, 2 foxes, 1 cat, and 1 bobcat. Only 2 of the 20 imported cases involved a RABV variant not associated with a domestic animal species. Only 9 animals involved in exposures were available for diagnostic testing, though all tested positive for RABV antigen. With respect to seasonality, rabies cases associated with bats (either by exposure or by identification of a bat RABV variant) were more likely to have onset of illness during the fall when compared to all other cases excluding those acquired through transplantation (OR 3.30; 95% CI 1.45–7.54). Exposures to bats were also more likely to occur during fall months when compared to exposures to other

Sixteen patients received PEP prior to the onset of symptoms. All of these cases occurred prior to 1980 and before the introduction of modern cell culture vaccines. Only two cases completed the PEP regimen according to recommended guidelines and can be considered true failures. The failure of PEP to prevent disease in the remaining 14 patients was attributed to either a delay in administration (i.e. administered greater than 72 hours after exposure), receipt of too few doses of vaccine, or failure to administer rabies immune globulin. Two patients had received rabies vaccine prior to their exposure: patient 22

**3.2 Source of infection** 

the exposing animal.

**3.3 Exposure history** 

animals (OR 16.50; 95% CI 1.83–148.61).

**3.4 Prophylaxis** 
