**3.9 Rabies virus diagnostic testing**

#### **3.9.1 Antemortem test results**

Results of RABV laboratory diagnostic testing were collected and summarized for 62 patients where specimens were collected prior to death (Table 3). When available, the number of days after illness onset the sample was collected (i.e. the sample collection date – illness onset date) is reported in parentheses following the test result. The cutaneous nerves of nuchal skin biopsies were tested for RABV antigen in 41 cases. A positive result was obtained in 24 cases (59%) with the first positive sample occurring a median of 7 days (range 2-12 days) after illness onset. RABV was detected in corneal impressions in 9 of 19 cases (47%) with 3 (16%) reporting inconclusive results and 7 (37%) reporting negative results. RABV antigen was first detected in corneal impressions a median of 11 days (range 2-13 days) after onset of rabies. Samples of brain were tested in 6 cases; 1 was inconclusive, 1 was negative, and 4 were first found positive a median of 12 days (range 7-21 days)




Table 3. Summary of Antemortem Diagnostic Test Results for 62 Patients with Human Rabies in the United States, 1960–2010

Human Rabies Epidemiology and Diagnosis 263

following onset of illness. Virus isolation from saliva was attempted in 23 cases and successful in 11 (48%) in samples collected a median of 8 days (range 3-13 days) after illness onset. RABV RNA was detected in 27 of 32 cases tested (84%) with the first positive results obtained from samples a median of 7 days (range 2-14 days) after the appearance of symptoms. Serum was tested for antibodies in 48 patients with no history of vaccination at the time of sample collection and was positive in 33 cases (69%). Antibodies in serum were first detected a median of 10.5 days (range 4-21 days) from the onset of illness in these unvaccinated patients. In contrast, antibodies in CSF were found in only 17 of 40 cases tested (43%) and first appeared a median of 14 days (range 5-28 days) following illness onset.

Confirmation of rabies through laboratory testing of postmortem samples was reported in 81 cases. The confirmation came shortly after death in the majority of cases. However, in at least 14 cases rabies was not suspected prior to death and was diagnosed as part of a postmortem investigation. Notable among these cases were all of the cases involving transplantation of infected organs or tissue (patients 30-31 and 89-93), 2 cases (patient 71 and patient 72) initially evaluated for suspected Creutzfeldt-Jakob disease, 1 case (patient 76) referred for testing of an undiagnosed encephalitis to the California Encephalitis Project, 1 case (patient 96) referred for testing to CDC's Unexplained Deaths Project, and 1 case (patient 59) originally attributed to disseminated candidiasis and mucormycosis in a pregnant female. The delay in diagnosis for these cases ranged from 1 to 6 months after

Despite being indigenous in wildlife, human rabies was a rarely reported disease in the United States during 1960–2010. An average of 2 cases per year was recorded during this time period, a trend that remained largely stable throughout each decade. While this should be considered a great success of public health efforts towards prevention and control, the true incidence of human rabies is likely higher due to under recognition. The disease occurs so infrequently that most clinicians have little direct experience with rabies and therefore may not consider it in the differential diagnosis of patients presenting with encephalitis. The lack of a clear animal exposure preceding illness in increasing numbers of cases leaves clinicians without a significant clue that might prompt consideration of rabies. The myriad of other possible etiologies of encephalitis further compounds this problem. For these reasons, a clear diagnosis cannot be found in the majority of cases of encephalitis even when extensive testing to identify the cause of illness is employed (Glaser, 2003). In fact, between 1979 and 1998 there was an average of over 1,000 deaths per year from encephalitis due to unknown cause reported in the United States (Khetsuriani, 2007). It is interesting to note that 1 of the 334 cases of encephalitis (0.3%) investigated by the California encephalitis project was diagnosed with rabies (Glaser, 2003). Rabies was not previously suspected in this patient and would almost certainly have remained unrecognized without the extensive testing performed as part of the study. Applying this rate of unrecognized rabies cases to the annual number of reported deaths due to encephalitis of unknown cause suggests there could be over 3 cases of human rabies that go unrecognized each year in the United States alone. This review provides a comprehensive summary of epidemiologic and clinical data of

**3.9.2 Postmortem test results** 

death.

**4. Conclusion**

Table 3. Summary of Antemortem Diagnostic Test Results for 62 Patients with Human

Rabies in the United States, 1960–2010

following onset of illness. Virus isolation from saliva was attempted in 23 cases and successful in 11 (48%) in samples collected a median of 8 days (range 3-13 days) after illness onset. RABV RNA was detected in 27 of 32 cases tested (84%) with the first positive results obtained from samples a median of 7 days (range 2-14 days) after the appearance of symptoms. Serum was tested for antibodies in 48 patients with no history of vaccination at the time of sample collection and was positive in 33 cases (69%). Antibodies in serum were first detected a median of 10.5 days (range 4-21 days) from the onset of illness in these unvaccinated patients. In contrast, antibodies in CSF were found in only 17 of 40 cases tested (43%) and first appeared a median of 14 days (range 5-28 days) following illness onset.

#### **3.9.2 Postmortem test results**

Confirmation of rabies through laboratory testing of postmortem samples was reported in 81 cases. The confirmation came shortly after death in the majority of cases. However, in at least 14 cases rabies was not suspected prior to death and was diagnosed as part of a postmortem investigation. Notable among these cases were all of the cases involving transplantation of infected organs or tissue (patients 30-31 and 89-93), 2 cases (patient 71 and patient 72) initially evaluated for suspected Creutzfeldt-Jakob disease, 1 case (patient 76) referred for testing of an undiagnosed encephalitis to the California Encephalitis Project, 1 case (patient 96) referred for testing to CDC's Unexplained Deaths Project, and 1 case (patient 59) originally attributed to disseminated candidiasis and mucormycosis in a pregnant female. The delay in diagnosis for these cases ranged from 1 to 6 months after death.

#### **4. Conclusion**

Despite being indigenous in wildlife, human rabies was a rarely reported disease in the United States during 1960–2010. An average of 2 cases per year was recorded during this time period, a trend that remained largely stable throughout each decade. While this should be considered a great success of public health efforts towards prevention and control, the true incidence of human rabies is likely higher due to under recognition. The disease occurs so infrequently that most clinicians have little direct experience with rabies and therefore may not consider it in the differential diagnosis of patients presenting with encephalitis. The lack of a clear animal exposure preceding illness in increasing numbers of cases leaves clinicians without a significant clue that might prompt consideration of rabies. The myriad of other possible etiologies of encephalitis further compounds this problem. For these reasons, a clear diagnosis cannot be found in the majority of cases of encephalitis even when extensive testing to identify the cause of illness is employed (Glaser, 2003). In fact, between 1979 and 1998 there was an average of over 1,000 deaths per year from encephalitis due to unknown cause reported in the United States (Khetsuriani, 2007). It is interesting to note that 1 of the 334 cases of encephalitis (0.3%) investigated by the California encephalitis project was diagnosed with rabies (Glaser, 2003). Rabies was not previously suspected in this patient and would almost certainly have remained unrecognized without the extensive testing performed as part of the study. Applying this rate of unrecognized rabies cases to the annual number of reported deaths due to encephalitis of unknown cause suggests there could be over 3 cases of human rabies that go unrecognized each year in the United States alone. This review provides a comprehensive summary of epidemiologic and clinical data of

Human Rabies Epidemiology and Diagnosis 265

The increasing number of cases associated with bats illustrates the growing importance of this species in the epidemiology of rabies (Figure 2). The advancement and increased utilization of RABV variant typing methods has led to a greater understanding of rabies epidemiology, particularly with regards to the role of bats (Smith, 1992). Excluding cases acquired through transplantation of infected tissue, a total of 42 RABV variants associated with bats were identified in this series, yet 16 (38%) of these cases had no reported exposure history. Without the availability of antigenic and molecular typing, this epidemiologic link to bats likely would have remained unrecognized in these cases. Many times, possible contact between the patient and a bat was recalled only after a bat RABV variant had been identified. Excluding cases acquired through transplantation, a total of 41/46 (89%) indigenous cases have been associated with bats either through a reported bat exposure or identification of a bat variant. The most common species of bats associated with human cases through variant typing were the silver-haired bat and the tricolored bat (*Lasionycteris noctivagans* and *Perimyotis subflavus*). This is a somewhat curious finding considering that these species of insectivorous bats are considered to be solitary tree-dwelling bats and are infrequently submitted to public health laboratories for rabies diagnostic testing (Krebs, 2000; Messenger, 2002; Blanton, 2010). However, there is some evidence suggesting that the RABV variants in these species have evolved genetic changes that may increase their infectivity (Morimoto, 1996; Dietzschold, 2000). Additional studies of bat RABV will undoubtedly improve understanding of the emergence, perpetuation, and epizootiology of

Classically, one expects human rabies cases to be preceded by an animal bite exposure. However, a definite history of a recent animal bite was reported in less than half of the cases in this series. The epidemiologic shift in the source of infection of human rabies in the United States provides one possible explanation for this observation. Bites from dogs or other carnivores are more likely to be recognized simply due to the size of the animal. A larger bite wound is also more likely to require medical treatment at which time consideration can be given to the need for rabies PEP. In contrast, North American bats are small and produce bite wounds that are superficial by comparison and less likely to require medical attention. With the advent of modern cell culture vaccines in 1980 and rabies immune globulin, no PEP failures have been reported in the United States. Unless the victim was previously aware of the risk of rabies from a bat bite, the opportunity for lifesaving PEP may be lost. A survey among cavers (spelunkers) in the United States found that 15% indicated a bat bite was not a risk for rabies, suggesting that a significant proportion of the public at large may also be unaware of the risk posed by bats (Gibbons, 2002). Some bat bites go unnoticed entirely because bat teeth are small and sharp (Feder, 1997). If the victim were engaged in an outdoor activity it is not hard to imagine a bat bite being confused for an insect bite or other minor trauma if the bat remained hidden in a tree or crevice (Gibbons, 2002). Furthermore, due to the extended incubation period a patient may not link the symptoms of the disease with an exposure that occurred months before the onset of illness. The median incubation in this series was nearly six weeks with evidence suggesting that long incubation periods on the order of years are possible (Smith, 1991). Thus, it is essential that the public be informed of the risk of rabies from all mammals and to remain vigilant for potential exposures, particularly when engaging in activities that put them at risk for contact with bats and other wildlife animals. Clinicians must also recognize that rabies should not be excluded from the differential diagnosis of a

this disease (Streicker, 2010).

all of the known cases within the last 5 decades which can be used as an aid in recognizing and identifying rabies among patients with encephalitis.

The epidemiology of human rabies in the United States has undergone striking changes over the last century. Prior to the 1960s, the vast majority of human rabies cases in the United State were due to bites from an infected dog. Worldwide, dogs remain the primary source of human rabies (WHO, 2004; Knobel, 2005). However, a dramatic decline in human rabies cases in the United States occurred during the 1940s and 1950s as illustrated in Figure 2. This decrease in cases paralleled the improvement and application of new rabies vaccines for both humans and animals. Key to this approach was the targeting of domestic animals for vaccination to create a barrier of protection from RABV transmission to humans, a strategy that was ultimately successful in eliminating the canine RABV variant from the United States (Velasco-Villa, 2008). Throughout the 1940s and 1950s the number of human rabies cases attributed to domestic animals (primarily dogs) far exceeded those involving wildlife animals. However, a reversal in this relationship took place in the 1960s when the number of human rabies cases associated with wildlife surpassed those due to domestic animals, a trend that continues to this day. Currently the most frequently reported rabid animals in the United States are raccoons, bats, skunks, and foxes (Blanton, 2010). Clinicians should be aware that these species can therefore be considered high risk though must also be careful not to discount exposures to other known vector species.

Fig. 2. Human Rabies Cases in the United States, 1946–2008

all of the known cases within the last 5 decades which can be used as an aid in recognizing

The epidemiology of human rabies in the United States has undergone striking changes over the last century. Prior to the 1960s, the vast majority of human rabies cases in the United State were due to bites from an infected dog. Worldwide, dogs remain the primary source of human rabies (WHO, 2004; Knobel, 2005). However, a dramatic decline in human rabies cases in the United States occurred during the 1940s and 1950s as illustrated in Figure 2. This decrease in cases paralleled the improvement and application of new rabies vaccines for both humans and animals. Key to this approach was the targeting of domestic animals for vaccination to create a barrier of protection from RABV transmission to humans, a strategy that was ultimately successful in eliminating the canine RABV variant from the United States (Velasco-Villa, 2008). Throughout the 1940s and 1950s the number of human rabies cases attributed to domestic animals (primarily dogs) far exceeded those involving wildlife animals. However, a reversal in this relationship took place in the 1960s when the number of human rabies cases associated with wildlife surpassed those due to domestic animals, a trend that continues to this day. Currently the most frequently reported rabid animals in the United States are raccoons, bats, skunks, and foxes (Blanton, 2010). Clinicians should be aware that these species can therefore be considered high risk though must also be careful

and identifying rabies among patients with encephalitis.

not to discount exposures to other known vector species.

Fig. 2. Human Rabies Cases in the United States, 1946–2008

The increasing number of cases associated with bats illustrates the growing importance of this species in the epidemiology of rabies (Figure 2). The advancement and increased utilization of RABV variant typing methods has led to a greater understanding of rabies epidemiology, particularly with regards to the role of bats (Smith, 1992). Excluding cases acquired through transplantation of infected tissue, a total of 42 RABV variants associated with bats were identified in this series, yet 16 (38%) of these cases had no reported exposure history. Without the availability of antigenic and molecular typing, this epidemiologic link to bats likely would have remained unrecognized in these cases. Many times, possible contact between the patient and a bat was recalled only after a bat RABV variant had been identified. Excluding cases acquired through transplantation, a total of 41/46 (89%) indigenous cases have been associated with bats either through a reported bat exposure or identification of a bat variant. The most common species of bats associated with human cases through variant typing were the silver-haired bat and the tricolored bat (*Lasionycteris noctivagans* and *Perimyotis subflavus*). This is a somewhat curious finding considering that these species of insectivorous bats are considered to be solitary tree-dwelling bats and are infrequently submitted to public health laboratories for rabies diagnostic testing (Krebs, 2000; Messenger, 2002; Blanton, 2010). However, there is some evidence suggesting that the RABV variants in these species have evolved genetic changes that may increase their infectivity (Morimoto, 1996; Dietzschold, 2000). Additional studies of bat RABV will undoubtedly improve understanding of the emergence, perpetuation, and epizootiology of this disease (Streicker, 2010).

Classically, one expects human rabies cases to be preceded by an animal bite exposure. However, a definite history of a recent animal bite was reported in less than half of the cases in this series. The epidemiologic shift in the source of infection of human rabies in the United States provides one possible explanation for this observation. Bites from dogs or other carnivores are more likely to be recognized simply due to the size of the animal. A larger bite wound is also more likely to require medical treatment at which time consideration can be given to the need for rabies PEP. In contrast, North American bats are small and produce bite wounds that are superficial by comparison and less likely to require medical attention. With the advent of modern cell culture vaccines in 1980 and rabies immune globulin, no PEP failures have been reported in the United States. Unless the victim was previously aware of the risk of rabies from a bat bite, the opportunity for lifesaving PEP may be lost. A survey among cavers (spelunkers) in the United States found that 15% indicated a bat bite was not a risk for rabies, suggesting that a significant proportion of the public at large may also be unaware of the risk posed by bats (Gibbons, 2002). Some bat bites go unnoticed entirely because bat teeth are small and sharp (Feder, 1997). If the victim were engaged in an outdoor activity it is not hard to imagine a bat bite being confused for an insect bite or other minor trauma if the bat remained hidden in a tree or crevice (Gibbons, 2002). Furthermore, due to the extended incubation period a patient may not link the symptoms of the disease with an exposure that occurred months before the onset of illness. The median incubation in this series was nearly six weeks with evidence suggesting that long incubation periods on the order of years are possible (Smith, 1991). Thus, it is essential that the public be informed of the risk of rabies from all mammals and to remain vigilant for potential exposures, particularly when engaging in activities that put them at risk for contact with bats and other wildlife animals. Clinicians must also recognize that rabies should not be excluded from the differential diagnosis of a

Human Rabies Epidemiology and Diagnosis 267

and dysphagia in a patient with acute progressive encephalitis should alert the clinician to

The age and sex of a patient in addition to the month of illness onset may also provide clues to the diagnosis of rabies in patients with encephalitis. Globally, human rabies is most likely to be seen in children less than 15 years old (WHO, 2004). However, patients in this series were more likely to be older with a median age of 29 years. The disproportionate number of cases linked with bats and other wildlife species as opposed to dogs is likely responsible for this discrepancy. Children are more likely to unintentionally incite a dog attack through provoking behaviors and inexperience or because their size and movements may be similar to prey. With respect to wildlife, one might expect older age groups to be more likely to engage in outdoor activities or other behaviors that would put them at higher risk of exposure to bats and wildlife animals. Similarly, the predominance of males in this case series may also be due in part to males engaging in these activities more frequently than females. Limited data on the administration of PEP suggests that males may have higher rates of PEP (Helmick, 1983) though this trend was not confirmed in other studies of state level data (Blanton, 2005; O'Bell, 2006). Furthermore, males may be less likely to seek PEP after an exposure occurs. In support of this claim is evidence suggesting that the perception and awareness of health-related risks may be lower in males compared to females (Naslund, 1997; Gustafson, 1998). The observation that most cases occurred during fall months corresponds well with the observation that most contact with wildlife, particularly bats, occurs during the late summer months (Messenger, 2002). Assuming an incubation period of 1-3 months, exposures occurring during summer would then be expected to become ill during the fall. This pattern was particularly significant when analyzing cases associated with bats. Most bat exposures occurred during the early fall months and is consistent with previous research demonstrating that the incidence of rabies-infected bats peaks in August (Constantine, 1979; Pape, 1999). Furthermore, the illness onset month in cases associated with bats was most common during the late fall months. Both exposures and onset of illness were more likely to occur in fall months in cases associated with bats compared with those not associated with bats. Based on these findings, a typical human rabies case in the United States would be a young adult male presenting with signs and symptoms of encephalitis in

When human rabies is suspected it is important to initiate diagnostic testing as early as possible. Current guidelines for antemortem testing of human patients recommend the collection of samples of skin, serum, saliva, and CSF. In this series, the earliest positive results were found with detection of viral RNA in saliva and viral antigen in skin after a median of 7 days following onset of illness. The earliest diagnosis occurred in patient 98 with both saliva and skin collected 2 days after illness onset reported positive by PCR and DFA respectively on the sixth day of illness. Virus isolation from saliva was also fairly rapid with positive results obtained as early as 3 days after illness onset (median 8 days). The development of a humoral immune response lags behind the secretion of virus in saliva and antibodies were detected in serum and CSF a median of 10 and 14 days respectively following the appearance of symptoms. The earliest detection of rabies specific antibodies after illness onset was 4 days in serum and 5 days in CSF. With respect to sensitivity, identification of viral RNA in saliva was the most sensitive testing modality with 84% of rabies cases tested giving positive results. Detection of antibodies in serum and viral antigens in brain and cutaneous nerves were also relatively sensitive with positive results in 69%, 67%, and 59% of cases tested respectively. Though isolation of RABV from saliva was

the possibility of rabies.

the fall.

patient with acute progressive encephalitis due to the absence of a recent animal exposure history.

The initial signs and symptoms of human rabies are largely nonspecific and include fever, malaise, headache, nausea, and vomiting. Not surprisingly, almost half of the patients in this series were evaluated for their symptoms at least once prior to being admitted to the hospital without the diagnosis of rabies being considered. Thus, it may be very difficult to diagnose rabies during the prodromal phase in the absence of an animal exposure to alert one to the possibility. However, the progression of illness is rapid with most patients seeking medical care within one day of the appearance of symptoms and requiring hospitalization within 4 days of illness onset. Furthermore, most patients succumbed to their illness within 2 weeks of the onset of symptoms. These data suggest that rabies would be less likely in patients who do not require hospitalization within one week of developing symptoms or in hospitalized patients surviving longer than 2 weeks. Prioritization of testing for other etiologies over rabies would be prudent in these situations. However, several signs and symptoms were found to occur more frequently in patients with rabies when compared to patients with encephalopathy who tested negative for rabies. Aerophobia and hydrophobia were the most specific findings associated with rabies cases despite the fact that these symptoms were found in relatively few cases. Intubation and sedation have become common components of modern medical management of critically ill patients, a practice that may mask these symptoms unless specifically noted by patients early in their course. The neurologic findings of paresthesias or localized pain, dysphagia, and focal weakness were also positively linked to rabies cases. In contrast, headache, malaise or fatigue, seizures, and confusion or delirium were observed more frequently in non-rabies cases than in rabies cases.

The clinical presentation of human rabies is often classified as either encephalitic (furious) or paralytic (dumb) rabies. The encephalitic form of the disease is characterized by overall hyperexcitability with episodes of confusion, agitation or aggressive behavior, and hallucinations. In the paralytic form, localized weakness and/or paralysis are the most prominent feature and are commonly accompanied by paresthesias, pruritis, or localized pain at the site of the bite and a normal mental status. The signs and symptoms associated with rabies cases in this series are those classically used to describe the paralytic rather than the encephalitic form. In general, 80% of human rabies patients are thought to manifest the encephalitic form while 20% have a paralytic form (Jackson, 2002)(Jackson, 2007). As such, clinicians may be more likely to submit cases for testing that resemble their preconceived notion of the more common furious form of human rabies and less likely to consider rabies with the more rare paralytic form. A connection between the paralytic form of rabies and transmission by vampire bats has been noted in several human rabies outbreaks (Nehaul, 1955; Hurst, 1959; Hurst, 1959; Pawan, 1959; Verlinde, 1975; da Rosa, 2006). However, vampire bats do not cause exclusively paralytic disease as the encephalitic form has been described with a vampire bat human rabies outbreak in Peru (Lopez, 1992). Host factors most certainly play a role also as two individuals bitten by the same dog were seen to develop encephalitic rabies in one and paralytic rabies in the other (Hemachudha, 1988). Nevertheless, it is unclear whether the overall preponderance of cases linked to bats may have affected the clinical syndrome observed in this series. Regardless of the source of infection, clinicians should be aware that the clinical presentation of human rabies is a spectrum that can include features of either the encephalitic or paralytic forms. Positive indicators such as hydrophobia, aerophobia, paresthesias or localized pain, focal weakness,

patient with acute progressive encephalitis due to the absence of a recent animal exposure

The initial signs and symptoms of human rabies are largely nonspecific and include fever, malaise, headache, nausea, and vomiting. Not surprisingly, almost half of the patients in this series were evaluated for their symptoms at least once prior to being admitted to the hospital without the diagnosis of rabies being considered. Thus, it may be very difficult to diagnose rabies during the prodromal phase in the absence of an animal exposure to alert one to the possibility. However, the progression of illness is rapid with most patients seeking medical care within one day of the appearance of symptoms and requiring hospitalization within 4 days of illness onset. Furthermore, most patients succumbed to their illness within 2 weeks of the onset of symptoms. These data suggest that rabies would be less likely in patients who do not require hospitalization within one week of developing symptoms or in hospitalized patients surviving longer than 2 weeks. Prioritization of testing for other etiologies over rabies would be prudent in these situations. However, several signs and symptoms were found to occur more frequently in patients with rabies when compared to patients with encephalopathy who tested negative for rabies. Aerophobia and hydrophobia were the most specific findings associated with rabies cases despite the fact that these symptoms were found in relatively few cases. Intubation and sedation have become common components of modern medical management of critically ill patients, a practice that may mask these symptoms unless specifically noted by patients early in their course. The neurologic findings of paresthesias or localized pain, dysphagia, and focal weakness were also positively linked to rabies cases. In contrast, headache, malaise or fatigue, seizures, and confusion or delirium were observed more frequently in non-rabies

The clinical presentation of human rabies is often classified as either encephalitic (furious) or paralytic (dumb) rabies. The encephalitic form of the disease is characterized by overall hyperexcitability with episodes of confusion, agitation or aggressive behavior, and hallucinations. In the paralytic form, localized weakness and/or paralysis are the most prominent feature and are commonly accompanied by paresthesias, pruritis, or localized pain at the site of the bite and a normal mental status. The signs and symptoms associated with rabies cases in this series are those classically used to describe the paralytic rather than the encephalitic form. In general, 80% of human rabies patients are thought to manifest the encephalitic form while 20% have a paralytic form (Jackson, 2002)(Jackson, 2007). As such, clinicians may be more likely to submit cases for testing that resemble their preconceived notion of the more common furious form of human rabies and less likely to consider rabies with the more rare paralytic form. A connection between the paralytic form of rabies and transmission by vampire bats has been noted in several human rabies outbreaks (Nehaul, 1955; Hurst, 1959; Hurst, 1959; Pawan, 1959; Verlinde, 1975; da Rosa, 2006). However, vampire bats do not cause exclusively paralytic disease as the encephalitic form has been described with a vampire bat human rabies outbreak in Peru (Lopez, 1992). Host factors most certainly play a role also as two individuals bitten by the same dog were seen to develop encephalitic rabies in one and paralytic rabies in the other (Hemachudha, 1988). Nevertheless, it is unclear whether the overall preponderance of cases linked to bats may have affected the clinical syndrome observed in this series. Regardless of the source of infection, clinicians should be aware that the clinical presentation of human rabies is a spectrum that can include features of either the encephalitic or paralytic forms. Positive indicators such as hydrophobia, aerophobia, paresthesias or localized pain, focal weakness,

history.

cases than in rabies cases.

and dysphagia in a patient with acute progressive encephalitis should alert the clinician to the possibility of rabies.

The age and sex of a patient in addition to the month of illness onset may also provide clues to the diagnosis of rabies in patients with encephalitis. Globally, human rabies is most likely to be seen in children less than 15 years old (WHO, 2004). However, patients in this series were more likely to be older with a median age of 29 years. The disproportionate number of cases linked with bats and other wildlife species as opposed to dogs is likely responsible for this discrepancy. Children are more likely to unintentionally incite a dog attack through provoking behaviors and inexperience or because their size and movements may be similar to prey. With respect to wildlife, one might expect older age groups to be more likely to engage in outdoor activities or other behaviors that would put them at higher risk of exposure to bats and wildlife animals. Similarly, the predominance of males in this case series may also be due in part to males engaging in these activities more frequently than females. Limited data on the administration of PEP suggests that males may have higher rates of PEP (Helmick, 1983) though this trend was not confirmed in other studies of state level data (Blanton, 2005; O'Bell, 2006). Furthermore, males may be less likely to seek PEP after an exposure occurs. In support of this claim is evidence suggesting that the perception and awareness of health-related risks may be lower in males compared to females (Naslund, 1997; Gustafson, 1998). The observation that most cases occurred during fall months corresponds well with the observation that most contact with wildlife, particularly bats, occurs during the late summer months (Messenger, 2002). Assuming an incubation period of 1-3 months, exposures occurring during summer would then be expected to become ill during the fall. This pattern was particularly significant when analyzing cases associated with bats. Most bat exposures occurred during the early fall months and is consistent with previous research demonstrating that the incidence of rabies-infected bats peaks in August (Constantine, 1979; Pape, 1999). Furthermore, the illness onset month in cases associated with bats was most common during the late fall months. Both exposures and onset of illness were more likely to occur in fall months in cases associated with bats compared with those not associated with bats. Based on these findings, a typical human rabies case in the United States would be a young adult male presenting with signs and symptoms of encephalitis in the fall.

When human rabies is suspected it is important to initiate diagnostic testing as early as possible. Current guidelines for antemortem testing of human patients recommend the collection of samples of skin, serum, saliva, and CSF. In this series, the earliest positive results were found with detection of viral RNA in saliva and viral antigen in skin after a median of 7 days following onset of illness. The earliest diagnosis occurred in patient 98 with both saliva and skin collected 2 days after illness onset reported positive by PCR and DFA respectively on the sixth day of illness. Virus isolation from saliva was also fairly rapid with positive results obtained as early as 3 days after illness onset (median 8 days). The development of a humoral immune response lags behind the secretion of virus in saliva and antibodies were detected in serum and CSF a median of 10 and 14 days respectively following the appearance of symptoms. The earliest detection of rabies specific antibodies after illness onset was 4 days in serum and 5 days in CSF. With respect to sensitivity, identification of viral RNA in saliva was the most sensitive testing modality with 84% of rabies cases tested giving positive results. Detection of antibodies in serum and viral antigens in brain and cutaneous nerves were also relatively sensitive with positive results in 69%, 67%, and 59% of cases tested respectively. Though isolation of RABV from saliva was

Human Rabies Epidemiology and Diagnosis 269

among 24 attempts to give an estimated 9% survival by intention-to-treat analysis (Willoughby, 2009). As knowledge is gained from both treatment failures and successes, further refinement of this approach may increase the rate of success. The recovery of patient 104 without application of the Milwaukee Protocol or even need for intensive care is the only well documented case of a presumed human abortive rabies infection. This case hints at the possibility that there may be a much larger spectrum of disease in rabies cases that has not been previously appreciated. It may be only recently recognized due to improved laboratory diagnostic techniques, greater availability of laboratory testing, or increased awareness of the possibility of rabies after several high profile cases in the United States including the survival of patient 95 and the cases transmitted through transplantation (patients 90-93). The association of both of these survivors with exposures to bats has also led to the hypothesis that canine RABV variants may be more virulent than bat variants (Lafon, 2005). However, one case of documented human rabies of canine origin in Equatorial Guinea purportedly survived infection with application of the Milwaukee Protocol, but later succumbed (Rubin, 2009). The patient's death on hospital day 22 was thought to be due to complications of malnutrition. Irrespective of the source of infection, the limited success of treatment attempts emphasizes the importance of prevention. Current treatment protocols require considerable expense and advanced medical facilities making practical application unrealistic in many countries. In contrast, rabies prevention strategies implementing safe, efficacious, and affordable human and animal vaccines have proven to be highly effective, particularly in combination with community outreach and education

The results of this study are subject to several limitations. Given the relative rarity of human rabies in the United States despite the abundance of the disease in nature, this case series represents a relatively small sample size and several cases had only limited data available. In addition, all of the data from cases were collected retrospectively and are therefore subject to recall bias. Histories of potential animal exposures may be particularly sensitive to poor memory given the lengthy incubation period seen with rabies; this may be exacerbated in cases where rabies was not initially considered or the diagnosis was delayed. In all cases there is a short window of opportunity to communicate directly with patients due to the rapid progression of disease and nearly universal fatal outcome. In the confirmed rabies cases additional clinical and laboratory data were gained through public health investigations. In contrast, data from non-rabies cases relied primarily on patient information forms completed at the time of submission for diagnostic testing. As such, those data represent only a snapshot of the clinical picture making comparisons of temporal patterns in rabies and non-rabies cases unfeasible. Moreover, no follow-up of clinical outcomes (i.e. survival or death) was available for non-rabies cases. Though statistically significant results were obtained, it is possible that other significant findings may have been

This review complements previous reports of smaller series of human rabies cases in the United States (Held, 1967; Anderson, 1984; Noah, 1998; CDC, 2006). Significant changes in the epidemiology of rabies include the increasing role of bats in human rabies and the concomitant rise in cases with no clear history of animal exposure. These findings underscore the need to increase public awareness of the risk of rabies from wildlife, particularly bats, and the importance of prompt medical evaluation if contact with such animals occurs. Advances in the treatment of human rabies have led to the first reported cases of recovery without immunization with rabies vaccine. These survivors raise hope

programs (Rupprecht, 2008; Cleaveland, 2010).

missed due to the small sample size.

successful in fewer than half of cases, the utility of virus isolates for variant typing, comparative analyses, investigations of pathogenesis, and other studies is invaluable. Similarly, while antibodies in CSF were found relatively infrequently, their detection is significant as this finding alone is considered diagnostic for rabies regardless of history of rabies vaccination (Moore, 2010). Examination of corneal impressions was positive in only 47% of cases tested and is no longer routinely used for antemortem diagnostic testing given the risk of damage to the eyes when performed by inexperienced practitioners and the relatively poor sensitivity. While the sensitivity of viral antigen detection in antemortem brain samples is high, routine testing of these samples is not recommended due to the invasiveness of the collection procedure. The best approach for maximizing the sensitivity and accuracy of antemortem diagnostic rabies testing is through submission of skin, saliva, serum, and CSF for evaluation with multiple modalities including detection of viral antigen, viral nucleic acid, and rabies specific antibodies, especially because some test results become available in less than 24 hours.

The diagnosis of human rabies can be essential in preventing further cases. Identifying human cases allows for a public health response to investigate the source of infection and provide risk assessments of contacts with potential exposures. If a suspected animal source infestation is discovered, these animals can be removed to avert any further exposures and tested to confirm whether additional animals are rabid. Any individuals with contact with potentially rabid animals can then be assessed for exposures and given PEP when indicated. Public education can also be targeted to increase awareness of the risk of rabies transmission from reservoir species and the importance of seeking medical attention promptly if an exposure does occur. Contacts of rabies patients should also be identified and evaluated for exposures. Although human-to-human transmission of rabies has only been well documented in cases of organ or tissue transplantation, transmission following exposure to human saliva or nervous tissue remains a theoretical possibility (Helmick, 1987). Rare anecdotal reports of direct human to human transmission of rabies through bites, kissing, lactation, intercourse, transplacental transmission, and delivery of healthcare have been reported though possible animal exposures were difficult to exclude and none of these cases were laboratory confirmed (Helmick, 1987; Gibbons, 2002). Healthcare providers can be reassured by the fact that no case of rabies has ever been confirmed in a caregiver of any of the estimated 55,000 cases occurring each year. Moreover, early recognition and contact isolation of suspected cases are effective means of limiting exposures in healthcare settings (Helmick, 1987). Given these facts, the median of 39 individuals receiving PEP per case found in this series may seem excessive. Though the higher rate of PEP of contacts among cases diagnosed postmortem compared to antemortem was not statistically significant, it is still likely that early diagnosis of human rabies will lead to fewer exposures and decrease unnecessary PEP. Lastly, identification of human rabies can avoid transmitting the virus through organ and tissue transplantation. Even if the diagnosis is made after transplantation has already taken place, removal of the transplanted tissue and administration of PEP has been successful in preventing infection in two recipients of corneas from an infected donor (Vetter, 2011).

The early diagnosis of rabies also provides the best opportunity for experimental therapy of victims. The 4 survivors reported in this series demonstrate that survival is rare but does occur. In addition, patient 95 was the first report of a patient recovering from rabies without receiving any vaccination against rabies through the use of the Milwaukee Protocol. Two subsequent survivors attributed to the application of this protocol have been reported

successful in fewer than half of cases, the utility of virus isolates for variant typing, comparative analyses, investigations of pathogenesis, and other studies is invaluable. Similarly, while antibodies in CSF were found relatively infrequently, their detection is significant as this finding alone is considered diagnostic for rabies regardless of history of rabies vaccination (Moore, 2010). Examination of corneal impressions was positive in only 47% of cases tested and is no longer routinely used for antemortem diagnostic testing given the risk of damage to the eyes when performed by inexperienced practitioners and the relatively poor sensitivity. While the sensitivity of viral antigen detection in antemortem brain samples is high, routine testing of these samples is not recommended due to the invasiveness of the collection procedure. The best approach for maximizing the sensitivity and accuracy of antemortem diagnostic rabies testing is through submission of skin, saliva, serum, and CSF for evaluation with multiple modalities including detection of viral antigen, viral nucleic acid, and rabies specific antibodies, especially because some test results become

The diagnosis of human rabies can be essential in preventing further cases. Identifying human cases allows for a public health response to investigate the source of infection and provide risk assessments of contacts with potential exposures. If a suspected animal source infestation is discovered, these animals can be removed to avert any further exposures and tested to confirm whether additional animals are rabid. Any individuals with contact with potentially rabid animals can then be assessed for exposures and given PEP when indicated. Public education can also be targeted to increase awareness of the risk of rabies transmission from reservoir species and the importance of seeking medical attention promptly if an exposure does occur. Contacts of rabies patients should also be identified and evaluated for exposures. Although human-to-human transmission of rabies has only been well documented in cases of organ or tissue transplantation, transmission following exposure to human saliva or nervous tissue remains a theoretical possibility (Helmick, 1987). Rare anecdotal reports of direct human to human transmission of rabies through bites, kissing, lactation, intercourse, transplacental transmission, and delivery of healthcare have been reported though possible animal exposures were difficult to exclude and none of these cases were laboratory confirmed (Helmick, 1987; Gibbons, 2002). Healthcare providers can be reassured by the fact that no case of rabies has ever been confirmed in a caregiver of any of the estimated 55,000 cases occurring each year. Moreover, early recognition and contact isolation of suspected cases are effective means of limiting exposures in healthcare settings (Helmick, 1987). Given these facts, the median of 39 individuals receiving PEP per case found in this series may seem excessive. Though the higher rate of PEP of contacts among cases diagnosed postmortem compared to antemortem was not statistically significant, it is still likely that early diagnosis of human rabies will lead to fewer exposures and decrease unnecessary PEP. Lastly, identification of human rabies can avoid transmitting the virus through organ and tissue transplantation. Even if the diagnosis is made after transplantation has already taken place, removal of the transplanted tissue and administration of PEP has been successful in preventing infection in two recipients of corneas from an infected donor

The early diagnosis of rabies also provides the best opportunity for experimental therapy of victims. The 4 survivors reported in this series demonstrate that survival is rare but does occur. In addition, patient 95 was the first report of a patient recovering from rabies without receiving any vaccination against rabies through the use of the Milwaukee Protocol. Two subsequent survivors attributed to the application of this protocol have been reported

available in less than 24 hours.

(Vetter, 2011).

among 24 attempts to give an estimated 9% survival by intention-to-treat analysis (Willoughby, 2009). As knowledge is gained from both treatment failures and successes, further refinement of this approach may increase the rate of success. The recovery of patient 104 without application of the Milwaukee Protocol or even need for intensive care is the only well documented case of a presumed human abortive rabies infection. This case hints at the possibility that there may be a much larger spectrum of disease in rabies cases that has not been previously appreciated. It may be only recently recognized due to improved laboratory diagnostic techniques, greater availability of laboratory testing, or increased awareness of the possibility of rabies after several high profile cases in the United States including the survival of patient 95 and the cases transmitted through transplantation (patients 90-93). The association of both of these survivors with exposures to bats has also led to the hypothesis that canine RABV variants may be more virulent than bat variants (Lafon, 2005). However, one case of documented human rabies of canine origin in Equatorial Guinea purportedly survived infection with application of the Milwaukee Protocol, but later succumbed (Rubin, 2009). The patient's death on hospital day 22 was thought to be due to complications of malnutrition. Irrespective of the source of infection, the limited success of treatment attempts emphasizes the importance of prevention. Current treatment protocols require considerable expense and advanced medical facilities making practical application unrealistic in many countries. In contrast, rabies prevention strategies implementing safe, efficacious, and affordable human and animal vaccines have proven to be highly effective, particularly in combination with community outreach and education programs (Rupprecht, 2008; Cleaveland, 2010).

The results of this study are subject to several limitations. Given the relative rarity of human rabies in the United States despite the abundance of the disease in nature, this case series represents a relatively small sample size and several cases had only limited data available. In addition, all of the data from cases were collected retrospectively and are therefore subject to recall bias. Histories of potential animal exposures may be particularly sensitive to poor memory given the lengthy incubation period seen with rabies; this may be exacerbated in cases where rabies was not initially considered or the diagnosis was delayed. In all cases there is a short window of opportunity to communicate directly with patients due to the rapid progression of disease and nearly universal fatal outcome. In the confirmed rabies cases additional clinical and laboratory data were gained through public health investigations. In contrast, data from non-rabies cases relied primarily on patient information forms completed at the time of submission for diagnostic testing. As such, those data represent only a snapshot of the clinical picture making comparisons of temporal patterns in rabies and non-rabies cases unfeasible. Moreover, no follow-up of clinical outcomes (i.e. survival or death) was available for non-rabies cases. Though statistically significant results were obtained, it is possible that other significant findings may have been missed due to the small sample size.

This review complements previous reports of smaller series of human rabies cases in the United States (Held, 1967; Anderson, 1984; Noah, 1998; CDC, 2006). Significant changes in the epidemiology of rabies include the increasing role of bats in human rabies and the concomitant rise in cases with no clear history of animal exposure. These findings underscore the need to increase public awareness of the risk of rabies from wildlife, particularly bats, and the importance of prompt medical evaluation if contact with such animals occurs. Advances in the treatment of human rabies have led to the first reported cases of recovery without immunization with rabies vaccine. These survivors raise hope

Human Rabies Epidemiology and Diagnosis 271

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#### **5. Acknowledgements**

The authors thank all of the clinicians and public health professionals who provided clinical, epidemiologic, and laboratory data on these cases. The authors also recognize the contributions of Jesse Blanton, Lizette Durand, Richard Franka, Felix Jackson, Ivan Kuzmin, Lillian Orciari, Dustyn Palmer, Sergio Recuenco, Andres Velasco-Villa, and Pam Yager.

#### **6. References**


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**Part 3** 

**Protozoan Pathogens** 


**Part 3** 

**Protozoan Pathogens** 

278 Non-Flavivirus Encephalitis

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Report, Vol.60, No.34, pp.1164-1166

**12** 

*Thailand* 

**Toxoplasmic Encephalitis** 

*Department of Protozoology, Faculty of Tropical Medicine, Mahidol University* 

Toxoplasmic encephalitis (TE), a life-threatening disease in HIV/AIDS infected individuals, is an inflammation of the brain caused by the reactivation of latent infection of the protozoa *Toxoplasma gondii*. Immunocompetent host when infected with *T. gondii* is almost always unnoticeable or develops mild and non-specific signs and symptoms, then tissue cysts are the consequence harbored in those infected persons life-long, quietly without any problem. However, when host immunity is suppressed by any cause but mostly by HIV/AIDS, the previously quiescent protozoa become active and the aggressive stage, tachyzoite, causes severe clinical manifestations in the Central Nervous System (CNS) such as encephalitis or

In the early 1980s, at the beginning of AIDS pandemic, there were many alarming casereports threatening the world medical community with increasing numbers of unknown causes and severe diseases presented in homosexual men, hemophiliacs and Haitian. TE was one of the most common opportunistic infections of this immunocompromised host. Huge efforts have been put on to combat with TE including budget, manpower and research on diagnostic methods, prophylaxis, treatment and prevention. The incidence of TE is now decreasing due to primary and secondary prophylaxis as well as immune restoration because of the HAART (Highly active antiretroviral therapy), but some old problems still

This chapter will focus on all aspects of TE including the etiologic organism, epidemiology, clinical manifestations, diagnostic methods, management and outcome as well as prophylaxis and prevention. Evidences from our research on *T. gondii* and literature review will be used as an input. With those frameworks, an extensive perspective on this

*T. gondii* was discovered since 1908 simultaneously by 2 groups of researchers. Firstly, Charles J. H. Nicolle (1866-1936) and Louis H. Manceaux (1865-1943) from the Pasteur Institute in Tunisia isolated a new parasite from the African rodent**,** *Ctenodactylus gundi*, and differentiated it from *Leishmania*. Secondly, the Italian researcher namely Alfonso Splendore (1871-1953) who worked at Sao Paulo, Brazil identified this protozoan from the liver of rabbit (Dubey et al., 1970; Sukthana, 2006). *T. gondii* was named a year later by Nicolle and Manceaux according to its bow-like shape (Toxoplasma is from a Greek word: toxos means bow or arc; plasma means life) and gondii may result from a misspelling of the scientific

**1. Introduction** 

abscess.

exist and new ones have surfaced.

fascinating disease will be forthcoming.

**2. Etiologic organism:** *Toxoplasma gondii*

Yaowalark Sukthana
