Endemic Angiostrongyliasis, Rio de Janeiro, Brazil

To the Editor: The nematode Angiostrongylus cantonensis (rat lung worm), a zoonotic parasite that can accidentally infect humans and cause eosinophilic meningoencephalitis, has the Norway rat (Rattus norvegicus) as one of its most frequent definitive vertebrate hosts (1). Adult worms live in the pulmonary arteries of the definitive hosts, which excrete first-stage larvae in their feces. Intermediate hosts, such as snails and slugs, are infected by first-stage larvae, which reach the infective third stage after 2 molts. Third-stage larvae are then ingested by rats as they feed on the intermediate hosts, thus closing the life cycle. Humans become infected by eating raw or undercooked snails and slugs and through paratemic hosts and vegetables contaminated with infected snail mucus (2). 
 
In Brazil, the first 3 documented cases of eosinophilic meningoencephalitis occurred in 2007 in 2 cities in the southeastern state of Espirito Santo (3). In 2009, a new case was reported in Pernambuco in the northeast region (4). Only intermediate hosts have been found naturally infected with rat lung worm in Brazil. Infected terrestrial and freshwater snails of the species Achatina fulica, Sarasinula marginata, Subulina octona, and Bradybaena similaris in Espirito Santo; A. fulica and Pomacea lineata in Pernambuco; and A. fulica in Rio de Janeiro and Santa Catarina have been reported (3,5,6). Thus, because of the recent cases of eosinophilic meningoencephalitis in Brazil and the occurrence of naturally infected A. fulica snails in Rio de Janeiro, we investigated the existence of potential natural reservoirs for the parasite in Sao Goncalo. 
 
Sao Goncalo (22°48′26.7′′S, 43°00′49.1′′W) is a densely populated city (≈1 million inhabitants) with a tropical Atlantic climate (14°C–35°C) that is part of the metropolitan region of Rio de Janeiro. Two collections were made in March and June 2010. Forty live traps (20 Tomahawk [Tomahawk Live Trap Company, Tomahawk, WI, USA] and 20 Sherman [H.B. Sherman Traps Inc., Tallahassee, FL, USA] traps) were placed along two 30-m transects for 4 consecutive nights (Brazilian Institute of Environment and Renewable Natural Resources license no. 2227–1/2010) in an urban area where A. fulica snails had been collected in high numbers. Twenty-seven Norway rats (16 males) were captured. We collected 265 adult lung worms from the pulmonary arteries of the captured animals, fixed the worms in 70% ethanol, and taxonomically identified them as A. cantonensis on the basis of the large size of the spicules and the patterns of the bursal rays (7). Voucher specimens have been deposited in the Helminthological Collection of the Oswaldo Cruz Institute (no. 35712). Nineteen (74%) rats were infected; mean intensity and mean abundance were 13.52 ± 2.36 and 9.81 ± 1.96, respectively. 
 
To confirm the morphologic identification of the Angiostrongylus specimens obtained, a DNA bar coding approach was used. DNA was extracted from 3 ethanol-preserved adult worms previously recovered from the pulmonary arteries of a naturally infected Norway rat, PCR-amplified, sequenced for a partial region of the COI gene (8), and subsequently compared with available GenBank Angiostrongylus spp. sequences. The three 360-bp COI sequences obtained (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"HQ440217","term_id":"330414605","term_text":"HQ440217"}}HQ440217) were Clustal-aligned (www.clustal.org) with homologous COI fragments of A. cantonensis (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GQ398121","term_id":"254941204","term_text":"GQ398121"}}GQ398121), A. vasorum (GenBank accession nos. {"type":"entrez-nucleotide","attrs":{"text":"EU493162","term_id":"187473267","term_text":"EU493162"}}EU493162, {"type":"entrez-nucleotide","attrs":{"text":"EU493163","term_id":"187473269","term_text":"EU493163"}}EU493163, {"type":"entrez-nucleotide","attrs":{"text":"EU493166","term_id":"187473275","term_text":"EU493166"}}EU493166, {"type":"entrez-nucleotide","attrs":{"text":"EU493167","term_id":"187473277","term_text":"EU493167"}}EU493167), and A. costaricensis (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"GQ398122","term_id":"254941217","term_text":"GQ398122"}}GQ398122) and subjected to phylogenetic analysis. Ancylostoma tubaeforme (GenBank accession no. {"type":"entrez-nucleotide","attrs":{"text":"AJ407940","term_id":"18077725","term_text":"AJ407940"}}AJ407940) was used as the outgroup. Haplotypes for A. vasorum isolates from Brazil (A. vasorum 5421, 5641, and 5642) were reconstructed from published information (9) and included in the alignment. We used MEGA4 (www.megasoftware.net) to construct a neighbor-joining phylogenetic tree based on Kimura 2-parameter (K2-p) distances (Figure). The 3 A. cantonensis specimens from Sao Goncalo, Rio de Janeiro, yielded a single haplotype, which formed a clade with the A. cantonensis haplotype from the People’s Republic of China with low genetic distance (K2-p 0.038) and high bootstrap support (98), thus confirming the morphologic identification. Comparisons with the other 2 Angiostrongylus species yielded higher genetic distance values (K2-p 0.120, with A. vasorum, and 0.149, with A. costaricensis). 
 
 
 
Figure 
 
Neighbor-joining phylogenetic tree based on Kimura 2-parameter (K2-p) distances that includes all Angiostrongylus COI sequences in GenBank and the sequences obtained from 3 Angiostrongylus specimens recovered from the pulmonary arteries of a naturally ... 
 
 
 
These results indicate that A. cantonensis lung worm infection is enzootic among the exotic Norway rat population in the region studied. The natural infection rate of 74% is the second highest reported among 14 severely A. cantonensis infection–endemic regions (2). These findings, together with the observation of dense populations of A. fulica snails in urban areas of the country (10), call attention to the risk for disease transmission to humans, given that Norway rats also are likely to be present in these areas. 
 
Local residents should be informed about disease transmission and prevention, and physicians should consider A. cantonensis lung worm infection in the differential diagnosis when appropriate. Although public health authorities should consider implementation of surveillance and control strategies to reduce the populations of snail and rat hosts, a better understanding is needed of the epidemiologic significance of these findings, which can be attained through studies to identify human cases of eosinophilic meningitis in the region.


Whether
C. hongkongensis belongs to the intestinal fl ora, as do Bifi dobacterium, Eggerthella, Eubacterium, and Lactobacillus spp., remains undetermined. Codony et al. recently investigated by real-time PCR the presence of Catabacteriaceae in 29 water samples in the vicinity of Barcelona, Spain. Four samples were positive, demonstrating presence of this organism in the European environment and its probable enteric origin (4).
Because our patient sought treatment with severe infection associated with isolation of other pathogenic bacteria, whether blood infection by C. hongkongensis may be responsible for such a fatal outcome is unknown. Nevertheless, we can exclude sample contamination by this anaerobic bacteria for the 2 following reasons. First, anaerobic contaminants are rare in blood cultures and generally involve Propionibacterium acnes. Furthermore, the rapid growth of the present isolate in blood cultures within 3 days suggested a relatively high bacterial load in the blood sample.
Our report confi rms that C. hongkongensis can be found in blood culture associated with gastrointestinal disease and may refl ect intestinal perforation. Identifi cation may be diffi cult. To confi rm the morphologic identifi cation of the Angiostrongylus specimens obtained, a DNA bar coding approach was used. DNA was extracted from 3 ethanol-preserved adult worms previously recovered from the pulmonary arteries of a naturally infected Norway rat, PCR-amplifi ed, sequenced for a partial region of the COI gene (8), and subsequently compared with available GenBank Angiostrongylus spp. sequences. The three 360-bp COI sequences obtained (GenBank accession no. HQ440217) were Clustal-aligned (www.clustal.org) with homologous COI fragments of A. cantonensis (GenBank accession no. GQ398121), A. vasorum (GenBank accession nos. EU493162, EU493163, EU493166, EU493167), and A. costaricensis (GenBank accession no. GQ398122) and subjected to phylogenetic analysis. Ancylostoma tubaeforme (GenBank accession no. AJ407940) was used as the outgroup. Haplotypes for A. vasorum isolates from Brazil (A. vasorum 5421, 5641, and 5642) were reconstructed from published information (9) and included in the alignment. We used MEGA4 (www.megasoftware. net) to construct a neighbor-joining phylogenetic tree based on Kimura 2-parameter (K2-p) distances (Figure). The 3 A. cantonensis specimens from São Gonçalo, Rio de Janeiro, yielded a single haplotype, which formed a clade with the A. cantonensis haplotype from the People's Republic of China with low genetic distance (K2-p 0.038) and high bootstrap support (98), thus confi rming the morphologic identifi cation. Comparisons with the other 2 Angiostrongylus species yielded higher genetic distance values (K2-p 0.120, with A. vasorum, and 0.149, with A. costaricensis).
These results indicate that A. cantonensis lung worm infection is enzootic among the exotic Norway rat population in the region studied. The natural infection rate of 74% is the second highest reported among 14 severely A. cantonensis infectionendemic regions (2). These fi ndings, together with the observation of dense populations of A. fulica snails in urban areas of the country (10), call attention to the risk for disease transmission to humans, given that Norway rats also are likely to be present in these areas.
Local residents should be informed about disease transmission and prevention, and physicians should consider A. cantonensis lung worm infection in the differential diagnosis when appropriate. Although public health authorities should consider implementation of surveillance and control strategies to reduce the populations of snail and rat hosts, a better understanding is needed of the epidemiologic signifi cance of these fi ndings, which can be attained through studies to identify human cases of eosinophilic meningitis in the region.

Acknowledgments
We thank the anonymous reviewers whose comments and suggestions helped improve an earlier version of the manuscript. During the 1980s, urban transmission of kala-azar became a major problem in Brazil. More than 3,000 cases are reported annually, and the disease has spread from northeastern Brazil westward to the Amazon region, as well as to the industrialized southeast. Several as yet unproven explanations for the urbanization of kala-azar in Brazil have been proposed (2), but whatever the reason, it is associated with proliferation of Lutzomyia longipalpis sand fl ies, which, in turn, are strongly associated with human environments. The vector can easily spread by entering buses or trains looking for food at night or for hiding places at dawn. Invasion of new areas by sand fl ies through transportation of ornamental plants has been observed (R. Brazil, pers. comm.), possibly by insect eggs or larvae being carried in organic matter.
Kala-azar has now reached the temperate Brazilian south and Argentina. This spread of the disease warns us of the danger of introduction in other temperate areas. Europe is particularly vulnerable because of the existing natural transmission of L. infantum. This risk is increased by recently created daily direct fl ights to Lisbon from Fortaleza, Natal, Brasília, and Belo Horizonte (Figure), Brazilian cities where epidemics of the disease have occurred. Lisbon is suitable to canine infection, and >10% of dogs may be infected (3). The climate is a barrier for the introduction of many vectors outside their normal range, such as Anopheles gambiae mosquitoes in temperate zones (4,5), but the threshold of change for L. longipalpis sand fl ies is minimal. The Mediterranean area is as dry as northeastern Brazil, where the disease is now highly endemic. Furthermore, the annual average temperature and