Leishmania (Viannia) guyanensis Infection, Austria

To the Editor: Infection with Leishmania spp. was diagnosed in an asymptomatic soldier during an explorative national cross-sectional serologic screening of soldiers volunteering for United Nations missions at the Military Hospital Vienna in 2009. Diagnosis was made by using a commercial ELISA kit (Ridascreen Leishmania; R-Biopharm, Darmstadt, Germany). One year later, the soldier was reassessed for persisting antibodies by using the same ELISA and for Leishmania DNA in a blood sample stored in EDTA by using the Leishmania OligoC-Test (Coris BioConcept, Gembloux, Belgium). (The study was approved by the Research Ethics Committee of the Austrian Armed Forces and written informed consent was obtained from the person investigated.) Because the results of both tests were positive, an additional PCR was performed for identification below genus level with the LITSR/L5.8S primer pair (1). To confirm the PCR results, we sequenced the amplicon in both directions in 2 independent setups and compared the obtained 299-bp sequence with published sequences from GenBank by performing a multiple sequence alignment. Our sequence (strain EN10) showed 100% (299/299 bp) identity with several strains from the Leishmania (Viannia) guyanensis complex, including the L. guyanensis strain MHOM/SR/87/TRUUS4 and the L. panamensis strains {"type":"entrez-nucleotide","attrs":{"text":"FJ948438","term_id":"227976404","term_text":"FJ948438"}}FJ948438, {"type":"entrez-nucleotide","attrs":{"text":"FJ948439","term_id":"227976405","term_text":"FJ948439"}}FJ948439, and {"type":"entrez-nucleotide","attrs":{"text":"FJ948446","term_id":"227976412","term_text":"FJ948446"}}FJ948446. Sequence homology to representatives of the L. (V.) braziliensis complex was ≈93%; to representatives of the L. (Leishmania) mexicana complex, 61%–68%; and to the L. (L.) donovani complex, 70%–71%. The L. (V.) guyanensis complex traditionally includes the species L. guyanensis, L. panamensis, and L. shawi, but L. panamensis seems to be a subspecies or even a synonym of L. guyanensis (2). We thus classified our strain as L. guyanensis. Sequence data were deposited at GenBank (accession no. {"type":"entrez-nucleotide","attrs":{"text":"JN671917","term_id":"399519285","term_text":"JN671917"}}JN671917). 
 
L. guyanesis/panamensis is found in 9 countries in Central and South America (3). It is a common cause of zoonotic cutaneous leishmaniasis in humans. The sloths Choloepus didactylus (L. guyanensis) and C. hoffmanni (L. panamensis) are believed to be the principal reservoir hosts and the sandfly species Lutzomyia umbratilis (L. guyanensis) and Lu. trapidoi (L. panamensis) to be the principal vectors (3). Also, dogs can act as reservoirs for the L. (V.) guyanensis complex (4). 
 
The infected soldier had never been to Central or South America and had no history of blood transfusions. His lifetime travel history included Italy, Spain, Greece, Germany, Croatia, New York City, and military assignments in Kosovo. Thus, how and where the infection had been acquired remain open for discussion. 
 
Although sandflies are not as robust as Anopheles spp., for example, the most plausible scenario is that either an L. guyanensis–infected sandfly or a noninfected but transmissive sandfly from a disease-endemic area was transported in a ship or airplane (comparable to the well-known “airport malaria” situation) to an area where the patient had traveled. In recent years, Lu. vexator has become widespread and abundant in upstate New York (5). Although, this is not a known vector for L. guyanensis, its spread in New York State shows that Lutzomyia spp. can rapidly adapt to new and distant areas. Of the areas where the infected person had traveled, at least in New York City and Spain, regular introduction of L. guyanensis by immigrants, travelers, or dogs from Central and South America is very likely. Moreover, Leishmania parasites are known to remain viable for a lengthy period in infected humans and animals and even in those that have received treatment. 
 
However, alternative scenarios with other sandfly species, possibly even those found in Europe, acting as vectors cannot be totally excluded. Approximately 25 sandfly species are found in Europe, of which at least 6 are vectors for Leishmania spp (6). Whether L. guyanensis can be transmitted by Phlebotomus sandfly species is unknown. When L. infantum, originally transmitted by Phlebotomus spp., was introduced from Europe to Central and South America in the post-Columbian era, it readily adapted to several vectors of the genus Lutzomyia (3). Adoption of new reservoir hosts and new vector species has also been observed in other species (7). Members of the Leishmania subgenus develop in the midgut, and representatives of the Viannia subgenus develop in the hindgut and the midgut. Nevertheless, several Lutzomyia species can transmit both, representatives of the Viannia and Leishmania subgenera. In general, most sandflies appear to be vector competent for >1 Leishmania spp. The New World species Lu. longipalpis and the Old World species Ph. argentipes, Ph. arabicus, Ph. halepensis, and Ph. perniciosus enabled the maturation of almost all Leishmania species tested under experimental conditions (8). The presence of sandflies in Austria was reported very recently (9), but the vector competence of the species found (P. mascittii) has still not been elucidated. Moreover, this finding likely reflects an increased population density rather than an introduction of a previously nonendemic species. 
 
Nonvector transmission is also a possibility. The infected person did not remember ever having received blood products; however, transmission is generally possible by all forms of blood contact, including through needle sharing among persons who use injection drugs and through sexual intercourse (10).


Rickettsia raoultii-like Bacteria in
Dermacentor spp. Ticks, Tibet, China To the Editor: Rickettsia raoultii is an obligate intracellular gramnegative bacterium belonging to the spotted fever group (SFG) of the genus Rickettsia. Genotypes RpA4, DnS14, and DnS28, originally isolated from ticks from Russia in 1999 (1), were designated as Rickettsia raoultii sp. nov. on the basis of phylogenetic analysis (2). R. raoultii has been found mainly in Dermacentor spp. ticks in several countries in Europe (3). It was detected in a Dermacentor marginatus tick from the scalp of a patient with tick-borne lymphadenitis in France (2), which suggests that it might be a zoonotic pathogen. We determined the prevalence of R. raoultii-like bacteria in Dermacentor spp. in highland regions in Tibet.
Ticks from sheep (Ovis aries) near Namuco Lake (a popular tourist destination 4,718 m above sea level) were collected and identifi ed morphologically as D. everestianus and D. niveus ticks (4). Genomic DNA was extracted from individual specimens by using the QIAamp DNA Mini Kit (QIAGEN, Hilden, Germany). All DNA samples were amplifi ed by using PCRs specifi c for the citrate synthase (gltA, 770 bp) gene (5) and the outer membrane protein A (ompA, 629 bp) gene (6). Some samples were amplifi ed by using a PCR specifi c for the ompB (2,479 bp) gene (7).
Of 874 tick specimens, 86 were D. everestianus ticks (13 male and 73 female), and 788 were D. niveus ticks (133 male and 655 female). Samples positive for gltA and ompA were considered SFG rickettsial species. Using this criterion, we found that 739 tick specimens (84.6%) were positive for Rickettsia spp. Of 86 D. everestianus ticks, 85 (98.8%) were positive for Rickettsia spp. and of 788 D. niveus ticks, 654 (83.0%) were positive. Infection rates for male and female D. niveus ticks were 87.9% and 82.1%, respectively. We found an overall prevalence of 84.6% for R. raoultii-like bacteria in Dermacentor spp. in the highland regions in Tibet.
Nucleotide sequence identities ranged from 99.2% to 100% (except for isolate WYG55, which had an identity of 98.6%) for the ompA gene and from 99.2% to 99.9% (except for isolate XG86, which had an identity of 98.5%) for the ompB gene. These results indicated that homology levels of most isolates were within species thresholds (ompA ≈98.8% and ompB ≈99.2%) (9). Isolate WYG55 showed the lowest identity (98.2%) among gltA gene sequences and the lowest identity (98.6%) among ompA gene sequences. Isolate XG86 showed lowest identity (98.5%) among ompB gene sequences. These results suggest that other Rickettsia spp. were among the investigated samples.
A BLASTn search (www. ncbi.nlm.nih.gov/) for the obtained sequences was conducted. The best matches (highest identities) detected were with sequences of R. raoultii. However, comparison of our sequences with corresponding sequences of R. raoultii in GenBank showed identity ranging from 98.0% to 99.0% for ompA and from 98.1% to 99.0% for ompB, which did not meet the threshold (9) for R. raoultii. We compared the new sequences with corresponding reference sequences of universally recognized SFG group Rickettsia spp. in Genbank and constructed 2 phylogenetic trees (Figure). The new sequences were placed into separate branches, which were closely related to R. raoultii branches.
Prevalence of R. slovaca and R. raoultii was 6.5% and 4.5% in D. silvarum ticks in Xinjiang Uygur Autonomous Region of China (10). In contrast, we found that the overall prevalence of R. raoultii-like bacteria might be ≤84.6% in D. everestianus and D. niveus ticks in Dangxiong County in Tibet.
Our fi ndings suggest that D. everestianus and D. niveus ticks are potential vectors of R. raoultii-like bacteria and indicate that spread of R. raoultii-like bacteria encompasses a large area in China. In the study sites, yak and Tibetan sheep are the major domestic animals, and rodents are the major wild animals. Rodents are also the major hosts of Dermacentor spp. ticks, which can transmit R. raoultii transstadially and transovarially (2). Animals bitten by infected ticks can acquire the pathogen and serve as natural reservoirs.
On the basis of phylogenetic analysis, we found that the Rickettsia spp. in ticks investigated represents a novel species, which can be designated Candidatus Rickettsia tibetani. However, additional phylogenetic studies are needed to obtain more information on the molecular biology of these bacteria. Figure. Unrooted phylogenetic trees inferred from comparison of A) outer membrane protein A (ompA) and B) ompB gene sequences of rickettsial species by using the neighbor-joining method. Sequences in boldface were obtained during this study. Numbers at nodes are the proportion of 100 bootstrap resamplings that support the topology shown.