LETTERS

DOI: http://dx.doi.org/10.3201/eid1801.102001 Address for correspondence: Andreas G. 12,000 persons. It is hypothesized that Nerlich, Division of Paleopathology, Institute throughout the Kingdom, the plague References of Pathology, Academic Clinic München- killed ≈1,250,000 persons (1,2). Bogenhausen, Englschalkingerstr 77 D-81925, Since the 14th century, noble 1. Schönian G, Mauricio I, Gramiccia M, München, Germany; email: andreas.nerlich@ families of Barletta had been buried in Canavate C, Boelaert M, Dujardin JC. Leishmaniases in the Mediterranean in extern.lrz-muenchen.de tombs in underground tunnels of Sant’ the era of molecular epidemiology. Trends Andrea church. During restoration of Parasitol. 2008;24:135–42. doi:10.1016/j. the church in 2009, more underground pt.2007.12.006 tunnels containing many skeletons 2. Zink AR, Spigelman M, Schraut B, Green- blatt CL, Nerlich AG, Donoghue HD. were discovered. It has been Leishmaniasis in ancient Egypt and upper hypothesized that the church had also Nubia. Emerg Infect Dis. 2006;12:1616–7. been used as a cemetery during the 3. Costa MA, Matheson C, Iachetta L, Lla- plague epidemic. During an inspection gostera A, Appenzeller O. Ancient leish- Plague Epidemic maniasis in a highland desert of north- of the skeletons, 5 skulls of young ern Chile. PLoS ONE. 2009;4:e6983. in the Kingdom of persons were identifi ed and collected. doi:10.1371/journal.pone.0006983 Naples, 1656–1658 For a negative control, the skull of a 4. Degrave W, Fernandes O, Campbell D, person buried in a tomb before the Bozza M, Lopes U. Use of molecular probes and PCR for detection and typ- To the Editor: In 1656, an epidemic was also collected. ing of Leishmania—a mini-review. Mem epidemic of plague occurred in the The skulls were radiographed Inst Oswaldo Cruz. 1994;89:463–9. Kingdom of Naples, Italy. Earlier the to identify unerupted teeth (Figure), doi:10.1590/S0074-02761994000300032 disease had spread from Algiers to which were then aseptically extracted. 5. Rodgers MR, Popper SJ, Wirth DF. Am- plifi cation of kinetoplast DNA as a tool Spain; in June 1647, it appeared in After classifi cation, each tooth was in the detection and diagnosis of Leish- Valencia, and in the spring of 1648, cut along a sagittal line to uncover the mania. Exp Parasitol. 1990;71:267–75. it appeared in Aragon and several dental pulp, which was then hydrated doi:10.1016/0014-4894(90)90031-7 other Spanish areas of Valencia, in sterile phosphate-buffered saline 6. Ferroglio E, Centaro E, Mignone W, Trisciuoglio A. Evaluation of an ELISA Andalusia, and Catalonia. In 1652, (pH 7.2) for 48 h at 37°C. The DNA rapid device for the serological diag- plague had spread to Sardinia and was extracted by using DNAeasy nosis of Leishmania infantum infec- then to the cities and territories of Blood and Tissue Kits (QIAGEN, tion in dog as compared with immuno- Naples, Rome, and Genoa. Within Hilden, Germany) and by modifying fl uorescence assay and Western blot. Vet Parasitol. 2007;144:162–6. doi:10.1016/j. the Kingdom of Naples, plague fi rst the fi rst step, which was conducted vetpar.2006.09.017 reached the town of Naples in the overnight at 56°C with 600 μL of 7. Tran T-N-N, Aboudharam G, Raoult D, spring of 1656. Despite measures ATL buffer (QIAGEN) and 50 μL of Drancourt M. Beyond ancient micro- restricting population movement, by proteinase K. To verify the presence of bial DNA: non-nucleotidic biomolecules for paleomicrobiology. Biotechniques. the summer of 1656, the disease had inhibiting substance, the control DNA 2011;50:370–80 10.2144/000113689. reached several provinces in southern extracts were screened by using a PCR 8. Pieraccini G. La stirpe de’ Medici di Caf- Italy (1,2). for human mitochondrial DNA (3). aggiolo: saggio di ricerche sulla trasmis- Historical records indicate that To investigate the cause of the sione ereditaria dei caratteri biologici. Vol II. Firenze (Italy): Bruno Nardini; 1986. p. the epidemic in Barletta, in southern deaths, we adopted a PCR suicide 32–49, 64–70, 112–118, 120–124. Italy, developed after the arrival of method and searched for Yersinia 9. Fornaciari G, Vitello A, Giusiani S, Giuf- a ship from Naples. On May 26, pestis. We amplifi ed the pla gene for Y. fra V, Fornaciari A, Villari N. The Medici 1656, the ship Sant’ Andrea arrived pestis by using Sybr green PCR in real Project: fi rst anthropological and paleo- pathological results of the exploration of from Naples at the port of Barletta. time with a modifi cation of a previous the Medici tombs in Florence. Med. nei However, after sanitary inspection, protocol (4) coupled with conventional Secoli. 2007;19: 521–44. the ship was prevented from landing PCR according to Drancourt et al. (5). 10. Bern C, Maguire JH, Alvar J. Complexi- and obliged to depart, but this measure Conventional PCRs were adopted ties of assessing the disease burden attrib- utable to leishmaniasis. PLoS Negl Trop was not suffi cient to prevent the for Bacillus anthracis by targeting Dis. 2008;2:e313. doi:10.1371/journal. disease from entering the port. The the pag and capC genes (6) and for pntd.0000313 Barletta epidemic peaked in October, Salmonella enterica serovar Typhi by after which the number of cases targeting the narG gene (7). To prevent diminished; and on June 22, 1657, cross-contamination, we conducted Barletta was declared free of plague. all PCRs with a negative control and Of this city’s original population of in the absence of positive controls. 20,000, the disease killed 7,000– Melting curve analysis and agarose

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Bolsi for their technical support. We also thank the EQADeBa project for allowing us to use some of the detection methods developed within this project. The EQADeBa project was funded by the Executive Agency for Health and Consumers (no. 2007 204). This project was funded by the Ministry of Health of Italy, Ricerca Corrente, 2010.

Silvia Scasciamacchia, Luigina Serrecchia, Luigi Giangrossi, Giuliano Garofolo, Antonio Balestrucci, Gilberto Sammartino, and Antonio Fasanella Author affi liations: Istituto Zooprofi lattico Sperimentale of Puglia and Basilicata, Foggia, Italy (S. Scasciamacchia, L. Serrecchia, L. Giangrossi, G. Garofolo, Figure. Radiograph of skull found under Sant’ Andrea church in Barletta, Italy, in 2009, A. Fasanella); and University Federico showing unerupted teeth (circled) that were later extracted aseptically. II, Naples, Italy (A. Balestrucci, G. Sammartino)

DOI: http://dx.doi.org/10.3201/eid1801.110597 gel electrophoresis of PCR products skeletons and suspected fi nding in indicated suspected positive samples. the remaining 3 suggests that these References All amplicons relative to conventional persons died of plague during the and suicide PCR were submitted for 1656–1658 epidemic in southern Italy. 1. Fusco I. The causes of the epidemic. In: Franco Angeli, editor. Plague, demog- sequencing analysis confi rmation. Although it has not been universally raphy and taxation in the Kingdom of The negative DNA was reanalyzed to agreed upon, several studies have Naples of XVII century [in Italian]. Milan confi rm the results. confi rmed that the agent of 16th to 18th (Italy); Franco Angeli; 2007. p. 32. From the 26 dental pulp samples century “plague” epidemics in Europe 2. Fusco I. The plague of 1656–58 in the Kingdom of Naples: diffusion and mor- analyzed from the 5 skulls of young were caused by Y. pestis. Different tality [in Italian]. Popolazione e Storia. persons, 7 samples were positive methods have documented Y. pestis 2009;1:115. for the pla gene of Y. pestis by the as the agent in 10 Black Death burial 3. Matheson CD, Vernon KK, Lahti A, Sybr green real-time PCR, and 2 of sites scattered over 5 countries (8). In Fratpietro R, Spigelman M, Gibson S, et al. Molecular exploration of the fi rst- these were positive for this gene by northern Italy, the presence of Y. pestis century tomb of the shroud in Akeldama, conventional PCR. All were negative has been confi rmed in Venice (14th– Jerusalem. PLoS ONE. 2009;4:e8319. for B. anthracis and S. enterica ser. 17th centuries) (8), Genoa (Bastione doi:10.1371/journal.pone.0008319 Typhi. GenBank BLAST (www.ncbi. dell’Acquasola) (14th century) (9), 4. Skottman T, Piiparinen H, Hyytiainen H, Myllys V, Skurnik M, Nikkari S. Simul- nlm.nih.gov/blast/Blast.cgi) results and Parma (16th–17th centuries) (10). taneous real-time PCR detection of Ba- of the 2 sequenced amplicons found This study confi rms that the plague cillus anthracis, Francisella tularensis, a 100% match with the reference that infected the Kingdom of Naples, and Yersinia pestis. Eur J Clin Microbiol sequences (GenBank accession no. which spanned almost all of southern Infect Dis. 2007;26:207–11. doi:10.1007/ s10096-007-0262-z AL109969.1); query coverage was Italy, was also caused by Y. pestis. 5. Drancourt M, Aboudharam G, Signoli M, 100%. The sequences obtained were Fozzati L, Dutour O, Raoult D. Detection deposited in the GenBank sequence Acknowledgments of 400-year-old Yersinia pestis in human database under accession nos. We thank Giovan Battista dental pulp: an approach to the diagnosis of ancient septicemia. Proc Natl Acad Sci JN208020–1. Pichierri,Saverio Pellegrino, and Giuseppe U S A. 1998;95:12637–40. doi:10.1073/ In conclusion, the confi rmed Paolillo for access to the 5 skeletons and pnas.95.21.12637 fi nding of DNA of Y. pestis in 2 Angela Aceti, Nicola Nigro, and Igor

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6. Fasanella A, Losito S, Trotta T, Adone recommended the implementation of a by determining incidence of leprosy, R, Massa S, Ciuchini F, et al. Detec- leprosy surveillance system in leprosy- following the disease’s evolution, and tion of anthrax vaccine virulence factors by polymerase chain reaction. Vaccine. endemic countries with indicators for characterizing the patients affected. 2001;19:4214–8. doi:10.1016/S0264- screening, treatment, and monitoring The surveillance system was 410X(01)00159-1 of patients (4). From these indicators, based on the notifi cation of every 7. Papagrigorakis MJ, Yapijakis C, Syn- WHO establishes an annual offi cial case by health professionals likely to odinos PN, Baziotopoulou-Valavani E. DNA examination of ancient dental pulp report on the global status of the diagnose and treat subjects according incriminates typhoid fever as a probable disease. According to offi cial reports to the WHO case defi nition (8), cause of the Plague of Athens. Int J In- received from 141 countries, the i.e., clinicians, private or hospital fect Dis. 2006;10:206–14. doi:10.1016/j. global registered prevalence of leprosy dermatologists, and infectious ijid.2005.09.001 8. Tran TN, Signoli M, Fozzati L, Aboud- was 211,903 cases in 2010 (5). disease specialists. The notifi cation haram G, Raoult D, Drancourt M. High La Réunion is a French overseas was realized through a standardized throughput, multiplexed pathogen de- department located in the Indian questionnaire that included tection authenticates plague waves Ocean 700 km east of Madagascar. sociodemographic, clinical, and in medieval Venice, Italy. PLoS One 2011;6:e1673. doi:10.1371/journal.pone. La Réunion’s health care system is microbiological data. Concurrently, 0016735 similar to that of continental France. the pathology laboratories were 9. Cerutti N, Marin A, Rabino Massa E. Although new cases of leprosy have consulted to detect any nondeclared Plague in ancient remains: an immuno- been punctually reported by health cases and to improve the completeness logical approach. In: Signoli M, Chevé D, Adalian P, Boetsch G, Dutour O, editors. professionals during the past few years, of data. Plague: epidemics and societies. Firenze which suggests that the disease is still This surveillance was (Italy): Firenze University Press. 2007; present, the situation in La Réunion retrospective for 2005–2010, then 238–241. is poorly documented because of the prospective for 2011. In total, 17 10. Haensch S, Bianucci R, Signoli M, Rajeri- son M, Schultz M, et al., Distinct clones lack of a specifi c surveillance system. patients responding to the case of Yersinia pestis caused the Black Death. Thus, the goal of eliminating leprosy defi nition of leprosy and given a PLoS Pathog. 2010;6:e1001134. as a public health problem (i.e., diagnosis during 2005–2010 were prevalence <1/10,000) (6) cannot be reported for an average population Address for correspondence: Antonio Fasanella, assessed because the goal requires a of 804,025 inhabitants in La Réunion Istituto Zooprofi lattico Sperimentale of Puglia good knowledge of the epidemiologic (data from the National Institute of and Basilicata, Via Manfredonia 20, Foggia status of the disease. Furthermore, Statistics and Economics Studies). 71100, Italy; email: [email protected] the risk of leprosy recrudescence The mean annual incidence during linked to a relapse of patients with this period was 3.4 cases/106 autochthonous cases or patients with inhabitants. The male:female sex leprosy migrating from neighboring ratio was 2.2, and the median age was leprosy-endemic countries, such as 54 years (range 8–77 years). More Madagascar, Comoros, and Mayotte than half the patients were born in La (5), is present. In 2009, a total of 1,572 Réunion (n = 9); the other patients’ Leprosy, Still new cases of leprosy were detected in birthplaces were Comoros Islands (n Madagascar, 319 in Comoros, and 37 = 4), Mayotte (n = 3), and Madagascar Present in in Mayotte (5,7). If one considers the (n = 1). Among the patients born La Réunion geographic proximity and the many in La Réunion, 6 had never left the tourist exchanges between La Réunion island, 3 had traveled but had always To the Editor: During recent and those neighboring islands, the resided in La Réunion, and 6 patents decades, a considerable and consistent risk of importation, although low, is resided in the same area of a city in decrease in worldwide incidence of constant. the southwestern part of the island. leprosy has been observed, mainly In that context, Cire Indian Ocean An active search for other because of the recommendation to (the Regional Offi ce of French Institute cases in this area was performed by introduce multidrug therapy in 1981 for Public Health Surveillance), in contacting all the health professionals (1) and the implementation of free collaboration with health professionals likely to diagnose leprosies; 1 therapy in 1994 (2) by the World involved in diagnosis and treatment of clinician reported a suspected case Health Organization (WHO). The the disease, has implemented a specifi c among his patients. That patient is prevalence rate of the disease has surveillance system for leprosy in currently being screened. Of the been reduced globally by >90% since La Réunion. The objectives are to patients overall, 15 were screened 1985 (3). Since 2000, WHO has guide potential preventive measures by skin biopsy or smear from the ear.

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