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We report first-time definitive evidence of human cutaneous leishmaniasis (CL) resulting from L. major infections in Ghana. The evidence is in the form of PCR testing and DNA sequence analysis of skin sample biopsies from residents of the Ho District in the forested Volta Region of southeastern Ghana (Figure 1
). The moist semi-deciduous forest of this region of Ghana is atypical for CL. Although two sand fly species with known or suspected potential to transmit L. major were present in communities where human cases of CL originated, they were found in such small numbers that their role in the transmission of CL is uncertain.
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For many years, leishmaniasis in Africa and Western Asia has been grossly underestimated; it now appears that the disease is much more prevalent than previously suspected.1 In Ghana, CL was considered a parasitic disease risk that was limited to the arid sahel-savannah region in the north, but no infections or evidence of transmission had been actually described. Thousands of man-hours of mosquito surveillance have been conducted in northern Ghana2 for the purpose of quantifying man-biting malaria vectors, but there is no record or report of sand flies in these collections. However, since 1999, an increasing number of suspected CL cases have been reported from the southern Volta Region of Ghana in a moist forest ecosystem where such skin lesions had not been previously reported. The assumption of CL was based subjectively on local microscopy, which identified Leishmania amastigotes in skin lesion biopsies taken from a cluster of local patients. Between 1999 and 2002, the Ghana Health Service recorded 2,426 suspected cases of CL in the Ho, Hohoe, and Kpando Districts (2,348, 2, and 76 cases, respectively). In 2003, the number of suspected cases rose to 6,450 (6,185, 174, and 91 in the same respective districts) with 116 villages affected (Ghana Ministry of Health (MoH), Annual Report, 2003). A limited survey of towns in the Ho district during 2002 identified suspected CL lesions in 12.2–32.3% of local school children. In an effort to determine the cause and extent of the outbreak, the Ghana MoH initiated an epidemiologic study in Ho district with assistance from the U.S. Naval Medical Research Unit No. 3 and the Noguchi Memorial Institute of Medical Research.
Initial efforts of our study intended to verify the presence of Leishmania parasites by applying newly developed PCR methods to 5 previously collected, archived biopsy samples embedded in paraffin and if possible, to identify the species of parasite and its genetic relatedness to a variety of reference strains of Leishmania. Entomological surveillance based on sand flies collected from oiled paper trapping and CO2-baited miniature CDC light traps conducted from May 2004–February 2005 sought to identify candidate vectors and their population trends in the affected region. Subsequently, the Ghana Health Service requested PCR-based diagnoses on newly collected needle biopsies from patients seeking diagnosis and treatment of skin lesions at local health clinics and the district hospital in Ho. Diagnostic reagents were provided and technology was transferred from NAMRU-3 to the Noguchi Memorial Institute for Biomedical Research to establish methods for PCR diagnosis of Leishmaniasis in Ghana by Ghanaian investigators and healthcare workers.
Verbal informed consent was obtained from villagers with suspected leishmaniasis. The lesion site was cleaned with Betadine solution. Using a 3-cc syringe, the biopsy area was anesthetized with 1% lidocaine and draped in a sterile fashion. A ~0.5 x 0.25 cm section of the lesion was excised with a #15 scalpel from subcutaneous fat. The elliptical section was removed and fixed in 100% ethanol for storage and transport. Adequate homeostasis was observed and a sterile dressing was applied. DNA was later extracted from paraffin-embedded tissue by first making thin slices of the paraffin and subsequently removing as much of the paraffin with a scalpel as possible. Remaining sample was sliced into small pieces and no more than 25 mg of tissue was mixed with 1 mL of xylene and extracted by a standard method.3 After paraffin-removal, DNA was extracted from the tissue using the Qiagen DNA mini kit (Qiagen, Valencia, CA) as per the manufacturer’s recommendations. DNA was resuspended in 100 µL of AE buffer and stored at 4°C until use.
A foot lesion from a BALB/c mouse experimentally infected with Leishmania major IPAP/EG/89/SI-177 4 was used as a positive control for genetic manipulations. The DNA was extracted from 25 mg of the mouse tissue using the Qiagen DNA mini kit. To avoid cross-contamination, the BALB/c mouse tissue was extracted in a separate room from the paraffin slices, on separate occasions, and a clean (non-infected) control was simultaneously prepared.
Target DNA was amplified from extracted DNA using previously described primers and conditions.5–7 Primers R174 (5'-ggt tcc ttt cct gat tta cg) and R798 (5'-ggc cgg taa agg ccg aat ag)6 were used to amplify a 603-bp product and primers LITSR (5' ctg gat cat ttt ccg atg) and L5.8S (5' tga tac cac tta tcg cac tt)5 were used to amplify a 336-bp fragment from the DNA extracted from paraffin-embedded tissue and control DNA. Six µl of paraffin-extracted and 2 µL of control BALB/c DNA, respectively, were used as a template for each PCR reaction. A reaction containing no DNA was used as a negative control for each assay. Amplicons were visualized after electrophoresis through 1% agarose containing a final concentration of 10 µg ml–1 ethidium bromide. Images were captured using a Kodak EDAS 290 image capture system.
PCR products were processed using the Qiagen PCR cleanup kit (Qiagen, Valencia, CA) to remove excess oligonucleotide primer and to concentrate the product. PCR products were subsequently directly sequenced using Big Dye Terminator Technology (ABI Biosystems, Foster City, CA) and primers R174, R798, LITSR, and L5.8S at 3.2 pmol per reaction, using a 3100 Avant Genetic Analyzer (ABI Biosystems, Foster City, CA). DNA sequences were manipulated and analyzed using the software program pDRAW32 1.1.87 (http://www.acaclone.com) and aligned using the program Clustal X.8 Amended sequences were sent to the BLAST nucleotide data base (BLAST 2.2.12) for interrogation. DNA accession numbers are as follows: ITS-1 DNA sequences from L. major IPAP/EG/89/SI-177, DQ295824; MHOM/GH/04/ HO-004, DQ295825; ssu rDNA from L. major, IPAP/EG/89/ SI-177, DQ295827 and from MHOM/GH/04/HO-004, DQ295826. Leishmania major ITS1 sequences from 3 Iranian isolates (MHOM/Ir/02/PIICC1, MHOM/Ir/02/PIIDT1, and MHOM/Ir/00/PII22; accession numbers AY260965, AY283793, and AY573188), a Kenyan isolate (MTAT/KE/??/ NLB089A; accession number AJ300482), a Sudanese isolate (MHOM/SD/90/SUDAN3; accession number AJ300481), a Russian isolate (MHOM/SU/73/5ASKH; accession number AJ000310), and an isolate from Turkmenistan (Vin; accession number AJ272383) were used for comparison. For purposes of DNA comparison, ITS-1 sequences from additional Old World Leishmania species were also included. These include 3 isolates of L. donovani (MHOM/ET/00/Hussen, AJ634360; MHOM/KE/83/NLB189, AJ634374; and MHOM/SD/68/1S, AJ000293), 2 isolates each of L. archibaldi (MHOM/ET/72/ GEBRE1, AJ634367 and MHOM/SD/97/LEM3429; AJ634358), and L. infantum (MOHM/SD/62/3S, AJ634361 and MHOM/TN/80/IPT1, AJ000289). A single isolate of L. tropica (MHOM/TN/88/TAT3, AJ300485) was also used. Phylogenetic and molecular evolutionary analyses were conducted using MEGA version 3.1.9 A phylogenetic tree (Figure 2) was constructed with the unweighted pair group method with arithmetic mean analysis.10 Evolutionary distances were calculated with the method of Jukes and Cantor.11 Bootstrap analysis12 was performed with 100 replicates.
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Over 2,200 sand flies were collected during 10 consecutive months of surveillance in the Ho District (May 2004–February 2005). Sand fly identifications were made using the taxonomic keys of Abonnenc13 and Niang et al.14 Microscopic dissection and examination of 722 individual female sand flies captured in light traps revealed no Leishmania infections. Phlebotomus duboscqi, a proven vector of L. major in Senegal, Burkina Faso, Nigeria, and Kenya had been previously reported from Ghana15 and was considered our primary suspected vector of CL in the Ho District. Surprisingly, however, P. duboscqi was near least abundant of 17 different sand fly species (including 6 new geographic records for sand flies in Ghana) that we collected and comprised only 0.1% of the total catch (Table 1). One other suspect sand fly species, P. rodhaini, was captured for the first time in Ghana, but at similarly low numbers (0.3%), insufficient to yield a profile of seasonal abundance. In Sudan, P. rodhaini was found to harbor an unknown Leishmania species and demonstrated a clear predilection for blood feeding on rodents.16
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Received January 26, 2006. Accepted for publication March 31, 2006.
* Address correspondence to David F. Hoel, Vector Biology Research Program, U.S. Naval Medical Research Unit No. 3, PSC 452 Box 141, FPO AE 09835. E-mail: hoeld{at}namru3.med.navy.mil 
Authors’ addresses: David J. Fryauff, U.S. Naval Medical Research Center, 503 Robert Grant Avenue, Silver Spring, MD 20910. Hanafi A. Hanafi, John D. Klena, and David F. Hoel, U.S. Naval Medical Research Unit No. 3, PSC 452 Box 141, FPO AE 09835. Maxwell Appawu, Naiki Puplampu, Kwadwo Koram, Michael D. Wilson, and Daniel Boakye, Noguchi Memorial Institute for Medical Research, P.O. Box LG 581, Legon Accra, Ghana. William O. Rogers, U.S. Naval Medical Research Unit No. 2, Box 3 Unit 8132, FPO AP 96520-8132. Shirley Odoom and Margaret Kweku, Ghana Health Service, Private Mail Bag, Ministries, Accra, Ghana. Gregory Raczniak, Ghana Detachment, U.S. Naval Medical Research Unit No. 3, C/O Department of State, 2020 Accra Place, Washington, DC 20158-2020.
Reprint requests: David F. Hoel, Vector Biology Research Program, U.S. Naval Medical Research Unit No. 3, PSC 452 Box 141, FPO AE 09835. E-mail: hoeld{at}namru3.med.navy.mil
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