Polymerase chain reaction-based differential diagnosis of Ancylostoma duodenale and Necator americanus infections in humans in northern Ghana Johanna M. de Gruijter 1 , Lisette van Lieshout 1 , Robin B. Gasser 2 , Jaco J. Verweij 1 , Eric A. T. Brienen 1 , Juventus B. Ziem 3 , Lawrence Yelifari 4 and Anton M. Polderman 1 1 Department of Parasitology, Leiden University Medical Center, Leiden, The Netherlands 2 Department of Veterinary Science, The University of Melbourne, Melbourne, Australia 3 University for Development Studies (UDS), Tamale, Ghana 4 Ministry of Health, Upper East Region, Bolgatanga, Ghana Summary We evaluated a two-step semi-nested polymerase chain reaction (PCR)-based approach for the specific detection of Ancylostoma duodenale DNA in human faeces. The test was used to determine to what extent this species of hookworm is present in the regions of Bolgatanga and Garu of northern Ghana. Initially, the sensitivity and specificity of the PCR were tested using a range of well-defined control samples. Subsequently, a total of 378 human faecal DNA samples from Bolgatanga and Garu were subjected to the PCR. The results were compared with those obtained using a previously established PCR for the specific detection of Necator americanus DNA in human faeces. Infection with A. duodenale was recorded in 74 (19.6%) samples and N. americanus in 278 (73.5%), of which 64 (16.9%), represented co-infections with both species. While A. duodenale was predominantly detected in the samples from Bolgatanga, infections in Garu related almost exclusively to N. americanus. The results showed that the present PCR approach is a valuable complementary tool for the diagnosis of A. duodenale infection in humans in Ghana, having implications for epidemiological studies and for the monitoring of the success of control programmes in regions in Africa. keywords Ancylostoma duodenale, differential diagnosis, Necator americanus, PCR Introduction Hookworms are among the most common intestinal parasitic nematodes, infecting an estimated 1.3 billion people worldwide (Chan et al. 1994; Chan 1997). Infec- tion causes iron-deficiency anaemia which may result in mental retardation, heart problems and growth deficit, particularly in children (Hotez & Pritchard 1995; Albonico et al. 1999; Crompton 2000). Two principal species of hookworm infecting humans are Necator americanus and Ancylostoma duodenale (see Chan et al. 1994). In northern Ghana, where presently extensive efforts are being under- taken to control hookworm disease and oesophagos- tomiasis in humans, N. americanus has been considered as the predominant species of hookworm, but it is unclear whether A. duodenale co-exist. To monitor the efficacy of mass treatment with ant- helmintics (i.e. albendazole) in northern Ghana, accurate identification and/or differentiation of the species involved is essential. Currently diagnosis of hookworm infection in humans relies on the detection of the eggs in the faeces. However, this is hampered by the fact that the eggs of A. duodenale are morphologically indistinguishable from those of other strongylid nematodes, including N. ameri- canus and Oesophagostomum bifurcum. Hence copro- culture is required to allow eggs to develop and hatch to release infective third-stage larvae (L3s) for subsequent identification or differentiation. Although there are some clear morphological characteristics to distinguish the L3s of A. duodenale from those of N. americanus (Skrjabin et al. 1952; Matsubayashi et al. 1965; Yoshida 1966; Lichtenfels 1980), reliable identification is time-consuming and requires skilled personnel. DNA methods for the identification of parasites can overcome some of the limitations of traditional copro- scopic techniques. In particular, polymerase chain reaction (PCR)-based methods using appropriate DNA target regions have proven to be useful alternatives. For instance, the first and second internal-transcribed spacer (ITS-1 and ITS-2, respectively) of ribosomal DNA (rDNA) provide genetic markers for the specific identification of a range of parasitic nematodes, including A. duodenale and Tropical Medicine and International Health volume 10 no 6 pp 574–580 june 2005 574 ª 2005 Blackwell Publishing Ltd
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