157 Am. J. Trop. Med. Hyg., 66(2), 2002, pp. 157–162 Copyright  2002 by The American Society of Tropical Medicine and Hygiene DETECTION BY POLYMERASE CHAIN REACTION OF SCHISTOSOMA MANSONI DNA IN HUMAN SERUM AND FECES LUI ´ S ANDRE ´ PONTES, EMMANUEL DIAS-NETO, AND ANA RABELLO Laboratory of Clinical Research, Centro de Pesquisas Rene ´ Rachou, Fundac ¸a ˜o Oswaldo Cruz—FIOCRUZ, Lima, Belo Horizonte, MG, Brazil; Ludwig Institute for Cancer Research, R. Prof. Antonio Prudente, Sa ˜o Paulo, SP, Brazil Abstract. A novel method for the detection of Schistosoma mansoni in human samples that is based on the amplification of a highly repeated DNA sequence has been developed. By use of simple DNA extraction techniques and a rapid 2-step polymerase chain reaction (PCR), it was possible to amplify S. mansoni DNA in human fecal and serum samples. The high sensitivity of the approach enabled the detection of the parasite DNA in fecal samples containing as few as 2.4 eggs per gram of feces, which makes it 10 times more sensitive than the Kato-Katz exam- ination. A detection limit of 1 fg of Schistosoma sp. DNA was determined when pure DNA was used as PCR template. The amplification reaction showed to be specific giving no cross-reaction with DNA from other helminths. The PCR assay developed in this study may constitute a valuable alternative for the diagnosis of the Schistosoma sp. infection. INTRODUCTION Schistosomiasis is a worldwide public health problem af- fecting  200 million people in the third world. Schistosoma mansoni infection is endemic in 54 countries and territories of South America, Caribbean, Africa, and the eastern Med- iterranean. 1 The study and development of new diagnostic techniques for schistosomiasis are still necessary in view of the difficulties to evaluate infection patterns accurately and to control the disease. Definitive diagnosis of S. mansoni infection requires the demonstration of eggs in feces. The Kato-Katz technique 2 is currently the method of choice for parasitological diagnosis of Schistosomiasis mansoni, as it is relatively inexpensive and simple. 1 However, it is observed that the sensitivity of parasitological methods diminishes when prevalence and in- tensity of infection are low, making these methods less ap- propriate for low-endemic areas and in posttreatment situa- tions. In addition, parasitological methods are not sufficient for diagnosing recent infections in which worms have not yet started to produce eggs (the prepatent period). To address these shortcomings, antibody detection meth- ods have been evaluated as adjuncts to fecal examinations. Comparative studies of parasitological and serological meth- ods confirmed higher sensitivity of the latter. 3 The existence of cross-reactivity with other helminthic infections, however, and its low specificity after treatment, due to the slow re- duction of specific antibody levels, constitute great disad- vantages of the imunodiagnostic methods. One possible solution to this problem could be the search for circulating antigens rather than antibodies. Several cir- culating antigens assays have been described in different lab- oratories. 4–6 The high specificity is the main advantage that the detection of circulating antigens offers as compared with the antibodies determination and the disadvantages are low sensitivity in light infections and dependence on the produc- tion of monoclonal antibodies. 7,8 Polymerase chain reaction (PCR) has shown its usefulness in the clinical approach of a wide variety of pathogenic in- fections, such as human immunodeficiency virus, 9,10 Legion- ella pneumophila, 11 Plasmodium falciparum, 12,13 and Try- panosoma cruzi. 14,15 In the study of Schistosoma sp., it has also been successfully used for sex determination of the cer- cariae, 16 for the cloning and sequencing of specific genes, 17,18 in the determination of genetic variability and population structure of Schistosoma sp. strains and species, 10,19 and in the development and application of new techniques to gen- erate expressed sequence tags. 10 Hamburguer and others 20 developed a PCR protocol that was based on the amplifica- tion of a highly repetitive DNA sequence of the parasite for monitoring S. mansoni infestation in water. 21 In the present article, we describe, for the first time, the usefulness of the PCR for detecting S. mansoni DNA in hu- man fecal and serum samples. METHODS Sample preparation. Schistosoma mansoni eggs were ob- tained from livers of mice (Swiss albinos) 45 days after in- fection with 100 cercariae and stored at -20°C in 0.9% sa- line until use. 22 Ten microliters of egg-saline solution con- taining  2,000 eggs were diluted in 90 L of water and vortexed for 5 min in order to break the eggs and release the miracidia, the larval form that infects the snail host. The entire volume was then used for DNA extraction. The DNA from other parasite species. The DNA from 4 related helminthic parasites (Ascaris lumbricoides, Ancy- lostoma duodenale, Taenia solium, and Trichiuris trichiuria) were provided by the Laborato ´rio de Esquistosomose— CPqRR, Belo Horizonte, Brazil. Artificial mixtures of eggs and feces. Four positive fecal samples were artificially prepared by the following proce- dure: 0.1 g of S. mansoni positive feces containing 216 eggs per gram of feces (determined by Kato-Katz stool exami- nation) were mixed to 0.9 g of negative feces, resulting in a preparation of  21.6 eggs per gram of feces. Tenfold dilutions were subsequently performed, producing 2 more samples with estimated concentrations of  2.16 and 0.216 eggs/g, respectively. The samples were then analyzed by the Kato-Katz method and stored at -70°C until use. Approxi- mately 100 mg of feces were diluted in 400 L of water and vortexed for 5 min to break the S. mansoni eggs and release the miracidia. Insoluble material and large debris were sep- arated by decantation, leaving the samples for 2 min at room temperature. One hundred microliters of the supernatant was used for DNA extraction. Patient samples. Patient samples were obtained from the endemic area of Comercinho, Minas Gerais, Brazil, or from