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Journal of Clinical Microbiology, October 2002, p. 3572-3576, Vol. 40, No. 10
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.10.3572-3576.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Evaluation of PCR for Diagnosis of American Cutaneous Leishmaniasis in an Area of Endemicity in Northeastern Brazil

Eduardo Henrique Gomes Rodrigues,¹ Maria Edileuza Felinto de Brito,¹ Mitzi Guedes Mendonça,² Roberto P. Werkhäuser,¹ Eridan M. Coutinho,¹ Wayner V. Souza,¹ Maria de Fátima P. Militão de Albuquerque,¹ Márcio L. Jardim,² and Frederico G. C. Abath^1*

Centro de Pesquisas Aggeu Magalhães, Fundação Oswaldo Cruz,¹ Departmento de Dermatologia, Universidade Federal de Pernambuco, Recife, Brazil²

Received 11 February 2002/ Returned for modification 15 May 2002/ Accepted 30 June 2002

Top Abstract Introduction Materials and Methods Results Discussion References

 
PCR-based approaches targeting kinetoplast DNA were evaluated for the diagnosis of American cutaneous leishmaniasis (ACL) in regions of endemicity in northeastern Brazil. A total of 119 cutaneous biopsy specimens from patients with ACL and nonleishmaniasis cutaneous lesions were studied. Two PCR-based systems were used; one was specific for the subgenus Viannia, and the other was specific for the genus Leishmania. The PCR specific for the subgenus Viannia had a sensitivity of 95.4%, whereas the genus-specific PCR detected the target DNA in 88.2% of the samples tested. The specificities of the assays, determined with samples from a group with nonleishmaniasis cutaneous lesions, was 100%. The results of the conventional tests indicate that the sensitivities of the PCR-based methods were significantly higher than those of smear examination, histological staining, and isolation by culture (P < 0.05). Antibodies specific for Leishmania braziliensis were detected by indirect immunofluorescence in 82.9% of the patients tested. Parasites were isolated from 40 of 86 patients (46.5%). Sixty-seven percent of dermal scrapings and 66.2% of stained tissue sections were positive by microscopy. Amplified products from the subgenus-specific PCR hybridized with the Leishmania panamensis minicircle, confirming infection consistent with L. braziliensis. The evidence available at present incriminates L. braziliensis as the only causative agent of ACL in the state of Pernambuco in Brazil.

Top Abstract Introduction Materials and Methods Results Discussion References

 
American cutaneous leishmaniasis (ACL) is an anthropozoonotic disease caused by protozoans of the genus Leishmania, which infect the vertebrate host after a bite by infected phlebotomus insects of the genus Lutzomya. Human infections may be inapparent or display a clinical spectrum ranging from localized, sometimes self-healing cutaneous lesions to severe mutilating mucocutaneous lesions to diffuse cutaneous leishmaniasis. In Brazil, ACL is widely distributed from the south of the Amazon Basin to the southeast of Brazil (Leishmaniasis Section, Special Programme for Research and Training in Tropical Diseases, World Health Organization, 2001 [http://www.who.int./tdr./index.html]), with most cases being caused by Leishmania (Viannia) braziliensis (17). However, Leishmania amazonensis is also important as a causative agent of ACL, having been reported in the northeast, southeast, and west-central parts of Brazil (3, 24, 29, 36). According to the National Health Foundation, the numbers of cases of ACL in 2000 were 27,502, 10,868, and 943 in Brazil, northeastern Brazil, and Pernambuco State, respectively (Fundação Nacional de Saúde, 2001 [http://www.funasa.gov.br]). In this context, cross-sectional epidemiological surveys developed in an area of endemicity in Pernambuco confirmed the high current rate of infection and an approximately 10-fold increase in rates of transmission during the last 10 years (5).

The diagnostic methods available at present are based on clinical and epidemiologic features, parasitologic detection (stained smears, culture, and histopathology), and immunological methods. These traditional laboratory methods have several limitations; prominent among them is low sensitivity (1, 38). The need for more sensitive methods has prompted the development of DNA-based diagnostic techniques. The kinetoplast, an organelle unique to the kinetoplastids, contains approximately 10,000 small circular DNAs, known as kinetoplast DNA (kDNA) minicircles, which are between 600 and 800 bp in members of the genus Leishmania. The abundance and other characteristics of these molecules have made them the target for a number of PCR-based techniques. However, the wide range of sensitivities (63 to 97%) and specificities (60 to 100%) that have been reported (1, 11, 12, 14, 22, 27, 32) clearly justify additional tests in regions of endemicity. In the study described in the present paper we evaluated PCR-based approaches that target kDNA for the diagnosis of ACL in regions of endemicity in Penambuco State, Brazil, and compared them to conventional diagnostic techniques. In addition, the use of specific PCR systems provided evidence that L. braziliensis is the only etiologic agent of ACL in Pernambuco.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Study area and patients. A total of 119 cutaneous biopsy specimens were studied from two groups of subjects: (i) 88 patients with confirmed ACL living in the Amaraji Municipality and neighboring regions (Pernambuco State), where L. braziliensis is endemic, and (ii) 31 patients with nonleishmaniasis cutaneous lesions (control group), including tuberculosis, sporotrichosis, epidermoid carcinoma, leprosy, tropical ulcer, zygomycosis, epidermal cyst, amyloidosis, and several types of nevi. The project was approved by the Ethical Committee of Centro de Pesquisas Aggeu Magalhães-FIOCRUZ, and all individuals enrolled in the study provided written consent. The definition of a confirmed case of ACL was based upon (i) the presence of typical lesions, a compatible epidemiological history, and a clinical response to specific treatment and (ii) positivity by at least one of the following tests: microscopic smear examination, histopathological examination, isolation by culture, or detection of circulating antibodies by indirect immunofluorescence (IIF). In the present study, ACL predominated in males between 10 and 29 years of age engaged in agricultural activity. The cutaneous form of the disease was predominant, with only 3 of 87 patients displaying mucocutaneous lesions. Single lesions were present in 45% of the patients, and the sizes of the lesions varied from 1 to 12 cm in diameter. Lesions were most frequently localized on the legs. The time of evolution of the lesions ranged from 15 days to 12 months (mean, 3 months).

Patient samples. Punch skin biopsy specimens of 4 to 6 mm were taken at the border of the lesion under sterile conditions and local anesthesia (3% prilocaine cloridrate). The specimens were divided into three samples. One of the samples was processed for PCR. The second sample was used for histopathological examination of tissue sections and microscopic smear examination. The third sample was used for in vitro and in vivo culture. Patient samples were collected from 1995 to 2000 in the field or at the outpatient facility of a reference hospital (Hospital das Clínicas, Universidade Federal de Pernambuco). Ten milliliters of blood was collected by venipuncture at the same time that the biopsy specimens were collected. It was then defibrinated, and the serum was collected after centrifugation. Serum samples were kept at -20°C until use.

Standard diagnostic procedures. Tissue scrapings for microscopic smear examination were collected by slitting the border of a skin lesion with a surgical blade, smearing the tissue onto a clean glass slide, fixation with methanol, and staining with Giemsa. The tissue sample for histopathological examination was fixed in 10% buffered formalin, and 5-µm sections were stained with hematoxylin-eosin. In vitro culture was performed by inoculation of tissue fragments into tubes containing culture medium (4% Bacto Blood Agar Base, 10% rabbit blood). The tubes were then incubated at 26°C. After preliminary microscopic observation for 5 days, the samples were transferred to fresh medium and observed carefully for at least 8 weeks before they were designated as negative by culture. For in vivo isolation, biopsy material was triturated and then inoculated into hamsters either intraperitoneally or intradermally (hind feet). Three months later the animals were killed and spleen and skin fragments were inoculated into culture medium. IIF was carried out as described previously (8).

PCR. DNA was purified for PCR with the GenomicPrep Cells and Tissue DNA isolation kit (Amersham Pharmacia Biotech) according to the instructions of the supplier. Frozen tissue samples of approximately 20 mg were used for each DNA isolation. After purification, the DNA was suspended in 100 µl of TE (10 mM Tris, 1 mM EDTA [pH 8.0]) and stored at -20°C until use. Two microliters of the DNA solution was added to the PCR mixture.

Two PCR-based systems were used. One was specific for the subgenus Viannia (primers 5'-GGGGTTGGTGTAATATAGTGG-3' and 5'-CTAATTGTGCACG-3') (11), and the other was specific for the genus Leishmania (primers 5'- (G/C)(G/C)(C/G)CC(A/C)CTAT(A/T)TTACACCAACCCC-3' and 5'-GGGGAGGGGCGT-3') (34). A 25-µl PCR mixture containing 10 mM Tris-HCl, 50 mM KCl, 0.1 mg of gelatin per ml, 1.5 mM MgCl, 0.2 mM each deoxynucleoside triphosphate, 25 pmol of each appropriate primer, and 2.5 U of Taq DNA polymerase (Amersham Pharmacia Biotech) was prepared. Amplification was carried out on a Perkin-Elmer model 4800 thermocycler. For the subgenus Viannia-specific PCR, programs were run for 35 cycles. Cycles consisted of annealing at 65°C for 1 min, extension at 72°C for 1 min, and denaturation at 94°C for 1 min. Thirty cycles were used for the genus Leishmania-specific PCR, consisting of 94°C for 30 s, 50°C for 30 s, and 72°C for 30 s. In both systems, the tubes were heated at 94°C for 4 min before cycling. Several negative controls (no DNA was added) and positive controls (100 or 10 pg of L. braziliensis genomic DNA was added) were included each time that PCR was undertaken to detect false-positive results due to contamination or variations in sensitivity.

Products (10 µl) were separated by electrophoresis in agarose gels, and the bands on ethidium bromide-stained gels were visualized and photographed over UV light with the Polaroid MP4+ system (33). The genus-specific PCR amplifies a 120-bp band and is able to detect 1 pg of promastigote genomic DNA. In contrast, the subgenus Viannia-specific PCR amplifies a 750-bp band and is able to detect 10 fg of promastigote genomic DNA. This amplicon is unique to this subgenus, representing a single linearized minicircle. The larger products are dose dependent and have been interpreted as read-through products (11).

PCR-hybridization. PCR-hybridization was performed on only a limited number of samples. Products amplified by the PCR specific for the genus Leishmania were subsequently hybridized with cloned Leishmania (Viannia) panamensis minicircles, allowing the typing of the products as members of the subgenus Viannia. Briefly, DNA was vacuum transferred from gels to positively charged nylon membranes (Hybond N+; Amersham Pharmacia Biotech) with a VacuGeneXL vacuum blotting system (Amersham Pharmacia Biotech). Hybridization was carried out with probes labeled with alkaline phosphatase, which allows visualization by chemiluminescence, with the AlkosPhos direct kit (Amersham Pharmacia Biotech). Both the labeling of the probes and detection of the label were performed according to the instructions of the manufacturer. Probed membranes were exposed to radiographic films (Biomax; Kodak).

Calculation of test performance indices and statistical analysis. The sensitivities and specificities of the diagnostic tests were calculated as described by Feinstein (15). The sensitivities of the PCR tests were assessed with samples from the patients with confirmed ACL, whereas the specificities were calculated on the basis of the results for patients without leishmaniasis living in leishmaniasis-free regions. These indices were compared by significance tests by using normal approximation, assigning statistical significance to differences with P values <0.05. EPI Info (version 6.04b) software and Statistica for Windows software (Statsoft, Tulsa, Okla.) were used. The sample size used for calculation of these indices was not necessarily the same because some diagnostic tests could not be performed with samples from all individuals selected for the study.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Evaluation of sensitivities and specificities of PCR-based methods. Eighty-eight patients met the diagnostic criteria for ACL described above and were therefore considered to have confirmed cases of ACL. These cases were used as the "gold standard" in calculation of the sensitivity. The subgenus Viannia-specific PCR detected DNA in 84 of 88 of the patients with confirmed cases, resulting in a sensitivity of 95.4%, whereas the genus-specific PCR detected the target DNA in 60 of 68 patients (88.2%) (Table 1). The difference between sensitivities was not significant (P > 0.05). The specificities calculated on the basis of the results for the 31 patients with nonleishmaniasis cutaneous lesions were 100% for both PCR systems used. Figures 1 and 2 present representative results obtained by the two PCR approaches. Spurious bands larger than 120 bp could be seen for some samples (Fig. 2).

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TABLE 1. Results of currently recommended methods for detection of ACL for patients with confirmed cases of disease

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FIG. 1. Representative agarose gel of PCR products obtained from biopsy samples with primers specific for the subgenus Viannia. Lanes: ACL, biopsy samples from patients with ACL; scars, specimens of scars from clinically cured patients; -, negative control; +, positive control; M, molecular size markers (bacteriophage DNA digested with HindIII) of 23.1, 9.4, 6.6, 4.4, 2.3, and 0.56 kb. The arrow indicates the 750-bp diagnostic band.

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FIG. 2. Representative agarose gel of PCR products obtained from biopsy samples with primers specific for the genus Leishmania. Lanes: ACL, biopsy samples from patients with ACL; scars, specimens of scars from clinically cured patients; -, negative control; +, positive control; M, molecular size markers (bacteriophage DNA digested with HindIII). The arrow indicates the 120-bp diagnostic band.

Comparison of sensitivities of PCR-based techniques with sensitivities of classical diagnostic methods. Because of the low sensitivities of the conventional tests for detection of Leishmania, there is no gold standard of general acceptance to be used for the evaluation of new diagnostic tests. Thus, the definition of a confirmed case of ACL was based upon a combination of elements (see Materials and Methods). Compared with confirmation of a case by use of the designated combination of elements (Table 1), the sensitivities of the PCR-based methods were significantly higher than those of smear examination, histological staining, and isolation by culture (P < 0.05). Parasites were isolated from 40 of 86 patients (46.5%). Microscopy of dermal scrapings and examination of stained tissue sections were positive for Leishmania for 66.7 and 66.2% of the patients, respectively. Antibodies specific for L. braziliensis were detected by IIF in 82.9% of the patients tested, yielding a sensitivity significantly lower than that observed for the subgenus Viannia-specific PCR (P < 0.05).

Preliminary evaluation of PCR-hybridization with nonradioactive probes for diagnosis of leishmaniasis. The primers specific for the genus Leishmania (34) enable the amplification of a 120-bp fragment for all Leishmania species, and the product can be typed as a member of the subgenus Viannia after hybridization with L. panamensis minicircles (30). For this preliminary experiment, samples from 11 patients with confirmed ACL and 4 patients with other cutaneous diseases were tested. Amplicons that hybridized with the L. panamensis minicircle were detected in all patients previously diagnosed with ACL by the subgenus Viannia-specific PCR, confirming infection with organisms consistent with L. braziliensis (Fig. 3). The analysis of the bands amplified by PCR suggested that some of them were spurious, being derived from host DNA. In fact, the hybridization corroborates this idea, showing that most of the nonspecific products were derived from DNA not related to kDNA sequences and possibly derived from human DNA. However, some products >120 bp hybridized with the probe, indicating that, although they are nonspecific, they are related to minicircle sequences.

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FIG. 3. (A) PCR products obtained by using genus-specific primers. (B) Hybridization of the amplification products, transferred to nylon membranes, with nonradioactive probes. Lanes: ACL, biopsy samples from patients with ACL; OD, samples from patients with other diseases with nonleishmanial ethiologies -, negative control; +, positive control; M, molecular size markers (bacteriophage DNA digested with HindIII). The arrows indicate the 120-bp diagnostic bands.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Parasites of the subgenus Viannia produce chronic cutaneous and mucocutaneous lesions, but the organisms that cause the lesions are very difficult to detect and treat. Indeed, the importance of establishing control strategies makes diagnosis and typing of the cause of leishmaniasis critical (28). This is particularly relevant for two reasons: (i) increased human mobility and long-distance travel and (ii) the possibility of coexistence of AIDS and leishmaniasis (20). Increased human mobility and long-distance travel may contribute to the spread of leishmaniasis to areas where the disease was not endemic, whereas AIDS may increase the severity of the parasitic disease. In addition, accurate diagnosis is important because lesions caused by epidermoid carcinomas, Paracoccidioides brasiliensis, tuberculosis, syphilis, and leprosy, which are all common in Brazil, have some resemblance to those found in patients with ACL (30, 32). Direct methods of identification of leishmania in tissue samples by microscopic smear examination or histological staining of tissue sections, which are simple and cheap, have limited sensitivities (11, 22, 30, 32, 37). Because of its low sensitivity, histopathology is more useful for the discrimination of ACL from other diseases causing similar macroscopic lesions. Isolation methods such as in vitro culture of parasites from punch biopsy specimens or lesion aspirates can be sensitive but are time-consuming and costly and have variable success rates (1, 37). Indirect tests for circulating antibodies against Leishmania, such as IIF, are sensitive but fail to distinguish between past and present disease, are unable to detect early infections, and have significant cross-reactions to antibodies against other pathogens, including some which resemble Leishmania in terms of clinical presentations (19, 37). Nevertheless, serological diagnosis is expected to improve with the identification of new relevant antigens and the possibility of designing tests for the detection of antibodies against the purified molecules (8, 9). PCR is claimed to be very sensitive and specific, but there are conflicting results from different groups. The technique was found to be not very sensitive (63 to 80%) or specific (60%) with biopsy specimens from patients with cutaneous leishmaniasis (11, 23, 25). In other studies, PCR showed detection rates of 97% with samples from patients in the New World (31). A possible explanation for these discrepancies is a difference in the quality of the DNA samples used for PCR (the samples may contain different amounts of DNA polymerase inhibitors). Thus, to optimize the PCR results, we worked with high-quality DNA preparations obtained with a commercial DNA purification kit. Ten femtograms of total DNA (equivalent to approximately 1 fg of kDNA) was the detection limit of the subgenus Viannia-specific PCR. Thus, this assay could theoretically detect DNA corresponding to that from less than one parasite per reaction tube (16), in agreement with the sensitivities reported previously (11). Thus, use of this method for the diagnosis of ACL is appropriate, since it is known that parasites are scarce in New World cutaneous leishmaniasis caused by Viannia parasites. The detection limit for the genus-specific PCR was 1 pg of total DNA.

The sensitivity of PCR was significantly higher than those of the conventional direct parasite detection tests used (histopathological examination, microscopic smear examination, and culture). This is in line with the results of other studies showing that PCR is consistently more sensitive than conventional methods (1, 12, 30). The present study was based upon the analysis of 119 cutaneous biopsy specimens (with 88 samples coming from patients with confirmed cases of ACL). This sample is larger than the ones used in several previous studies (1, 12, 29).

Although it is possible that four confirmed cases of ACL were not identified by the PCR system proposed by de Bruijn and Barker (11) due to infection with non-Viannia species (Table 1), a more reasonable explanation is that the PCR has suboptimal sensitivity, since the genus-specific PCR was also not able to detect one of these four confirmed cases. The PCR systems used were negative for lesions with nonleishmanial etiologies, resulting in 100% specificities.

The PCR primers used in the PCR described by de Bruijn and Barker (11) were based on sequences present in World Health Organization reference strains (12). Although they also anneal to DNA from strains from Colombia, Venezuela, Ecuador, Peru, and Brazil (11), this had not been formally demonstrated in isolates from regions of endemicity in Pernambuco. This is particularly relevant, since several L. braziliensis variants from this region have recently been characterized (7, 10; M. E. F. Brito et al., unpublished data).

The potential confusion between the clinical presentations of ACL caused by L. amazonensis and L. braziliensis coupled with the overlapping distribution of L. amazonensis and L. braziliensis in some regions poses diagnostic problems. Although patients with ACL caused by species of the subgenera Viannia and Leishmania generally have similar clinical presentations, the clinical evolution and response to chemotherapy may differ for ACL caused by the two subgenera (24). Thus, approaches allowing the discrimination between the New World dermotropic leishmanias (subgenus Viannia and subgenus Leishmania) are relevant (12, 14, 18, 30, 32). In the state of Minas Gerais, Brazil, clinical epidemiological and molecular evidence supported a conclusion (29) that L. (Viannia) braziliensis and L. (Leishmania) amazonensis were the species present, although L. (Viannia) braziliensis was the predominant species. This species is also predominant in the states of São Paulo, Rio de Janeiro, Espírito Santo, and Bahia (24). Studies characterizing Leishmania species in Pernambuco are relatively scanty. The previous studies had shown that Leishmania isolated from patients with ACL and rodents in areas of endemicity in Pernambuco were of the species L. braziliensis, suggesting that this species is the only causative agent of ACL in this region (4, 7, 10; Brito et al., unpublished). The results described herein support this hypothesis, as DNA products specific for the subgenus Viannia were amplified from 95.4% of the samples from patients with confirmed cases of ACL. Genus-specific PCR followed by hybridization with specific minicircles confirmed the presence of Leishmania organisms of the L. braziliensis complex in 11 patients. Although we did not formally rule out the possibility of mixed infections in all patients, the parasite was isolated and characterized from 40 patients with ACL, confirming infection with L. braziliensis on the basis of analysis with monoclonal antibodies and isoenzyme analysis (Brito et al., unpublished). Thus, typing of Leishmania by PCR yielded results in agreement with those obtained by typing of cultured parasites. The evidence available at present incriminates L. braziliensis as the only causative agent of ACL in Pernambuco. In line with this, Lutzomya whitmani, the local vector in Pernambuco (6), has not been incriminated in the transmission of L. amazonensis (21, 35).

The genus-specific primers described by Schubach et al. (34) enable the amplification of a 120-bp fragment for all Leishmania species. The specificity of the assay can be guaranteed by molecular hybridization experiments performed with three distinct cloned minicircle molecules from Leishmania (Viannia) (L. panamensis minicircle), Leishmania mexicana complex (L. amazonensis minicircle), and Leishmania chagasi complex (L. chagasi minicircle) (13, 29, 30). The use of hybridization after PCR improves the sensitivity and specificity of the approach. However, most of these studies have used probes labeled with radioactive material (29, 30), which is not convenient for work in areas where leishmaniasis is endemic, as the disposal of radioactive waste in the environment is difficult and expensive. Thus, we adapted a PCR-hybridization assay (34) so that probes labeled with nonradioactive material could be used. This approach confirmed the presence of Leishmania (Viannia) in 100% of the samples from 11 patients with confirmed cases of ACL tested.

PCR is an extremely sensitive approach, having the potential to provide specific results in less than 1 day. PCR systems for detection of different species of Leishmania are likely to be valuable tools not only for differential diagnosis of ulcer-forming skin diseases but also for investigation of relationships between causative agents and the clinical manifestations and epidemiology of the disease. The possibility of PCR automation, the simplification of sample collection and processing, as well as the in-house preparation of reagents can make this technique economically attractive for the processing of large numbers of samples in regions of endemicity (2, 26).

In conclusion, the present paper reports on the use of the PCR method and a hybridization method adapted by using nonradioactively labeled probes for the diagnosis of ACL in a setting of endemicity. The study shows that PCR is more sensitive than the conventional methods of diagnosis and provides evidence that the primers specific for the subgenus Viannia described by de Bruijn and Barker (11) are suitable for the detection of Leishmania isolates in Pernambuco State in northeastern Brazil. Finally, our results, in conjunction with previous findings, incriminate L. braziliensis as the only causative agent of ACL in Pernambuco.

 
We are indebted to Siddharta Mahanti and Octavio Fernandes for critical reading of the manuscript. We thank Valdir Bandeira and Cristiane V. Oliveira (recipient of a PIBIC/FIOCRUZ scholarship) for collaboration in the study.

Financial support for this work was provided by FIOCRUZ and CNPq. Eduardo H. Rodrigues was the recipient of a CAPES MSc scholarship during the development of the study.

 
* Corresponding author. Mailing address: Departamento de Imunologia, Centro de Pesquisas Aggeu Magalhães, Av. Prof. Moraes Rego s/n, Cidade Universitária, 50670-420, Recife, Brazil. Phone: 55-81-33012558. Fax: 55-81-34532449. E-mail: fabath{at}cpqam.fiocruz.br.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Journal of Clinical Microbiology, October 2002, p. 3572-3576, Vol. 40, No. 10
0095-1137/02/$04.00+0     DOI: 10.1128/JCM.40.10.3572-3576.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

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