Diagnostic Microbiology and Infectious Disease 74 (2012) 1115

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Diagnostic Microbiology and Infectious Disease

j o u r n a l h o m e p a g e : w w w. e l s e v i e r. c o m / l o c a t e / d i a g m i c r o b i o

Leptospirosis diagnosis by immunocapture polymerase chain reaction: a new tool for early diagnosis and epidemiologic surveillance
Ilana Teruszkin Balassiano a, b,, Juliana Magalhes Vital-Brazil a, Martha Maria Pereira a
a b

Laboratrio de Zoonoses Bacterianas, Centro de Referncia Nacional para Leptospirose, WHO/PAHO Centro Colaborador para Leptospirose, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil Coleo de Leptospira, Instituto Oswaldo Cruz, Fiocruz, Rio de Janeiro, Brazil

a r t i c l e

i n f o

a b s t r a c t
The aim of this study was to develop an immunocapture polymerase chain reaction (IC-PCR) protocol for leptospirosis. For the standardization of IC-PCR, polyclonal (AS) and monoclonal (MAb) antibodies against different serogroups and serovars of Leptospira were coupled to polystyrene plates. Human sera were articially contaminated with leptospires and incubated on plates. The bacterial DNA was obtained and used in a multiplex PCR. Sensitivity was tested using sera contaminated with crescent concentrations of leptospires, while specicity was established using sera contaminated with different bacterial genera and sera obtained from patients positive for viral infections. IC-PCR using AS was able to recognize specic serogroups, although some cross-reactions have been observed. No cross-reactions were observed when MAbs were used; however, the sensitivity in this case was lower than that of IC-PCR using AS. IC-PCR proved to be specic to Leptospira and is a promising tool for early diagnosis of leptospirosis, providing additional information about the infecting serovar or serogroup. 2012 Elsevier Inc. All rights reserved.

Article history: Received 14 March 2012 Accepted 31 May 2012 Available online 7 July 2012 Keywords: Leptospirosis Immunocapture-PCR Diagnosis Epidemiology

1. Introduction Leptospirosis is recognized as an important zoonotic disease widespread worldwide, being most common in tropical regions (Levett, 2001; Ko et al., 2009). The infection is considered a public health problem in many countries, including Brazil, where most reports are focused on urban slums, especially due to exposure to rainwater contaminated with the urine of infected animals (Ko et al., 1999). The tropical climate, which provides periods of intense rainfall, resulting in oods, combined with a dense population living in poverty and poor sanitation conditions makes this country susceptible to the maintenance and spread of leptospirosis (Ko et al., 1999; Fonseca et al., 2006b). The disease varies from a subclinical infection to a severe illness with multi-organ involvement, leading to fatal forms in some cases (Mrien et al., 1995; Levett, 2001). Early diagnosis is important and complicated owing to the wide spectrum of clinical symptoms of the disease, and the infection is frequently misdiagnosed as inuenza, hepatitis, hemorrhagic fever, or dengue fever. Therefore, diagnosis

This work was supported by grants from Fundao Carlos Chagas de Amparo Pesquisa do Estado do Rio de Janeiro (FAPERJ) and Instituto Oswaldo Cruz (Fundao Oswaldo Cruz). Corresponding author. Tel.: + 55-21-2562-1643; fax: + 55-21-2562-1610. E-mail address: ilana@ioc.ocruz.br (I.T. Balassiano). 0732-8893/$ see front matter 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.diagmicrobio.2012.05.028

should be based primarily on laboratory tests rather than on clinical aspects by themselves (Mrien et al., 1992; Branger et al., 2005). The gold standard methodology for leptospirosis diagnosis, the microscopic agglutination test (MAT), is based on the antibody response of the host, which can occur only in a period of 810 days after the onset of symptoms, named seroconversion (Mrien et al., 1995). MAT presents the inconvenience of being laborious and requires the maintenance of living cultures (Fonseca et al., 2006a; Perez and Goarant, 2010). The diagnosis can also be based on the cultivation of leptospires, using blood collected from patients in the acute phase of the disease. However, the bacterial culture presents low sensitivity, about 30%, and may be a retrospective diagnosis, since it can take up to 2 months (Mrien et al., 1995; Fonseca et al., 2006a). Nevertheless, the isolation of Leptospira is extremely valuable, especially in epidemiologic studies, since it is possible to identify the infective serogroup/serovar. Polymerase chain reaction (PCR) has been successfully used for the amplication of different DNA sequences of leptospires (Gravekamp et al., 1993; Levett et al., 2005; Kositanont et al., 2007; Slack et al., 2007; Bomm et al., 2008) and is considered an important technique for the early detection of the microorganism, while other methods either failed or proved to be unreliable (Brown et al., 1995; Ooteman et al., 2006). PCR is an extraordinarily useful tool for the detection of infectious agents that are difcult to cultivate, such as leptospires; however, it often restricts data available for epidemiologic surveillance, especially concerning the infective strain (Perez and Goarant, 2010).

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Immunocapture-PCR (IC-PCR) has already been described as a rapid and efcient tool for detection and identication of Salmonella, Shigella , and Mycobacterium species (Luo et al., 2002; Peng et al., 2002; Warren et al., 2007; Chui et al., 2010; Katsuda et al., 2010). Generally, this assay is based on an initial step of concentration and immunologic capture of the pathogen, and a second step in which the microorganism is identied following the amplication of specic regions of its DNA. Even though this method has not been described for detecting Leptospira yet, it seemed to be a promising possibility. The main goal of this study was to develop a new IC-PCR that would combine the early diagnosis with the presumptive identication of Leptospira at the serovar or serogroup level. 2. Materials and methods 2.1. Bacterial strains and growth conditions Leptospira interrogans serovars Icterohaemorrhagiae (RGA strain reference no. CLEP 0001), Copenhageni (M20 strainreference no. CLEP 0002), and Canicola (Hond Utrecht IV strainreference no. CLEP 0003) were obtained from the Collection of Leptospira (CLEP, Oswaldo Cruz Foundation, Rio de Janeiro, Brazil) and grown in Ellinghausen-McCullough-Johnson-Harris medium (Difco, Isre, France) at 28 C. Escherichia coli, Salmonella enterica, Shigella sonei, and Yersinia enterocolitica were kindly provided by the Collection of Bacteria of Importance to Health (Oswaldo Cruz Foundation) and grown in brain heart infusion medium (Difco) at 37 C. 2.2. Sera samples Blood from human healthy donors was collected to provide serum. Twenty-nine sera samples were collected from patients diagnosed with a disease usually misdiagnosed as leptospirosis, such as dengue fever, hantaviruses, and viral hepatitis. All samples were provided by Dr. Marluce Aparecida Assuno Oliveira from the Fundao Ezequiel Dias (Minas Gerais, Brazil). 2.3. IC-PCR: Standardization for serogroup identication Twenty-one rabbit antisera containing polyclonal antibodies against reference strains (Table 1), obtained from the Royal Tropical Institute (Amsterdam, The Netherlands), were separately coupled to
Table 1 Rabbit antisera used in the standardization of IC-PCR for leptospirosis. Antisera AS-1 AS-2 AS-3 AS-4 AS-5 AS-6 AS-10 AS-11 AS-12 AS-13 AS-14 AS-15 AS-16 AS-17 AS-19 AS-20 AS-21 AS-22 AS-23 AS-24 AS-25 Serogroup Icterohaemorrhagiae Icterohaemorrhagiae Canicola Grippotyphosa Pomona Australis Bataviae Celledoni Cynopteri Djasiman Hebdomadis Javanica Panama Pyrogenes Sejroe Shermani Tarassovi Autumnalis Autumnalis Autumnalis Ballum Serovar Copenhageni Icterohaemorrhagiae Canicola Grippotyphosa Pomona Australis Bataviae Celledoni Cynopteri Djasiman Hebdomadis Poi Panama Pyrogenes Saxkoebing Shermani Tarasovi Rachmati Bangkinang Carlos Kenya Reference strain M20 RGA Hond Utrecht IV Moskva V Pomona Ballico Van Tienen Celledoni 3522C Djasiman Hebdomadis Poi CZ214 Salinem Mus 24 1342K Perepelitsin Rachmat Bangkinang I C3 Njenga

polystyrene 96-well plates at 4 C for 18 h. Sera were diluted 1:100 with 0.05 mol/L carbonic acid buffer (pH 9.6). The wells were washed with a solution (0.2 mol/L phosphate-buffered saline; 0.05% Tween 20; pH 7.6) and subsequently incubated with a block solution (0.2 mol/L phosphate-buffered saline; 2% bovine serum albumin; pH 7.6) at 37 C for 1 h. The wells were washed again with the same buffer and incubated at 37 C for 1 h with 100 L of sera from healthy donors and articially contaminated with 10 7 leptospires/mL. L. interrogans serovar Copenhageni M20 strain was used in this step. Sera without bacteria were used for the control wells. Following washing with 0.2 mol/L phosphate-buffered saline (pH 7.6), the wells were incubated with sterile Milli-Q water and heated at 100 C for 10 min to obtain the bacterial DNA. The PCR mixture consisted of 10 mmol/L TrisHCl buffer (pH 8.3) (Sinapse Biotecnologia, So Paulo, Brazil), 2 mmol/L MgCl2 (Sinapse Biotecnologia), 200 mol/L of each deoxynucleotide (Promega, So Paulo, Brazil), 2 U of Taq DNA polymerase (Sinapse Biotecnologia), 1 mol/L of aB (Kawabata et al., 2001) and lipL41 (Ahmed et al., 2006) primer pairs, and 5 L of the DNA template obtained in the previous step. PCR was performed in a thermocycler (Gene Amp PCR System 9700, PE Applied Biosystems, Carlsbad, CA, USA), and the rst amplication cycle consisted of denaturation at 95 C for 5 min, 35 cycles of 95 C for 30 s (denaturation), 54 C for 30 s (annealing) and 72 C for 1 min (extension), and a nal extension at 72 C for 7 min. Positive (PCR mix with DNA of L. interrogans serovar Copenhageni M20 strain) and negative (PCR mix without DNA) controls were also included in the reactions. The amplication products (10 L) were mixed with 1 L Blue Green Loading Dye (LGC Biotecnologia, So Paulo, Brazil) and subjected to electrophoresis on 1.5% agarose gel prepared with TAE (2 mol/L Trizma base; Sigma, St. Louis, MO, USA; 1.2 mol/L acetic acid; 0.5 mol/L ethylenediaminetetraacetic acid) buffer. The molecular size marker 100-bp DNA ladder (Sinapse Biotecnologia) was also included. The gels were analyzed under UV light. 2.4. IC-PCR for serovar identication The procedure described above was used to couple monoclonal antibodies (MAbs) to polystyrene 96-well plates. In this step, specic MAbs against serovars Icterohaemorrhagiae (F70C14), Copenhageni (F70C24), and serogroup Icterohaemorrhagiae serovar non-Icterohaemorrhagiae (F89C12) were used. Sera samples were articially contaminated with 10 7 cells/mL with the following reference strains: L. interrogans serovars Copenhageni (M20 strainreference no. CLEP 0002), Icterohaemorrhagiae (RGA strainreference no. CLEP 0001), and Canicola (Hond Utrecht VI strainreference no. CLEP 0003). 2.5. Sensitivity of IC-PCR The sensitivity of IC-PCR was established by using sera articially contaminated with increasing concentrations of L. interrogans serovar Copenhageni M20 strain (10 0 to 10 7 cells/mL), in plates coated with

Rabbit antisera were obtained from the Royal Tropical InstituteKIT Biomedical Research (Amsterdam, The Netherlands).

Fig. 1. IC-PCR protocol standardization using reference antisera (AS), showing specic recognition of leptospires by polyclonal antibodies of serogroup Icterohaemorrhagiae and cross-reactions with heterologous serogroups represented by PCR products of lower intensity. Amplication reactions were disposed on the gel in order to compare control and test wells of each AS. M, 100-bp DNA ladder. A) a, Positive control for the PCR; b, negative control for the PCR; c/d, AS-1; e/f, AS-2; g/h, AS-3; i/j, AS-4; k/l, AS-5; m/n, AS-6; o/p, AS-10; q/r, AS-11; s/t, AS-12; u/v, AS-13; w/a (B), AS-14. B) b/c, AS-15; d/e, AS-16; f/g, AS-17; h/i, AS-19; j/k, AS-20; l/m, AS-21; n/o, AS-22; p/q, AS-23; r/s, AS-24; t/u, AS-25.

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3.2. IC-PCR is sensitive and specic Wells were coupled with AS-1 and MAb F70C24, separately, and the test was performed with sera articially contaminated with crescent concentrations of serovar Copenhageni M20 strain (10 0 10 7/mL). The results showed that IC-PCR with reference antiserum was 10 times more sensitive (limit of detection: 10 5 leptospires/mL; Fig. 3) than with monoclonal antibodies (limit of detection: 10 6 leptospires/mL; Fig. 4). In order to compare IC-PCR and conventional PCR sensitivities, thermal lysis was applied directly to sera articially contaminated with crescent concentrations of leptospires. In this case, the limit of detection by PCR was 10 3 leptospires/mL (data not shown). In order to test the specicity of the protocol, wells were coupled with the same antisera and monoclonal antibodies used in the standardization step. The test was performed with sera articially contaminated with other Gram-negative bacterial species presenting agella and with sera obtained from patients previously conrmed for viral infections that are commonly misdiagnosed as leptospirosis. Any cross-reactions were detected in this step, conrming that the IC-PCR protocol standardized by our group is specic to Leptospira (data not shown). 4. Discussion Leptospirosis is widespread around the world, being particularly common in tropical and subtropical regions where environmental conditions favor the survival and transmission of leptospires. The World Health Organization considers leptospirosis a neglected tropical disease and estimates the median global incidence of this infection to be at least 5.1 cases per 100,000/year in endemic areas and 14 cases per 100,000/year during epidemics (Lau et al., 2012). Early detection of leptospirosis demands rapid and sensitive diagnostic tests (Fonseca et al., 2006b), and, usually, PCR-based approaches are the methods of choice for this purpose. A single positive sample provides a true result before seroconversion; however, this can lead to the loss of serology-based identication of the infective serovar, which hampers epidemiologic studies (Perez and Goarant, 2010). In order to address this problem, Perez and Goarant (2010) proposed a direct multilocus sequence typing (MLST) scheme from clinical samples. Leptospira could be identied in sera using real-time PCR, and, subsequently, MLST was conducted to identify the infective serovar. Our proposal, based on IC-PCR, follows the same line of reasoning; however, the identication step is based on serologic characteristics rather than on genotypic ones. Although the genotypic classication of leptospires has increased in recent years, the phenotypic characterization is still important, since the molecular classication is incompatible with the system of serogroups, which has been established for decades (Doungchawee et al., 2007). The IC-PCR protocol established by our group allowed the specic detection of leptospiroses in serum samples, since no amplication products were observed when sera contaminated with other

Fig. 2. IC-PCR protocol standardization using monoclonal antibodies (MAbs) and sera articially contaminated with serovars Copenhageni (A) or Icterohaemorrhagiae (B). Amplication reactions were disposed on the gel in order to compare control and test wells of each MAb. M, 100-bp DNA ladder; a, positive control for the PCR; b, negative control for the PCR; c/d, MAb F70C14; e/f, MAb F70C24; g/h, MAb F89C12.

reference antiserum AS-1 (specic to serogroup Icterohaemorrhagiae) and MAb F70C24 (specic to serovar Copenhageni). 2.6. Specicity of IC-PCR The specicity of IC-PCR was veried using sera articially contaminated with Gram-negative bacterial species, but not Leptospira sp., for instance, E. coli, Salmonella enterica, Shigella sonei, and Y. enterocolitica. In this step, sera obtained from patients with previously diagnosed viral hepatitis, hantaviruses, and dengue fever, but negative for leptospirosis, were also used.

3. Results 3.1. IC-PCR protocol allows serogroup and serovar identication The protocol was initially established using a polystyrene 96-well plate coupled with 21 reference antisera in each well, separately. The test was performed with serum articially contaminated with serovar Copenhageni M20 strain. Specic recognition of leptospires by serogroup Icterohaemorrhagiae antisera AS-1 and AS-2 (Fig. 1A) was observed but also some cross-reactions represented by PCR products of lower intensity with heterologous serogroups (AS-3, AS-4, AS-10, AS-13, and AS-16) compared to the homologous serogroups (AS-1 and AS-2) (Fig. 1A and B). Following this initial standardization, wells were coated with monoclonal antibodies and the test was performed with sera articially contaminated with serovars Copenhageni (Fig. 2A), Icterohaemorrhagiae (Fig. 2B), and Canicola (data not shown), separately. The results showed that PCR products were detected only in the wells coated with MAbs of homologous serovars, and cross-reactions were not observed. Sera contaminated with serovar Canicola, which served as a control, did not produce any positive amplication when tested with the plates coupled with MAbs of serovars Copenhageni and Icterohaemorrhagiae (data not shown).

Fig. 3. IC-PCR sensitivity determination using reference antiserum AS-1 and human sera articially contaminated with increasing concentrations of serovar Copenhageni. Amplication reactions were disposed on the gel in order to compare control and test wells of each leptospira concentration. M, 100-bp DNA ladder; a, positive control for the PCR; b, negative control for the PCR; c/d, 107; e/f, 106; g/h, 105; i/j, 104; k/l, 103; m/n, 102; o/p, 101; q/r, 100.

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Fig. 4. IC-PCR sensitivity determination using monoclonal antibody F70C24 and human sera articially contaminated with increasing concentrations of serovar Copenhageni. Amplication reactions were disposed on the gel in order to compare control and test wells of each leptospira concentration. M, 100-bp DNA ladder; a, positive control for the PCR; b, negative control for the PCR; c/d, 107; e/f, 106; g/h, 105; i/j, 104; k/l, 103; m/n, 102; o/p, 101; q/r, 100.

microorganisms were used. This result reinforces the value of this methodology, especially because the symptoms of leptospirosis during the acute phase are almost indistinguishable from other bacterial and viral febrile infections (Srimanote et al., 2008). The specicity of the IC-PCR protocol was also observed as regards the detection of particular serogroups and serovars. When sera articially contaminated with serogroup Icterohaemorrhagiae were tested on plates coupled with different reference antisera, the specic recognition of M20 strain by polyclonal antibodies of serogroup Icterohaemorrhagiae occurred. However, as expected, cross-reactions were also found when plates were coupled with reference antisera of heterologous serogroups, corroborating previous observations (Levett, 2003; Doungchawee et al., 2007). This can be explained by the fact that polyclonal antibodies are able to recognize a wide range of epitopes found in different serogroups of Leptospira. Nevertheless, it is important to emphasize that cross-reactions occurred as lowintensity PCR products, being distinguishable from the products related to the specic serogroup recognition. We also noticed the specic serovar identication when MAbs were coupled to the polystyrene plates, and, in this case, no cross-reactions were observed. It is well established that monoclonal antibodies enable epitope-specic recognition (Doungchawee et al., 2007). In the acute phase of the disease, also known as leptospiraemia, leptospires may reach 10 610 7 microorganisms/mL in the blood of the patient (Ko et al., 2009). For this reason, we chose 10 7 leptospires/mL as the ideal concentration to articially contaminate the serum used in the test, since it could mimic real conditions. We noticed that the IC-PCR protocol for serogroup identication was 10 times more sensitive (limit of detection: 10 5 leptospires/mL) than the protocol standardized to serovar identication (limit of detection: 10 6 leptospires/mL). This suggests that perhaps polyclonal antibodies can recognize a higher diversity of epitopes than monoclonal antibodies, enabling the recognition of a greater quantity of leptospires in the immunologic step of the protocol. When thermal lysis was applied directly to sera, the limit of detection by PCR was 10 3 leptospires/mL. The problem of sensitivity of PCR applied to the diagnosis of leptospirosis was previously discussed by Bal et al. (1994). They concluded that different DNA extraction methods directly affect reproducibility and sensitivity, especially due to the presence of different types of inhibitors. Based on this premise, we can assert that the present IC-PCR protocol needs improvements in order to minimize the loss of leptospires during the immunologic concentration or the DNA extraction steps. Taken together, our results indicate that, although adjustments are necessary, we successfully standardized an IC-PCR protocol suitable for use as a new tool for the early diagnosis of leptospirosis, providing additional information about the infecting serovar or serogroup, which is crucial for epidemiologic surveillance. Our next step will be the use of clinical samples collected from patients in the early days of disease and presenting clinical symptoms of leptospirosis. Acknowledgments The authors thank Tatiane Mendes Varela Ramos for laboratory assistance, Dr. Marluce Aparecida Assuno Oliveira for providing sera

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