Correspondence Clinical Letter
Correspondence Paraffin-embedded tissue: an alternative to Bartonella sp. infection diagnosis
DOI: 10.1111/ddg.13607
Dear Editors, We read with interest the article “Molecular diagnosis of skin infections using paraffi n-embedded tissue - review and interdisciplinary consensus” by Sunderkötter et al. 2018 [1]. We agree with the authors’ views regarding the application of nucleic acid amplification techniques (NAATs), such as PCR in paraffi n-embedded tissue, if a potentially infectious etiology was not considered at the time of the biopsy. We also agree that there are limitations to interpreting the results. The following factors relate to potential false-negative results: low initial quantity of pathogen nucleic acids, poor DNA quality control during the paraffi nization/deparaffi nization processes and irregular distribution of pathogens in the tissue. Contamination during pre-analytical phases may induce false-positive results. On the other hand, application of NAATs using paraffi n-embedded tissue may increase the potential for more stable results. Our group has studied Bartonella sp. infection for some years. These bacteria are neglected and re-emerging
pathogens. The clinical spectrum of human infection has expanded quickly. We have used NAATs not only in bacillary angiomatosis patients for Bartonella sp. diagnosis, but also in fresh and paraffi n-embedded skin fragments as described by the authors. We recently initiated a study of Bartonella sp. coinfections in leprosy patients. We selected 12 lepromatous or borderline-lepromatous patients who were followed up at the University of Campinas Outpatient Center and from whom skin biopsies had been collected for the diagnosis of diseases or relapses. We performed conventional PCR targeting the Bartonella sp. 16S-23S internal transcribed spacer (ITS) region, nested PCR targeting the ftsZ gene (specific for Bartonella henselae) and real-time PCR, targeting the gltA gene (specific for B. henselae), as previously described in deparaffi nized tissue [2, 3]. These samples were obtained before or after multi-drug therapy. Infection with Bartonella sp. was not suspected when the skin samples were obtained. One to seven years after the biopsy procedures, patients were invited to participate in a Bartonella sp. infection survey of leprosy patients (Ethics Committee CAAE: 44670015.4.0000.5404). Of the 12 patients, four showed positivity for Bartonella sp. with PCR; three of these were also positive for Bartonella sp. with blood PCR, suggesting previous infection with Bartonella sp. One patient only showed positivity for Bartonella sp. with PCR in deparaffi nized tissue (Figure 1). All four patients were offered treatment for the potential Bartonella sp. infection, including the fourth patient, since he had recurrent type 2 leprosy reactions and subclinical infection can trigger
Figure 1 Results of Bartonella sp. positive samples from leprosy patients.
© 2018 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd. | JDDG | 1610-0379/2018/1609
1147
Correspondence Clinical Letter
leprosy reactions. Nevertheless, we recognize the possibility of a false-positive result due to pre-analytical contamination. It is known that false-negative PCRs are frequent with Bartonella sp. infections, either in paraffi n-embedded cutaneous fragments or fresh samples, for reasons well discussed by Sunderkötter et al. We have also obtained false-negative results with blood PCR for Bartonella sp. Potential interfering factors were analyzed in depth by Wilson et al. 1997 and Drummond et al. 2018 [4, 5]. Despite these unresolved factors, molecular diagnosis based on paraffi n-embedded cutaneous samples is a potential tool for the diagnosis of Bartonella sp. infection as we previously described [1]. Luciene Silva dos Santos, Marina Rovani Drummond, Andrea Fernandes Eloy da Costa França, Maria Letícia Cintra, Paulo Eduardo Neves Ferreira Velho Bibliography 1
2
3
4
5
Sunderkötter C, Becker K , Kutzner H et al. Molecular diagnosis of skin infections using paraffin-embedded tissue - review and interdisciplinary consensus. J Dtsch Dermatol Ges 2018; 16(2): 139 –47. Silva MN, Vieira-Damiani G, Ericson ME et al. Bartonella henselae transmission by blood transfusion in mice. Transfusion 2016; 56(6 Pt 2): 1556 – 9. Staggemeier R , Pilger DA , Spilki FR , Cantarelli VV. Multiplex SYBR® green-real time PCR (qPCR) assay for the detection and differentiation of Bartonella henselae and Bartonella clarridgeiae in cats. Rev Inst Med Trop Sao Paulo 2014; 56(2): 93 – 5. Drummond MR , Lania BG, Diniz P et al. Improvement of Bartonella henselae DNA Detection in Cat Blood Samples by Combining Molecular and Culture Methods. J Clin Microbiol 2018; 56( 5). Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 1997; 63(10): 3741 – 51.
Authors’ reply Dear Editors, We thank dos Santos and colleagues for their positive comments on our interdisciplinary consensus regarding molecular diagnosis of skin infections using paraffi n-embedded tissue. We appreciate that they agree with us on the select indications for nucleic acid amplification techniques (NAATs) using paraffi n sections, and on the caveats that need to be taken into consideration during the pre- and post-analytical phase. Their fi ndings on possible subclinical coinfections with Bartonella spp. in patients with borderline or lepromatous leprosy are intriguing. As we have stated in our consensus report (1), NAAT analysis of paraffi n-embedded tissues is useful for confi rmation of a histologically suspected diagnosis of Bartonella infection, because bacillary angiomatosis is characterized by clinical variability and encompasses several differential diagnoses (such as pyogenic granuloma, Kaposi‘s sarcoma, senile angioma, angiokeratoma, angiosarcoma, verruga peruana [Peruvian warts] and mycobacterial furunculosis). Molecular diagnostic methods also help to distinguish between bartonellosis caused by B. henselae (cat-scratch disease) and B. quintana. Finally, as illustrated by the work of dos Santos et al., retrospective NAT analyses of paraffi n-embedded tissues may help to discover Bartonella spp. as co-infecting pathogens in leprosy (or other skin diseases). Cord Sunderkötter, Karsten Becker, Walter Geißdörfer, Christian Bogdan Correspondence to Prof. Dr. med. Cord Sunderkötter Universitätsklinik und Poliklinik für Dermatologie und Venerologie Universitätsklinikum Halle (Saale) Martin-Luther-Universität Halle-Wittenberg Ernst-Grube-Straße 40 06120 Halle/Saale E-mail: [email protected]
Bibliography 1
1148
Sunderkötter C, Becker K , Kutzner H et al. Molecular diagnosis of skin infections using paraffin-embedded tissue - review and interdisciplinary consensus. J Dtsch Dermatol Ges 2018; 16(2): 139 –47.
© 2018 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd. | JDDG | 1610-0379/2018/1609
Correspondence Paraffin-embedded tissue: an alternative to Bartonella sp. infection diagnosis
DOI: 10.1111/ddg.13607
Dear Editors, We read with interest the article “Molecular diagnosis of skin infections using paraffi n-embedded tissue - review and interdisciplinary consensus” by Sunderkötter et al. 2018 [1]. We agree with the authors’ views regarding the application of nucleic acid amplification techniques (NAATs), such as PCR in paraffi n-embedded tissue, if a potentially infectious etiology was not considered at the time of the biopsy. We also agree that there are limitations to interpreting the results. The following factors relate to potential false-negative results: low initial quantity of pathogen nucleic acids, poor DNA quality control during the paraffi nization/deparaffi nization processes and irregular distribution of pathogens in the tissue. Contamination during pre-analytical phases may induce false-positive results. On the other hand, application of NAATs using paraffi n-embedded tissue may increase the potential for more stable results. Our group has studied Bartonella sp. infection for some years. These bacteria are neglected and re-emerging
pathogens. The clinical spectrum of human infection has expanded quickly. We have used NAATs not only in bacillary angiomatosis patients for Bartonella sp. diagnosis, but also in fresh and paraffi n-embedded skin fragments as described by the authors. We recently initiated a study of Bartonella sp. coinfections in leprosy patients. We selected 12 lepromatous or borderline-lepromatous patients who were followed up at the University of Campinas Outpatient Center and from whom skin biopsies had been collected for the diagnosis of diseases or relapses. We performed conventional PCR targeting the Bartonella sp. 16S-23S internal transcribed spacer (ITS) region, nested PCR targeting the ftsZ gene (specific for Bartonella henselae) and real-time PCR, targeting the gltA gene (specific for B. henselae), as previously described in deparaffi nized tissue [2, 3]. These samples were obtained before or after multi-drug therapy. Infection with Bartonella sp. was not suspected when the skin samples were obtained. One to seven years after the biopsy procedures, patients were invited to participate in a Bartonella sp. infection survey of leprosy patients (Ethics Committee CAAE: 44670015.4.0000.5404). Of the 12 patients, four showed positivity for Bartonella sp. with PCR; three of these were also positive for Bartonella sp. with blood PCR, suggesting previous infection with Bartonella sp. One patient only showed positivity for Bartonella sp. with PCR in deparaffi nized tissue (Figure 1). All four patients were offered treatment for the potential Bartonella sp. infection, including the fourth patient, since he had recurrent type 2 leprosy reactions and subclinical infection can trigger
Figure 1 Results of Bartonella sp. positive samples from leprosy patients.
© 2018 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd. | JDDG | 1610-0379/2018/1609
1147
Correspondence Clinical Letter
leprosy reactions. Nevertheless, we recognize the possibility of a false-positive result due to pre-analytical contamination. It is known that false-negative PCRs are frequent with Bartonella sp. infections, either in paraffi n-embedded cutaneous fragments or fresh samples, for reasons well discussed by Sunderkötter et al. We have also obtained false-negative results with blood PCR for Bartonella sp. Potential interfering factors were analyzed in depth by Wilson et al. 1997 and Drummond et al. 2018 [4, 5]. Despite these unresolved factors, molecular diagnosis based on paraffi n-embedded cutaneous samples is a potential tool for the diagnosis of Bartonella sp. infection as we previously described [1]. Luciene Silva dos Santos, Marina Rovani Drummond, Andrea Fernandes Eloy da Costa França, Maria Letícia Cintra, Paulo Eduardo Neves Ferreira Velho Bibliography 1
2
3
4
5
Sunderkötter C, Becker K , Kutzner H et al. Molecular diagnosis of skin infections using paraffin-embedded tissue - review and interdisciplinary consensus. J Dtsch Dermatol Ges 2018; 16(2): 139 –47. Silva MN, Vieira-Damiani G, Ericson ME et al. Bartonella henselae transmission by blood transfusion in mice. Transfusion 2016; 56(6 Pt 2): 1556 – 9. Staggemeier R , Pilger DA , Spilki FR , Cantarelli VV. Multiplex SYBR® green-real time PCR (qPCR) assay for the detection and differentiation of Bartonella henselae and Bartonella clarridgeiae in cats. Rev Inst Med Trop Sao Paulo 2014; 56(2): 93 – 5. Drummond MR , Lania BG, Diniz P et al. Improvement of Bartonella henselae DNA Detection in Cat Blood Samples by Combining Molecular and Culture Methods. J Clin Microbiol 2018; 56( 5). Wilson IG. Inhibition and facilitation of nucleic acid amplification. Appl Environ Microbiol 1997; 63(10): 3741 – 51.
Authors’ reply Dear Editors, We thank dos Santos and colleagues for their positive comments on our interdisciplinary consensus regarding molecular diagnosis of skin infections using paraffi n-embedded tissue. We appreciate that they agree with us on the select indications for nucleic acid amplification techniques (NAATs) using paraffi n sections, and on the caveats that need to be taken into consideration during the pre- and post-analytical phase. Their fi ndings on possible subclinical coinfections with Bartonella spp. in patients with borderline or lepromatous leprosy are intriguing. As we have stated in our consensus report (1), NAAT analysis of paraffi n-embedded tissues is useful for confi rmation of a histologically suspected diagnosis of Bartonella infection, because bacillary angiomatosis is characterized by clinical variability and encompasses several differential diagnoses (such as pyogenic granuloma, Kaposi‘s sarcoma, senile angioma, angiokeratoma, angiosarcoma, verruga peruana [Peruvian warts] and mycobacterial furunculosis). Molecular diagnostic methods also help to distinguish between bartonellosis caused by B. henselae (cat-scratch disease) and B. quintana. Finally, as illustrated by the work of dos Santos et al., retrospective NAT analyses of paraffi n-embedded tissues may help to discover Bartonella spp. as co-infecting pathogens in leprosy (or other skin diseases). Cord Sunderkötter, Karsten Becker, Walter Geißdörfer, Christian Bogdan Correspondence to Prof. Dr. med. Cord Sunderkötter Universitätsklinik und Poliklinik für Dermatologie und Venerologie Universitätsklinikum Halle (Saale) Martin-Luther-Universität Halle-Wittenberg Ernst-Grube-Straße 40 06120 Halle/Saale E-mail: [email protected]
Bibliography 1
1148
Sunderkötter C, Becker K , Kutzner H et al. Molecular diagnosis of skin infections using paraffin-embedded tissue - review and interdisciplinary consensus. J Dtsch Dermatol Ges 2018; 16(2): 139 –47.
© 2018 Deutsche Dermatologische Gesellschaft (DDG). Published by John Wiley & Sons Ltd. | JDDG | 1610-0379/2018/1609
