No evidence of Borrelia in cutaneous infiltrates of B-cell lymphomas with a highly sensitive, semi-nested real-time polymerase chain reaction targeting the 5S-23S intergenic spacer region.

BACKGROUND: The role of Borrelia in the development of skin lymphomas has been under discussion for decades. A similar association has been shown for Helicobacter pylori and gastric lymphomas (MALT type). Nevertheless, few molecular studies investigated Borrelia in skin lymphomas and the results are controversial. METHODS: We analysed 46 formalin-fixed, paraffin-embedded skin specimens of clincopathologically confirmed B-cell lymphomas (15 marginal zone lymphomas; 20 follicular lymphomas; three diffuse large B-cell lymphomas; eight secondary cutaneous infiltrates) taken from 36 patients from Northern Germany, an endemic area for Borrelia. Fifteen pseudolymphomatous lesions of cutaneous Borreliosis served as the control. Both groups were examined with a real-time (rt) PCR and a semi-nested PCR targeting the 5S-23S intergenic spacer region (IGS). A multiplex PCR was used to investigate B-cell clonality in all lymphomatous infiltrates (Biomed Primers). RESULTS: With both assays no Borrelia burgdorferi-specific DNA was identified in any of the B-cell lymphomas, while all 15 Borreliosis specimens gave a positive PCR result in the semi-nested PCR protocol, 12 were also positive in the rt PCR (P < 0.01). All B-cell lymphomas showed monoclonal IgH-Rearrangement. Analysis of cutaneous B-cell lymphomas from available studies including ours (n = 334) reveals an odds ratio <1. CONCLUSION: While some previous studies suggested an association between B. burgdorferi and the development of cutaneous B-cell lymphomas in endemic areas, we were unable to confirm this in our patients, despite a highly sensitive Borrelia PCR assay. Our results including meta-analysis of previous studies question the need for antibiotic therapy in patients with cutaneous B-cell lymphomas.

Cutaneous pseudolymphoma-A review on the spectrum and a proposal for a new classification.

Cutaneous pseudolymphomas (PSLs) belong to a group of lymphocytic infiltrates that histopathologically and/or clinically simulate lymphomas. Different causative agents (e.g., Borrelia sp., injected substances, tattoo, arthropod bite) have been described, but in many cases no cause can be identified, hence the term idiopathic PSL. Clinicopathological correlation is important to make the diagnosis. Four main groups of cutaneous PSL can be distinguished based on histopathologic and/or clinical presentation: (a) nodular PSL; (b) pseudo-mycosis fungoides (pseudo-MF) and simulators of other CTCLs; (c) other PSL (representing distinct clinical entities); and (d) intravascular PSL. This article gives an overview of the histopathologic and clinical characteristics of cutaneous PSLs and proposes a new classification.

Annular Lichenoid Dermatitis (of Youth) Immunohistochemical and Serological Evidence for Another Clinical Presentation of Borrelia Infection in Patients of Western Austria.

Annular lichenoid dermatitis of youth (ALDY) is a more recently described inflammatory disease of the skin of unknown etiology with clinical similarities to morphea. The authors clinically, histopathologically, and immunohistochemically investigated 14 biopsies from 12 patients in western Austria with this disease. There were 6 female and 6 male patients with solitary (n = 7) and multiple lesions (n = 5) affecting the trunk (n = 11), upper arm (n = 2), thigh (n = 1), and calf (n = 1). Clinically, early lesions were erythematous in nature leading to central paleness, scaling, wrinkling, dermal atrophy, slight pigmentation, and telangiectasia later on. Histopathologically, all specimens showed the typical features of ALDY with a superficial lichenoid process with sprinkling of lymphocytes along the basal cell layer and within the epidermis accompanied by mild fibrosis. Pigment incontinence, superficial fibrosis, and dilatation of superficial capillary vessels are prominent features in more advanced stages of disease. Immunohistologically, using a polyclonal antibody against Borrelia, 11/14 specimens revealed spirochetes, either vital (n = 4) or degenerated (n = 7), in close proximity to collagen bundles. Thirteen of 14 specimens in addition showed focal (n = 4) or clustered (n = 9) positivity for CD20 in the papillary dermis. Nine of 12 sera tested for Borrelia with an enzyme-linked immunosorbent assay were positive. Lichen sclerosus et atrophicus and morphea have previously been reported to be possibly related to Borrelia infection. We postulate that a similar relationship to Borrelia infection may be true for ALDY implying that ALDY may be an early superficial stage of morphea.

Histopathology and immunophenotype of acrodermatitis chronica atrophicans correlated with ospA and ospC genotypes of Borrelia species.

BACKGROUND: Chronic cutaneous borreliosis (acrodermatitis chronica atrophicans, ACA) is a relatively rare manifestation of borreliosis attributed mainly to Borrelia afzelii. Chronic borreliosis has been associated with ospA and ospC genotypes. Literature on molecular investigations of Borrelia in lesions of ACA is scant. METHODS: Histopathological and immmunohistochemical features in 22 biopsies of ACA (16 patients) were examined. Paraffin-embedded biopsies were analyzed with polymerase chain reaction (PCR) assays targeting ospA and ospC genes, sequencing and phylogenetic analysis. RESULTS: Genotyping of ospA identified B. afzelii, serotype 2, in 12 of 16 patients. ospC-PCR was positive in seven patients revealing genotypes Af5 (n = 4), Af2 (n = 2) and Af6 (n = 1). Histopathologically, interstitial granulomatous infiltrates (CD68 positive) were common, combined with thickened collagen bundles and band-like infiltrates of CD4 positive T lymphocytes. Plasma cells were sparse/absent in 9 of 22 specimens even on staining with CD138. On CD34-staining, interstitial fibroblasts were often reduced akin to the situation in morphea. CONCLUSIONS: With assays targeting ospA and ospC genes we confirmed from paraffin-embedded biopsies that B. afzelii, serotype 2, osp C groups Af5, Af2 and Af6 is the main cause of ACA. Specimens commonly showed a combination of band-like T-cell-rich infiltrates with interstitial granulomatous features, a pattern previously under-recognized in ACA. This finding was particularly typical for lesions infected with ospC genotype Af5.

Electrochemotherapy as a novel treatment for primary cutaneous marginal zone B-cell lymphomas.

In the present study, we describe the use of electrochemotherapy as alternative therapy for primary cutaneous marginal zone B-cell lymphomas in patients unsuitable for surgery or radiotherapy. Our experience refers to three patients with primary cutaneous marginal zone B-cell lymphomas related to Borrelia burgdorferi infection, treated with specific antimicrobial therapy and electrochemotherapy.

Primary cutaneous marginal zone lymphoma associated with juxta-articular fibrotic nodules in a teenager.

Primary cutaneous marginal zone lymphoma (PCMZL) has rarely been reported in teenagers and is occasionally associated with Borrelia burgdorferi infection. Juxta-articular fibrotic nodules represent a unique, localized fibrosing response to spirochete infections, namely Borreliosis. Herein, we report a 15-year-old healthy boy who presented with a 4-year history of progressive acquisition of asymptomatic, erythematous nodules, ≤ 3 cm, beginning with his right forearm (3), then right arm (1) and lastly his right inner thigh (1). Biopsy showed PCMZL in three of five samples, and inflamed, fibrotic nodules, near the elbow in two. The bottom heavy lymphomatous nodules consisted of mostly small CD20+ CD43+ lymphocytes, some with plasmacytoid features. Mature plasma cells were lambda light chain restricted by in situ hybridization. The juxta-articular fibrotic nodules were located in the deep dermis and subcutis, had peripheral plasma cell-rich infiltrates, and showed nodular sclerosis (morphea profunda-like) in one, and lamellar and angiocentric sclerosis in the other reminiscent of quiescent lesions of chronic localized fibrosing leukocytoclastic vasculitis. Immunohistochemistry for B. burgdorferi revealed rare positive organisms; however, polymerase chain reaction (PCR) and serology were negative for B. burgdorferi as were serologic and/or PCR assays for Bartonella henselae, Ba. quintana, Ehrlichia chaffeensis, Treponema pallidum, Helicobacter pylori and Babesia microti. No evidence of extracutaneous disease was found by the review of systems and imaging studies. A 4-week trial of doxycycline therapy failed, whereas intralesional (IL) corticosteroid therapy induced rapid regression of his nodules. After two local recurrences, also treated with IL corticosteroids, he is well, without cutaneous disease, 20 months later. A literature review of 19 pediatric cases PCMZL reveals a similar natural history as adult PCMZL. Despite negative serology and PCR for B. burgdorferi, the occurrence of ipsilateral juxta-articular fibrotic nodules, positive B. burgdorferi immunohistochemistry and rapid response to IL corticosteroids implicate the presence of a replicative or non-replicative infectious (spirochetal) antigen in the initiation and promotion of this teenager's PCMZL.

Acrodermatitis chronica atrophicans with pseudolymphomatous infiltrates.

In this study, we describe the clinicopathologic features of pseudolymphomatous infiltrates found within lesions of acrodermatitis chronica atrophicans (ACA). We studied 11 patients (10 females, 1 male, age range 60-88 years). The diagnosis of ACA in all cases was confirmed by clinicopathologic correlation and positive serology for Borrelia. Histopathologic examination revealed prominent, pseudolymphomatous inflammatory cell infiltrates in all cases, with 2 distinct patterns. Eight of 11 cases showed a band-like lymphocytic infiltrate, exocytosis of lymphocytes and a fibrotic papillary dermis, similar to features seen in mycosis fungoides. The other 3 cases showed dense, nodular-diffuse dermal infiltrates with many plasma cells and without germinal centers. The plasma cells expressed both kappa and lambda immunoglobulin light chains with a polyclonal pattern in all 3 cases. In conclusion, ACA may present with pseudolymphomatous infiltrates showing both a T-cell and, less frequently, a B-cell pattern. These lesions need to be distinguished from a cutaneous lymphoma. In the context of the knowledge of Borrelia-associated cutaneous lymphomas, follow-up seems advisable in these cases.

The coenzyme A disulphide reductase of Borrelia burgdorferi is important for rapid growth throughout the enzootic cycle and essential for infection of the mammalian host.

In a microarray analysis of the RpoS regulon in mammalian host-adapted Borrelia burgdorferi, bb0728 (cdr) was found to be dually transcribed by the sigma factors σ(70) and RpoS. The cdr gene encodes a coenzyme A disulphide reductase (CoADR) that reduces CoA-disulphides to CoA in an NADH-dependent manner. Based on the abundance of CoA in B. burgdorferi and the biochemistry of the enzyme, CoADR has been proposed to play a role in the spirochaete's response to reactive oxygen species. To better understand the physiologic function(s) of BbCoADR, we generated a B. burgdorferi mutant in which the cdr gene was disrupted. RT-PCR and 5'-RACE analysis revealed that cdr and bb0729 are co-transcribed from a single transcriptional start site upstream of the bb0729 coding sequence; a shuttle vector containing the bb0729-cdr operon and upstream promoter element was used to complement the cdr mutant. Although the mutant was no more sensitive to hydrogen peroxide than its parent, it did exhibit increased sensitivity to high concentrations of t-butyl-hydroperoxide, an oxidizing compound that damages spirochetal membranes. Characterization of the mutant during standard (15% oxygen, 6% CO(2)) and anaerobic (< 1% O(2) , 9-13% CO(2)) cultivation at 37°C revealed a growth defect under both conditions that was particularly striking during anaerobiosis. The mutant was avirulent by needle inoculation and showed decreased survival in feeding nymphs, but displayed no survival defect in unfed flat nymphs. Based on these results, we propose that BbCoADR is necessary to maintain optimal redox ratios for CoA/CoA-disulphide and NAD(+) /NADH during periods of rapid replication throughout the enzootic cycle, to support thiol-disulphide homeostasis, and to indirectly protect the spirochaete against peroxide-mediated membrane damage; one or more of these functions are essential for infection of the mammalian host by B. burgdorferi.

A case of Borrelia-associated cutaneous pseudolymphoma in a horse.

This case report describes a 10-year-old horse that developed multiple dermal papules over the right masseter area following removal of a tick from the same site 3 months earlier. Histological examination of a biopsy from a papule was suggestive of either a T-cell-rich B-cell lymphoma or cutaneous lymphoid hyperplasia, a form of pseudolymphoma sometimes associated with a tick bite. Positive serological testing and PCR of the biopsy sample for Borrelia in conjunction with immunohistochemical testing of the skin biopsy, the clinical history and response to treatment with doxycycline strongly supported the diagnosis of Borrelia-associated cutaneous pseudolymphoma.

Prevalence of Borrelia burgdorferi infection in a series of 98 primary cutaneous lymphomas.

Borrelia burgdorferi has been variably associated with different forms of primary cutaneous lymphoma. Differences in prevalence rates among reported studies could be a result of geographic variability or heterogeneity in the molecular approaches that have been employed. In the present study, we investigated the prevalence of Borrelia burgdorferi sensu lato DNA in diagnostic tissue samples from fresh cutaneous biopsies of 98 primary cutaneous lymphomas and 19 normal skin controls. Three different polymerase chain reaction (PCR) protocols targeting the hbb, flagellin, and Osp-A genes were used. Direct sequencing of both sense and antisense strands of purified PCR products confirmed the specificity of the amplified fragments. Sequence specificity was assessed using the Basic Local Alignment Search Tool, and MultAlin software was used to investigate the heterogeneity of target gene sequences across the different samples. Borrelia DNA was not detected in 19 controls, 23 cases of follicular lymphoma, 31 cases of extranodal marginal zone lymphoma, or 30 cases of mycosis fungoides. A single case of 14 diffuse large B-cell lymphoma cases was positive for B. burgdorferi. This study does not support a pathogenic role of B. burgdorferi in primary cutaneous B- and T-cell lymphomas from areas nonendemic for this microorganism and the consequent rationale for the adoption of antibiotic therapy in these patients.

Testicular damage by microcirculatory disruption and colonization of an immune-privileged site during Borrelia crocidurae infection.

The agent of African relapsing fever, Borrelia crocidurae, causes reversible multiple organ damage. We hypothesize that this damage is caused when the spirochete forms aggregate with erythrocytes in vivo, creating rosettes that plug the microcirculatory system. To test this hypothesis, we compared testicular microcirculation over an extended time period in two groups of rats: one experimentally inoculated with B. crocidurae, the other with the nonerythrocyte rosette-forming Borrelia hermsii. In the B. crocidurae group, erythrocyte rosettes formed during spiro-chetemia blocked precapillary blood vessels and reduced the normal pattern of microcirculatory blood flow. After spirochetemia, erythrocyte rosettes disappeared and flow was normalized. Decreased blood flow and focal vascular damage with increased permeability and interstitial bleeding adjacent to the erythrocyte microemboli induced cell death in seminiferous tubules. Interestingly, we found that B. crocidurae could penetrate the tubules and remain in the testis long after the end of spirochetemia, suggesting that the testis can serve as a reservoir for this bacteria in subsequent relapses. The group infected with B. hermsii displayed normal testicular blood flow and vasomotion at all selected time points, and suffered no testicular damage. These results confirmed our hypothesis that the erythrocyte rosettes produce vascular obstruction and are the main cause of histopathology seen in model animal and human infections.

IL-10 helps control pathogen load during high-level bacteremia.

During relapsing fever borreliosis, a high pathogen load in the blood occurs at times of peak bacteremia. Specific IgM Abs are responsible for spirochetal clearance so in absence of B cells there is persistent high-level bacteremia. Previously, we showed that B cell-deficient mice persistently infected with Borrelia turicatae produce high levels of IL-10 and that exogenous IL-10 reduces bacteremia. This suggested that IL-10 helps reduce bacteremia at times of high pathogen load by a B cell-independent mechanism, most likely involving innate immunity. To investigate this possibility, we compared B. turicatae infection in RAG2/IL-10(-/-) and RAG2(-/-) mice. The results showed that IL-10 deficiency resulted in significantly higher bacteremia, higher TNF levels, and early mortality. Examination of the spleen and peripheral blood showed markedly increased apoptosis of immune cells in infected RAG2/IL-10(-/-) mice. Neutralization of TNF reduced apoptosis of leukocytes and splenocytes, increased production of IFN-gamma by NK cells, increased phagocytosis in the spleen, decreased spirochetemia, and rescued mice from early death. Our results indicate that at times of high pathogen load, as during peak bacteremia in relapsing fever borreliosis, IL-10 protects innate immune cells from apoptosis via inhibition of TNF resulting in improved pathogen control.

Borrelia in granuloma annulare, morphea and lichen sclerosus: a PCR-based study and review of the literature.

BACKGROUND: Morphea, granuloma annulare (GA) and lichen sclerosus et atrophicans (LSA) have also been suggested to be linked to Borrelia infection. Previous studies based on serologic data or detection of Borrelia by immunohistochemistry and polymerase chain reaction (PCR) reported contradictory results. Thus, we examined skin biopsies of morphea, GA and LSA by PCR to assess the prevalence of Borrelia DNA in an endemic area and to compare our results with data in the literature. METHODS: Amplification of DNA sequences of Borrelia burgdorferi sensu lato by nested PCR from formalin-fixed and paraffin-embedded skin biopsies of morphea, GA and LSA, followed by automated sequencing of amplification products. PCR-based studies on Borrelia species in these disorders published until July 2009 were retrieved by a literature search. RESULTS: Borrelia DNA was detected in 3 of 112 skin biopsies (2.7%) including one of 49 morphea biopsies (2.0%), one of 48 GA biopsies (2.1%) and one of 15 LSA biopsies (6.6%). Amplification products belonged to B. burgdorferi sensu stricto in two cases available for sequence analysis. CONCLUSIONS: The results of our and most of other PCR-based studies do not argue for a significant association of B. burgdorferi sensu lato with morphea, GA, LSA.

[Cutaneous marginal zone lymphoma (SALT) and infection with Borrelia burgdorferi]. / Kutanes Marginalzonenlymphom (SALT) bei chronischer Borrelia-burgdorferi-Infektion.

A relationship between Borrelia burgdorferi and the development of cutaneous B-cell lymphoma (CBCL) has been long discussed. B. burgdorferi DNA has been detected in patients with CBCL and a response of CBCL to antibiotics has been observed. In our patient with a Borrelia infection, a marginal zone lymphoma (SALT) regressed after ceftriaxone therapy. This further case of a combined appearance of CBCL and B. burgdorferi underlines a possible relationship as an example of an infectious trigger in tumorigenesis.

New concepts on erythema annulare centrifugum: a clinical reaction pattern that does not represent a specific clinicopathological entity.

BACKGROUND: Erythema annulare centrifugum (EAC) is considered an inflammatory skin disease with unknown aetiology. In most textbooks it is assigned to the incoherent conglomeration of figurate or gyrate erythemas. OBJECTIVES: To re-evaluate a large cohort of patients with EAC and to assess the evidence for infection with Borrelia. METHODS: We retrospectively investigated 90 cases with the diagnosis of EAC. Haematoxylin and eosin sections were re-examined and diagnoses were specified; these were then confirmed by clinicopathological correlation. Infection with Borrelia was assessed by focus-floating microscopy and by a Borrelia-specific polymerase chain reaction (PCR). RESULTS: Besides a miscellaneous group of annular disorders at times confused with EAC such as urticaria, leucocytoclastic vasculitis and psoriasis (20 of 90; 22%), EAC appeared to serve as a collective term for three main clinicopathological reaction patterns: (i) (tumid) lupus erythematosus (29 of 90; 32%), (ii) spongiotic dermatitides (25 of 90; 28%) and (iii) pseudolymphoma (16 of 90; 18%). In 13 of 16 (81%) cases with a pseudolymphomatous reaction pattern spirochaetes stained positive but were negative in other reaction patterns of EAC as well as in negative controls. These findings were confirmed by a Borrelia-specific PCR which was positive in two of three (67%) of these pseudolymphomatous EAC cases but was negative in all other variants of EAC (none of five) as well as 20 controls. CONCLUSIONS: We conclude that 'EAC' is a clinical reaction pattern that does not represent a specific clinicopathological entity and should lead to consideration of mainly lupus erythematosus, dermatitis and, in some cases, cutaneous Lyme disease.