Focus Floating Microscopy: "Gold Standard" for Cutaneous Borreliosis?

Klaus Eisendle, MD, PhD; Tanja Grabner, MD; Bernhard Zelger, MD, MSc

Disclosures

Am J Clin Pathol. 2007;127(2):213-222. 

In This Article

Abstract and Introduction

Borrelia burgdorferi is difficult to detect in routine biopsy material from patients with skin lesions of borreliosis. In this study, a new immunohistochemical method, focus floating microscopy (FFM), was developed to detect B burgdorferi in tissue sections and was compared with polymerase chain reaction (PCR). By using standard histologic equipment, tissue sections stained with a polyclonal B burgdorferi antibody were simultaneously scanned through 2 planes: horizontally in serpentines and vertically by focusing through the thickness of the section.

Borrelia were detected in 47 of 71 ticks, 34 of 66 tick bites, 30 of 32 erythema chronicum migrans cases, 41 of 43 borrelial lymphocytomas, and 50 of 51 acrodermatitis chronica atrophicans cases. FFM proved to be more sensitive than PCR (96.0% vs 45.2%) and nearly equally specific (99.4% vs 100%). All 169 control cases, except 1 false-positive case of secondary syphilis, were negative with FFM. FFM is an easy, quick, and inexpensive method to reliably detect Borrelia in cutaneous tissue sections.

The spirochetal origin of borreliosis (Lyme disease) was first demonstrated by Burgdorfer et al[1] in 1982. The microorganisms (Borrelia burgdorferi) are transfected by ticks, in Europe usually Ixodes ricinus, and cause characteristic disease. Similar to syphilis, borreliosis has been separated into 3 stages. In stage 1, erythema chronicum migrans (ECM) (Figure 1) and borrelial lymphocytoma (BL) (Figure 2) appear weeks after the tick bite; in stage 2, borrelial arthritis, lymphocytic meningoradiculoneuritis (Bannwarth syndrome), and borrelial carditis appear after months; and in stage 3, acrodermatitis chronica atrophicans (ACA) (Figure 3) appears after years.[2,3,4] Stages may overlap or be skipped. Many other tissues such as muscle, soft tissues, and internal organs may be involved, and unusual clinical manifestations may be seen.[4,5]

A, Characteristic clinical manifestations of erythema chronicum migrans. B, Histologic examination (H&E, ×20) reveals superficial and middermal dense perivascular infiltrate of (C) lymphocytes, some plasma cells, and an increase of fibroblasts and mucin between collagen bundles (H&E, ×200).

A, Borrelial lymphocytoma with characteristic histologic features of dense infiltrates of lymphocytes (B, H&E, ×10) with follicle formation (C, H&E, ×200).

A, Acrodermatitis chronica atrophicans of left leg characterized by ill-defined, hyperpigmented, and atrophic patch (note prominent veins). B, Histologic examination (H&E, ×10) reveals a dense lichenoid and middermal perivascular infiltrate with hints of follicle formation (C, H&E, ×100) composed of lymphocytes, some plasma cells, and an increase of fibroblasts between fibrosclerotic collagen bundles (D, H&E, ×200).

Histologic examination of borrelial infection reveals an infiltrate of lymphocytes, macrophages, plasma cells, and fibroblasts with variable fibrosclerosis.[2] After initial enthusiasm,[6] detection of microorganisms has turned out to be difficult, frequently unreliable, and almost always extremely time-consuming by different procedures, including histochemical stains (Gram, Wright, Wright-Giemsa, and polychromes), fluorochromes (thioflavine-T, acridine orange, and rhodamine), silver impregnation techniques (Warthin-Starry, modified Dieterle, modified microwave-Dieterle, and Bosma-Steiner) in the 1980s,[7,8,9,10,11] and immunohistochemical analysis in the 1990s.[7,8,12,13,14] In our opinion and that of others, there is need for a reliable and easy method of direct detection of Borrelia in tissue.[12] To our knowledge, no company supplies positive control sections, making assessment of methods difficult.

Serologic techniques (immunofluorescence, enzyme-linked immunosorbent assay [ELISA], and immunoblot) are similarly unsatisfying, with false-negative (20%-80%) and false-positive results occasionally due to cross-reactions with Treponema pallidum or, more commonly, to a positive endemic background of 20% to 50% in many parts of Europe.[8,15,16] Cultures with specified media such as modified Pettenkofer-Kelly or Barbour-Stoenner-Kelly can detect Borrelia in all clinical forms, but these techniques are not generally available and are unreliable, with less than 50% sensitivity.[8,17] Molecular techniques initially seemed to solve the riddle,[18,19,20] but in due course, it became clear that sensitivity varies (30%-90%) according to the Borrelia strains, the material (fresh frozen tissue or paraffin material), and the applied primers.[8,21,22,23,24] Borreliosis remains a diagnosis based on circumstantial evidence combining clinicopathologic and laboratory information and clinical response to therapy.

To the best of our knowledge, this article describes for the first time a new immunohistochemical detection method that is highly sensitive and reliable, works on routinely formalin-fixed, paraffin-embedded tissue samples, and, according to performance, is called focus floating microscopy (FFM).

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