Histopathological diagnosis of Japanese spotted fever using formalin-fixed, paraffin-embedded skin biopsy specimens

Clinical specimens

In the period 2004–2010, we collected specimens from 50 patients clinically suspected of having JSF. The clinics and hospitals supplying samples included Mahara Clinic (Anan, Tokushima, n = 17), Yamada Red Cross Hospital (Ise, Mie, n = 13), Myojin Clinic (Kozagawa, Wakayama, n = 9), Uwajima Municipal Hospital (Uwajima, Ehime, n = 7), Hyogo Prefectural Awaji Hospital (Sumoto, Hyogo, n = 1), Shinano Hospital (Tomi, Nagano, n = 1), Yasu Hospital (Yasu, Shiga, n = 1) and Notogawa Hospital (Higashiomi, Shiga, n = 1).

Biopsy samples were taken from 49 cases, and autopsy material from one. The skin samples (n = 61) included eschar (n = 42), eruptions (n = 12) and scabs (n = 7). All but the autopsied tissues, fixed in formalin, were sent to our department within 24–72 h. Normal skin sampled at autopsy served as a negative control. For serological assays, sera were sent to Ohara Research Laboratory, Fukushima, or Prefectural Institutes of Public Health. In total, 89 sera (acute 46 and convalescent 43) were analysed serologically, as described earlier [12]. The cut-off value was set at <×40 for both IgM and IgG. The Rj Aoki strain was used as the antigen. Rickettsia was isolated in limited cases in Ohara Research Laboratory, as reported previously [12].

Cultivation of rickettsial strains and preparation of cell blocks

Rickettsiae (Aoki and Katayama strains of Rj and Kato, Karp and Gilliam strains of Ot) were sent from Ohara Research Laboratory. All strains were passed in cultured L929 cells (fibroblast-like cells of a C3H/An mouse) at a biosafety level 3 containment laboratory in the Department of Microbiology, Wakayama Medical University, Wakayama.

The cells were grown at 32°C in 25 cm² plastic cell culture flasks containing Dulbecco’s modified Eagle’s minimal essential medium (Nissui, Tokyo, Japan) supplemented with 5% fetal calf serum (Hyclone, Logan, UT, USA). The cells harvested 5 to 7 days after inoculation were fixed in 10% formalin overnight. Cell blocks of uninfected and infected L929 cells were prepared by a gelation method using sodium alginate membranes [13]. Cell blocks were also prepared from R. conorii (Malish strain)-infected monkey Vero cells (Fuller Laboratories, Fullerton, CA, USA) fixed in 10% formalin.

Monoclonal antibodies and IHC

Mouse IgM monoclonal antibodies, clones S3 and X1, directed to the Rj Aoki strain were a gift from Dr Yosaburo Oikawa, Department of Parasitology, Kanazawa Medical University, Kanazawa. Both clones react with the epitope common to SFG rickettsiae, but do not cross-react with Ot [14,15].

Sections, 4 μm thick, were prepared from cell blocks and tissue specimens. After inactivating endogenous peroxidase with 0.3% H₂O₂ in methanol for 20 min, sections were heat-retrieved in 10 mM citrate buffer, pH 7.0 for 10 min with a pressure cooker. Incubation with the monoclonals (dilution: 1:100) at room temperature overnight and then amino acid polymers (Simple Stain MAX-PO, Nichirei, Tokyo, Japan) for 30 min at room temperature followed [10,11]. Antigen localization was visualized in 50 mM Tris–HCl buffer, pH 7.6 containing 1 mM 3,3′-diaminobenzidine and 0.006% H₂O₂. Nuclei were counterstained with haematoxylin.

DNA preparation

Five 4-μm-thick FFPE sections were collected in Eppendorf’s tubes. After deparaffinization, DNA was extracted using a QIAamp DNA FFPE Tissue kit (#56404; Qiagen, Hilden, Germany). At sample processing, microtome blades were renewed to prevent sample-to-sample contamination.

Real-time PCR

The 17k genus common antigen gene of SFG rickettsia origin was amplified by RT-PCR, according to the previous reports [9,16,17]. Primer pairs for Rj consisted of 5′-ATG AAT AAA CAA GGT ACA GGA ACA-3′ (forward: 24mer) and 5′-AAG TAA TGC ACC TAC ACC TAC TC-3′ (reverse: 23mer), generating products of 114 bp length (GenBank D16515). Both primers were 100% homologous to R. conorii (GenBank M28480) and R. rickettsii (M28479), while three bases (forward) and one base (reverse) were mismatched with R. typhi (M28481) and R. prowazekii (M28482). Signals were detected with a TaqMan^® hybridization probe FAM-GGT GGC GCA TTA CTT GGT TCT CAA TTC GGT AAG GG-TAMRA for Rj (Applied Biosystems, Foster City, CA, USA). The number of bases mismatched with the TaqMan^® probe (35mer) was one base for R. conorii, two bases for R. rickettsii, three bases for R. prowazekii and four bases for R. typhi.

Assays were carried out in 20 μL final volume containing 1.5–3.0 μL of sample DNA, 2× reaction mixture (10 μL, Premix Ex Taq™; TaKaRa Bio, Otsu, Shiga, Japan), 10 pmol primers, and 10 pmol TaqMan^® probe. RT-PCR was performed using the DNA Engine Opticom^® System (Bio-Rad, Berkeley, CA, USA), with initial holding temperature of 95°C for 30 s, followed by 50 cycles with two-step PCR at 95°C for 5 s and at 60°C for 30 s with fluorescence monitoring on 6-carboxy fluorescein aminohexyl amidite (FAM) channel.

β2-microglobulin (β2m) served as an internal control for effective DNA extraction [18]. Primers designed with Primer3 software (SourceForge, Mountain View, CA, USA) consisted of 5′-TGC TGT CTC CAT GTT TGA TGT ATC T-3′ (forward) and 5′-TCT CTG CTT CCC CAC CTC TAA GT-3′ (reverse) for human/monkey β2m (GenBank NM_004048), and 5′-CAG TGT GAG CCA GGA TAT AG-3′ (forward) and 5′-GAA GCC GAA CAT ACT GAA CTG CTA C-3′ (reverse) for mouse β2m (GenBank NM_009735). The product sizes were 86 bp for human/monkey and 152 bp for mouse.

Sequencing analysis

For sequencing, the SYBR Green method (Qiagen) using the same primer pairs was employed. RT-PCR was performed using Rotor-Gene Q (Qiagen) according to the QIAGEN SYBR-Green PCR Handbook (2009), with initial holding temperature of 95°C for 15 min, followed by 45 cycles with four-step PCR at 95°C for 20 s, at 55°C for 30 s, at 72°C for 30 s and at 57°C for 15 s. The melting curve was checked in the respective reactions. DNA from FFPE sections of L929 cells infected with Rj (Katayama strain), Vero cells infected with R. conorii and skin biopsies from five cases (eschar: A4, A10, A14 and B1, and scab: B8) were examined. When the plateau was not obtained in the amplification curve in the first run, the second PCR was performed by adding, as a template, 1.5 μL of 1:1000 diluted PCRed aliquot to reaction mixture. After electrophoresis in 1% Agarose gel, the amplified products were extracted with the QIAquick gel extraction kit (Qiagen). Direct sequencing analysis with the dye terminator method [19] was performed in FASMAC Co. (Atsugi, Kanagawa, Japan). The comparison was done using the BLAST program (National Center for Biotechnology Information, Bethesda, MD, USA).

Clinical features of JSF

Table 1 summarizes 31 seropositive cases and one autopsy case (group A) and nine seronegative cases but with positivity of the SFG rickettsial antigen and/or genome (group B). All group B cases exhibited clinical features of JSF, response to antibiotic therapy, and positivities of IHC and/or RT-PCR. Case distribution in groups A and B (n = 41) is shown on Japan’s map (Fig. 1). The male to female ratio was 23:18. The mean age was 62.0 years (range, 28–87). Infection occurred during April–December with a peak in September (n = 10). Serologically, Rj antibodies of IgM and/or IgG types got elevated in group A, except for case A32 who died acutely. Rj was isolated from two cases (A1 and A6) [12].

Nine (18%) of 50 cases were judged as non-JSF disorders (group C, detailed data not shown), based on clinical follow-up features, negative serology and negative IHC/real-time PCR findings in 11 biopsies (eschar 8, eruptions 2 and scab 1). Final clinical diagnoses included tick bite fever of unknown nature (n = 4), Tsutsugamushi disease (Irie/Kawasaki type), streptococcosis, pneumonia, herpes and allergy (n = 1, respectively).

IHC and RT-PCR

Fifty skin biopsy specimens (eschar 34, eruptions 10 and scabs 6) were obtained from group A (n = 38) and group B (n = 12). Rickettsial antigens were immunolocalized as coarse dots in the cytoplasm of endothelial cells and macrophages in the lesions. Both monoclonals consistently gave comparable results. Representative IHC features are demonstrated in Fig. 2. Autopsy tissues (spleen, liver, intestine, salivary gland, kidney and testis) showed positivity of the rickettsial antigens and DNA [11].

Positivity rates of IHC and RT-PCR were 94% (32/34) and 62% (21/34) for eschar, 80% (8/10) and 30% (3/10) for eruptions, and 33% (2/6) and 50% (3/6) for scabs. For IHC, eschar was most suitable, while scabs were insufficient. For scab samples, RT-PCR functioned better than IHC.

Unexpectedly, 18 biopsies sampled in Mahara Clinic happened to be fixed in 100% formalin, and this evidently lowered the detection rate of RT-PCR, whereas IHC was tolerant of such harsh conditions. In samples fixed in 10% formalin, RT-PCR gave positive results in 73% (16/22) for eschar and in 60% (3/5) for eruptions, while for the samples fixed in 100% formalin, the rates were 42% (5/12) for eschar and 0% (0/5) for eruptions (Table 2).

Both IHC and RT-PCR were concordantly positive in 51% (26/51) of 50 skin biopsies plus one autopsy sample. IHC was positive but RT-PCR negative in 33% (17/51). Two (4%) scab samples were IHC negative and RT-PCR positive. In six (12%), both methods gave negative results. On a patient basis, only three (7%) of 41 cases in groups A and B were negative in both methods, and in two of them, only tiny scabs were submitted (cases A27 and A28). In 26 (84%) of 31 seropositive cases, the diagnosis of JSF was made earlier by IHC and/or RT-PCR than by serology.

Cell blocks of Rj-infected L929 cells served as positive controls for both IHC and RT-PCR. Positive signals were also obtained from R. conorii-infected monkey Vero cells with both methods. No positivity of IHC and RT-PCR was seen in uninfected or Ot-infected cells and in normal skin. Human/monkey or mouse β2m DNA was consistently amplified from all DNA samples. Cycle threshold values varied from sample to sample (Tables 1 and 2). The mean cycle values were 26.4 for Rj-infected L929 cells, 36.8 for regularly fixed eschar (n = 15; median, 35.6; range, 30.5–48.0), and 42.6 (n = 3; range, 40.5–44.7) for regularly fixed eruptions and scabs. Eschar fixed in 100% formalin required 39.4 cycles (n = 4; range, 37.1–41.3).

Sequencing analysis

An exactly homologous sequence of the 114 bp stretch in the Rj 17k genus common antigen gene was confirmed in the positive control cells and five skin biopsy specimens. As expected (GenBank M28480), the DNA sequence of R. conorii showed three bases difference in the amplified fragment.