The first reported case of Blaptica dubia cockroach allergy

Background Periplaneta americana and Blattella germanica cockroaches are widespread, and risk of sensitization increases in urban environments where these roaches thrive as household pests. There are no prior reports of Blaptica dubia cockroach allergy, though human exposure to B. dubia is increasing through commercial breeding as feeder insects. Case presentation A 50-year-old B. dubia cockroach breeder presented with progressively worsening upper and lower respiratory symptoms in recent years. Symptoms were worse with exposure to her B. dubia roach colony. Skin prick testing (SPT) to B. dubia cast skin, internal organs, and feces was performed in both the subject and a human control. Testing for P. americana and B. germanica sensitization was also performed in the subject. SDS–Polyacrylamide gel electrophoresis (PAGE), immunoblots, and enzyme-linked immunosorbent assays (ELISA) studies were performed using the subject and control serums to explore for specific IgE binding to B. dubia as well as P. americana. Our results showed SPT was positive to B. dubia internal organs in the subject and negative in the control. In the subject, SPT was negative to P. americana though intradermal (ID) testing was positive and serum specific IgE (sIgE) testing was negative to B. germanica. Immunoblotting of the subject's serum to B. dubia internal organ extract showed several distinct bands of IgE binding at 47 kilodaltons (kD), 68 kD, 74 kD, 83 kD, and 118 kD. The strongest band was at 118 kD on B. dubia immunoblotting, which was absent in P. americana on SDS-PAGE. ELISA studies showed an increased IgE response to both B. dubia and P. americana in the subject versus the control. Conclusions This case confirmed the first reported allergy to B. dubia cockroaches. There may be cross-reactivity between B. dubia and P. americana, though our case suggests SPT and sIgE testing using P. americana and B. germanica extract has potential to miss a B. dubia cockroach allergy. This allergy is likely underreported, and further study is needed to explore the natural history of B. dubia cockroach allergy.


Background
Cockroaches are widely abundant insects worldwide, though only a small fraction of the approximately 4500 species in existence are adapted to human habitats [1].
Of the domiciliated cockroach species, the American Cockroach (Periplaneta americana) and German Cockroach (Blattella germanica) are the most pervasive pests [1,2]. Exposure to both P. americana and B. germanica have demonstrated potential to evoke IgEmediated allergic sensitization in humans [2,3]. It is estimated that 15-60% of atopic individuals are sensitized to one of these cockroaches, and the risk of sensitization increases significantly among atopic

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Allergy, Asthma & Clinical Immunology individuals with heightened cockroach exposure as may occur in urban dewllings [2,[4][5][6][7] Among sensitized individuals, P. americana or B. germanica cockroach exposure may exacerbate symptoms associated with both allergic rhinitis and asthma [4,8,9]. To date, 12 allergens have been associated with B. germanica and 13 with P. americana as listed on the World Health Organization and International Union of Immunological Societies (WHO/IUIS) Allergen Nomenclature Database [3]. While not confirmed on the WHO/IUIS database, other domiciliated cockroach species throughout the world and are hypothesized to have varying degrees of cross-reactivity to B. germanica and P. americana [1].
The Orange-Spotted Cockroach/Argentinian Wood Roach (Blaptica dubia) is a tropical cockroach species that is not endemic to human dwellings, though human exposure to B. dubia is increasing with their widespread commercial breeding as feeder insects [3,10]. Feeder insects are bred with the purpose of being used as food for other animals, typically reptiles. While no reports of B. dubia allergy exist in medical literature, websites for B. dubia breeders have described the potential to develop allergic symptoms with B. dubia exposure [11,12]. In this report, we present the first confirmed case of B. dubia allergy in a Dubia cockroach breeder.

Case presentation
The subject was a 50-year-old female who presented with progressively worsening symptoms of dyspnea, wheezing, cough, and nasal congestion. She had been breeding B. dubia cockroaches in her home for commercial sale to pet shops for the past several years. Initially, she had no symptoms with exposure to the B. dubia cockroaches, though recently she had noticed marked worsening of her upper and lower airway symptoms with exposure to her B. dubia roach colony. She reported that once a B. dubia cockroach ran across her arm and she broke out in an urticarial rash where it had touched her skin. She did not reside in an inner city dwelling nor was there evidence of a cockroach infestation in her home.

Skin testing
Skin testing was performed to multiple aeroallergens including P. americana using commercial extract (GreerLaboratories GB26A03) and to non-standardized B. dubia cockroach cast skin, internal organs, and feces particles. Each B. dubia sample for skin prick testing (SPT) was mixed with saline and SPT conducted in both the subject and a non-cockroach allergic atopic human control (Table 1). Serum specific IgE (sIgE) testing was performed to B. germanica in the subject as this extract was not available at the time of skin testing.

Cockroach protein extraction for in vitro studies
Cockroach protein extracts were made in lab for in vitro studies. Briefly, 2.5 g of material isolated from B. dubia cast skins, internal organs, and P. americana whole body extract (WBE) were separately incubated with 25 ml of extraction buffer for 18 h at 4 ℃. Samples were

Enzyme-linked immunosorbent assay (ELISA)
The subject's serum and a non-atopic human control's serum were added to ELISA plate wells coated with cockroach protein extracts and incubated overnight at room temperature. These plate wells had been coated with 100 µg of the different cockroach protein extracts in 100µl of carbonate coating buffer and then incubated overnight at 4 °C (set up in triplicates) and then washed. This was blocked with 200 µL 3% bovine serum albumin (BSA)/tris-buffered saline (TBS) for 1 h at 37 °C and then removed. Samples were then added to ELISA plate wells, incubated overnight at room temperature, then washed. 100 µl of monoclonal mouse IgG to human immunoglobulin E (anti-IgE) (1:300 dilution in 3% BSA/ TBS) conjugated to alkaline phosphatase was then added followed by 100 µL of p-Nitrophenyl Phosphate (pNPP) alkaline phosphatase substrate. An average of triplicate optical densities (OD) were obtained. OD ratios (subject/ negative control) were used to determine the subject's "IgE response" to various cockroach protein extracts.

Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and immunoblot
The SDS-PAGE ran for 1.5 h at 130 V with 50 µg, 25 µg, and 12.5 µg of each denatured protein extract (B. dubia cast skins, B. dubia internal organs, and P. americana WBE) as shown in Fig. 1. A marker/ladder was also loaded, as well as a denature buffer only for blank wells. The gel was stained with Coomassie Blue R250, de-stained, and then photographed and the concentration with the best resolution of protein bands was chosen for downstream immunoblot.
The immunoblot was performed to B. dubia internal organ protein extract. 25 µg of B. dubia internal organ denatured protein extract was loaded in three separate lanes of gel (Fig. 2). The gel ran for 1.5 h at 130 V and then transferred onto a 0.45 micron nitrocellulose membrane for 2 h at 36 V. The membrane was removed and cut into strips, placed into reaction troughs, and washed. Strips were blocked for 1 hour at room temperature with 5% non-fat dry milk (NFDM) then aspirated. 0.3 ml of each sample in 2.7 ml of 5% NFDM were added to reaction troughs, incubated overnight at room temperature, aspirated, and then washed. 3 ml of a 1:250 monoclonal, mouse immunoglobulin-G (IgG) to anti-human immunoglobulin-E (IgE) (AP conjugated) in 5% NFDM was then added and incubated for 4 h at room temperature then washed. 3ml of development buffer was added to each trough and observed for color development, then washed with 5ml of deionized H2O for 10 minutes with one change of water at 5 min (Fig. 2).

Results
SPT and intradermal (ID) skin testing results are shown in Table 1. Results were notable for a negative SPT in the subject to P. americana though ID testing was positive. B. dubia SPT was positive to internal organs in the subject and negative in the control (  Fig. 1. Immunoblotting of the subject's serum with B. dubia internal organ extract detected five bands of IgE binding at 47 kD, 68 kD, 74 kD, 83 kD, and a strong band at 118 kD (Fig. 2). Positive and negative control reagents performed as expected (Fig. 2). The 118 kD denatured protein was present in B. dubia cast skin and internal organ extracts but not P. americana WBE on SDS-PAGE (Fig. 1). ELISA of the subject's serum versus B. dubia internal organs, cast skin, and P. americana WBE found IgE OD detected at a ratio of 7.2×, 8.6×, and 6.8× greater than the negative control, respectively.

Discussion and conclusions
Cockroaches produce potent allergens, though the vast majority of human exposure to cockroaches are to domiciliated house pests such as P. americana and B. germanica. This case represents the first confirmed B. dubia cockroach allergy in a cockroach breeder with evidence of IgE-mediated sensitization to multiple B. dubia allergens. This sensitization almost certainly contributed to the subject's allergic rhinitis and asthma that developed through her occupational exposure to B. dubia.
Intradermal and in vitro studies suggest the subject was sensitized to P. americana but not B. germanica cockroach. The false negative SPT we observed to commercial P. americana extract was likely related to the variability of protein concentrations within commercially available cockroach extracts [13]. We suspect the subject also had false negative SPT to B. dubia cast skin, as in-vitro studies suggested the subject was sensitized to B. dubia cast skin and her clinical history of contact urticaria when a roach ran across her arm also suggest sensitization to external cockroach allergens. We hypothesize this probable false negative SPT to B. dubia cast skin likely occurred because the saline mixed with cast skin for SPT did not elicit significant amount of protein. This was a limitation of our investigation, as were unable to utilize the B. dubia cockroach extracts made in lab for the subject's SPT. The subject's IgE sensitization to P. americana may be due to unbeknownst prior exposure and sensitization to this cockroach species. Is also possible that cross-reactivity between B. dubia and P. americana resulted in positive P. americana testing. It is noteworthy that the most robust IgE binding on immunoblot to B. dubia internal organ extract occurred at the high molecular weight 118 kD allergen. By comparison, this 118 kD protein was absent in P. americana WBE on SDS-PAGE. These findings may suggest that at least one of the several B. dubia allergens identified were unrelated to potential cross-reactivity with P. americana cockroach.

Conclusion
We confirmed the first known allergy to B. dubia cockroaches with evidence of IgE sensitization to B. dubia and symptoms of allergic rhinitis, asthma, and contact urticaria with B. dubia exposure. This allergy