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Skinmed. Author manuscript; available in PMC 2016 March 11. Published in final edited form as: Skinmed. ; 13(5): 345–346.

Western Blotting of Human Sera—Can It Help Diagnose Bed Bug Bites? Jerome Goddard, PhD1, Amanda C. Tardo, MS2, and Monica E. Embers, PhD2 1Department

of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, Mississippi State, MS

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2Division

of Bacteriology and Parasitology, Tulane National Primate Research Center, Tulane University, Covington, LA Reports of bed bug (Cimex lectularius) infestations and bite reactions have dramatically increased over the past decade and should be included in the differential diagnosis in cases of unexplained insect bites.1 Proteins in bed bug saliva are readily recognized by the human immune system, leading to various humoral and cellular responses, including production of cytokines/chemokines.2

IMPORTANCE OF INSECT BITE LESION IDENTIFICATION

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Cutaneous reactions to bed bug bites were once considered little more than a nuisance; however, we recently showed that bed bugs may sometimes cause severe cutaneous vasculitis similar to Churg-Strauss syndrome.3 Even though the sialome of bed bugs has been published,4 there are almost no studies attempting to link particular salivary proteins to corresponding cutaneous reactions. In addition, lawsuits may be filed against commercial or rental properties for alleged negligence or failure to follow a “standard of care or best management practice” for bed bug prevention or control.5 In such litigation, plaintiffs often claim that they have been bitten by bed bugs even if no bugs were ever seen or collected. Physicians and other healthcare providers may sometimes get involved in these cases and be asked to testify as to whether bites and eruptions are caused by bed bugs.

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The problem is, no one can accurately determine whether a particular insect bite lesion is caused by bed bugs, or any other arthropod for that matter. Predaceous insects may bite defensively, leading to immediate pain; however, blood-feeding insects usually produce painless, almost imperceptible bites, which do not become apparent until hours or days later, when the patient's own sensitivity may or may not lead to itching and other manifestations. In the case of bed bugs, as many as half of people bitten develop no reaction at all,6 while the others may develop a range of reactions from itchy macules (or maculopapules) to large urticarial wheals, indurations, or bullous lesions.7 There may be bite patterns that are suggestive of particular arthropods. For example, chigger or flea bites are often multiple, occurring on the ankles or legs; spider or tick bites are often singular, occurring on the arms

Address for Correspondence: Jerome Goddard, PhD, Department of Biochemistry, Molecular Biology, Entomology and Plant Pathology, Mississippi State University, 100 Twelve Lane, Clay Lyle Entomology Building, Mississippi State, MS 39762 [email protected].

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or legs; and black flies often bite in great numbers around the head or at the posterior cervical region at the base of the hair. Bed bugs are anecdotally reported to bite in a line or in groups of three, but only one or a few bites are possible in light infestations. If, for example, a patient has a few bed bug bites on the ankle, how would a healthcare provider know if they are from a bed bug, chigger, or any other arthropod? They all look the same (Figure 1). From a histopathologic perspective, there is nothing pathognomonic about these lesions, and most fall into the generic diagnosis of “probable arthropod assault.” Furthermore, these lesions can be confounded by scratching and resulting excoriation of the skin surface. Considering the widespread variability in bite reactions and biting patterns of arthropods, healthcare providers might/should—at least at this time—avoid proclaiming mysterious pruritic lesions as “bed bug bites.”

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DEVELOPING A TEST FOR BED BUG BITES

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New information is needed to help differentiate bed bug bite reactions from other reactions, especially those with an allergic component. In a recent study in our laboratory, we showed that Western blot assays (WBAs) might help identify patients with sensitivity to bed bug bites. In the experiment, Western blotting was conducted twice with the same six human sera, all obtained via university institutional review board study #12-343, including two patients with known bed bug bite reactivity, two frequently bitten with no bite reactivity, and two controls (persons never bitten by bed bugs). For antigen, salivary glands of 100-200 C lectularius were dissected and removed to produce a crude extract as previously reported.8 The enzyme-linked immunosorbent assays were performed in 96-Well Costar high-binding EIA plates (Sigma-Aldrich, St. Louis, MO). In the first run, 17 μg of salivary gland extract (SGE) was added to each well and sera diluted 1:100. In the second run, 30 μg of SGE was used and sera diluted 1:50. Blots were developed with goat anti-human IgG (H+L) at 1:1000 dilution.

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The best sensitivity in our WBAs was obtained when we used 30 μg of SGE antigen (run #2) (Figure 2). All samples were run on the same gel with the same SGE preparation, therefore differences in size were likely legitimate. Both WBA runs revealed the same bands in approximately the same places for patients 3 and 6, whereas immunoreactive bands at ~35 kD and ~75 kD were seen in patient 1. All of these three patients had repeated exposure to bed bug biting, but only patients 1 and 3 routinely develop cutaneous reactions when bitten. Interestingly, patient 6, who is frequently bitten without reaction, showed the same ~37 kD band. Patient 1, who had the most striking cutaneous reactions, exhibited reactivity to two antigens. The 75 kD band in patient 1 seems unique among the others and could perhaps be diagnostic for persons with intense cutaneous reactions. No bands were observed in the two sera from persons never bitten (controls, patients 4 and 5).

CONCLUSIONS This work provides the first results of western blot profiles of people with varying degrees of cutaneous reactions to bed bug bites and reveals possible unique immunoreactive bands for further analysis. If this work can be cost-effectively confirmed by other laboratories, it

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could be a valuable tool for clinical diagnosis as well as for cases in the legal profession (lawsuits). Follow-up studies in our laboratory are underway to: (1) identify antigenic targets, and (2) develop a sensitive and specific enzyme-linked immunosorbent assay for detection of bed bug bite responses. Soon, perhaps, new diagnostic tests will help demystify arthropod bite reactions.

ACKNOWLEDGMENTS Mark Feldlaufer (USDA, Beltsville, MD), Kristine Edwards (Mississippi State University, Starkville, MS), Andrea Varela-Stokes (MSU, Starkville, MS), and Mr Frank Meek (Orkin Inc, Atlanta, GA), provided clinical samples for this study. Dr Harold Harlan (Crownsville, MD) provided the live bed bugs. This contribution has been approved for publication as a journal article No. J-12523 of the Mississippi Agriculture and Forestry Experiment Station, Mississippi State University.

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1. Goddard J, de Shazo RD. Bed bugs (Cimex lectularius) and clinical consequences of their bites. JAMA. 2009; 301:1358–1366. [PubMed: 19336711] 2. Goddard J, Hasenkampf N, Edwards KT, de Shazo R, Embers ME. Bed bug saliva causes release of monocytic inflammatory mediators: plausible cause of cutaneous bite reactions. Int Arch Allergy Immunol. 2013; 161:127–130. [PubMed: 23343710] 3. de Shazo RD, Feldlaufer MF, Mihm MC, Goddard J. Bullous reactions to bed bug bites reflect cutaneous vasculitis. Am J Med. 2012; 125:688–694. [PubMed: 22560811] 4. Francischetti IM, Calvo E, Andersen JF, et al. Insight into the sialome of the bed bug, Cimex lectularius. J Proteome Res. 2010; 9:3820–3831. [PubMed: 20441151] 5. USATODAY. Woman sues Catskills resort for $20M over bedbug attacks: USA Today online. Mar 8. 2006 http://www.usatoday.com/travel/hotels/2006-03-08-bedbugs-catskills_x.htm 6. Goddard J, de Shazo RD. Multiple feeding by the common bed bug, Cimex lectularius, without sensitization. Midsouth Entomol. 2009; 2:90–92. 7. Reinhardt K, Kempke RA, Naylor RA, Siva-Jothy MT. Sensitivity to bites by the bedbug, Cimex lectularius. Med Vet Entomol. 2009; 23:163–166. [PubMed: 19292820] 8. Goddard J, Edwards KT. Effects of bed bug saliva on human skin. JAMA Dermatol. 2013; 149:372–373. [PubMed: 23552994]

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Known bed bug bites (left) and known chigger bites (right). (Photos courtesy of Kristine T. Edwards and Audrey Sheridan, both at Mississippi State University, Starkville, MS.)

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Figure 2.

Western blot analysis of human sera using bed bug salivary gland proteins. Patients 1 and 3 displayed cutaneous reactions when bitten by bed bugs, patients 2 and 6 have been bitten frequently but never show skin reactions, and patients 4 and 5 have never knowingly been bitten by bed bugs.

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Western Blotting of Human Sera-Can It Help Diagnose Bed Bug Bites?

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