Description of the rare localization of Dirofilaria repens in human in the right inguinal lymph node

Highlights

• •Rare localization of Dirofilaria repens in human.
• •Description of dirofilariasis caused by D. repens in the inguinal lymph node.
• •Difficulties in the diagnosis of D. repens in the inguinal lymph node.

Abstract

D. repens is the causative agent of subcutaneous dirofilariasis in canine animals. In recent years, human invasion by this vector-borne helminthiasis has been recorded in many countries with temperate climate, but the localization of this helminth in the cavities of the human body is extremely rare. Cases of atypical localization of D. repens are described in the organs of the chest, cervical lymph node, in the spermatic cord and epididymis. It is usually requires differential diagnosis with neoplastic or metastatic processes. We report a case of the detection of Dirofilaria repens in the right inguinal lymph node in a 56-year-old woman. Helminth was identified by parasitological and molecular biological methods. A morphological study of cross sections of the nematode in histological preparations and molecular biological method, the polymerase chain reaction (PCR) of scrapings of histological material, were used in parallel.

Introduction

Dirofilaria repens is a causative agent of subcutaneous dirofilariasis in canine animals, more rarely, feline [1,2]. This transmissible helminthiasis is widespread in areas where high level of damage to the final hosts (canine) is registered, and high level of transmission by mosquitoes of the genera Aedes, Culex, Anopheles takes place. Such regions include the countries of southern Europe and the south of the Russian Federation [[3], [4], [5]]. In these territories Dirofilaria repens infection in humans for whom dirofilaria is “inadvertent human pathogens” are recorded [6].

Annually, in the south of Russia, up to 10 cases of autochthonous transmission of dirofilaria are reported during the period of maximum mosquito activity [7], usually from May to October. However, there are frequent cases of transportation of this nematode to the northern regions of the Russian Federation. The clinical feature of human D. repens invasion is the predominant localization of the nematode subcutaneously in the periorbital area, subconjunctival or under the skin of head or neck (up to 70 % of cases) [[8], [9], [10]]. Therefore, the cases of rare localization of this helminth are of significant clinical interest.

The aim of this paper is to describe a rare case of Dirofilaria repens in human in the right inguinal lymphatic node, that caused problem in diagnosis of dirofilariasis.

Case description

On August 27, 2019, patient S., 53 years old, sought medical attention for bulging and pain in the inguinal area in the medical organization in the city of Tyumen in the North of Russia. She had complaints of mild fever 37.8C0, hyperemia and edema of the skin and pain in the right inguinal area. Palpation revealed a painful, mobile lymph node, enlarged up to 1,5−2 cm in diameter. The patient was examined to exclude infectious diseases (HIV, felinosis), neoplastic and autoimmune processes. Also she received supportive treatment (antibiotics, non steroid anti-inflammatory drugs, H1 histamine blockers). During the treatment, the patient's condition slightly improved, however, a right inguinal lymph node remained enlarged. The results of laboratory and instrumental studies allowed to exclude infectious diseases, the neoplastic and autoimmune processes. Ultrasound examination revealed single inguinal heterogeneous lymphatic node, up to 1.5 cm in diameter.

To exclude the cancer «in situ» right inguinal lymphatic node was removed. Histological picture did not conform any types of neoplasma, but fragments of the helminth were found

The study of histological sections discovered two fragments of a cross section of the nematode in the speciments from the center of the lymph node, with a cuticular membrane with surrounding severe chronic purulent inflammation, and the development of granulation tissue (Fig. 1).

Fig. 1.

Histological specimen of the right inguinal lymph node of the patient. ((Description of the histological specimen: in the center of the lymph node there are two transverse sections of dirofilaria, having a cuticular membrane with surrounding pronounced neutrophilic infiltration, macrophage stromal infiltration with chronic purulent inflammation, with an accumulation of neutrophils, giant multinuclear cells such as foreign bodies with the development of granulation tissue. Stained with hematoxylin-eosin. × 100).

The parasite was identified as Dirofilaria repens by the size of the cross-sectional diameters and characteristic comb-shaped outgrowths (Fig. 2).

Fig. 2.

Cross section of a nematode with comb-shaped cuticular outgrowths.

Later, it was established that patient visited Southeast Asia (India) in the past year. To exclude the tropical anthroponous lymphatic filariasis (brugiosis) as well as the invasion of D. immitis we studied the blood by a concentration method to discover microfilaria, but this test was negative.

To identify the species of parasite in histological samples we used polymerase chain reaction (PCR). The material was scraped off with a sterile scalpel from a slide and placed in a sterile 1.5 ml tube. DNA was isolated from histologic sample using a commercial DNA IQ reagent kit (Promega, USA) according to the instructions in the original modification [11]. Using a DNA IQ reagent kit (Promega), highly purified DNA products were obtained.

DNA amplification was carried out by PCR using a GeneAmp PCR System 9700 thermal cycler (Applied Biosystems). To assess the specificity of the amplification reaction, we used positive (a sample with control DNA MK1 — a DNA sample from a person of a known genotype from the COrDISPlus reagent kit (GORDIZ LLC) and negative (a sample without DNA) controls. Electrophoresis was carried out using an ABI PRISM DNA analyzer 3130xl in accordance with the instructions. In the process of electrophoresis, information about laser gel scanning and fluorescence detection was automatically transferred to the control computer and processed by ABI PRISM 3130 DataCollection v. 3.0. The processing of the results and identification of alleles was carried out automatically using the GeneMapper ID program (version 3.2). Alleles corresponding to the standard ones were identified in the control MK1 DNA sample. Alleles were identified using the S450 size standard and the allelic ice probe attached to the COrDISPlus test system. Studies have identified the presence of degraded DNA belonging to a female person.

In the obtained DNA samples, nematodes were identified by enzymatic amplification on a DT-Prime 5 instrument (DNA Technology, Moscow). Original specific primers were selected and synthesized for PCR for the mitochondrial dirofilaria COI gene fragment (they are at the patenting stage). The final concentration of magnesium ions was 2.5 mm. Amplification modes: initial denaturation at 95 °C for 7 min, then 40 cycles, including 95 °C 10 s, 52 °C 15 s, 72 °C 30 s. Morphologically confirmed samples containing D. repens and D. immitis DNA were used as a positive control, and a sample without DNA as a negative control. Amplification products were visualized on a 2% agarose gel (Fig. 3).

Fig. 3.

DNA amplification products of samples.

Species of D. repens helminths were verified by the morphological study of nematode cross section and confirmed by PCR.

Discussion

Localization of D. repens inside the cavities of the human body is extremely rare. Only isolated cases of atypical localization of this nematode are presented. In 2015 A.M. Bronstein et al. [12] described a case of pleuropulmonary

Dirofilariasis caused by D. repens. In 1981, scientists from Israel described a case of D. repens in an enlarged, painless cervical lymph node removed from a 29-year-old patient in Israel [13]. In 1997, D. repens was found in the spermatic cord in a 52-year-old resident of the province of Ravenna in northern Italy [14]. Also in 2013 in Italy and France, 2 cases of autochthonous dirofilariasis with damage to the spermatic cord and epididymis were described, which led to the formation of false tumors [15]. In a 29-year-old man from St. Tropez (Italy), the formation that appeared in the left epididymus progressed in the form of erythema with damage in the both testicles and spread to the inguinal region, accompanied by leukocytosis. And the 66-year-old man, a resident of the Var department (France) had an inflammatory, firm and mobile nodule attached to the spermatic cord. Histological examination in both cases established D. repens invasion. Publications about the rare localizations of this helminth can expand knowledge of the rare localizations of D. repens and reduce the time for the establishment of the correct diagnosis.

Conclusion

This case is of significant interest to physicians of various specialities (surgeons, oncologists, infectious disease specialists and pathologists in primarily in the differential diagnosis of malignant neoplasms of the lymphatic system.

CRediT authorship contribution statement

Larisa Ermakova: Conceptualization, Investigation, Methodology, Writing - original draft. Sergey Nagorny: Investigation, Methodology, Supervision. Igor Kornienko: Investigation, Methodology. Julia Kiosova: Investigation, Methodology. Sergey Todorov: Writing - original draft. Natalia Pshenichnaya: Writing - review & editing, Supervision. Ainash Kuandykova: Writing - review & editing.

Declaration of Competing Interest

None.